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Iontophoretic delivery of selected antiparkinsonian agents in vitro Tomasz Giller

A thesis submitted for the degree of Doctor of Philosophy

University of Bath

Department of Education

March 2009

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Attention is drawn to the fact that copyright of this thesis rests with its author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognize that its

copyright rests with the author and they must not copy it or use material from it except as permitted by law or with the consent of the author.

This thesis may be made available for consultation within the University Library and May be photocopied or lent to other libraries for the purposes of consultation

I would like to dedicate this thesis

To my parents and my brother

For their constant support

Acknowledgements

The author would like to acknowledge The Parkinson’s Disease Society of the United Kingdom for funding.

Contents

1

Contents Abstract .......................................................................................................... 2

List of abbreviations .................................................................................. 3

Objectives and organization of the thesis ........................................... 5

Introduction ................................................................................................. 7

Management of Parkinson’s disease ....................................................... 10

Dopamine agonists ................................................................................... 12

The challenges in treatment of PD ........................................................... 13

Transdermal formulations in PD ............................................................... 14

Iontophoresis ............................................................................................ 18

Iontophoretic drug delivery in PD ............................................................. 22

Iontophoretic target flux assessment and drug selection for

iontophoretic delivery .............................................................................. 26

Bibliography .............................................................................................. 28

Chapter I – Selegiline ............................................................................... 43

Chapter II – Pramipexole ........................................................................ 65

Chapter III – Piribedil .............................................................................. 92

Chapter IV – Pergolide ........................................................................... 112

Chapter V – Trihexyphenidyl ............................................................... 125

Chapter VI – Entacapone ....................................................................... 144

Chapter VII – Human Skin .................................................................... 158

General Conclusions ............................................................................... 176

Appendix I ................................................................................................. 188

Appendix II ................................................................................................ 203

Appendix III .............................................................................................. 215

Appendix IV .............................................................................................. 222

Appendix V ................................................................................................ 227

Appendix VI .............................................................................................. 237

Abstract

2

Abstract Pharmacological treatment of Parkinson’s disease involves frequent dose

adjustment, complex dose regimes. Also, oral antiparkinsonian drugs suffer from the

first pass effect, and a variable absorption in the gastrointestinal tract. The

transdermal route is an advantageous alternative, as shown by the recent

commercialization of a passive patch containing rotigotine (Neupro®). In this work,

transdermal iontophoretic delivery of six drugs: pramipexole (PMP), piribedil (PIR),

selegiline (SEL), trihexyphenidyl (THP), entacapone (ETC) and pergolide (PER) was

performed, using side-by-side diffusion cells. Dermatomed pig skin, and human full-

thickness skin were employed as membrane models. Samples of receptor solution

were analyzed for the drug and electro-osmotic marker content, via HPLC and LC-

MS. The influence of formulation parameters on the transdermal drug flux was

studied. Namely, the effects of: donor solution pH; donor drug concentration in both,

single-ion and co-ion situation; current intensity, drug ionization; and electroosmosis

were investigated. Also, the water mobility of ionized drugs and their octanol-water

distribution coefficient was measured. Iontophoresis significantly enhanced the

transport of all the drugs, with respect to passive diffusion. Iontophoretic fluxes were

proportional to the intensity of the current applied for all the substances examined.

This confirms that iontophoresis could allow easy dose individualization. Single-ion

fluxes were independent of the drug molar concentration for all the drugs. Drug fluxes

dropped markedly with the pH; this was probably due to the increased competition

offered by the very mobile hydrogen ions (H+), and reduced skin cation

permselectivity. Iontophoretic fluxes usually decreased as competing co-ions were

introduced in the donor. However, two distinct behaviours were observed: for one

group of drugs fluxes and transport numbers were linearly proportional to mole

fraction; while for the other group, only the initial raise in mole fraction resulted in

increasing flux. Iontophoretic fluxes took longer to stabilize across full thickness

human skin, and were lower than across dermatome pig skin. Briefly, the best

candidates for iontophoretic delivery were pramipexole, selegiline, and piribedil.

Trihexyphenidyl, entacapone and pergolide are poor candidates and probably would

require patches of impractical size.

Abstract

3

List of abbreviations 5FU – 5-fluorouracil

AcN – acetonitrile

ALAAD – aromatic L-amino acid decarboxylase inhibitor

ANOVA – analysis of variance

AUC – area under the curve

AV – average

C12EO3 – laureth-3 oxyethylene ether

C12EO7 – laureth-7 oxyethylene ether

CALM-PD – comparison of the the agonist pramipexole vs. levodopa on motor

complications in Parkinson’s Disease

CDS – continuous dopaminergic stimulation

Cl – clearance

COMT – catechol-O-methyl-tranferase

Css – steady-state concentration

D – dose

DA – dopaminergic agonist

DATATOP – Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism

DP – degree of permselectivity

EM – electromigration

EMEA – European Medicine Agency

EO – electroosmosis

ETC – entacapone

FDA – Food and Drug Administration

HDS – human dermatomed skin

HSC – human stratum corneum

IF – inhibition factor

iP – isoelectric point

LID – lidocaine

LRRK2 – leucine rich repeat kinase 2

MAO-B – mono amino oxidase type B

MeOH – methanol

Abstract

4

MPTP - 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

NDMA – N-methyl-D-aspartate

PAR – paracetamol

PD – Parkinson’s disease

PER – Pergolide

PINK1 – PTEN induced putative kinase 1

PIR – piribedil

PPX – pramipexole

PRP – propranolol

QIN – quinine

ROP – ropinirole

RSD – relative standard deviation

SC – subcutaneous

SD – standard deviation

SEL – selegiline

SNc – substantia nigra pars compacta

t½ – half life

TD – transdermal

THF – tetrahydrofuran

THP – trihexyphenidyl

UK‐PDRG – Parkinson’s Disease Research Group-United Kingdom

UPRDS – unified Parkinson’s disease rating scale

Vd – volume of distribution

Objectives

5

Objectives and organization of the thesis Aims of the thesis

The investigations described in this thesis focus on the transdermal

iontophoretic delivery of six selected antiparkinsonian drugs. Namely, iontophoretic

experiments with pramipexole, piribedil, pergolide, selegiline, entacapone and

trihexyphenidyl were carried out. The aims of these experiments were, specifically:

1. To verify the feasibility of the iontophoretic transdermal delivery of

abovementioned antiparkinsonian agents.

2. To investigate the possibility of delivering therapeutic doses of these drugs.

3. To investigate the role of formulation and current intensity on transdermal

transport of these drugs.

4. To look into the impact of several physicochemical properties (ie Log P, water

mobility) of selected drugs, on the transdermal transport thereof.

5. To study the role of mechanisms governing iontophoretic transdermal

transport.

Organization of the thesis

The thesis is organised in nine sections followed by six appendices. The first

section, Introduction, describes shortly Parkinson’s disease (PD), current therapeutic

strategies and emerging problems with PD treatment. Also, the Introduction depicts

iontophoresis as a technique of drug delivery, and reviews the transdermal therapies

applied in PD. Finally, it provides the basis for selection of drugs for subsequent

transdermal experiments.

In the following six chapters (I-VI), iontophoretic experiments on selected

drugs are systematically described and discussed, one drug in each chapter. Every

section devoted to one drug contains a brief description of drugs clinical,

physicochemical and pharmacological properties; materials and methods used in

experimental work; results, discussion and conclusions. Although the experimental

techniques used in experiments were similar for all the drugs, the materials and

Objectives

6

methods section was written separately for every chapter. This was done because

the experimental conditions varied from drug to drug, and also to facilitate the

reading of the thesis. Following chapter (VII) describes the experiments with human

skin and compares them to the corresponding ones, carried out with pig skin. Finally,

the General conclusions section summarizes and discusses the entire thesis. In this

part, the thesis conclusions and future perspectives are presented.

The appendices contain the raw data obtained in the course of experimental

work. The data are organised in sections of tables, with the experimental conditions

and investigated effects indicated.

Introduction

7

Introduction Parkinson’s disease (PD) was first characterized by James Parkinson in 1817

(1). In his report on “shaking palsy”, he described the cardinal features of the

disease: resting tremor, instability of posture and gait, bradykinesia, which were

completed forty years later by adding muscular rigidity to give a more compelling

clinical picture (2). In the 20th century it was discovered that PD is a

neurodegenerative progressive disorder, resulting from a neuronal atrophy of

substantia nigra, pars compacta (SNc), combined with formation of intraneuronal

inclusions called Lewy bodies (3). This entails an impaired dopaminergic innervation

of basal ganglia, and produces all the specific clinical symptoms (Table 1). This led

to levodopa employment as a therapeutic for PD (4).

Currently, the prevalence of the Parkinson’s disease varies from 0.08% to

0.8% in general population, depending on the classification criteria and population

studied; in the population over the age of 65, 3% is affected worldwide (5). That

makes PD the second most frequent neurodegenerative disorder, after Alzheimer’s

disease. Most of the cases, around 90%, are the idiopathic PD, and the remaining

10% accounts mainly for familial forms of PD (6). The incidence of PD is slightly

higher in men than in women (5). In early onset of Parkinson’s disease (between 25

and 39 years of age) the mean life expectancy is 38±5 years, compared to 49±5 in

healthy population; In the late onset of the disease (between 40 and 64 years of

age), the mean life expectancy is of 21 years, in comparison to 31 years; For

patients of 65 years and older the mean life expectancy is 5 years, compared to 9 for

healthy subjects (7).

Despite an early description of the disease, the aetiology of PD still remains

unrevealed. Up to this date, several factors implicated in development of the disease

have been identified. The oxidative stress was the first pathogen regarded as

contributing to neurodegeneration of dopamine cells. This hypothesis was supported

by the ability of dopamine to create reactive oxygen species, such as: O2-, H2O2,

hydroxyl radicals, or reactive quinones (8). Also, this evidence was backed up by a

post mortem examination of brains of PD patients. In SNc of PD patients levels of

glutathione were significantly reduced, and high levels of iron (an oxidant causing

Introduction

8

hydroxyl radicals formation) were detected (9). Other than the oxidative stress, a

mitochondrial dysfunction is mentioned as another factor implicated in PD (10; 11).

Histological findings revealed that in SNc of patients affected by the disease, there is

a defect in the respiratory-chain complex I, decreasing its activity by roughly 35%.

This was further confirmed by the studies that discovered, that substances blocking

mitochondrial complex I induce Parkinson-like syndrome in both, humans and

animals (12; 13). Also, mutations in several genes related to mitochondria are now

appreciated to play a major role in PD. Mutations in PINK1 gene, encoding

mitochondrial kinase were found to be responsible for a familial form of early-onset

parkinsonism (14). Moreover, the mutations in LRRK2, a protein bound to

mitochondrion membrane, are now regarded as the most common cause of familial

form of PD (15). Nonetheless, the malfunctioning mitochondrial complex I was found

only in around 40% of sporadic PD patients (no PD in family history), suggesting the

coexistence of other pathogenic mechanisms (9; 16).

Another mechanism involved in PD is inflammation (17). The presence of

activated microglial cells (the main type of active immune-defence cells in the central

nervous system, CNS), and high levels of inflammatory-associated factors, was

demonstrated in cerebrospinal fluid and striatum of parkinsonian patients (18). It was

also demonstrated in tissue cultures and animal models, that the agents inducing

production of inflammation-mediating cytokines, can induce a dopamine cell death.

In the same time, the anti-inflammatory agents have neuroprotective properties in 1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) PD model (19). Finally, there is

strong evidence that the malfunctioning of the protein-degradation process plays a

key role at the basis of PD, particularly in familial forms (9). The ubiquitin-

proteasome system serves as the major mean of degradation of intracellular

unwanted proteins. The excessive production of faulty proteins or their impeded

degradation process, or both of them, can result in the proteolytic stress (20). This in

turn, can result in accumulation and aggregation of abnormal proteins, and further in

mutilation of various cellular functions and apoptosis. Mutations in the parkin gene

have been correlated with juvenile onset of recessive Parkinson’s disease (21). The

parkin protein is an ubiquitin ligase, whose main function is to attach ubiquitin to

proteins and target them for degradation. Indeed, an augmented expression of the

parkin protein has the abilities of preventing apoptosis, induced by the

Introduction

9

overexpression of its substrate proteins in vitro (22). Furthermore, a protein

commonly found in Lewy bodies, α-synuclein, is susceptible to misfold, and to

aggregate. It has been reported, that its mutant forms have been associated with the

familial form of PD (23).

Although a variety of processes engaged in PD development have been

discovered, it remains doubtful that any of those mechanisms have a direct effect on

dopaminergic cell death. In fact, all of the four processes described above can

induce one another. The oxidative stress could damage mitochondria and

proteasomes (24), and reversely, the mitochondrial fault can lead to oxidative stress,

inflammation, and proteasomal damage (9; 25). Likewise, inhibition of proteasomal

function can provoke inflammatory reaction (26), and mitochondrial dysfunction (27).

While none of them is crucial, it is possible, that only the coexistence and synergy of

these processes, could cause the disease.

Table 1 Main symptoms of Parkinson’s disease.

Motor symptoms Non-motor symptoms

Resting tremor

Rigidity

Bradykinesia

Gait disturbance

Postural instability

Autonomic dysfunction

Cognitive impairment

Depression

Dementia

Dysphagia

Dysarthria

Olfactory dysfunction

Pain and sensory disturbances

Sleep disturbances

Sialorrhea

Introduction

10

Management of Parkinson’s disease

As the cause of PD still remains elusive, current treatment strategies are

mainly symptomatic. Nonetheless, there has been a major interest in neuroprotective

therapies, aiming to stop or slow down the degeneration of SNc. After promising

results were obtained in vitro and in animal models, several substances have been

put into the clinical trials. Among them are the antiapoptotic agents, like rasagiline or

TCH346 (28; 29), and promitochondrial substances, like coenzyme Q10 and creatine

(30; 31). Unfortunately, none of these drugs was found to have positive effect on PD

symptoms. Also, two dopamine agonists, pramipexole and ropinirole, have been

tested in clinical trials for their neuroprotective potential (32; 33). Both studies have

demonstrated that the tested drugs can slow down the disease progression; however

the methodology applied in these studies remains under debate (34; 35).

The treatment of Parkinson’s disease can be divided into two general

categories: pharmacological treatment and surgical interventions. The latter are

based on the strategy to suppress the pathological neural activity in basal ganglia

circuits. This is achieved by lesioning implicated brain structures or by their constant

electrical stimulation by means of implanted electrodes (deep brain stimulation) (36;

37). The targets of these interventions are: globus pallidus, thalamus, subthalamic

nucleus. However, lesion interventions often lead to high incidence of serious side

effects (speech impairment, cognitive problems) in comparison to the deep brain

stimulation, while being similarly effective. Also, the electrode implantation is

adjustable and reversible, thus it became the procedure of choice. In terms of the

effectiveness, these procedures are particularly effective in treatment of tremor,

while less effective in treatment of akinesia, rigidity and posture instability (38).

Surgical treatment is mostly applied for patients, who continue to be disabled

regardless of the best available pharmacological therapy.

Today, the pharmacological treatment is the first choice treatment for PD. The

therapeutic strategy is based on reestablishment of balance in dopaminergic and

cholinergic neurotransmission in basal ganglia, disrupted by the degeneration of SNc

(39), and maintaining constant and sufficient dopamine receptors stimulation (40).

There are three main therapeutical groups involved: levodopa and drugs acting on

Introduction

11

levodopa/dopamine metabolism, dopamine agonists, and to lesser extent,

anticholinergics.

Dopamine depletion in striatum lays at the basis of motor features of PD.

However, dopamine itself cannot be employed in treatment, as it is unable to cross

the blood-brain barrier. In contrast, levodopa, dopamine’s metabolic precursor, is

actively transported to cerebrospinal fluid. The introduction of levodopa to PD

treatment ameliorated the quality of life, and reduced the mortality in comparison to

pre-levodopa era, in virtually all patients (41). It is by far the most effective drug on

the market and sooner or later, its employment in treatment is unavoidable (42).

There is vast clinical evidence that levodopa is very efficient drug in treatment of

parkinsonian symptoms (42). Nowadays it is appreciated as a golden standard in PD

treatment.

Nonetheless, levodopa treatment is also associated with various problems.

After an initial period of good clinical response to the drug (honeymoon), the use of

levodopa results in the development of side effects, such as motor fluctuations and

dyskinesias. In fact, after five years of treatment, 50% of patients develop these

highly disabling side effects (43). For the young onset of the disease, this proportion

is as high as 90%. It is now well established, that the cause of development of motor

side effects is the replacement of physiological constant dopamine-tone, by pulsatile

levels of dopamine in the cerebrospinal fluid (44; 45; 46; 40). The latter is the effect

of levodopa therapy. Many strategies have been developed to improve the levodopa

clinical effect. The drug is now routinely administered with aromatic-L-amino-acid-

decarboxylase (ALAAD) inhibitors, like carbidopa (Sinemet®) or benserazide

(Madopar®), to prevent the peripheral conversion of the drug to dopamine. The

usage of ALAAD inhibitors results in longer half-life of levodopa, and allows a higher

proportion of the dose to reach the cerebrospinal fluid (47). Also, addition of ALAAD

inhibitor provides better control of parkinsonian symptoms, than levodopa alone. To

further potentiate this effect, inhibitors of another enzyme metabolizing levodopa,

catechol-O-methyl transferase (COMT), have been introduced to PD treatment.

Entacapone in combination with levodopa and ALAAD inhibitor (Stalevo®) was

shown to provide even greater clinical benefit, without increasing the development of

motor complications (48).

Introduction

12

In another attempt to reach more constant dopaminergic tone, two

slow-release levodopa formulations (Madopar HBS® and Sinemet SR®) were

introduced to the treatment. They resulted in similar changes in levodopa

pharmacokinetics: levodopa tmax was 2-3h longer; Cmax and variability in levodopa

plasma levels were significantly reduced, in comparison to immediate release

formulations (49; 50; 51). The five-year trial showed however, that there was no

difference in dyskinesia incidence, and only a small improvement in the control of

parkinsonian symptoms, between the slow-release and immediate-release

formulations (52).

Intravenous levodopa infusions resulted in stable drug concentrations in

plasma, and virtual disappearance of motor side effects in patients experiencing

severe motor fluctuations (53). Furthermore, it has been postulated that continuous

levodopa infusion not only improves motor complications, but also widens the

levodopa therapeutic window (54). Similar effects were obtained with duodenal

infusions of the drug (55; 56). However, both of these administration techniques

require hospital care and are normally reserved for patients with severe motor

fluctuations (57). Nonetheless, the observation, that by continuous dopaminergic

stimulation (CDS) it is possible to stop, or even reverse the development of

dyskinesias, has given the basis to the new treatment paradigm in Parkinson’s

disease (58).

Dopamine agonists

Dopamine agonists (DA) directly stimulate dopamine receptors. This group is

composed of two main categories: the ergot derivatives (cabergoline, pergolide,

bromocriptine, lisuride) and the non-ergot compounds (piribedil, pramipexole,

rotigotine, ropinirole, and apomorphine). DAs offer several advantages over

levodopa. Firstly, all of dopamine agonists, except of apomorphine, have longer half-

life than levodopa (Table 3), thus they provide more constant dopaminergic

stimulation, than this dopamine precursor (59). Secondly, they act directly on

dopaminergic receptors, independently of the degenerating dopaminergic neurons.

Finally, in contrast to levodopa, DAs do not undergo an oxidative metabolism, what

is a putative pathomechanism of PD. As a result, it is unlikely, that they contribute to

the disease progression.

Introduction

13

The clinical effectiveness of DAs has been investigated in many controlled

clinical trials (60). When compared to levodopa as a monotherapy, they generally

provide worse control of parkinsonian symptoms, however DAs do not cause as high

incidence of dyskinesias and motor fluctuations (61; 62). To postpone the

development of the motor side effects, DA monotherapy has become a common

strategy at the beginning of treatment of PD, particularly in its early onset. Also,

several clinical trials demonstrated that DAs in combination with levodopa provide a

levodopa sparing effect (63; 64; 65). The latter has recently been attributed to

neuroprotective action of dopamine agonists. Two clinical studies have sought for

neuroprotective properties in DAs. In CALM-PD trial (66) it was demonstrated, that

over four-year period pramipexole reduced the rate of loss of dopamine transporter

by roughly 40% in comparison to levodopa. Similar effects were obtained for

ropinirole in REAL study, with 34% reduction (33). The studies were however

criticised for the applied methodology (34; 67).

Nevertheless, the use of dopamine agonists is not free of problems. Some of

them are subject of extensive first pass metabolism, which decreases their

bioavailability (Table 3). Ergot derived DAs were reported to cause pleural fibrosis

and fibrotic valvular heart disease (68; 69); sudden irresistible sleeping attacks have

been reported with ropinirole and pramipexole (70); commonly reported side effects

were also: hallucinations, psychosis, daytime sleepiness, and confusion (2).

The issue, whether the de novo therapy should be initiated with levodopa or

dopamine agonist has lately been widely discussed (71). Recent data shows

however, that the DAs provide enough control over parkinsonian symptoms, while

the progress of the disease is slowed down in comparison to levodopa therapy. It

has also been postulated, that the neuroprotective properties ascribed to DAs, might

be in fact a result of more constant dopaminergic tone provided by these drugs (34).

The challenges in treatment of PD

The biggest goal in PD therapy is finding an effective neuroprotective factor,

possible to significantly slow down or stop the progress of the disease. Levodopa still

remains the most effective drug for symptomatic treatment of PD. Nonetheless, its

chronic administration is associated with severe limitations, mainly due to developing

Introduction

14

motor complications. Continuous IV delivery of levodopa offers good clinical

outcome, however up to date is restricted to hospital treatment. There is a clear

requirement for “patient-friendly” levodopa formulation, able to deliver the drug at

constant rate.

The use of dopamine agonists reduces, although not eliminates, the incidence

of motor fluctuations. Also, it provides worse control of the PD symptoms. It has

been suggested, that the means of delivery of dopaminergic drug is highly important,

and that it does impact the ability of given compound to produce dyskinesias (94).

The importance of constant drug delivery was underlined by transdermally delivered

rotigotine, which has shown promising results in PD treatment (95; 96). Although

there is an insufficient evidence to support a notion, that Neupro reduces long term

motor complications, transdermal rotigotine was shown to have a very low

predisposition to induce dyskinesia in rat model of PD (97). This underscores the

necessity of improvement in antiparkinsonian drug delivery techniques.

Finally, there is a large group of symptoms, particularly non-motor (Table 1),

that do not respond well to dopaminergic treatment, i.e. freezing, sleep disturbances,

autonomic dysfunction. These are the target for future therapies.

Transdermal formulations in PD

Transdermal route seems ideal for continuous dopaminergic stimulation.

Starting from the pharmaceutical benefits, as an easy, once-daily application of a

patch; through the pharmacokinetic reasons: avoidance of gastric drug degradation

and the first pass effect, a steady and continuous delivery, and stable plasma levels

of a drug; to finish with pharmacodynamic reasons, that is the potential for limiting

motor complications (40; 94), or avoidance of stomach and liver enzymes inhibition

(98). However, transdermal passive delivery is possible only for molecules with

certain physicochemical properties. Generally, a good candidate should have

relatively low molecular mass (M<400Da), water octanol distribution coefficient

between 10 and 1000, good solubility characteristics (low melting point), and usually

should be neutral in pH range from four to seven (99). Also, because of limited rate

of transdermal delivery, only the most potent drugs can be effectively transported

this way. There are several passive patches available on the market (Table 2):

Introduction

15

So far, two of the commercially available transdermal patches, Neupro and

Emsam, deliver drugs used in PD (73). In 2006, rotigotine was approved by the

EMEA for PD therapy (95). As this drug is subject to heavy gastrointestinal

metabolism (100; 101), it was formulated as a transdermal patch. Transdermal

rotigotine was demonstrated as an efficient antiparkinsonian treatment, in several

clinical trials. Parkinson Research Group has conducted a placebo-controlled, dose-

ranging trial, where rotigotine was assessed in 225 de novo PD patients (102). Clear,

dose-dependent clinical effect of the drug (measured by Unified Parkinson’s Disease

Rating Scale, UPRDS) was observed for dose ranging from 4.5 to 13.5mg/day, with

a plateau between 13.5 and 18.0mg/day. In another study from this group, the

efficiency of rotigotine was compared to that of oral pramipexole (96). 506 patients

with PD history of at least three years, with marked motor fluctuations, and on stable

levodopa dose of no less than 300mg/day, were randomly assigned to receive

rotigotine, pramipexole or placebo. Rotigotine was shown to be non-inferior in

reducing the off-time, and similarly efficacious to pramipexole in other response

parameters (such as levodopa dose reduction, responders rate, or severity of side

effects). Finally, Neupro efficacy was also compared with that of ropinirole, in a large,

placebo-controlled, multicentre clinical trial (103). Although it was found that

rotigotine is effective in comparison to placebo, non-inferiority to ropinirole was not

confirmed by the study. However, the study was later criticized for non-equivalence

of rotigotine and ropinirole doses.

Oral selegiline, in a dose of 10mg/day, is also licensed for Parkinson’s

disease treatment, as monotherapy and as levodopa adjunct. Selegiline transdermal

system (Emsam) has now been approved by Food and Drug Administration (FDA)

for the treatment of major depressive disorder (98; 104). Treatment of major

depressive disorder requires doses three to six times higher, than PD therapy. Up to

date, there are few studies investigating the effectiveness of transdermal selegiline in

PD. Nonetheless, this formulation seems very promising for several reasons. Firstly,

to provide continuous dopaminergic stimulation, constant drug plasma-levels are

essential. Selegiline transdermal patch provides constant-rate delivery, resulting in

stable plasma concentrations. The plasma-levels achieved by 6mg Emsam patch are

comparable with Cmax of 10mg oral formulation (93). Secondly, the absolute

bioavailability of selegiline is increased from 0.04 (105) to 0.78 (93), which allows for

Introduction

16

significant dose reduction. Thirdly, the area under the plasma concentration curve

(AUC) of potentially neurotoxic amphetamine metabolites of selegiline, is significantly

lower for Emsam than for oral formulation.

Table 2 The examples of commercially available drugs delivered transdermally (73).

Drug Commercial name Highest available

dose [mg/24h]

Buprenorphine BuTrans® 0.48

Clonidine Catapres-TTS® 0.3

Estradiol Estraderm®, Estradot® 0.1

Ethinylestradiol Evra® 0.15

Fentanyl Durogesic DTrans® 2.4

Lidocaine Lidoderm® 42

Nicotine Nicopatch® 21

Nitroglycerine Trintek®, Minitran® 15

Oxybutynin Kentera® 3.9

Rivastigmine Exelon® 9.5

Rotigotine Neupro® 16

Scopolamine Scopoderm TTS® 0.33

Selegiline Emsam® 12

Among the dopamine agonists, the passive transdermal delivery of

apomorphine, lisuride, pergolide, piribedil and bromocriptine has also been

investigated. Apomorphine is the dopamine agonist with the target receptor spectrum

close to dopamine (Table 3), and the only DA of potency similar to levodopa (82).

However, apomorphine is rather difficult to manage: it is easily oxidised in presence

of the air or light (75); administered orally it is subject to heavy first pass metabolism,

while elevating the oral dose is nephrotoxic (106); it is commonly applied

subcutaneously and intravenously, which is frequently associated with ulceration and

nodules formation in the administration site (39). The attempts with sublingual, nasal,

Introduction

17

or rectal apomorphine formulations did not find practical implementation (107; 108;

109). Priano et al. managed to deliver apomorphine transdermally in vitro and in vivo

from microemulsions (110). The transdermal apomorphine was demonstrated as

being effective in reducing off time, in the manner comparable to continuous

apomorphine infusion. However, the UPRDS scores were less affected. The use of

permeation enhancers and consequent skin irritation was the factor limiting usage of

this application. Also, in two patients, this transdermal formulation did not produce

detectable drug levels in plasma.

Pergolide transdermal delivery from surfactant-base elastic vesicles was

tested in vitro (111; 112). The results underscored the role of vesicle morphology

and elasticity, drug concentration inside the vesicle, the pH of media, and occlusion

in effectiveness of vesicular system. Nonetheless, even in the optimal conditions, the

pergolide delivery rate was not able to assure the therapeutical doses.

Piribedil delivery through the skin was also investigated in a couple of studies.

Smith et al. (113) examined the effect of piribedil patch on the chemically induced

motor disability in marmosets. It was demonstrated that the drug transdermal

delivery results in measurable and dose-dependent piribedil concentrations. These,

in turn, were associated also in dose-dependent manner, with reversal of all

components of the parkinsonian motor deficits. However, in a randomized and

double-blind study in humans (114), the use of 60cm2 patch, containing 100mg of

micronized piribedil, did not result in any beneficial effect. The inefficacy of treatment

was attributed to sub-therapeutical plasma levels of the drug.

One study assessed the efficacy of transdermal lisuride in early Parkinson’s

disease (115). The study reported, that the application of lisuride patch resulted with

some beneficial effect on patients’ motor functions (measured by appropriate test),

however no data regarding the amount of drug delivered was provided. Similarly,

only one report describes transdermal delivery of bromocriptine (116). In the in vivo

study carried out on rabbits, bromocriptine plasma-concentrations achieved by

transdermal application, were higher than those obtained after oral administration of

commercially available Parlodel® tablet. This suggests that it might be possible to

effectively deliver bromocriptine in humans via transdermal dosage forms. However,

Introduction

18

in the bromocriptine delivery study, the permeation enhancers were employed, which

might limit the use in human subjects, due to potential skin irritation.

The case of rotigotine has demonstrated that transdermal formulations can be

effectively used in the course of everyday PD therapy. Nevertheless, for many other

drugs, this type of delivery does not assure fast enough delivery to guarantee clinical

benefit, or is associated with application site irritation, therefore limiting the usage.

More studies are needed to achieve better understanding of transdermal drug

delivery processes, and further, to develop more refined transdermal delivery

systems.

Iontophoresis

Transdermal iontophoresis is the drug delivery technique that uses a small

electric current (I<0.5mA/cm2), to deliver charged and polar, neutral-molecules

through the skin. The current is applied via two electrodes, where one is immersed in

a medium containing a charged drug, and the other is positioned in an inert

conducting medium, i.e. sodium chloride solution (Figure 1). There are three major

phenomena responsible for drug transport (117; 118): electromigration,

electroosmosis and passive diffusion. The first one is characterised by the

movement of charged particles in an applied electric field. In general, this is the

principal mechanism by which the charged molecules are transported. Equation 1

describes the dependency of the transdermal flux of each ion, on the current applied

(119):

Equation 1 IFztJi

ii =

where ti is the transport number of species i; F is the Faraday’s constant; zi is the

valence of i; and I is the current intensity. Equation 1 shows, that current adjustment

allows for direct control over the drug penetration through the skin. The transport

number ti, is defined as the fraction of the total passing current, carried by a specific

ion. It is the key parameter describing the efficiency of drug delivery by means of

iontophoresis. ti is dependent on the charge-carrying properties of the given ion, and

the presence and properties of the co- and counter-ions involved in the iontophoretic

process. The conservation of charge implies, that the sum of the electrical currents

Introduction

19

carried by each of the ions, must be equal to the total current applied. In other words,

the sum of all the transport numbers must be equal to 1. This means that all the ions

present in the system compete amongst themselves for charge carrying.

DH+

DH+Cl -Cl-

Cl-

Cl -Cl-

Cl-

Na+

Na+

Na+

Na+

Na+

Cl-+ +Ag0 AgCl e- Cl-++ Ag0AgCl e-

Ag/AgCl electrodes

Anode +

Cathode _

SKIN

<10 cm

Stratum Corneum

Figure 1 Mechanism of iontophoretic drug delivery for cationic drugs. At the anode, positively charged drug ions are repelled, and travel through the skin to the interior of body. In exchange, most common internal anions – Cl- move the opposite way. At the cathode, chlorides are pushed through the skin, and the internal cations (like Na+) are extracted from the deeper parts of the skin. Electroosmotic flow enhances the cation transport in both electrodes.

In practice, two situations can be distinguished: first, when the only ionic

species taking part in the iontophoretic process are the delivered drug and the

counter-ions, from beneath the skin, i.e. as in hydromorphone experiments (120); in

this experimental setup the delivered drug was the only cation in donor solution and

the delivery was found to be independent of the drug concentration in formulation.

The second situation, where there is more than one competing ion species present

in the donor, i.e. when the donor solution contains buffers, as in rotigotine

experiments (84). The first situation has been approximated by Nernst-Planck

electrodiffusion theory with electroneutrality assumption (121). This theory predicts

that, when a drug ion competes only with a counter-ion for current transport, the

transport number value is defined by Equation 2:

Introduction

20

Equation 2 −+

+

−+

+

+

+=

+=

AC

C

AC

CC µµ

µDD

Dt

In this equation, D is the diffusion coefficient, and µ is the ionic mobility of the

cation C+, and the anion A-, in the skin. It is noteworthy, that the in Equation 2

transport number does not depend on the concentration of cationic drug, allowing for

high fluxes even with low drug concentration. However, the application of

Nernst-Planck theorem to transdermal iontophoresis has some limitations, as it was

derived for homogenous, uncharged membranes and for constant voltage

conditions. Also, particularly in the in vivo situations, there is more than one counter-

ion taking part in iontophoretic process. These differ from the conditions of

transdermal iontophoresis experiments, for the skin is not a homogeneous

membrane, and it is charged at neutral pH. Furthermore, constant current rather than

constant voltage is preferably used in iontophoretic experiments. Nevertheless,

validity of this approach was experimentally confirmed for several drug molecules,

such as: lidocaine (122) or hydromorphone (120) and also for small inorganic ions

(123) Figure 2.

In reality, the maximum transport number achievable for a drug delivered by

iontophoresis, whether a cation or an anion, is limited by the body’s main and highly

mobile electrolytes: Cl- or Na+ respectively. Thus, the value of transport number

usually does not surpass 0.2 for organic cations (124) i.e. 0.12 for lidocaine (122) or

0.10 for ropinirole (125).

In the second situation mentioned above, where there are two or more ionic

species in the donor compartment, the transport number is described by Equation

3 (126).

Equation 3 ∑∑ ⋅⋅

⋅⋅==

jjjj

iii

jj

ii zµC

zµCtσσ

,

where Ci is the concentration of ion i, µi its mobility inside the membrane and zi its

valence. In this situation the ionic flux is dependent on the relative concentrations of

all the ions present in the donor solution as well as their ionic mobilities in the skin

Figure 2).

Introduction

21

Single-ion delivery

10 30 100 400 8000

500

1000

1500

Hydromorphone concentration [mM]

Flux

[µg/

h]

Co-ion delivery

7.5 15 22.5 27 300

1

2

3

4

Lidocaine concentration [mM]

Lido

cain

e Fl

ux [µ

mol

/h]

A B

Figure 2 Iontophoretic drug delivery for single-ion (A), and co-ion (B) situation. Graph A depicts hydromorphone delivery rate from a variety of drug content in water. Graph B presents the delivery rate of lidocaine from solutions containing different drug concentrations and the background electrolyte (NaCl). The data was taken from references (127; 120)

Another transport mechanism is the electroosmosis. This phenomenon occurs

because the skin is charged at physiological pH. When a voltage is applied across a

charged membrane, a bulk solvent flow occurs. The direction of this volume flow is

the same as the movement of the ions of the charge opposite to the membrane

(128). The isoelectric point (iP) of the skin is around 4.3 (129) so the skin is

negatively charged at the physiological pH. Thus, electroosmotic flow occurs from

the anode to cathode direction. This impairs the transport of anions and assists that

of cations. The electroosmotic flow carries any substance dissolved in the solvent,

and for that reason allows for the transdermal delivery of neutral and polar

molecules. The main factors controlling the electroosmotic flow are: the pH that

controls the charge density on the skin (129); and the ionic strength, which is

responsible for screening of the skin’s charge (130). The electroosmotic flow of

compound i, can be assessed by the Equation 4 (131):

Equation 4 J V CiEO

i= ⋅

where V is the volume flow and Ci is the concentration. This approach was exploited

to separate the electroosmosis and electromigration, in several previous publications

(132; 122). It was based on two assumptions: 1) that the transport of electroosmotic

marker and the delivered drug follow the same pathways; and 2) that the

electroosmotic transport of the marker-molecule is directly proportional to its

concentration in the donor solution. While the second condition has been tested

(122), the first one can be discussed upon, as some of the drug molecules,

Introduction

22

particularly the more lipophilic, can partition into the skin, or can be generally

transported in different ways, other than polar electroosmotic markers. In following

chapters, the fluxes of the electroosmotic marker will be only used to conclude on

changes of magnitude of electroosmotic flow, and the quantitative contribution of

electroosmotic flow will be understood as the upper limit, rather than the actual

amount of drug transported by electroosmosis.

Iontophoretic drug delivery in PD

Iontophoresis broadens the spectrum of transdermally delivered drugs. By

enhancing the transport through the skin, it is possible to deliver medicines at higher

rate than with passive penetration. This potentially allows the drugs, which reach

only sub-therapeutical plasma concentrations when delivered passively, to be

efficiently delivered. As discussed previously, some of antiparkinsonian medications

can be delivered by passive transdermal patch (85; 104), however, for many drugs

form this group this way of administration appeared to be inefficacious, or there is

lack of transdermal studies.

Up to date, four PD drugs: rotigotine, 5-OH-DPAT, ropinirole and

apomorphine, have been studied in iontophoretic formulations. Ropinirole was

investigated in both, in vitro across full thickness piglet skin, and in vivo in hairless

rats (125; 133). In the in vitro study, the highest efficiency of ropinirole transport

achieved was 13%, in the absence of background electrolyte. The drug flux was

proportional to the intensity of the applied current. The presence of competing ions in

a donor solution influenced the ropinirole transport number in a complicated manner.

No clear dependency of t# was found on ropinirole concentration, neither on its molar

fraction. The cited explanation was that both processes, electromigration and

electroosmosis, had a significant input to the total ropinirole transport; the

electromigrative component of the transport was rising with the drug concentration,

while the electroosmotic component was limited by it. As a result, the summary flux

was hard to predict. In subsequent in vivo study, the effect of ropinirole concentration

in donor, in the absence of competing ions was studied. For the range of 25 to

250mM ropinirole, no differences in the input rates were observed. Also, the drug

profiles in the plasma were statistically indistinguishable. The in vivo study resulted

Introduction

23

in higher drug transport numbers, than the in vitro. Both studies strongly indicated,

that ropinirole could be efficiently delivered in humans.

The iontophoretic delivery of rotigotine and 5-OH-DPAT was studied in vitro,

in similar conditions (84; 134; 135). The studies investigated the influence of several

different factors on transdermal transport. Firstly, it has been shown for both drugs,

that the type of membrane: human stratum corneum (HSC) or human dermatomed

skin (HDS), has a significant impact on the flux profile and the maximum flux value.

The use of the HDS reduced the maximal flux of rotigotine roughly by half, in

comparison to the HSC. In case of 5-OH-DPAT the reduction was only 16%. The

concentration of rotigotine and 5-OH-DPAT in donor solution, and also the intensity

of applied current, were found to have significant impact on the transdermal transport

of both drugs. The increase in current or donor concentration resulted in proportional

increase in fluxes for both substances. Also, the effect of the donor pH on the drug

fluxes was similar. Both molecules were delivered more efficiently from solutions with

higher pH values, with their fluxes roughly doubling from pH 4.0 to 5.0. Moreover, the

rotigotine study revealed that the maximal flux did not depend on the receptor pH;

however the latter altered the drug-flux profile. Furthermore, the temperature of

experiment had a positive effect on the drug transport, as investigated for rotigotine.

The increase of temperature from 20°C to 32°C, resulted in increase of the drug flux

by around 60%. This was explained by a possible increase in rotigotine diffusivity in

the skin, due to the change of lipid organization between given temperatures (136).

Another phenomenon examined, was the influence of different competing ion. The

observations indicated that the replacement of sodium with bigger, thus less mobile

triethylamine ion resulted in higher rotigotine fluxes. Nonetheless, further increase in

mass of the co-ion, resulted in a drop of the drug flux.

Finally, apomorphine among the antiparkinsonian agents, is probably the

most studied drug for iontophoretic delivery. The earliest in vitro studies explored the

effects of current intensity, temperature, apomorphine concentration and pH of donor

(137). HSC and DHS were employed as membrane models. The results were similar

to those observed with rotigotine transdermal transport. Factors enhancing

apomorphine flux were: temperature, current intensity, and drug concentration in

donor. Replacement of isolated stratum corneum by dermatomed skin resulted in

Introduction

24

drop of drug flux. In a follow up study, the optimal in vitro conditions were used to

deliver apomorphine to idiopathic PD patients (138). During the study, apomorphine

was iontophoretically delivered to ten patients, using two current intensities. The

delivery was closely regulated by the applied current. Also, a high correlation with in

vitro experiments was observed. Nonetheless, the apomorphine plasma levels did

not reach the therapeutic conditions.

Apomorphine iontophoresis was further studied in series of publications by Li

et al., investigating the impact of physicochemical conditions (139), and the use of

chemical permeation enhancers on the drug delivery (140; 141; 142; 143). The

physicochemical study demonstrated, that under the experimental conditions

applied, elevating pH resulted in higher drug fluxes. A small effect of concentration of

sodium as a competing ion was also observed. In addition, similarly to the rotigotine

study (134), replacement of sodium co-ion to triethylamine or tributylamine resulted

in raise of flux. The studies addressing the applicability of permeation enhancers,

evaluated several substances and systems: elastic vesicles (140); laureth-3

oxyethylene ether (C12EO3), laureth-7 oxyethylene ether (C12EO7), and sodium

sulphosuccinate (141); C12EO3, C12EO7 and cholesterol sulphate (142); lauric acid,

dodecylammonium bromide, C12EO3 and propylene glycol (143). The use of elastic

vesicles resulted in moderate, 40-50% increase in the drug flux and significant drop

of skin resistance during iontophoresis. The best enhancing effect was obtained with

C12EO3 alone, which increased the flux 2.3 times, and by combination of C12EO3/

C12EO7/cholesterol-sulphate in proportions 70/30/5, with 2.0 increase of flux. Also,

the effect enhancer was more accentuated in the experiments with dermatomed

human skin, than in human stratum corneum. In subsequent study (144), the effect

of enhancer pretreatment was studied in 16 PD patients. Eight patients were treated

with surfactant prior to iontophoresis, while the remaining patients created the control

group. The application of enhancers resulted in significantly higher apomorphine

bioavailability (10.6±0.8% vs. 13.2±1.4%) and input rate (75.3±6.6 vs. 98.3±12.1

nmol/cm2·h). Also, in five patients in the study group versus three in the control,

clinical improvement was observed.

Introduction

25

Table 3 Drugs used in Parkinson’s disease with pharmacokinetic properties and target sites. *D1-D5 – dopamine receptor subtype (1-5) agonist. M1-M4 – acetylcholine muscarinic receptor subtype (1-4) antagonist. NMDA – N-methyl-D-Aspartate receptor antagonist. COMT – Catechol-O-methyl transferase inhibitor. MAO-B – Mono amino oxidase B inhibitor.** The reported value is an apparent clearance (Cl/F)h. *** Transdermal bioavailability of rotigotine. SC – subcutaneous.

Drug Total daily oral

dose (mg)

Daily doses

Oral bioavail-

ability F (%)

Half-life (h)

Clearance (L/h)

Target iontophoretic

flux (µmol/h)

Target receptor* Reference

Levodopa (associated with benserazide or with carbidopa)

400-800 3-4 80-98 1.5-2 25 76-150 D1 – D5 (73; 74; 75) Dopamine agonists

Apomorphine 3-30 SC 3-10 100 SC 0.5-1 168 4.7 D1 – D5 (74; 73) Piribedil 50-250 1-5 10 12.1 78.6 12.0 D2,D3,α2A,α2c (76; 77; 78; 79; 80)Pramipexole 0.375 – 4.5 3 90 12.9±3.3 25 0.6 D2,D3,D4 (78; 81; 82; 73) Ropinirole 3-24 3 50 6 47 2.0 D2,D3 (73; 74; 75) Bromocriptine 10-40 3 3-6 3-8 55 0.2 D2,D3,D4,5HT (73; 74; 75; 82) Cabergoline 2-6 1 50-80 63-110 192 0.4 D2,D3,D4 (73; 74; 75) Lisuride 1-5 3 10-20 1.2 – 2.5 48 0.1 D2,D3,D4 (73; 74; 75) Pergolide 0.15 – 5.0 3 20-60 21 <457** 0.4 D1–D5,5HT,α1 (78; 73; 72; 83; 74)Rotigotine 4.5-18 TD 1 37*** 2-3 630 2.0 D1,D2,D3 (84; 85; 86)

AnticholinergicsTrihexyphenidyl 5-15 3-4 -- 5.3-32.7 20** 0.5 M1, NMDA (87; 73; 88) Procyclidine 10-30 3 75 8-16 4.1 3.3 M1,M2,M4 (89; 73; 88; 74) Biperiden 3-12 3 29-33 18-24 56 0.5 M1 (89; 73; 88; 74) Benztropine 2-16 2-4 ~ 60 - - 1.3 M1 (89; 73; 88; 74) Orphenadrine 150-400 2-4 95 30-40 - 59 M1, NMDA (89; 73; 88; 74)

Other categories

Entacapone 200-2000 1-6 30-46 0.3-3.4 45 30-150 COMT (73; 90; 91) Selegiline 10 1-2 4-10 0.5-2 93 0.9 MAO-B (73; 92; 93) Amantadine 100-400 2 86-94 10 - 45 7 - 22 25-100 NMDA (73; 74; 75)

Introduction

26

Iontophoretic target flux assessment and drug selection for iontophoretic

delivery

To assess the best drugs suitable for iontophoretic delivery there are several

factors to be considered. First, the physicochemical properties such as molecular

mass, ionisation, water solubility and lipophilicity must be taken into account. Big

molecules are less mobile and tend to have small transport numbers. This limits their

employment in this administration route. Poor water solubility makes it difficult to

provide enough drug in the donor solution for effective iontophoretic delivery. The

pKa of a compound decides on its ionisation properties. Too high or too low of a

value for this parameter would constrain extreme pH values in donor solution and

make the iontophoretic delivery impractical. The type of salt used is another factor

that matters. Because the most commonly used are Ag/AgCl electrodes,

hydrochloride salts are the most suitable for iontophoretic applications.

Furthermore, pharmacokinetic properties have to be considered when

selecting the right drug candidate. Particularly important are the oral bioavailability,

plasma half-life, and total clearance. Drugs with high oral bioavailability and long

half-life are suitable for oral administration, and in such case, formulating expensive

iontophoretic dosage forms is less justified. Also, drugs with high total clearance

require high delivery rates, to maintain constant therapeutic level of the drug in

blood, sometimes unachievable for iontophoresis. On the other hand, the plasma

profile of the drugs with higher clearance can be more effectively adjusted. Finally,

the most suitable candidates are the potent drugs requiring low doses for clinical

effect.

Two approaches were used to assess the target iontophoretic flux (Table 3).

First one was based on Equation 5. To maintain drugs steady state concentration in

plasma, its elimination must be compensated by absorption. Hence the rate of

elimination must be equal to the rate of absorption. Equation 5 describes the rate of

elimination as a function of drugs clearance and concentration in plasma. This must

be equalized by the iontophoretic input of delivered substance to the body. The

values of clearance and steady state therapeutical concentration for each drug were

taken from appropriate references (Table 3).

Introduction

27

Equation 5 ssCClK ⋅=0

For the drugs whose clearance or required plasma concentration was

unavailable in the literature, the second approach was used to estimate the

iontophoretic flux. It consisted of multiplying of the delivered daily dose of a drug by

its bioavailability, and dividing it by 24 hours to obtain the hourly rate of delivery.

Transdermal iontophoretic administration is limited by the maximal current

density that can be applied: 0.5mA/cm2, and the size of a patch that is acceptable for

patient – 50cm2. Thus, Equation 1 predicts the maximum 200µmol per hour of

hypothetical drug can be delivered, providing it would have the transport number

t#=20%. That, unfortunately, makes impossible iontophoretic delivery of levodopa,

because of too high delivery rate required (Table 3). However most of the other PD

drugs do not require such high doses and it might be possible to deliver them in

therapeutic doses by means of iontophoresis.

For this study, six drug were selected on the basis of their physicochemical

and pharmacokinetic properties. Selegiline, trihexyphenidyl, pramipexole, pergolide

and piribedil all had estimated target fluxes within the capacity of iontophoretic

delivery (Table 2). They had at least one pKa’s over 6, which allowed for a total drug

ionisation in acceptable pH. All of these drugs had low molecular mass (<310Da).

There was a limited information available regarding the solubility of these

compounds, however the in silico estimations (146) indicated the sufficient degree of

water solubility. In terms of delivery of entacapone, it was rather unlikely to be able to

deliver this drug iontophoretically due to large required fluxes (Table 2).

Nonetheless, as opposed to other selected drugs entacapone was a weak acid and it

was interesting to investigate the delivery of an anionic substance, in similar

conditions as the other cationic compounds. Finally, no or limited research has been

previously done on iontophoresis of those drugs.

The drugs are presented as follows: The first two are those with the best

outcome: selegiline and pramipexole. The latter is followed by pergolide and

piribedil, to group the three dopaminergic agonists. Next drug, trihexyphenidyl,

rounds off the cationic drugs. The last presented study describes entacapone, the

only anionic substance.

Introduction

28

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Introduction

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90. Inhibition of soluble catechol-O-methyltransferase and single-dose

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93. Pharmacokinetics and absolute bioavailability of selegiline following

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97. Continuous administration of rotigotine does not induce dyskinesia in a rat

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101. Extensive gastrointestinal metabolic conversion limits the oral

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104. Transdermal selegiline. Patkar, A A, Pae, C U and Zarzar, M. 2007,

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105. A new low-dose formulation of selegiline: clinical efficacy, patient

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106. Studies of renal function in animals chronically treated with apomorphine.

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108. Intranasal apomorphine. Nastech Pharmaceutical. Kendirci, M and Hellstrom, W J. 2004, IDrugs: The Investigational Drug Journal, Vol. 7, pp. 483-488.

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109. A novel sublingual apomorphine treatment for patients with fluctuating

Parkinson's disease. Ondo, W, et al. 1999, Movement Disorders, Vol. 14, pp. 664-

668.

110. Transdermal apomorphine permeation from microemulsions: a new

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111. The in vitro transport of pergolide from surfactant-based elastic vesicles

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112. Transdermal delivery of pergolide from surfactant-based elastic and rigid

vesicles: characterisation and in vitro transport studies. Honywell-Nguyen, P L, et al. 2002, Pharmaceutical Research, Vol. 19, pp. 991-997.

113. Transdermal administration of piribedil reverses MPTP-induced motor

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114. A randomized, double-blind study of a skin patch of a dopaminergic

agonist, piribedil, in Parkinson's disease. Monastruc, J L, et al. 1999, Movement


115. Transdermal lisuride delivery in the treatment of Parkinson's disease.

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116. Transdermal administration of bromocriptine. Degim, I T, et al. 2003,

Biological and Pharmaceutical Bulletin, Vol. 26, pp. 501-505.

117. Iontophoretic drug delivery. Kalia, Y N, et al. 2004, Advanced Drug

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118. Scott, E R, et al. Electrotransport systems for transdermal delivery. A

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Introduction

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119. Transdermal ion migration. Phipps, B and Gyory, R. 1992, Advanced

Drug Delivery Reviews, Vol. 9, pp. 137-176.

120. In vitro and in vivo evaluation of transdermal iontophoretic delivery of

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of Controlled Release, Vol. 11, pp. 123-135.

121. Theoretical models of iontophoretic delivery. Kastings, G B. 1992,

Advanced Drug Delivery Rewievs, Vol. 9, pp. 177-199.

122. Contributions of electromigration and electroosmosis to iontophoretic

drug delivery. Marro, D, et al. 2001, Pharmaceutical Research, Vol. 18, pp. 1701-

1708.

123. Electromigration of ions across the skin: Determination and prediction of

transport numbers. Mudry, B, Guy, R H and Delgado-Charro, M B. 2006, Journal

of Pharmaceutical Sciences, Vol. 95, pp. 561-569.

124. Quantitative structure-permeation relationship for iontophoretic transport

across the skin. Mudry, B, et al. 2007, Journal of Controlled Release, Vol. 122, pp.

165-172.

125. Iontophoretic delivery of ropinirole hydrochloride: effect of current density

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126. Iontophoretic delivery of model inorganic and drug ions. Phipps, J B, Padmanabhan, R V and Lattin, G A. 1989, Journal of Pharmaceutical Sciences,

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127. Optimizing iontophoretic drug delivery: identification and distribution of

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pp. 1710-1713.

128. The role of electroosmotic flow in transdermal iontophoresis. Pikal, M J. 2001, Advanced Drug Delivery Reviews, Vol. 46, pp. 281-305.

Introduction

41

129. Characterisation of the iontophoretic permselectivity properties of human

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Controlled Release, Vol. 70, pp. 213-217.

130. Reverse iontophoresis — Parameters determining electroosmotic flow: I.

pH and ionic strength. Santi, P and Guy, R H. 1996, Journal of Controlled Release,

Vol. 38, pp. 159-165.

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134. Transdermal iontophoresis of rotigotine: influence of concentration

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Journal of Controlled Release, Vol. 96, pp. 159-167.

135. Transdermal iontophoresis of the dopamine agonist 5-OH-DPAT in

human skin in vitro. Nugroho, A K, et al. 2005, Journal of Controlled Release, Vol.

103, pp. 393-403.

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281-286.

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validation. van der Geest, R, Danhof, M and Bodde, H E. 1997, Pharmaceutical

Research, Vol. 14, pp. 1798-1803.

Introduction

42

138. Iontophoretic delivery of apomorphine II: an in vivo study in patients with

Parkinson's disease. van der Geest, R, et al. 1997, Pharmaceutical Research, Vol.

14, pp. 1804-1810.

139. Iontophoretic delivery of apomorphine in vitro: physiochemic

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Research, Vol. 18, pp. 1509-1513.

140. Effect of elastic liquid-state vesicles on apomorphine iontophoresis. Li, G L, Danhof, M and Bouwstra, J A. 2001, Pharmaceutical Research, Vol. 18, pp.

1627-1630.

141. Iontophoretic R-apomorphine delivery in combination with surfactant

pretreatment: in vitro validation studies. Li, G L, et al. 2003, International Journal of

Pharmaceutics, Vol. 266, pp. 61-68.

142. Pretreatment with a water based surfactant formulation affects

transdermal iontophoretic delivery of R-apomorphine in vitro. Li, G L, et al. 2003,


143. In vitro iontophoresis of apomorphine across human stratum corneum.

Structure-transport relationship of penetration enhancement. Li, G L, et al. 2002,


144. Transdermal iontophoretic delivery of apomorphine in patients improved

by surfactant formulation pretreatment. Li, G L, et al. 2005, Journal of Controlled

Release, Vol. 101, pp. 199-208.

145. Smith, D A, van de Waterbeemd, H and Walker, D K. Pharmacokinetics and metabolism in drug design. Hamburg : Wiley-VCH, 2001.

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Chapter I Selegiline

43

Chapter I – Selegiline Introduction

Selegiline (Figure 1) is a selective,

irreversible monoamine oxidase type B (MAO-B)

inhibitor used in major depressive disorders (1),

and in the treatment of Parkinson’s disease;

alone or as an adjunct to levodopa therapy (2; 3).

Its clinical effectiveness was assessed in

many clinical trials, as DATATOP (4),

SELEDO (5), UK-PDRG (6; 7) or FSTM 1993 (8; 9). Recently, meta-analysis of ten

clinical trials, which assessed the efficacy of selegiline therapy in early Parkinson’s

disease (PD), was issued (10). In total, the data from 2422 patients were analysed.

The study revealed, that introduction of selegiline to therapy provides significant

benefit in comparison to placebo, and mild benefit in comparison to levodopa alone.

In addition, there was a marked levodopa sparing effect, which in turn was

associated with lower incidence of motor fluctuations. Also, the analysis has shown,

that selegiline provides some neuroprotective effect, that is statistically significant,

although clinical importance of this effect is minor. Despite previous reports (11),

the study did not confirm the significant increase mortality among patients treated

with selegiline.

The pharmacokinetics of oral selegiline (Deprenyl, Eldepryl, 5mg tablet) are

characterised by a fast absorption and a marked first-pass effect. The latter limits

the bioavailability to 4-10% (12; 13; 14). Rapid absorption, combined with short half-

life of no more than two hours, produces serrated drug time-profiles in plasma. The

newly developed formulations, like the transdermal passive patch

(Emsam, 6, 9, 12mg/24h), or the rapidly soluble sublingual form (Zelapar, 1.25mg),

greatly increased selegiline bioavailability, to 78% and 30% respectively. This

compares to the bioavailability of oral tablet, 4-10% (15). Transdermal and

sublingual formulations, by circumventing the first-pass effect, reduce significantly

selegiline metabolites levels in plasma. Furthermore, transdermal patches produce

N

Figure 1 Selegiline molecular structureM=187.2Da


44

stable plasma levels of the drug, in contrast to the oral formulation which produces

a rapidly declining drug peak in plasma. So far, however, transdermal selegiline has

not been licensed for management of Parkinson’s disease, as opposed to

sublingual Zelapar, although the doses required in PD therapy are significantly

lower, than these used for treatment of depression.

Transdermal iontophoresis has recently been recognized as a promising

method of drug delivery (16). It bypasses the liver, thus avoids the first pass effect;

offers constant and patient-adjustable drug absorption; and is applicable to wider

spectrum of drugs, than the formulations based on transdermal passive transport.

As a means of drug-delivery it induces only minor side effects such as redness and

itching, which quickly subside. The method is easy to use and is well tolerated by

patients (17).

A preferred candidate for iontophoretic delivery would be a potent drug of

relatively small molecular mass, soluble in water; with a pKa between 5.0 and 10.0.

Iontophoretic drug delivery is relatively expensive compared to oral delivery, its

usage has to be well justified. Iontophoresis is then most beneficial when delivering

drugs of low oral bioavailability, short half-life, and when the dosage has to be

tailored to the patient.

Selegiline seems to be well suited for transdermal iontophoretic delivery. It

has a relatively short half life t1/2=0.5-2h; volume of distribution of Vd=780L; and

clearance of Cl=93 L/h (14). The common oral dosage is 10mg once daily (2; 18)

and reported bioavailability ranges from 4 to 10% (12; 13; 14). The average steady

state plasma concentration obtained in clinical trial, after intravenous infusion of

10mg selegiline to 12 healthy subjects was Css=9.8nM (13). Relatively low

therapeutical concentration and bioavailability make selegiline a good candidate for

iontophoretic delivery from pharmacokinetic point of view. Also the drugs

physicochemical properties (molecular weight 187.2Da, in silico obtained

logP=3.0±0.6 (19), tertiary amine pKa=7.4 (20)) make it a good candidate for

transdermal iontophoresis. From the pharmaceutical point of view, selegiline

iontophoretic application could have several advantages over the other available

selegiline dosage forms. Iontophoretic patch could achieve reduced patch loading

(amount of drug contained in the patch), in comparison to the passive transdermal


45

patch. Furthermore, iontophoresis offers a possibility of dosage profiling to suit

patients requirements. Also, an iontophoretic selegiline application could resolve

the problem of the unspecific inhibition of intestinal MAO-A by the oral form, in

gastrointestinal tract. MAO-A is an important enzyme in tyramine metabolism and

its inhibition can impose cardiovascular safety risk, following the ingestion of

tyramine-rich foods. Iontophoretic application could avoid this problem, through

circumvention of the gastrointestinal tract and lower administered doses.

Iontophoretic target flux, which would assure steady state plasma concentrations at

9.8nM (13), was estimated as product of selegiline clearance and this

concentration: JSEL=Cl*Css=0.91 µmol/h.

The aim of this study was to evaluate selegiline as a candidate for

transdermal iontophoresis, and to assess the feasibility of iontophoretically

delivering therapeutic doses of this drug. Analysis of several factors, potentially

affecting iontophoretic flux (such as pH, presence of background electrolyte, drug

concentration in formulation, or applied current) was performed to explain the role

of the mechanisms governing selegiline transdermal transport. Finally, selection of

the best conditions was made to indicate to optimal iontophoretic formulation for the

best delivery.

Materials and Methods

Materials

Selegiline hydrochloride, sodium chloride, paracetamol (PAR), silver wire

(99.99% pure), silver chloride (99.999% pure) were obtained from

Sigma Aldrich UK; hydrochloric acid, sodium hydroxide, acetonitrile HPLC grade

and n-octanol were supplied by Fisher Scientific UK; triethylamine was supplied by

Acros Organics UK. Deionised water (resistivity≥18.2MΩ/cm) was used to prepare

all solutions.

Skin preparation

Full thickness porcine skin, obtained from a local abattoir, was rinsed under

cold running water. The skin slices were taken from the epidermal side with an

electric dermatome (Zimmer, Dover, OH) set to 750µm thickness and then were


46

wrapped in Parafilm® and stored at -20°C for no longer than four months. Both,

dorsal and abdominal skin from eight different pigs was used in the experiments.

Iontophoresis experiments

On the day of the experiment skin was thawed at the room temperature.

Subsequently, it was clamped between two standard, side by side diffusion cells

with the epidermal side facing the donor chamber (Figure 2). The volume of both

cells was 3.3ml and the transport area 0.78cm2. Silver-silver chloride electrodes

(16; 21) were placed in both chambers. A constant current of 0.4mA was applied for

six hours using a Kepco APH 1000DM (Flushing, NY) power supply, such that the

donor chamber had a higher potential. Both chambers were magnetically stirred

throughout the permeation experiment. The receptor solution contained 0.9% NaCl

solution to provide the main counter-ion, Cl–. The whole receptor solution was

removed and sampled hourly. The unbuffered donor solution (see Table 1 and

Table 2) was replaced with fresh one every two hours (unless otherwise stated) to

provide enough Cl– ions for correct electrode electrochemistry.

Figure 2 The setup for iontophoretic experiments consists of the side-by-side diffusion cell, with the skin clamped in between, stratum corneum facing the donor solution. The constant current is applied through two Ag/AgCl electrodes. Whole volume of the receptor solution is sampled hourly and analysed by HPLC for selegiline and paracetamol concentration.

Single-ion experiments. Selegiline concentration effect

In this study the impact of selegiline hydrochloride concentration on

transdermal transport was investigated. Three selegiline concentrations (30mM,

90mM and 120mM) were tested for transdermal delivery. The pH of the donor

solution was 4.4±0.2, which assured total ionisation of the drug (pKa=7.4). 15mM


47

paracetamol was also added to the donor solution as a marker of electroosmosis.

Current intensity of I=0.4mA was applied.

Effect of competing ions

Iontophoresis was performed from solutions containing selegiline and

sodium ions. 15mM paracetamol was also used as a marker of electroosmosis. In

the first series of experiments, the molar fraction of selegiline was kept constant

(10, 50, 75%) while the total cation concentration in donor solution was varied. In

second series, donor solutions containing various selegiline molar fractions (10-

75%), with the total cation molarity constant (120mM), were investigated. Table 1

summarizes the donor solutions tested.

Table 1 Composition of the donor solution (in rows), used in experiments with background electrolyte. *Number of replicates for selegiline/paracetamol data.

SEL Mole Fraction

Total Molarity

[mM] SEL Conc.

[mM] NaCl Conc.

[mM] Paracetamol

Conc. [mM]

pH N*

75% 40 30 10

0 4.56/0

120 90 30 6/0

50% 60 30 30 0

4.517/0

120 60 60 15 12/11

25% 120 30 90 0 4.6 10/0

20% 100 20 80 0 4.7 5/0

10% 50 5 45 0

4.86/0

120 12 108 15 12/12

Effect of the pH

The effect of the pH of donor solutions was studied in two series of

experiments: for single ion situation; and for binary combination with sodium. For

the first series, the donor solution contained 30mM of selegiline on its own. The

second series contained 60mM selegiline and 60mM of NaCl. The pH of the

solutions was adjusted to a series of values with 0.1M HCl. Table 2 outlines the

exact compositions of donor solutions used in experiments.


48

Current intensity effect

The effect of the applied current was investigated in a set of single-ion

experiments. The concentration of selegiline ions was 30mM, and the pH was 4.8.

Paracetamol was introduced to the donor in concentration of 15mM. The selegiline

flux was measured for 0.1, 0.2 and 0.4mA current intensity, and the passive control

experiment.

Passive control

Passive flux of selegiline was assessed using the same experimental setup,

except that no current was applied and that the donor solution, containing 30mM

selegiline HCl, was not replaced. The entire receptor solution volume was sampled

every two hours.

Table 2 Formulations (in rows) used in iontophoretic experiments to investigate the role of pH in selegiline delivery.

SEL Molar Fraction

Total Molarity

Selegiline Concentration

Na+ Concentration

Paracetamol Concentration pH n

100% 30mM 30mM 0

0 2.0 6 0 3.0 6

15mM 3.5 6 15mM 4.0 6

0 4.5 12 15mM 4.8 6

50% 120mM 60mM 60mM 15mM 3.0 6 4.0 12 4.5 6

Electroosmotic control

To assess the reference electroosmotic flow, in absence of selegiline,

iontophoresis was carried out with donor solutions containing 120mM NaCl and

15mM paracetamol at the pH 4.5 and 6.5. Whole receptor solutions were sampled

hourly and tested for paracetamol content.


49

HPLC analysis Selegiline

Selegiline was quantified using a Jasco HPLC system (composed of PU-980

pump, autosampler AS-1595, and UV-VIS detector UV-975) by means of the

selegiline external standard calibration curve in corresponding media, of at least

seven standards, ranging from 20µM to 700µM of selegiline. Isocratic separation

was performed using Dionex Acclaim™ 120, C18 HPLC column (150 mm x 4.6

mm, 5µm). The mobile phase consisted of 55% acetonitrile, 45% H2O, spiked with

200µl/l of triethylamine and acidified to pH=2.5 with perchloric acid. The flow rate

was set to 1 ml/min; the column oven (JetStream 210) temperature was 20C; and

the injection volume was set to 5µl. In some experiments, where the selegiline

concentration was particularly low, the injection volume was increased to 50µl, and

the corresponding calibration curve was done, ranging from 0.5µM to 50µM

selegiline. UV detection was performed at 204nm. The limit of detection was 0.1µM

0.01µM and the limit of quantification was accepted as 1µM and 0.1µM,

respectively, for the 5µL and 50µL injection volume. The precision and accuracy of

the method was assessed as 3% and 7% respectively.

Paracetamol

Paracetamol was quantified using the same HPLC system with the same

analysis parameters except for: the injection volume was set to 10µl; the mobile

phase consisted of 5%AcN and 95%H2O with 200µl/l triethylamine acidified to the

pH=2.5 with perchloric acid; UV detection was performed at 243nm. The limit of

detection was 10nM and the limit of quantification was 100nM. The method

precision and accuracy was assessed as 2% and 4% respectively. The range of

calibration curve standards was from 1µM to 50µM.

Determination of the distribution coefficient of selegiline

Ten milligrams of selegiline HCl was added to a) 4mL of water and b) 4mL

0.025M HCl solution. Then, 1mL of n-octanol was added to both solutions.

Subsequently, the mixtures were shaken for 48 hours to achieve equilibrium at

room temperature. After that time the mixtures were left for 5min to separate,

collected and analysed by HPLC. The pH of water phases was measured. The

distribution coefficient was calculated as in Equation 1 (22).


50

Equation 1 water

oloc

CCD tanloglog =

LogP was estimated using Equation 2 (22).

Equation 2 )101log(loglog pHpKaPD −+−=

Conductivity measurements and selegiline water mobility estimation

The specific conductivity of a range of selegiline water solutions (10, 5, 2, 1,

0.5mM) was measured using a conductimeter Metrohm T-120 at 22C. The

cell-constant of the probe (K) was 0.85. The values of molar conductivities (specific

conductivity normalized by concentration) were calculated and plotted against

square root of concentration. The molar conductivity at infinite dilution was

estimated by extrapolating the curve to concentration of 0 (infinite dilution

conductivity). Subsequently, using Kohlraush law (Equation 3) of independent ion

migration (with mobility of chloride ion taken from literature (23)) the water mobility

of selegiline ion was calculated as in Equation 4. Next, selegiline water transport

number was estimated using Equation 5 (24).

Equation 3 −∞

−+∞

+∞ Λ+Λ=Λ CC

Equation 4 −−Λ

=∞

Clsel Fµµ

Equation 5 −+

=Clsel

selselt µµ

µ

To validate the method, the mobility of sodium ions was assessed. The

obtained value of infinite-dilution molar-conductivity for NaCl was

128.0±0.5·10-8Si·m2/mol, which is only around 1% different from the literature value,

(126.4·10-8Si·m2/mol) (23) proving validity of our approach.

Data analysis and statistics

Statistical analysis was carried out using Prism 5.0 (GraphPad Software,

San Diego, CA, USA). Statistical differences within multiple data sets were


51

assessed by a non-parametric Kruskal-Wallis test, followed by Dunns multiple

comparison sets. For binary data sets, a two-tailed, unpaired t-test was employed.

The level of statistical significance was fixed at p < 0.05. The reported fluxes for

each replicate were obtained as an average value of the three final hours of

experiment. Transport numbers were calculated using measured fluxes and

equation t#=J·z·F/I, where J is drug transdermal flux, z – drug valence, F – faraday

constant, I – current intensity, and t# – transport number.

Results and Discussion

Single ion experiments

The primary aim of this study was to determine the highest transdermal flux

of selegiline and its corresponding maximal transport number, during constant-

current transdermal iontophoresis. According to the Nernst-Planck theorem (25),

the optimum approach to achieve the maximal delivery, is when the delivered ion is

the only ion of its kind, in the donor compartment. In such case, it competes for the

charge carrying only with counter ions from subdermal compartment; and its flux is

independent of concentration in donor. The validity of this model has been verified

for several small inorganic ions (26), but only few large organic molecules were

examined. It has been shown for some of the organic molecules, like lidocaine or

quinine (27), that single ion delivery flux did not depend on drug donor

concentration. For some others, like propranolol (27), it did. It was then important to

investigate, whether selegiline flux is a function of its donor concentration.

Figure 3 represents the selegiline and paracetamol fluxes corresponding to

the series of experiments, containing 30-120mM selegiline as a single ion. It was

clear, that a four fold increase in concentration did not produce a significant change

in flux. Although Kruskal-Wallis analysis of variance, followed by multiple-

comparison Dunn’s test, found statistically significant differences between the

delivery from 90mM and 120mM selegiline solutions, fluxes do not exhibit any clear

trend, as the lowest flux value was observed for a 90mM solution. It is also

noteworthy, that no statistical differences were noted between 30mM and 120mM

solutions.


52

30mM n=16/6 90mM n=22/6 120mM n=11/60.0

0.5

1.0

1.5

2.0

0

5

10

15

Selegiline concentration

Sele

gilin

e Fl

ux [µ

mol

/h]

Paracetamol Flux [nm

ol/h]

0 1 2 350

70

90

110

130

SelegilineNaCl

Λ∞NaCl

Λ∞Sel

C1/2 [mM]1/2

Mol

ar C

ondu

ctiv

ity[S

i*cm

2 /mol

]

Figure 3 The effect of selegiline concentration on its own and paracetamol iontophoretic transport. Values are average with standard deviations for 4-6th hour flux. Number of replicates is given as selegiline replicates/paracetamol replicates.

Figure 4 Molar conductivities of selegiline hydrochloride and NaCl as a function of square root of concentration. The Y intercepts of plotted curves are the infinite-dilution conductivities of NaCl and SEL: Λ∞

NaCl=128.0±0.5·10-4Si·cm2/mol, Λ∞

Sel=105.1±0.5·10-4Si·cm2/mol.

Such changes of flux may be due to several factors. One of the most likely

mechanisms to influence the transport is the alteration of electroosmosis. Namely,

high concentration of positively charged drug ions may cause the drug molecules

partition to stratum corneum and interact closely with the negatively charged skin

proteins. This could neutralize the skins negative charge and attenuate

electroosmosis, thus limit the transport. To evaluate the electroosmotic effect in

selegiline delivery, paracetamol was added to formulations as a marker of

electroosmosis and its fluxes were measured (Figure 2). With minimal passive

penetration and a pKa of 9.5, which assures its neutral character at the investigated

pH, it can be only transported by electroosmosis. Statistical analysis with Kruskal-

Wallis test found no differences in paracetamol penetration from the three

formulations. Thus, change in electroosmosis cannot explain the differences in

selegiline fluxes for different selegiline concentrations. Another factor, that could

influence the selegiline are minor changes in the pH of the donor solutions. The

more concentrated selegiline formulations had lower pH (4.2) than the diluted ones

(4.5). If that was the case, however, the clear trend of rising or falling flux with pH

should be observed. Finally, the factor, that was not identical in every experiment,

and that can have the effect on the selegiline transdermal transport, is the skin

itself. The variability due to the skin inhomogeneity could produce the changes in

drug flux, as shown on Figure 3. Similar variability in single-ion delivery was

observed before, in several studies (28; 29).


53

Selegiline water mobility

Recent work by Mudry et al. (30), suggests the use of ion mobility in water as

a predictive factor for maximal transdermal transport number. It was demonstrated,

that there is a strong positive correlation between transport number measured in

water and in the skin (26). Selegiline ionic mobility in water was estimated using

conductivity measurements. The value of the infinite-dilution molar conductivity of

selegiline hydrochloride was obtained from the plot on Figure 4. The selegiline ionic

mobility in water, µSEL+=3.0·10-8m2/V/s, was calculated as in Equation 3 and

Equation 4, with chloride ion mobility µCl-=7.9·10-8m2/V/s taken from the literature

(23). Selegiline water transport number, tw=27%, computed as in and Equation 5

appeared 2.7 times higher than experimental transdermal transport number:

10.1±1.41%. This suggests that in the case of selegiline, water mobility is a poor

predictive factor for transdermal transport number. The correlation introduced by

Mudry has however purely empirical character; it is then unclear what are its

limitations or other implicated factors. Table 3 represents physicochemical

properties of several drug molecules of similar molecular weight. Although

selegiline has the highest water mobility, it is clear that it is not the most efficient

charge carrier. This observation suggests, that factors other than water mobility

play an important role in iontophoretic transdermal transport of molecules of this

size.

Table 3 Physicochemical properties of drug molecules with corresponding transdermal transport number. t#TD – transdermal transport number.

Drug Mw LogP Water

mobility [108·m2/V/s]

t#TD

Lidocaine (30) 234 2.3 1.5 0.16

Propranolol (30) 259 3.2 1.5 0.03

Quinine (30) 324 3.4 0.9 0.02

Ropinirole (28) 262 3.3 1.6 0.10

Selegiline 187 3.6 3.0 0.10

Hydromorphone (29) 285 0.3 – 0.10


54

Co-ion delivery

The secondary objective was to determine, whether the selegiline delivery, is

a linear function of selegiline molar fraction in a binary formulation, and also, to

investigate if the nominal drug concentration in such formulations, has an effect on

drug transport rate.

Recent reports have shown that iontophoretic delivery of some drugs, like

lidocaine (31) or hydromorphone (29), is proportional to the drugs mole fraction in

donor solution, while the nominal drug concentration in donor is of less importance.

On the other hand, it was reported that iontophoretic transport of ropinirole does not

follow this pattern. Namely, in work of Luzardo-Alvarez et al. (28) ropinirole delivery

varied with the total ion concentration, for constant mole fraction of the drug in

donor. In case of ropinirole the explanation invoked was the limitation of

electroosmosis by high drug concentration; while for lidocaine, the predominant role

of electromigration in electroosmotic transport.

It was then important to determine the factors governing transdermal

iontophoretic delivery of selegiline during co-ion delivery. In the first subset of

experiments, the [SelegilineH+]/[Na+] ratio was kept constant, while the total

molarity was modified. The results are presented in Figure 5. For none of the

constant [SelegilineH+]/[Na+] molar ratio values (10, 50, 75%), has t-test found a

significant difference in selegiline transport between various total molarities.

According to Equation 6 and Equation 7, electromigration is dependent on the ionic

relative concentrations. These experiments might suggest that selegiline transport

is mainly governed by electromigration. Namely, if selegiline was transported by

electromigration, then according to Equation 6 and Equation 7, its transport number

would be dependent on relative concentrations of ions in the donor solution, what is

shown on Figure 5.

Equation 6 IFztJi

ii =

Equation 7

∑∑ ⋅⋅

⋅⋅==

jjjj

iii

jj

ii ZµC

zµCtσσ


55

50 120 60 120 40 1200.00

0.25

0.50

0.75

1.00

1.25

10%50%75%

Selegilinemole fraction

Total cation concentration [mM]

Sele

gilin

e Fl

ux [µ

mol

/h]

Figure 5 Comparison of selegiline fluxes for different selegiline concentrations and the same selegiline/sodium mole fractions.

These experiments established, that for binary ion combinations selegiline

molar fraction, and not its nominal concentration, is the key parameter determining

the drug flux. The next step was to verify whether the flux mole fraction dependency

has a predictable character. This would be most convenient, to be able to predict

the drug’s maximal transport-number on the basis of the transport numbers

obtained only for small mole fractions. Physicochemical properties of some drugs

(ex. negligible water solubility) do not allow for single-ion delivery, what makes the

measurement of their maximal transport number very difficult.

The selegiline fluxes were studied for a series of mole fractions, from 10% to

75% (Figure 6a). Total cationic concentration was kept constant at 120mM.

Following the approach of Mudry et al. (32) the data was analysed by

approximating the points on the plot by a straight line. This approach makes no

assumption on the mathematical dependence of iontophoretic flux of a drug on its

mole fraction in donor, and is only a way to predict the flux for the single ion

situation. The obtained straight line (R2=0.68, p<0.05), allowed for the accurate

prediction of selegiline flux for 100% molar fraction – 1.42±0.15 µmol/h (transport

number of 9.5±1.0%). This compares well with the experimental value of

1.26±0.32 µmol/h (transport number of 8.5±2.1%), measured for the selegiline in

single ion experiment.

The behaviour of flux as a function of donor composition can be interpreted

as an indicator of what processes take predominant role in drug transport. While

keeping a total molarity of donor solution at 120mM, selegiline mole fraction was

varied from 10 to 75%. The transdermal fluxes of paracetamol and selegiline were

studied. In Figure 6b it is visible, that the rising selegiline content in donor produces

sharp drop of paracetamol flux. Even the smallest 10% selegiline mole fraction


56

causes the significant 3.5 drop in the transport of electroosmotic marker, as

compared to the flux in absence of the drug. Given, that the drug flux increases,

these observations further suggest the minor role of electroosmosis in selegiline

transdermal transport.

0% 25% 50% 75% 100%0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

0%

2%

4%

6%

8%

10%

Selegiline Mole Fraction

Sele

gilin

e Fl

ux [µ

mol

/h]

Selegiline Transport Num

ber

0% 25% 50% 75% 100%0

20

40

60

0

1

2

3

4

Selegiline Mole FractionPa

race

tam

ol F

lux

[nm

ol/h

]

Volume Flow

[µl/h]

a b

Figure 6 a) Influence of selegiline mole fraction on the drug transdermal transport. Total ion concentration was equal to 120mM, pH≈4.6. Values are means with standard deviations (n>6). Regression curve (p<0.05, R2=0.68) is extrapolated to 100% selegiline mole fraction. “” represents the value predicted by the curve for 100% selegiline (nudged by 2%) and “” is experimentally obtained value for 100% selegiline. Dotted lines limit the 95% confidence band for regression line. b) Paracetamol flux (AV±SD) as a function of selegiline mole fraction in donor solution. Total cation concentration is 120mM.

A similar inhibiting effect of drug donor concentration on the electroosmotic

flow was observed before for ropinirole (28), lidocaine, quinine and propranolol (31).

It is interesting, that the least lipophilic drug, lidocaine (logP=2.26) limited mannitol

electroosmotic effect only at concentration of 100mM, while for more lipophilic

quinine and propranolol (logP equal 3.5 and 3.2 respectively) 10mM concentration

had the same effect. Selegiline was then assessed for lipophilicity and the logP

value obtained was 3.6. The fact that selegiline is more lipophilic than lidocaine

might explain why it limits the electroosmotic flow at lower donor concentrations.

This is supported by the study of Tashiro et al. (33), who have found that the

compounds of high lipophilicity have much higher absorption in the skin, than the

hydrophilic ones. Also, these observations stay in accordance with the work of

Hivronen and Guy (34), investigating the impact of lipophilicity of several β-blockers

on their transdermal transport. The experiments has shown that, at concentration of

1mg/ml, the most lipophilic drugs strongly inhibit the electroosmosis (measured

from-anode-to-cathode direction), as opposed to the least lipophilic substances,

that did not inhibit the electroosmotic effect at all. Furthermore, the increasing donor


57

concentration of every β-blocker caused drop in the flux of electroosmotic marker,

similarly to selegiline (Figure 6b)

The pH effect

The aim of this set of experiments was to investigate the effect of the pH of

the selegiline formulation on the drug’s transdermal transport, and elucidate the pH

value for maximal delivery. First, it has to be mentioned that, for 99% of selegiline to

be ionised, the pH of the donor solution cannot exceed 5.4 (selegiline pKa=7.4).

Another limiting factor is the water solubility of selegiline, which at pH 5.9 is

7.2±0.2 mM. To assure the total selegiline ionisation as well as sufficiently high

selegiline HCl concentration, assuring good electrochemistry at the electrodes, the

pH range of 2.0 to 4.9 was selected to investigate. The pH of donor solutions was

adjusted with 0.1M HCl to the selected values (Table 2), and then iontophoretic

experiments were carried out. Figure 7 represents the corresponding selegiline (a)

and paracetamol (b) fluxes measured.

2 3 4 50.0

0.5

1.0

1.5

a pH

Sele

gilin

e Fl

ux [µ

mol

/h]

2 3 4 50

5

10

15

20 100%50%

b pH

Para

ceta

mol

Flu

x [n

mol

/h]

Figure 7 Selegiline (a) and paracetamol (b) flux as a function of pH. Values are plotted as average (over last three hours of experiment) flux with standard deviations for two situations: selegiline is the only cation in donor solution (100% mole fraction) and its concentration is 30mM, selegiline/NaCl mole fraction is 50% and total ion concentration is 120mM. The plot for 50% mole fraction on figure a) is nudged by 0.1pH unit for better visibility.

In the pH range, from 4.9 to 4.0, selegiline average fluxes are relatively

stable, for both 50% and 100% mole fraction. A significant drop of selegiline flux is

visible from pH 4 to 3. In terms of paracetamol fluxes it is similar, except for 50%

mole fraction where the flux of the electroosmotic marker drops considerably from


58

pH 4.5 to 4.0. A similar effect was observed before (27; 35) and explained by the

reduction in electroosmosis and competition of hydronium ions.

The direction and magnitude of the electroosmotic flow depend

predominantly on the charge fixed on the skin (IPskin≈4.3). Decreasing the pH below

the value of pig skin’s IP causes the neutralisation and reversal of the charge fixed

on it, changing the direction of electroosmotic flow, hence the drop in paracetamol

and selegiline flux in the anode to cathode direction. The limitation of electroosmotic

flow certainly explains the drop of the paracetamol flux, which as a neutral molecule

is transported mainly by this mechanism. However, it is not evident, whether it can

account for the total reduction in selegiline flux.

As discussed before, electroosmosis most probably does not play major role

in selegiline transport. In previous set of experiments, presented at Figure 6b, it is

visible that the electroosmotic flow for 100% selegiline mole fraction at pH=4.9 is

reduced 7.3 times in comparison to the control experiment (no selegiline,

100% NaCl) and for 4.6 times for 50% mole fraction. This could mean that when

selegiline is present in donor as a single ion, or in high molar proportion, it already

significantly limits the electroosmotic flow through the skin, regardless of the pH.

Further reduction due to the drop of the pH, visible on Figure 7b, might not be of

great importance. Also, it is worth to compare paracetamol and selegiline fluxes at

Figure 7 for 50% series. It is clear, that while paracetamol flux drops from pH 4.5 to

4.0 there is no corresponding decrease in selegiline flux.

Although it is hard to completely separate the effects of electromigration and

electroosmosis, above observations further support the suggestion that

electroosmotic effect for selegiline is relatively small.

Another explanation for the drop in selegiline fluxes at a low pH can be the

competition from H+ ions. However, at pH 3, their concentration is only 1mM, which

might seem quite low in comparison to 30mM selegiline. Nonetheless, hydronium

ions are highly mobile (water mobility µH+=35·10-8m2/Vs in comparison to

µSEL+=3.0·10-8m2/V/s) and according to Equation 3 can carry large portion of

current, reducing drug transport.


59

Current intensity effect

Subsequently, a set of experiments was devised to test, whether it is

possible to control the selegiline flux by current manipulation. The donor solution

was always 30mM selegiline hydrochloride and 15mM paracetamol. Iontophoresis

experiments were carried out using different current intensities (0.1mA, 0.2mA,

0.4mA) and a passive experiment was performed as a control. The results,

summarized in Figure 8, show a linear relationship between the flux and applied

current (R2=0.99, p=0.05), as predicted by Equation 6. This suggests that simply by

adjusting the current it is possible to change selegiline transport in a controllable

manner. Selegiline delivery for a current intensity of 0.4mA was 1.35±0.27µmol/h,

which ensures sufficient drug delivery even with as small transport area as used in

experiment (0.78cm2). The value of y intercept represents selegiline passive

penetration of 0.022±0.02µmol/h/cm2.

0.0 0.1 0.2 0.3 0.40.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Current Intensity [mA]

Sele

gilin

e Fl

ux [µ

mol

/h]

Figure 8 Effect of current intensity on selegiline transport as a single-ion. Values are the average ± SD for last three hours of experiments. Dotted lines represent the 95% confidence interval. P<0.0001, R2=0.84.

Furthermore, the plot of selegiline flux against the applied current can be

used as an alternative way to estimate the selegiline transport number. According

to Equation 6, the transport number of selegiline can be obtained from the slope of

the curve on Figure 8 (slope a = t# / F·z = 3.30±0.15), after appropriate conversion

of units. This yields the value of transport number t#=8.85±0.40%. This value is

lower (not significantly) than an average transport number calculated for each data


60

point separately: t#=9.30±1.67%. The latter is in fact a transference number, that is

the number of moles of molecule per mole of electrons transported. It includes also

the neutral part of drug species transported via electroosmosis and the input of

passive diffusion, hence it is slightly higher.

Conclusions

Selegiline is relatively good charge carrier and the transdermal iontophoresis

of selegiline HCl can easily achieve clinically relevant rate of delivery, even with

very small transport area. The drug transdermal flux is a linear function of current

intensity, and allows drug input profiling. The highest transport number is

predictable with good accuracy from its linear dependency on selegiline mole

fraction in the donor, and can be achieved from low concentration solutions. The

electromigration is suggested as a predominant process ruling selegiline transport

with limited role of electroosmosis. The pH range of the donor solution, which

allows efficient selegiline delivery, is limited from pH 4.0 to pH 5.0. This limit is

determined by H+ ions competition, and selegiline pKa assuring high ionisation and

solubility.


61

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62

9. Symptomatic effect of selegiline in de novo parkinsonian patients. Allain, H, Pollak, P and Neukirch, H C. Suppl. 1, 1993, Movement disorders, Vol. 8, pp.

S36-S40.

10. Monoamine oxidase B inhibitors for early Parkinson’s disease. Macleod, A D, et al. 2005, Cochrane Database of Systematic Reviews, Vol. 3, pp. 1-38.

11. Comparison of therapeutic effects and mortality data of levodopa and

levodopa combined with selegiline in patients with early, mild Parkinson's disease.

Lees, A J, et al. 1995, BMJ, Vol. 311, pp. 1602-1607.

12. Clinical pharmacokinetics and pharmacodynamics of selegiline. An

update. Mahmood, I. 1997, Clinical Pharmacokinetics, Vol. 33, pp. 91-102.



comparison with oral selegiline capsules. Azarro, A J, et al. 2007, Journal of

Clinical Pharmacology, Vol. 47, pp. 1256-1267.



pp. 23-27.






17. Lidocaine iontophoresis versus eutectic mixture of local anesthetics

(EMLA®) for IV placement in children. Galinkin, J, et al. 2002, Anesthesia and

Analgesia, Vol. 94, pp. 1484-1488.

18. Joint Formulary Committee. British National Formulary. 56 ed.

London : British Medical Association and Royal Pharmaceutical Society of Great

Britain, 2008.


63

19. Tetko, I V, et al. ALOGPS 2.1 home page. Virtual Computational

Chemistry Laboratory. [Online] [Cited: 10 May 2006.] http://www.vcclab.org.

20. Potentiometric and spectrophotometric pKa determination of water-

insoluble compounds: validation study in a new cosolvent system. Volgyi, G, et al. 2007, Analytica Chimica Acta, Vol. 583, pp. 418-428.

21. Iontophoretic delivery of amino acids and amino acid derivatives across

the skin in vitro. Green, P G, et al. 1991, Pharmaceutical Research, Vol. 8, pp.

1113-1120.

22. Partition coefficients and their uses. Albert, L, Corwin, H and David, E. 1971, Chemistry Reviews, Vol. 71, pp. 525-616.

23. Young, T F. Electrochemical Information. [book auth.] Bruce H Billings,

et al. American Institute of Physics Handbook. 2nd. New York : McGraw-Hill, Inc,

1963, 5, pp. 5-263.

24. Atkins, P W. Molecules in motion: ion transport and molecular diffusion.

Physical Chemistry. 6th ed. Oxford : Oxford University Press, 1978, pp. 723-759.


Advanced drug delivery rewievs, Vol. 9, pp. 177-199.





and pig skin. Marro, D and Delgado-Charro, M B. 2001, Journal of Controlled

Release, Vol. 70, pp. 213-217.




64






165-172.

31. Contributions of electromigration and electroosmosis to iontophoretic

drug delivery. Marro, D, et al. 2001, Pharmaceutical Research, Vol. 18, pp. 1701-

1708.

32. Prediction of iontophoretic transport across the skin. Mudry, B, Guy, R H and Delgado-Charro, M B. 2006, Journal of Pharmaceutical Sciences, Vol. 111,

pp. 362-367.

33. Effect of lipophilicity on in vivo iontophoretic delivery. II. B-blockers.

Tashiro, Y, et al. 2001, Biological and Pharmaceutical Bulletin, Vol. 24, pp. 671-

677.

34. Iontophoretic delivery across the skin: electroosmosis and its modulation

by drug substances. Hirvonen, J and Guy, R H. 1997, Pharmaceutical Research,

Vol. 14, pp. 1258-1263.

35. Transdermal iontophoresis of rotigotine across human stratum corneum

in vitro: influence of pH and NaCl concentration. Nugroho, A K, et al. 2004,


36. Transdermal delivery of peptides by iontophoresis. Hirvonen, J, Kalia, Y N and Guy, R H. 1996, Nature Biotechnology, Vol. 14, pp. 1710-1713.

37. Symptomatic effect of selegiline in de novo Parkinson’s patients. Allain, H, Pollack, P and Neukirch, H C. Suppl. 1, 1993, Movement Disorders, Vol. 8, pp.

S36-S40.


Chapter II Pramipexole

65

Chapter II Pramipexole Introduction

Pramipexole (PPX) (Mirapex®, Sifrol®) is a

potent, synthetic dopamine agonist, with selective

agonist activity on D2, D3 and some on D4

dopamine receptor subtypes; very limited agonist

properties on D1, D5, or serotonin receptors

subtypes has been reported (1; 2). It is used in

treatment of Parkinson’s disease and restless leg

syndrome (3; 4).

Clinical effectiveness of pramipexole in Parkinson’s disease (PD) has been a

subject of extensive studies. Two large multicentre studies investigated the

efficiency of PPX as monotherapy, in de novo PD patients (5; 6). Both of them have

shown, that pramipexole is effective in initial short and long-term therapy in

comparison to placebo significantly reducing the disease symptoms. Also,

pramipexole therapy of 0.5mg three times daily was compared to

levodopa/carbidopa 100/25mg treatment (7). Despite smaller efficiency than

levodopa in controlling parkinsonian symptoms, pramipexole was shown to induce

significantly less motor fluctuations in comparison to levodopa (28% versus 51%

after 2 years of treatment). As an adjunct therapy with levodopa, pramipexole was

assessed in large, 360-patients, clinical trial (8). Pramipexole reduced the time

spent in "off" periods, ameliorated motor function of patients during "on" and "off"

periods, reduced the severity of "off" periods, decreased disability and PD severity

during "on" and "off" periods, as assessed by the Unified Parkinson Disease Rating

Scale, and permitted for reduction in levodopa dosage. Eventually, PPX

effectiveness was compared to other dopamine agonists. Study of Poewe et al. (9)

compared pramipexole (up to 4.5mg/day) with transdermal rotigotine (up to

16mg.day) and placebo, involving 506 patients with advanced Parkinson’s disease.

Rotigotine and pramipexole were found to be of similar efficacy in reducing the

Figure 1 Molecular structure of pramipexole. Mw=211.4

S

NNH2

NH


66

symptoms of the disease. Another study (10; 11) showed equal efficiency of the

PPX and bromocriptine, however underlined the safer profile of pramipexole.

Several studies investigated pramipexole pharmacokinetic parameters (12;

13). The study on 16 healthy participants revealed, that pramipexole has a half-life

of t½=12.9±3.3h, clearance of Cl=25L/h, and volume of distribution of Vd=486±96L.

Pramipexole was mainly excreted in urinary tract: its renal clearance accounted for

80% of total clearance. The latter was significantly correlated with creatinine

clearance. Bioavailability of oral formulation was F=0.9 (14). Peak plasma

concentration was achieved in 1 to 3 hours and the Cmax linearly depended on the

dose, and ranged from 1.8 to 21.3nM for the doses of 0.5 to 6.3mg respectively (2).

The commonly applied dosage is three times a day, initially at 0.125mg single dose,

up to 4.5mg daily, in three separate doses (4; 15).

In terms of physicochemical properties, pramipexole has relatively small

molecular weight of 211.4Da. It is a weak double-base, with dissociation constants

of pKa1≈5.0, pKa2≈11.0 (secondary and primary amine group respectively, as in

Figure 1, due to mesomeric effect), and log P=-0.2 (16). Pharmacokinetic and

physiochemical properties make pramipexole a good candidate for transdermal

iontophoresis. The benefit from this type of formulation would be a controllable and

steady drug input to the patient’s body; one daily application instead of three daily

oral doses; steady drug levels in plasma. The latter are particularly important in

conditions like Parkinson’s disease, where it is well-established, that in a long term

therapy, varying dopamine activity can lead to very severe side effects. In addition,

iontophoretic pramipexole delivery could permit for drug titration adjusted to the

individual patient’s requirements.

The desired transdermal flux of PPX, needed to achieve the therapeutic

range of the drug in plasma, can be obtained from pharmacokinetic data provided

by Wright et al. (12). At the maximum steady state concentration of Css=25.7nM

(obtained at 1.5mg of PPX, three times daily, measured on the third day) (12; 17),

and with the clearance of Cl=25L/h the flux that would allow to maintain this

concentration steady is J=C·Cl=0.64µmol/h. This value is well within the abilities of

iontophoretic delivery.


67

The aim of this study was to in vitro evaluate pramipexole as a candidate for

transdermal iontophoresis, and to assess the feasibility of delivering therapeutic

doses of this drug. Analysis of several factors potentially affecting iontophoretic flux

(such as pH, presence of background electrolyte, drug concentration in formulation,

or applied current) was performed to explain the mechanisms governing

pramipexole transdermal transport. Finally, selection of the best conditions was

made to indicate the optimal formulation for the best delivery with clinical potential.

Materials and Methods

Materials

Pramipexole base was obtained from Chemos GmbH, Germany, sodium

chloride, silver wire (99.99% pure), silver chloride (99.999% pure) were obtained

from Sigma Aldrich UK, hydrochloric acid and sodium hydroxide, acetonitrile HPLC

grade (AcN), mannitol, n-octanol were supplied by Fisher Scientific UK,

triethylamine was supplied by Acros Organics UK. Deionized water

(resistivity≥18.2MΩ/cm) was used to prepare all the solutions.

Skin preparation

Full thickness porcine skin obtained from a local abattoir was rinsed under



wrapped in Parafilm® and stored at -20°C for no longer than four months. The

dorsal as well as the abdominal side of porcine skin from four different pigs was

used in experiments.

Iontophoretic experiments

Skin sample was thawed at room temperature and clamped between two standard,

side by side diffusion cells with the epidermis side facing the donor chamber.

Volume of both cells was 3.3ml and transport area 0.78cm2. Silver/silver chloride

home-made electrodes were placed in both chambers (18). Constant current of

0.4mA was applied for 6 hours by Kepco APH 1000DM (Flushing, NY) power

supply in the way that donor chamber contained the anode. Both chambers were


68

magnetically stirred throughout permeation experiment. Receptor solution

contained 0.9% NaCl solution, to provide the main counter ion – Cl-, in all

experiments. The pH of the receptor solution was 5.8. Whole receptor solution was

sampled hourly. The donor solution (Table 1 and Table 2) and was replaced with

fresh, every two hours to provide enough Cl- ions for correct electrode

electrochemistry. The specific composition of donor solutions for individual studies

is outlined below in Table 1 and Table 2.


Single ion experiments had pramipexole as the only cation in donor solution.

This study was set up to investigate the effect of a) pramipexole concentration on

its transdermal delivery; b) applied current on pramipexole transdermal transport; c)

the pH on pramipexole transdermal transport. 30mM mannitol was present in all

solutions as a marker of electroosmosis. The detailed donor solution formulations

for this study are presented in Table 1. The initial pH of pramipexole free-base

water-solution was roughly 11.4, and it was decreased to desired values, using the

1M and 0.1M HCl solutions.

Table 1 Donor solutions used in single-ion experiments. Number of replicates, n, is given as pramipexole/mannitol replicates.

PPX Conc. [mM]

Mannitol Conc. [mM] pH

Current intensity

[mA] Average valence n

Concentration effect

15

30 8.0 0.4

1.0 5/5 30 1.0 18/6 60 1.0 12/6 90 1.0 12/6

pH effect 30 30

2.5

0.4

2.0 6/0 4.0 1.9 5/5 5.0 1.5 5/5 6.0 1.1 5/5 7.0 1.0 5/5 8.0 1.0 18/6

Current intensity

effect

30 30 8.0

0.0 1.0 6/0 0.1 1.0 6/6 0.2 1.0 6/6 0.4 1.0 18/6

30 30 5.0 0.0 1.5 6/0 0.2 1.5 6/5 0.4 1.5 5/5


69


Iontophoresis was carried out with pramipexole and sodium ions in donor

solutions. 30mM mannitol was used as a marker of iontophoresis. Total

concentration of positive ions was equal to 30mM. The initial pH of pramipexole

free-base water-solution was roughly 11.4, and it was subsequently adjusted with

1M HCl to 8.0 or to 5.0. Table 2 summarizes the formulations used in these

experiments.

Table 2 Donor formulations used in co-ion experiments. Number of replicates, n, is given as pramipexole/mannitol replicates.

Mole Fraction

PPX conc. [mM]

NaCl conc. [mM]

Total cation conc. [mM] pH

Average PPX

valence

Mannitol Conc. [mM]

Current intensity

[mA] n

10% 3.0 27.0

30 5.0 1.5 30 0.4

6/5 25% 7.5 22.5 11/550% 15.0 15.0 6/0 75% 22.5 7.5 6/0

100% 30.0 0 5/5 10% 3.0 27.0

30 8.0 1.0 30 0.4

5/6 25% 7.5 22.5 6/6 50% 15.0 15.0 6/6 75% 22.5 7.5 6/6

100% 30.0 0 18/6

Passive control

Passive flux of pramipexole was assessed using the same experimental

setup, except for no current was applied, and the donor solution was never

replaced. The original pH of the free pramipexole base was 11.4, and it was

adjusted to 5.0, 8.0 and 11.0 with 1M HCl.

EO control

To assess the reference electroosmotic flow of mannitol, iontophoresis

experiments were carried out with donor solutions containing 30mM NaCl and

30mM mannitol at pH of 8.0 and 5.0 adjusted with 0.01M NaOH and 0.01 HCl.

Subsequently, receptor solution was tested for mannitol content.

To investigate the blank mannitol flow (originating from the skin)

iontophoresis experiment was carried out with donor solution containing 30mM


70

NaCl and no mannitol, at pH of 8.0 adjusted with 0.01M NaOH. Subsequently,

receptor solution was tested for mannitol content.

Analytical Methods

Pramipexole

Pramipexole was quantified using Jasco HPLC system (composed of PU-

980 pump, autosampler AS-1595, and UV-VIS detector UV-975) by means of

pramipexole external-standard calibration-curve in corresponding media, of at least

seven standards ranging from 50µM to 1mM. Isocratic separation was performed

using Dionex Acclaim™ 120, C18 HPLC column (250 mm x 4.6 mm, 5µm) with flow

rate of 1 ml/min and injection volume 10µl with column temperature of 20C. The

mobile phase consisted of 100%H2O spiked with 200µL/L of triethylamine and

acidified to pH=2.5 with perchloric acid. Detection was performed at wavelength of

260nm. Limit of detection was 0.1µM and limit of quantification was accepted as

1µM. The method precision and accuracy was 2% and 4%, respectively.

Mannitol

Mannitol was quantified using Shimadzu LCMS 2010 EV system, with

electrospray ionisation in the following conditions: a gradient separation (H2O:AcN

10:90 to 95:5 in 10min) was performed using a Phenomenex Luna™ HILIC, HPLC

column (150 mm x 4.6 mm, 5µm) with flow rate of 0.5 ml/min and injection volume

10µl with column temperature of 25C. Detection was performed at m/z=218.85 in

negative mode with detector voltage of 2.0kV. Data collection rate was 0.2s. The

quantification was made by means of mannitol external standards, prepared in

corresponding media. The calibration curve consisted of at least nine standards

ranging from 0.1 to 40µM. Limit of detection was 20nM and limit of quantification

was 100nM. The accuracy and precision of the applied method was assessed as

6% and 7%, respectively.

Determination of distribution coefficient of pramipexole

4ml of n-octanol was added to 4ml of 1mM pramipexole solution of pH

adjusted to 8.0 or to 5.0, with 1M HCl. Mixtures were shaken for 48 hours to

achieve equilibrium in 25°C. After that time, mixtures were left to separate for


71

10min, and then water and octanol phases were collected to separate vials. The pH

of water phase was rechecked, and subsequently both phases were analysed by

HPLC for pramipexole content. The Distribution coefficient was calculated from

Equation 1 (19).

Equation 1 water

oloc

CCD tanloglog =

LogP and pKa was estimated using Equation 2 (19).

Equation 2 )10101log(loglog 2211 pHpKapKapHpKaDP −+− +++=

Conductivity measurements and pramipexole water mobility estimation

The specific conductivity was measured for range of pramipexole water

solutions (10, 5, 2, 1, 0.5mM) using conductimeter Metrohm T-120 at 22C with the

probe of cell constant K=0.85. The solutions were made of pramipexole base and

equimolar amount of HCl. The measured pH of prepared solutions was 7.8, what

assured that 99.9% of pramipexole molecules were monovalent. Then, values of

molar conductivities (specific conductivity normalized by concentration) were

calculated, and plotted against square root of concentration. Conductivity at infinite

dilution was estimated by extrapolating the curve to zero. Subsequently, using

Kohlraush law of independent ion migration Equation 3 (with mobility of chloride ion

taken from literature) water mobility of pramipexole ion was calculated (20).

Afterwards, water transport numbers were estimated as in Equation 4 and Equation

5 (20). The mobility of the chloride ion, µCl-=7.9·10-8 m2/s/V was taken from literature

(21).

Equation 3 −

∞−+

∞+∞ Λ+Λ=Λ CC


72

Equation 4 −−Λ

=∞

ClPPX Fµµ

Equation 5 −+

=ClPPX

PPXPPXt µµ

µ


Statistical analysis was carried out using Prism 5.0 (GraphPad Software,

San Diego, CA, USA). Statistical differences within multiple data sets were

assessed by one-way ANOVA followed by Bonferroni multiple comparison sets. For

binary data sets, a two-tailed, unpaired t-test was employed. The level of statistical

significance was fixed at p < 0.05. The reported fluxes for each replicate were

obtained as an average value of the three final samplings. Transport numbers for

each replicate were calculated as in equation t#=J·F/z·I, where J is transdermal drug

flux, F is Faraday’s constant, z is pramipexole valence, and I is the current intensity.

Transdermal flux J, was the average of fluxes for last three hours of experiment.

The data on graphs is presented as mean value of all replicates with standard

deviation. All the regressions were performed using the least square method.

Results and discussion

Primary objective of this work was establishing the feasibility of delivering

therapeutic doses of pramipexole, and investigating the factors that influence the

iontophoretic transport. All the iontophoretic experiments led to great increase in

pramipexole transdermal transport, in comparison to the most efficient passive

pramipexole delivery (62.0±21.9nmol/h/cm2 at pH 11.0).

It has been theoretically predicted, that to achieve the highest flux during

anodal delivery, the delivered drug must be the only cation in donor solution This

was verified for several drugs (22; 23) and small inorganic ions (24). The set of

experiments was established to study the pramipexole flux from single-ion

formulations; and to verify, if the drug concentration has an impact on its


73

transdermal transport, in single-ion situation. Initially, three formulations containing

30, 60 and 90mM pramipexole were studied (Table 1). To determine the input of

electroosmosis to the drug transdermal transport, each formulation contained

30mM mannitol as a marker of electroosmotic effect. The pH of the solutions was

adjusted to 8.0 with 1M HCl to assure pramipexole being monovalent cation (PPX

pKa1≈5, pKa2≈11) (16). The results are presented in Figure 2.

15mM 30mM 60mM 90mM0

1

2

3

Pramipexole FluxMannitol Flux

0

5

10

15

20

25

Pramipexole Concentration

Pram

ipex

ole

Flux

[µm

ol/h

] Mannitol Flux [nm

ol/h]

Figure 2 Pramipexole () and mannitol () delivery from formulations containing different concentration of the drug as a single-ion. Values are means with standard deviations. The pH for all formulations was equal to eight and the current applied was 0.4mA.

Unexpectedly, one-way ANOVA, followed by Bonferroni test, found

differences between drug fluxes from the three formulations; the flux originated from

formulation containing 30mM pramipexole, was significantly higher (p<0.05) than

the fluxes from formulations of 60 and 90mM drug concentration. No difference was

found between the latter two. Potentially responsible, different electroosmotic

contribution, was ruled out, as mannitol fluxes were non-significantly different for all

three formulations (Figure 2). The average PPX flux from 30mM pramipexole

solution was by 10% higher, than the lowest flux in the group, from 90mM

pramipexole solution. The relative standard deviations in both donor solutions were

8.9% and 9.4% respectively. This means, that the differences, however statistically

significant, are relatively small. It has to be mentioned, that the result of one-way

ANOVA statistic might be due to the fact, that the skin samples used in these

experiments were acquired from different pigs. In order to explain, whether

observed differences are a result of variability in the skin, or rather the systematic


74

effect of concentration, another donor solution was tested. The 15mM pramipexole

solution at the pH adjusted to eight, generated fluxes statistically not different to any

other formulation (Figure 2). From this fact it can be concluded, that the

concentration of pramipexole has no effect on its transdermal transport, when

delivered from single-ion formulation, in the range of concentrations investigated.

This seems to stay in good accordance with the theoretical model which predicts,

that transmembrane transport is only dependent on the ratio of membrane

diffusivities of the drug and counter-ion, in single-ion situation. Several experimental

data seems to further confirm presented results. The transdermal flux of ropinirole,

in vitro and in vivo (25; 26), and of hydromorphone (22) was found to be constant

for a span of concentrations, ranging from 2.5 to 250mM and from 10 to 800mM,

respectively for ropinirole and hydromorphone. Also, selegiline fluxes, studied in

previous chapter, were found to be relatively stable over concentration range from

30 to 120mM. Similar effect was found for quinine and lidocaine, however small

effect of concentration was found for propranolol (27).

Mannitol fluxes measured in these experiments were stable for

concentrations from 30 to 90mM, and the value of flux obtained for 15mM is

significantly smaller (one-way ANOVA followed by Bonferroni test, p<0.05). This

indicates that electroosmosis decreases with decreasing molarity. Different

behaviour of electroosmotic flux was observed in previous works (28; 29; 30). Santi

and Guy denoted the drop of electroosmotic marker transport with increasing

molarity of receptor solution, during reversed iontophoresis. Also in the work of

Pikal (28), water transference numbers dropped with increasing square root of

electrolyte concentration of NaCl.

It is also not clear, how the flux of this electroosmotic marker translates to

the flux of the drug. Several papers (27; 31; 32) presented the approach, where the

electroosmotic contribution to iontophoretic transdermal transport of a drug can be

expressed as in:

Equation 6 DrugEO CVJ ⋅=


75

where JEO is the electroosmotic drug flux, CDrug is the drug concentration in donor,

and V is the volume flow. The latter was estimated, using the flux of mannitol, as:

mannitolmannitol CJV = . This approach is based on two assumptions: 1) mannitol and the

drug are transported in identical way by convective solvent flow; 2) that the

transport of both, marker molecule and drug molecule is proportional to their

concentration in donor. The second assumption was tested for, in range of mannitol

concentrations 1-100mM (27). The first one, however, can be criticised, particularly

in case of delivery of large organic molecules. The first condition assumes, that the

relative concentration of drug to mannitol is the same in the skin, and in the donor

solution. Depending on the chemical structure of delivered drugs, some molecules,

in particular the more lipophilic ones, can partition into the skin, which is unlikely for

mannitol. Also, if delivered molecules are charged, they can interact with the skin

differently, than neutral mannitol. Hence, it is likely, that in case of large drug

molecules the transdermal pathways of drug and mannitol might be different.

Moreover, the understanding of electroosmotic flow contribution to the total

flux, as in Equation 6, leads to certain conclusion which seems to be false. That is,

if Equation 6 was true, elevating concentration of the drug in single-ion situation

should result in higher electroosmotic contribution. As the total flux is the sum of

electroosmosis and the electromigration, and the latter does not depend on the

concentration, the raise in total flux should be observed. It has been however

reported for several compounds, that the total drug fluxes are not significantly

different, for donor concentrations differing as much as two orders of magnitude

(22; 27; 33). These observations might indicate that electroosmotic contribution to

total drug flux is not always directly proportional to its concentration in donor

solution. It is possible, that although electroosmosis contributes to the transport of

both, mannitol and pramipexole, the magnitude of this contribution might be

different to each compound.

It is also possible, that electroosmotic contribution is small, in comparison to

total transport and experimental error. It has been shown previously that

electroosmotic effect can be effectively employed in drug delivery. Merino et al. has

shown that 5-fluorouracil (5FU) can be delivered with similar rate by

electromigration and electroosmosis (32). However, the average flux of 5FU was


76

roughly 200nmol/h, what is comparable to the experimental error in pramipexole

delivery. If the electroosmotic effect in pramipexole iontophoresis was similar or

smaller, it would be very hard to detect it at all with employed experimental setup.

For above reasons, in this work it will not be concluded on the extent of

electroosmotic flow as in Equation 6. The changes in mannitol transdermal flow will

rather be understood as limitations or enforcement of electroosmotic effect, without

concluding on the actual magnitude of electroosmotic contribution to the total

transdermal drug flux.

The effect of pH

The following issue was to study the effect of the donor-solution pH on

pramipexole transdermal transport. The limits to the examined range of pH were

established, taking into account the drug pKa values (pKa1=5.0, pKa2=11.0) (16),

and the tolerability of the skin to the applied pH. Apart from the delivery

optimisation, the effect of the pH on drug transport was interesting to investigate, as

pramipexole has two ionisable amine groups (Figure 1) and, depending on the pH

of the donor, its molecule can bare double or single charge. Several values of the

pH were chosen to investigate (Table 1). Varying the pH from 8.0 to 2.5

2.5 4 5 6 7 80

1

2

3

a pH

Pram

ipex

ole

Flux

[µm

ol/h

]

2.5 4.0 5.0 6.0 7.0 8.00.00

0.05

0.10

0.15

0.20

0.25

b pH

Pram

ipex

ole

Tran

spor

t No.

2.5 4.0 5.0 6.0 7.0 8.00

5

10

15

20

c pH

Man

nito

l Flu

x [n

mol

/h]

Figure 3 PPX flux (a), transport number (b), and mannitol flux (c) for different values of pH. The molarity for all formulations was 30mM, the current applied was 0.4mA, and no background electrolyte was present. Values are means with standard deviations.

allowed studying the effect of different average valences of pramipexole, on its

transdermal transport number. Figure 3 summarizes the pramipexole fluxes (a),

pramipexole transport numbers (b), and mannitol fluxes (c) measured.


77

As pictured in the graph a, in Figure 3 that PPX flux decreases when the pH.

The observed change of flux is a result of number of effects: First, the skins cation

permselectivity decreases with the decreasing pH (34). The limitation of a flux in

low pH values has been observed before for a number of compounds (35).

Secondly, particularly for the small values of the pH, the competition of hydronium

ions decreases the drug transport. And finally, the valence of pramipexole rises,

which was shown to have a significant, positive effect on the drug transdermal

transport (31). It is noteworthy, that the increasing valence enhances the transport,

while the decreasing cation permselectivity and H+ competition impede it. All these

processes take place simultaneously, and are not easy to separate.

Figure 4 The pH gradient in the applied experimental setup. The values of pH for the stratum corneum and epidermis were taken from the reference (36).

Let’s assume that the pH in stratum corneum, in steady-state iontophoresis,

is the same as the pH of donor. There are three phenomena involved in

transdermal transport: electromigration, electroosmosis and passive diffusion. The

latter effect is relatively small in the range of pH applied (Equation 7); hence the two

remaining processes will be used to explain the results obtained (Figure 3). For the

two smallest pH values, 4.0 and 2.5, sharp fall of both, PPX flux and transport

number, as well as limitation of mannitol transport was observed, in comparison to

higher pH values. First, it has to be noted that the concentration of H+ ions is

becoming significant at these pH values (0.1 and 3.2mM, respectively). It is still


78

relatively small in comparison to PPX concentration, but with very high ionic mobility

(water mobility µH+=35·10-8m2/sV), H+ ions, even in small concentration, can carry a

large portion of charge (21). Then, with the pH below the value of isoelectric point of

the pig skin (iP=4.3), the charge fixed on the skin changes, from negative to

positive. This results in switch in permselectivity of the skin, from the positive to the

negative ions (25), which can contribute to limitation of the PPX flux. The restraint

electroosmotic flux from-anode-to-cathode direction is represented by drop of

mannitol flux at Figure 3c.

Further increase of the pH to 5.0, results in a dramatic rise of both, PPX flux

and transport number, while the mannitol transport remains unchanged. With

negligible competition of H+ ions at the pH 5.0, the flux increase can be attributed to

change in the skin permselectivity acting in favour of cation (PPX+) transport

(iPskin=4.3). Furthermore, at the pH 2.5 and 4.0 most of the PPX molecules are

double charged. In the work of Abla et al. (31), it has been shown, that the

increasing valence of delivered dipeptides from one to two, doubled its transdermal

flux. It is then noteworthy, that despite of negative effect of diminishing PPX

average charge (zpH4=1.9, zpH5=1.5), the flux has increased due to the enhanced

permselectivity of the skin and the minimal H+ competition. Similarly, the same flux

for the pH 5.0 and 6.0, can be explained by the effects of reduced average PPX

valence (zpH5=1.5, zpH6=1.1) and increasing negative charge on the skin, mutually

cancelling each other out. Subsequently, from pH 6 to pH 8 the PPX valence

changes only slightly, its flux rises which might be due to gradually growing

negative charge of the skin and cation permselectivity.

It must also be noted, that the pH of donor solution might not have the same

value, as the pH inside the skin. This is an important observation, as the pH

determines the valence of the delivered drug. The latter, in turn, decides upon the

cation transport number in single-ion situation, which is defined as:

Equation 7 −−−+++

+++

+=

CzCzCztµµ

µ#


79

where µ, z, and C stand for ion mobility, valence, and concentration inside the skin;

while subscripts “+” and “-” refer to cation and anion. In all the experiments, the pH

of receptor solution was 5.8; this implies that for different pH values of donor, a

gradient of pH would occur through the skin. The ionisation degree of pramipexole

molecules is determined by the pH. It is then possible, that the valence of

pramipexole inside the skin is different from its valence in donor.

Another interesting observation from Figure 3b, is that the PPX transport

number changes only slightly in range of pH from 5.0 to 8.0. In fact, one way

ANOVA followed by Bonferroni multiple comparison test found no differences

between PPX transport number measured at pH 5.0, pH 7.0 or pH 8.0 (p<0.05),

and for the pH 6.0 the significant difference was only found in comparison to the pH

8.0. The considerations regarding transport numbers are similar, to those regarding

fluxes, however valence is of a double importance. Whit pH rising from 5.0 to 8.0

average valence drops from 1.5 to 1.0. In according with Equation 8, this impedes

the flux Ji, (Figure 3a), and furthermore the PPX molecules carry less charge zi,

which limits the transport number. This is yet compensated by the increase in skin

cation permselectivity and higher PPX flux.

Equation 8 i

iii

i

ii I

zFJtIzFtJ ⋅⋅

=⇔⋅

=

From the fact, that PPX transport number at pH 5.0 is equal to that at pH 8.0

some conclusions regarding the electroosmotic contribution can be drawn. In an

attempt to quantify electroosmotic contribution, lets employ obtained results in

following case (Figure 5). Assuming, that in stratum corneum the pH is equal to the

pH of donor, there are but the single-charged pramipexole ions at pH 8.0; and there

is equal amount of double and single-charged PPX ions at pH 5.0. The

concentration of PPX ions in stratum corneum at pH 8.0 is C1, while concentration

of double and single-charged ions at pH 5.0 is C2= ½·C1. The average speeds of

particles, due to the electric field E, are V1 and V2, for monovalent and bivalent

particles respectively. Also, it is assumed, on basis of mannitol data from Figure 8c,

and the data from Marro et al. (34), that at the pH 5.0 the net bulk solvent flow is


80

zero; and at pH 8.0 the net bulk solvent flow is directed along the electric field. The

velocity of the bulk solvent flow is VBF (28).

Figure 5 Movement of pramipexole ions in stratum corneum, for two different cases: at pH=5 and at pH8. E stands for electric field; V1, V2. VBF are the velocities of monovalent, bivalent pramipexole molecules and bulk volume flow, respectively.

The pramipexole transport number and flux for the pH 5.0 can be then expressed

as in Equation 9 and Equation 10:

Equation 9 JVCzVCzt pH 1212225

#⋅⋅+⋅⋅

≡

Equation 10 12225 VCVCj pH ⋅+⋅=

where 5#pHt is PPX transport number at pH 5.0; J is the total current density passed;

5pHj is the PPX flux; z1 and z2 valences of pramipexole equal 1 and 2, respectively.

At pH 8.0, relevant expressions for transport number and flux are:

Equation 11 ( )J

VVCzt BFpH +⋅⋅≡ 1118

#


81

Equation 12 ( )BFpH VVCj +⋅= 218

where 8#pHt and 8pHj are PPX transport number and flux respectively. Furthermore,

from the experimental data it is known, that with the accuracy of the experimental

error:

Equation 13 5

#8

#pHpH tt =

Equation 14 5.1/ 58 =pHpH jj

If we accept, that the input of electroosmosis is due to bulk volume flow, occurring

through the skin with net velocity VBF, the value of BFBF VCj ⋅= 1 would correspond

to the input of electroosmosis to the total flux. The set of Equations 9 to 14, allows

working out the relative values of V1 and VBF as BFVV ⋅= 21 . Then, the contribution of

electroosmotic flow can be represented as 58 pHpHBF jjj −= . This means, that

electroosmosis is entirely responsible for the increase of flux, from pH 5.0 to 8.0 at

given conditions.

To deeper investigate the influence of PPX valence on its transport number,

the impact of the applied current on transdermal pramipexole flux was studied.

Iontophoretic experiments were carried out for two series of donor solutions, at two

different pH values (Table 1). For the first one, the pH was adjusted to 8.0 to assure

PPX single ionisation, while for second series the pH was adjusted to 5.0 for the

average charge. Figure 6 depicts the obtained results.


82

0.0 0.1 0.2 0.3 0.40.0

0.5

1.0

1.5

2.0

2.5

3.0

αβ

Current intensity [mA]

Pram

ipex

ole

Flux

[µm

ol/h

]

0.0 0.1 0.2 0.3 0.40

5

10

15

20pH=8pH=5


Man

nito

l flu

x [n

mol

/h]

A B

Figure 6 Impact of current intensity on pramipexole (A) and mannitol (B) transdermal flux (AV±SD for last three hours of experiment).

As expected by Equation 8, flux-current relationship is linear for both values

of the pH (r2>0.95, p<0.0001, for both lines). The regressions were found to be

significant (p<0.0001), and the slopes of the lines were statistically different

(p<0.0001):

α µ β µ=⋅

= ± =⋅

= ±tz F

mol h mAtz F

mol h mApH

pH

pH

pH

8

8

5

5

6 44 016 4 20 0 22. . [ / / ] , . . [ / / ]

The corresponding transport numbers are tpH8=17.4±0.6% and tpH5=17.0±0.9%. This

shows, that with tolerance to the experimental error, tpH8=tpH5. Furthermore, it can

be concluded one of the following: 1) the mobility of the pramipexole in the stratum

corneum does not depend on the valence of pramipexole molecules, and is

constant from pH=8 to pH=5 (Equation 5); or 2) higher cation permselectivity of the

skin for pH 8.0 compensates for decreased pramipexole electromigration effect,

keeping the transport number on the same level as for the pH 5.0. Again, it has to

be underlined, that this is only true under the condition, that the pH inside the skin is

equal to that of the donor solution.

Several other studies investigated the role of the pH and drug valence in

transdermal transport. Abla et al. (31) investigated the effect of valence in peptide

iontophoresis. It was demonstrated, that bivalent dipeptides were transported more

efficiently than monovalent ones of similar molecular weight. In particularly, flux of

bivalent peptides H-Tyr-d-Arg-NH2, H-Tyr-Lys-NH2 or H-Lys-Lys-NH2 was

approximately twice higher than the similar monovalent peptide H-Tyr-d-Arg-OH.


83

There are however several major differences in experimental setup applied

by Abla et al. and the study presented here. Firstly, the increase of valence is

attained by chemical modification of peptides, rather than changing the pH, as it is

in case of PPX study. This allows for constant skin permeability in case of peptide

experiments, while in pramipexole ones lowering the pH alters the skin

permselectivity. Secondly, the peptides are delivered from donor solutions

containing high concentration (170mM Na+ and K+) of background electrolyte, while

pramipexole was transported as a single ion. The competition between the peptide

and other donor ions might cause different effect of charge than observed for

pramipexole, which competes only with the counter ion. Tertiary, the compared

peptides are different chemical species and can have different other properties than

valence, which could affect their transport.

Another study (32) investigated the effect of pH on transdermal transport of

5-fluorouracil (5FU). Also in this work, the pH profile of 5FU transdermal transport

looks different to the pramipexole one. The flux of 5FU, when delivered from

cathode compartment, reaches the maxima at pH 8.5 and 3.0, and is almost zero at

pH 6.0. Again here, the experimental setup is quite different to PPX experiments.

Similarly to the peptides from Abla et al. study (31), 5FU was delivered from

solutions containing high concentration of background electrolyte, what limits its

electromigration. The fluxes obtained when delivering 5FU (of around 0.2µmol/h)

are an order of magnitude lower, than pramipexole fluxes (of around 2 µmol/h).

Also, for some conditions applied, 5FU is delivered as a neutral species. In such

case, the electroosmotic effect plays an important role, and in fact at pH 3.0 is the

main transport mechanism for 5FU. On the contrary, for pramipexole

electromigration is the process most contributing to its transport. As an effect, the

transport of 5FU is resultant of electroosmotic flow and electromigration, while the

transport of pramipexole is mainly due to electromigration and is hampered with

decreasing pH, by factors discussed earlier.

Passive delivery

Pramipexole passive delivery has been assessed as a control for the current

effect experiments. It was noted that the magnitude of passive flux for the most

basic formulation (pH 11.0) was relatively high (0.062±0.021µmol/h/cm2). The target


84

iontophoretic flux required to maintain the upper level of therapeutic drug-

concentration in blood, was calculated as:

Css·Cl=26nM·25l/h=640nmol/h=0.64µmol/h. Figure 8 shows that this rate of delivery

can be easily achieved by passive application. The size of the patch required to

achieve the target flux is of around 11cm2, even using very simple passive

formulation like these, applied in passive experiments. Worse penetration of

pramipexole from formulations of lower pH can be explained by higher degree of

pramipexole ionisation. In ionic state, pramipexole molecules become less

lipophilic, and thus are less prone to cross the lipid-rich stratum corneum.

0 1 2 3 480

85

90

95

100

105

110

Λ∝

C1/2 [mM]1/2

Mol

ar c

ondu

ctiv

ity [S

i*cm

2 /mol

]

5.0 8.0 11.00.00

0.02

0.04

0.06

0.08

pH

PPX

pass

ive

flux

[µm

ol/h

/cm

2 ]

Figure 7 Molar conductivity of pramipexole monohydrochloride solutions. The extrapolation of the fitted straight line to C1/2=0 indicates the infinite dilution molar conductivity Λ∞=102.3 Si·cm2/mol. This value is used to calculate the mobility of pramipexole in water as in Equation 5, with the mobility of chloride ion µCl-=7.9·10-8m2/V/s.

Figure 8 Passive pramipexole delivery as a function of pH of donor solution.

Pramipexole water mobility

Recently, it was found that water transport number can be a good predictive

factor for skin transport number (24). Indeed, a strong, positive correlation was

found between the two factors. Pramipexole water mobility was calculated as in

Equation 3, from the conductivity measurements presented in Figure 7. The

pramipexole water mobility obtained was 2.7·10-8m2/V/s, with the corresponding

transport number 0.25, at pH of the solutions equal 7.6. At this pH virtually all the

pramipexole molecules are monovalent. The water transport number is

considerably higher than the pramipexole transport number obtained in transdermal

experiments (0.17 for pH=8.0). This however, can be attributed to the

physicochemical properties of pramipexole. In comparison to the species used in


85

cited model (24), like Na+, K+, Cl- or CH3COO-, pramipexole is more lipophilic, it

limits the electroosmosis (Figure 9c) and can partition to the skin. These factors

might influence its skin transport number making it smaller than the PPX water

transport number, unlike for small inorganic ions.

Co-ion delivery

The goal of these experiments was to investigate the ionic competition

during pramipexole transdermal iontophoresis, and to study the factors implicated in

the transport. The competing ion was always sodium. Sodium was chosen, as it is a

common component of drug formulations, often used as buffering or stabilising

factor. Two series of experiments, for two different pH values (5.0 and 8.0), were

carried out, to look at the influence of valence on the pramipexole co-ion delivery.

According to Equation 7, it is the ionic mobility, valence and concentration

inside the skin, what determines the transport number. Practically, it was shown

that for the drugs like lidocaine or hydromorphone it is the mole fraction that

controls the transdermal transport (22; 23). For others, like ropinirole, the transport

number dependence on donor ionic composition was more complexed (25). There

is very limited information on the impact of valence on the co-ion transport, hence

one series of experiments was performed with monovalent pramipexole ions, while

the second with the equal proportion of mono and bivalent. For both series of

experiments, the range of PPX mole fraction was tested (Table 2). Figure 9 depicts

the fluxes obtained (a) with corresponding transport (b) numbers and mannitol

fluxes (c). It is clearly visible, that similarly to lidocaine (23), pramipexole delivery is

governed by its mole fraction in donor solution. Fluxes for both pH values can be

reasonably well approximated by linear regression (rpH52=0.88, rpH8

2=0.96, p<0.0001

for both regressions). It is noteworthy that the slopes of the lines generated by the

data points are statistically different (p<0.01) and in similar relation, as these in

Figure 6A (αpH8/αpH5=1.54). As a consequence, transport numbers plotted on graph

b in Figure 9, render lines of statistically not different slopes (p=0.49). In the light of

recent report (24), that the slope of transport number as a function of mole fraction

correlate well with the mobility of respective cation, this means that mobility of

pramipexole in stratum corneum at the pH 5.0 is similar to that at the pH 8.0.


86

0.00 0.25 0.50 0.75 1.000.0

0.5

1.0

1.5

2.0

2.5

3.0

a

αpH5

αpH8

Mole Fraction

Pram

ipex

ole

Flux

[µm

ol/h

]

0.00 0.25 0.50 0.75 1.000.00

0.05

0.10

0.15

0.20

b Mole Fraction

Tran

spor

t num

ber

0.00 0.25 0.50 0.75 1.000

50

100

150pH8.0pH5.0

c Molar fraction

Man

nito

l Flu

x [n

mol

/h]

Figure 9 Pramipexole fluxes (a) transport numbers (b) and relevant mannitol fluxes (c). The points for pH 5.0 at figure (b) are nudged by 0.05 along X axis to facilitate visualization.

The Figure 9c represents the mannitol fluxes measured for both series of

experiments. Clear difference in mannitol transport profile for each experiment

series is visible. The mannitol transport measured for pH 8.0 drops gradually to the

value of 15.0±1.4nmol/h, while for pH 5.0 the decrease is much sharper, and flux

drops below this level only at mole fraction of 0.25 to eventually reach value of

3.2±0.4nmol/h. So in fact, two sorts of changes can be observed: the drop of

mannitol flux due to rising mole fraction, and due to dropping pH. Previously, the

limitation of electroosmotic flow was usually explained by close interaction of

relatively lipophilic and positively charged molecules with skin proteins (25; 27). For

this reason the logD value of pramipexole for pH 5.0, 8.0 and 11.0 was measured

and the values obtained were, respectively -3.3, -1.6 and 1.2. This indicates that

PPX, particularly in it ionic form is rather hydrophilic molecule. It is then unlikely that

PPX+ ions accumulate in highly lipophilic stratum corneum, attenuating its negative

charge and limiting the electroosmotic flow.

However, it also can be argued, that pramipexole has certain buffering

properties, and entering the skin changes its pH. As the pKa of pramipexole is of

around five, at this pH its buffering capacity would be the highest. This could

explain much sharper decline of mannitol flux at pH 5.0 than at 8.0 as less amount

of drug would be needed to lower the pH of the skin. Furthermore, the Ip of pig skin

is around 4.3 (34), so at pH 5.0 the expected electroosmotic flux would be smaller

than the one at pH 8.0. It is noteworthy the fact, that the intercept of the linear

regressions for transport number (Figure 9b) are significantly different (p<0.05).

This might reflect the differences between the electroosmotic flows at two pH

values, as they presumably occur at low pramipexole concentration.


87

Increasing mole fraction of pramipexole, similarly to selegiline, reduces the

electroosmotic flow, while the increase in pramipexole flux seems to remain

unaltered Figure 9. This might suggest that electroosmosis does not significantly

contribute to total pramipexole transport. However, as discussed earlier, the

electroosmotic effect seemed responsible for as much as a third of total PPX

transport, for pH 8.0. These contradictory results leave unsolved the issue of the

role of electroosmosis in transdermal electrotransport.

Conclusions

Pramipexole is a very good charge carrier and can be effectively delivered by

means of iontophoresis, even for the very small application area, as used in

experiments described. Possibly, it can be delivered as well passively, this however

requires further study. Pramipexole iontophoretic influx can be easily and accurately

adjusted by current manipulation. When delivered as single-ion, PPX flux is

independent on donor concentration. Electroosmosis input to the total pramipexole

transport remains elusive. PPX effectiveness as a charge carrier is relatively stable

in range of pH from 5.0 to 8.0 and decreases below 5.0; the flux is however the

highest at pH 8.0 possibly because of higher skin cation permselectivity. Lower pH

values limit significantly the drug transport mainly due to the competition of

hydronium ions, reversed skin permselectivity. Addition of the background

electrolyte decreases the pramipexole transport number linearly with the drug mole

fraction in donor. The profile of this decrease is independent from the pH in range

5.0-8.0.

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Chapter III Piribedil

92

Chapter III ‐ Piribedil Introduction

Piribedil (Trivastal® 50mg) is a non-

ergot, centrally acting dopamine agonist

with high affinity to D2 and D3 receptors

(1). It possesses also the antagonist

action at the two adrenergic receptors α2A

and α2C (2; 3). It is used in long term

Parkinson’s disease treatment.

A number of clinical trials have found piribedil to be an efficient

antiparkinsonian agent as both an adjunct to L-dopa treatment, and in monotherapy.

Rascol et al. (4) demonstrated the effectiveness of piribedil in de novo Parkinson’s

disease patients in a double blind, placebo-controlled, multicentre trial. Piribedil

therapy up to 300mg/day appeared safe, well tolerated and effective in similar extent

to other dopamine agonists (ropinirole, pramipexole, pergolide or rotigotine). Other

clinical trials proved the usefulness of piribedil administered concomitantly with L-

dopa. Ziegler and co-workers (5) show the significant benefit of piribedil over placebo

in terms of control of motor symptoms in patients on stable dosage of L-dopa. Also,

the use of piribedil allowed for L-dopa dose reduction. 150mg/day of piribedil was

also proven to have similar efficacy in controlling PD symptoms to 25mg/day

bromocriptine, however patients on piribedil required less L-dopa (6).

Several studies investigated the piribedil pharmacokinetic parameters. Simons

et al. (7) demonstrated, that after single 15min intravenous infusion the piribedil

mean half-life was 12.1 hours ranging from 10.5 to 15.1h; the reported mean

clearance was 78.6L/h ranging from 50.8 to 115.6L/h; the average Cmax was linearly

dependent on the dose, and the measured values were 77.8, 155.9, 288.3 and

745.1nM, for the doses of 2,4,8,16mg respectively. Another study investigated the

oral route of administration of piribedil (8). It was shown that the drug is rapidly

absorbed from gastrointestinal tract; Cmax is reached within 1h; its oral bioavailability

is below 0.1 due to heavy liver metabolism. Allain and co-workers (9) reported that

Figure 1 Piribedil chemical structure. Mw=298.3

O

ON

NN

N


93

after a 30-day course of three times daily of Trivastal retard, 50mg, the plasma Cmax

and Cmin were 148.5 and 120.7nM, respectively. It was however reported previously

(10), that effective plasma concentrations range already from 33 to 100nM.

Physicochemical properties of piribedil (M=298Da, logP=2.84, pKa1=6.9 and

pKa2=1.3 (11)) along with its pharmacokinetic parameters, make it a good candidate

for transdermal delivery. The aim of such formulation would be not only reducing the

dose by bypassing first-pass effect and increasing bioavailability, but also achieving

the stable plasma levels of the drug in relatively short time. This is especially

important in a condition such as Parkinson’s disease, in which it is well-recognized

that the fluctuations in dopaminergic activity can produce severe side-effects in long

term therapy (12). Previous study with passive piribedil patch (13) did not show

expected results mainly because of sub-therapeutical levels achieved by formulation.

Transdermal iontophoresis seemed to be a natural choice of a delivery

method for piribedil to investigate. Similarly to passive patch it assures the

circumventing of the first pass effect, which might lead to the reduction of the dosage.

Furthermore, this active method of delivery is often able to deliver drugs at a higher

rate than passive patches. This could help overcome the low drug plasma-levels, the

main problem encountered in the piribedil passive patch study (13). Another

advantage of application of iontophoresis is the profiling of the drug input and

adjustment to patients requirements. This might be particularly beneficial in the case

of piribedil, as relatively high inter-individual variability has been reported after

chronic oral administration of piribedil (Trivastal Retard 50mg three times a day): Cmin

varied from 6.5 to 1500nM and Cmax from 9.2 to 1700nM (9). Iontophoretic

administration may aid a more accurate titration of the drug to a patient.

In this study, transdermal iontophoresis of piribedil on the pig skin model in

vitro was examined. In particularly, the feasibility of delivering therapeutic doses and

the effects of piribedil donor concentration, its mole fraction, pH of donor solution and

current intensity were examined. To investigate the magnitude of the electroosmotic

effect, contributing to the total piribedil transport, transdermal mannitol transport was

studied. To reach the upper range of concentrations obtained by prolonged

administration of Trivastal Retard 50mg three times daily, 148.5nM (9), at the


94

reported clearance 78.6L/h (7) the target rate of delivery needs to be of around

11.6µmol/h (148.5nM·78.6L/h=11.6µmol/h).

Materials and methods

Materials

Piribedil (PBD) free base was purchased from Tocris Bioscience UK. Sodium

chloride, mannitol, silver wire (99.99% pure), silver chloride (99.999% pure) were

obtained from Sigma Aldrich UK; hydrochloric acid, sodium hydroxide, acetonitrile

HPLC grade (AcN) and n-octanol were supplied by Fisher Scientific UK; triethylamine

was supplied by Acros Organics UK. Deionised water (resistivity≥18.2MΩ/cm) was

used to prepare all solutions.

Skin preparation



electric dermatome (Zimmer, Dover, OH) set to 750µm thickness and were then


dorsal and abdominal skin, from two different pigs was used in the experiments.


On the day of the experiment the skin was thawed at room temperature.

Subsequently, it was clamped between two standard, side-by-side diffusion cells with

the epidermal side facing the donor chamber. The volume of both cells was 3.3ml

and the transport area 0.78cm2. Homemade silver-silver chloride electrodes (14)

were placed in both chambers. A constant current of 0.4mA was applied for six hours

using a Kepco APH 1000DM (Flushing, NY) power supply, such that the donor

chamber had higher potential. Both chambers were magnetically stirred throughout

the permeation experiment. The receptor solution contained unbuffered 0.9% NaCl

solution of pH=5.8, to provide the main counter-ion, Cl-. The whole receptor solution

was removed and sampled hourly. The unbuffered donor solution was replaced with

a fresh one every two hours (unless otherwise stated) to provide enough Cl- ions for

correct electrode electrochemistry.


95


Single ion experiments had piribedil as the only cation in the donor solution.

This study was set up to investigate the effect of a) piribedil concentration on its

transdermal delivery, b) the applied current on piribedil transdermal transport and c)

the pH on piribedil transdermal transport. 30mM mannitol was present in all solutions

as a marker of electroosmosis. The pH of the solutions was adjusted by 0.1M HCl.

Donor solutions are detailed in Table 1.

Table 1 Iontophoretic formulations used in single-ion experiments. *Where the two values are given, the second refers to the number of replicates for mannitol transport.

PBD Conc. [mM]


Current Intensity

[mA] n*


15.0 30 5.0 0.4

6/6 22.5 6 30.0 5/5

pH effect 30 30

2.0

0.4

6 3.0 6 3.5 5 4.0 6/6 5.0 5/5

Current intensity

effect 30 30 5.0

0.0 6 0.1 6 0.2 6 0.4 18/5


Iontophoresis was carried out with donor solutions containing piribedil and

sodium ions. 30mM mannitol was used as a marker of electroosmosis. Total

concentration of positive ions was equal to 30mM and the pH was adjusted with 1M

HCl to 3.5 or to 5.0.

Passive control

Passive flux of piribedil was assessed using a similar experimental setup,

except that no current was applied and the donor solution was never replaced. The

donor solution was composed of a) 30mM piribedil with 30mM mannitol acidified to


96

pH 5.0 with 1M HCl and b) saturated piribedil solution at pH 12.0 with 30mM

mannitol.

Table 2 Iontophoretic formulations used in co-ion piribedil delivery. *Where the two values are given, the second refers to the number of replicates for mannitol transport.

Mole Fraction

PBD Conc. [mM]

NaCl Conc. [mM]

Total Cation Conc. [mM]

pH Average

PPX Valence

Current Intensity

[mA] n*

10% 3.0 27.0

30 5.0 1.0 0.4

12/6 25% 7.5 22.5 6/6 50% 15.0 15.0 17 75% 22.5 7.5 6

100% 30.0 0 5/5 10% 3.0 27.0

30 3.5 1.0 0.4 5

50% 15.0 15.0 6 100% 30.0 0 5

25% 7.5 22.5 30 5.0 1.0 0.4 6/6 20.0 60.0 80 6/6

EO control

The reference electroosmotic flow of mannitol (in the absence of drug), was

measured with donor solutions containing 30mM NaCl and 30mM of mannitol at pH

of 5.0 adjusted with 0.01 HCl.

To investigate the blank mannitol flow (that could originate from the skin) an

experiment was done with solution containing 30mM NaCl and no mannitol, at pH of

5.0 adjusted with 0.01M HCl. Subsequently mannitol fluxes were measured.

Analytical Methods

Piribedil

Piribedil was quantified using the Jasco HPLC system (composed of PU-980

pump, autosampler AS-1595, and UV-VIS detector UV-975) by means of piribedil

external standard calibration curve in corresponding media of at least seven

standards. Isocratic separation was performed using Dionex Acclaim™ 120, C18

HPLC column (250 mm x 4.6 mm, 5µm) with flow rate of 1 ml/min and injection

volume 10µl with column temperature of 25C. The mobile phase consisted of

55%H2O and 45%AcN spiked with 200µl/l of triethylamine and acidified to pH=2.5

with perchloric acid. Detection was performed at wavelength of 240nm. Limit of

detection was 0.1µM and the limit of quantification was accepted as 1µM. The


97

quantification was carried out by means of series of external standards (10 to

300µM). The precision and accuracy of quantification method was 3% and 4%,

respectively.

Mannitol

Mannitol was quantified using Shimadzu LCMS 2010 EV system with

electrospray ionisation in the following conditions: a gradient separation (H2O:AcN

10:90 to 95:5 in 10min) was performed using the Phenomenex Luna™ HILIC, HPLC

column (150 mm x 4.6 mm, 5µm) with flow rate of 0.5 ml/min and injection volume

10µl with column temperature of 25C. Detection was performed at m/z=218.85 in

negative mode with detector voltage of 2.0kV. Data collection rate was 0.2s. Limit of

detection was 20nM and limit of quantification was 100nM. The quantification was

made by means of external standards with use of at least nine standards (from

0.2µM to 40.0µM). The accuracy and precision of the applied method was assessed

as 6% and 7%, respectively.

Solubility of piribedil

373.9mg of piribedil base was added to 25ml of water and acidified with 0.1M

HCl to pH 4.0, 5.0 and 6.0. Then, solutions were shaken at the temperature of 50°C

stabilised by a water-bath for an hour. Subsequently, the solutions were let to cool

down to room temperature. The pH was rechecked and, if necessary, readjusted.

Supernatants were collected and analysed by HPLC for piribedil content.

Determination of distribution coefficient of piribedil

4ml of n-octanol was added to 4ml of 1mM piribedil solution of pH 5.0 and 4.0.

Mixtures were shaken for 48 hours to achieve equilibrium in 25°C. After that time,

water and octanol phases were collected manually to separate vials and were

analysed by HPLC. Distribution coefficient was calculated from Equation 1 (15).

Equation 1 water

oloc

CCD tanloglog =

LogP and pKa was estimated using Equation 2 (11):


98

Equation 2 )10101log(loglog 2211 pHpKapKapHpKaDP −+− +++=

Conductivity measurements and piribedil water mobility estimation

The specific conductivity was measured for range of piribedil water solutions

(10, 5, 2, 1, 0.5mM) using conductimeter Metrohm T-120 at 22C with the probe of

cell constant K=0.93. The measured pH of investigated solutions was 5.5. At this pH

virtually all piribedil molecules are monovalent, and the concentration of H+ or OH-

ions is negligible. Then, values of molar conductivities (specific conductivity

normalized by concentration) were calculated and plotted against square root of

concentration. Conductivity at infinite dilution was estimated by extrapolating the

curve to zero. Subsequently, using Kohlraush law of independent ion migration

Equation 3 (with mobility of chloride ion taken from literature), water mobility of

piribedil ion was calculated. Afterwards, water transport numbers were estimated as

in Equation 4 and Equation 5 (16). The mobility of the chloride ion, µCl-=7.9·10-8

m2/s/V was taken from literature (17).

Equation 3 −

∞−+

∞+∞ Λ+Λ=Λ CC

Equation 4 −−Λ

=∞

ClPBD Fµµ

Equation 5 −+

=ClPBD

PBDPBDt µµ

µ


Statistical analysis was carried out using Prism 5.0 (GraphPad Software, San

Diego, CA, USA). Statistical differences within multiple data sets were assessed by

one-way ANOVA followed by Bonferroni multiple comparison sets. For binary data

sets, a two-tailed, unpaired t-test was employed. The level of statistical significance

was fixed at p < 0.05. The reported fluxes for each replicate were obtained as an

average value of the last three hours of experiment. Transport numbers for each

replicate were calculated as in t#=z·F·J/I, form the average of fluxes for last three

hours of experiment. The data on the graphs is presented as mean value of all


99

replicateds with standard deviation. All regressions were performed using the least

square method, and regression significance was tested with the runs test.


Solubility study

The solubility of a drug is an important factor in iontophoretic formulation. This

property determines the applicable range of the pH and the maximal concentration of

the drug in the donor solution. Piribedil, unlike selegiline or pramipexole, is only

sparingly soluble in water. The mean solubility of piribedil for pH 6.0, 5.0 and 4.0

was, respectively, 1.4 (±0.8), 34.1 (±1.0), >50 mM.

Water mobility study

In order to estimate the piribedil water mobility, conductance measurements

were carried out. The extrapolation to zero of the molar conductance of piribedil HCl

as a function of a square root of its concentration, returned the value of Λ∞, the

infinite dilution conductivity, which can be used in Equation 4, in order to calculate the

piribedil water mobility (Figure 2). The calculated value was

µPBD+=3.92±0.19·10-8m2/V/s. The water transport number of piribedil calculated from

this value as in Equation 5 was tPBD=0.33. The pH of studied solutions was of 5.5,

which assured the single ionisation of piribedil.

Figure 2 Linear regression of NaCl and piribedil molar conductivity as a function of square root of concentration. The Y-intercepts are the infinite dilution molar conductivities. The data points are the means of three replicates with standard deviations.

To analyse piribedil applicability in transdermal iontophoresis a similar

approach was undertaken as with pramipexole and selegiline, in previous chapters.

Initially, the single ion experiments were carried out to determine the dependency of

0 1 2 3 480

100

140

Λ∝

PiribedilNaCl

C1/2 [mM]1/2

Mol

ar c

ondu

ctiv

ity [S

i*cm

2 /mol

]


100

the flux on concentration, pH, and current. Subsequently, the effect of competing ions

was investigated, by adding sodium to donor solutions.

Single-ion experiments – pH effect

The single-ion experiments were performed to estimate the highest transport

number for piribedil. The elimination of any competing ions from the donor solution

was crucial to achieve the highest piribedil transport. It has been proven theoretically

(18), and demonstrated experimentally for several substances (19; 20; 21) that

presence of competing ions hinders the transport number of a delivered drug. First,

the effect of the pH of donor was investigated. The upper limit of the pH range was

restricted to 5.0 by the piribedil solubility. For higher pH values, the concentration of

piribedil HCl was too low, to assure correct electrochemistry at the electrodes. The

investigated range of pH was from 2.0 to 5.0 (Table 1). The results are presented in

Figure 3. First, it has to be mentioned, that all of the experiments led to a higher drug

transport, than the passive control at pH 5.0 (14±6nmol/h). Generally, the decreasing

pH, as expected, caused a drop of the piribedil flux. There is no change in piribedil

transport between pH 4.0 and 3.0, while the significant drops of the flux can be

observed at the going down from 5.0 to 4.0; and 3.0 to 2.0. There are several

phenomena explaining the drop of the flux with a decreasing pH. The first one is the

competition of hydronium ions. As discussed in previous chapters, these highly

mobile ions may carry a large portion of current, even at small concentrations. This

mechanism could explain the drop of the flux from around 0.2µmol/h at pH 3.0, to 0.1

µmol/h at pH 2.0. At the latter value of the pH, the concentration of H+ ions is 10mM.

This consists a fourth of the total cation concentration. In these terms the experiment

at pH 2.0 is not a single-ion one.

Finally, the skin permselective properties can be altered by changing the pH,

that would result in change of the flux. The pH determines the total charge fixed on

the skin, as shown by Pikal and Shah (22; 23), which in turn determines the direction

and the magnitude of the electroosmotic flow. At the physiological pH, charge fixed

on the skin is negative, and the electroosmotic flow is directed from anode-to-

cathode, hence contributes to the cation transport. Lowering the pH value below the

skin isoelectric point (iP=4.3), reverses the charge fixed on the skin to positive, and

the direction of electroosmosis to the cathode-to-anode (24). As a consequence, the


101

drop in the pH impedes the cation transport. In several previous publications studying

the effect of pH on the iontophoretic drug delivery, the decline of the drug flux with

dropping pH was explained by limitation and reversal of electroosmotic effect (25; 26;

27). Following the work of Pikal (28) the total flux was understood as:

Equation 6 VCJJ EM 111 +=

where J1 is the total flux of compound 1, J1EM is its flux due to electromigration; C1 is

the concentration of compound 1 in the membrane; and V is the velocity of bulk

volume flow through the membrane. The value of C1V was understood as

electroosmotic contribution to the total transport. This understanding of

electroosmotic contribution will not be used in this work, for several reasons. First, in

previous works (25; 26; 27) drugs were usually delivered from donor solutions with

high content of salts. In such conditions, a significant competition of the buffer ions

reduces the drug ion concentration in the stratum corneum, thus the influence of a

drug on the skin was small (29). In contrast, piribedil is delivered as a single ion, and

greatly limits the transport of mannitol (Figure 3c). In fact, only for pH 5.0 the mannitol

fluxes were statistically different from the blank mannitol flow. Secondly, the

lipophilicity of piribedil (logD=0.9 at pH 5.0, and logP=2.8) is much higher than

mannitol, which promotes drug accumulation in the skin (30). Hence, it would be hard

to assume that piribedil concentration in the skin is the same as in the donor. Finally,

the conductivity experiments seem to indicate, that mobility of piribedil in the skin is

much lower than in water. This supports the idea that PBD is “slowed down” in the

skin media, hence its concentration might differ from the donor concentration.

2.0 3.0 3.5 4.0 5.00.0

0.1

0.2

0.3

0.4

0.5

a pH

Pirib

edil

Flux

[µm

ol/h

]

2.0 3.0 3.5 4.0 5.00

1

2

3

4

b pH

Piri

bedi

l Tra

nspo

rt N

umbe

r [%

]

Blank M

annito

l flow

PBD 30mM pH4.0

PBD 30mM pH5.0

NaCl 3

0mM pH5.0

01234

80100120140

C

Man

nito

l Flu

x [n

mol

/h]

Figure 3 The fluxes (a) and transport numbers (b) of piribedil for different values of donor pH. Figure 3c represents the mannitol transport measured for piribedil pH experiments, compared to the blank mannitol flow (originating from the skin, 30mM NaCl and no mannitol in donor), and the reference mannitol transport (for 30mM NaCl and 30mM mannitol donor solution).


102

Nonetheless, changes in mannitol flux certainly represent changes in volume

flow, and still can be used to explain the changing piribedil flux. As mentioned before,

for pH values lower than 5.0, mannitol flux was indistinguishable from the blank

mannitol flow (measured in absence of mannitol in the donor). This might suggest

that the volume flow is very limited during the piribedil iontophoresis at these pH

values. The EO flow is higher for pH 5.0, although it is still very small in comparison

to the control experiment (Figure 3c). The rise of mannitol flux from pH 4.0 to 5.0

corresponds to the rise in piribedil flux. It is then possible, that the increasing drug

flux is due to rising skin permselectivity. However, if that was the case, very limited

volume flow would require a significant piribedil concentration in the skin, to

compensate for the piribedil flux difference from pH 4.0 to 5.0. Certain accumulation

of the drug in the stratum corneum can be expected, for reasons mentioned above.

Also, accumulation of the iontophoretically delivered substances was previously

observed (31; 32), in the study of Sylvestre et al. over transdermal iontophoretic

delivery of dexamethasone phosphate (31). The reported concentrations of roughly

from 10 to 50mM (10-50µmol/cm3), were compared to 8.5mM of dexamethasone

phosphate in the donor solution. Further study is required to conclude on deposition

of piribedil in stratum corneum. Overall, the dependency of total iontophoretic flux of

piribedil, on the pH is determined by competition from H+ ions at low pH values, and

permselective properties of the skin at higher pH. The formulation of piribedil at pH

higher than 5.0 is difficult, due to rapidly dropping water solubility of the drug.

Single ion experiments – concentration effect

The following was to investigate the effect of piribedil concentration in the

single ion situation. Due to low piribedil solubility, a relatively narrow range of

concentrations, 15mM to 30mM, was investigated. The lower threshold was

determined by the minimal concentration of Cl- required, to keep the voltages on the

electrodes low. The upper threshold was limited by the piribedil solubility at pH 5.0.

As observed previously for selegiline and pramipexole, piribedil fluxes for all the

concentrations were relatively stable, although some variability was observed (Figure

4). Anova test found flux originating from 22.5mM piribedil solution to be significantly

higher, by roughly 25%, as compared to the other two. No difference was found

between the latter.


103

Figure 4 The effect of piribedil concentration in donor, as a single-ion, on transdermal its transdermal transport and on electroosmotic transport of mannitol.

The issue of stability of drug flux originating form single-ion solution was

already discussed in previous chapters. Shortly, hydromorphone (21) was found to

express stable fluxes for a concentration range of 10 to 800mM, with similar, 25%,

degree of variability, attributed to the inhomogeneity of the skin. Similar observations

were made for ropinirole (20), selegiline, and pramipexole.

As shown in Figure 4, the flow of the electroosmotic marker, mannitol, was

found higher for 30mM piribedil donor solution, as compared to 15mM. The latter was

significantly different from the blank mannitol flow (originating from the skin). Both

measured mannitol fluxes are very small (by two orders of magnitude) in comparison

to the reference mannitol flow, where piribedil was substituted with 30mM NaCl

(Figure 3c). This again, might be explained by accumulation of piribedil in the skin.

High levels of positively charged piribedil could enforce its interaction with negatively

charged proteins of the skin, attenuating the net negative charge. That would result in

limited electroosmotic transport observed in Figure 4.

Single ion experiments – current intensity effect

This case investigates the effect of applied current on the piribedil transdermal

transport. The study allows methodical assessment of piribedil transport number,

from the slope of the flux/current regression, as in the equation t#=J·F/z·I. The results

of the study are presented in Figure 5. The transport number calculated from the

slope was t#=0.0263±0.0011. This means that only 2.6% of total charge passing

through the skin during the experiment was carried by piribedil molecules. In

comparison to other drugs delivered as single ions, like hydromorphone (21),

15.0mM 22.5mM 30.0mM0.0

0.2

0.4

0.6

0.8

0

1

2

3

4

Mannitol

Piribedil

Piribedil Concentration

Piri

bedi

l Flu

x [µ

mol

/h] M

annitol Flux [nmol/h]


104

lidocaine (33), pramipexole or selegiline, piribedil is rather a mediocre charge carrier.

This study proves also that the current can be conveniently used to regulate the

transdermal drug flux of piribedil. For the maximal investigated value of current,

0.4mA, at the diffusion-cell transport area of 0.78cm2, the average flux was

0.41µmol/h. This compares to theoretically calculated flux of 11.6µmol/h, required to

reach the upper limits of therapeutic piribedil plasma levels. With 0.5mA/cm2 being an

acceptable current density for human applications, the required area A of

iontophoretic patch is given by: A=2·(11.6µmol/h·0.78cm2/0.41µmol/h)=44.1cm2. The

factor 2 reflects the necessity of having the same area of anode and cathode, to keep

the current density at acceptable levels. The area of 44.1cm2 is equal to that of two

circles with a diameter of 5.3cm each, which is a reasonable patch size.

However, this effective delivery would be only possible, given that there is

enough chloride ions in the donor for the entire delivery period. One of the

advantages of the iontophoretic dosage form is a one-a-day application. For the 24

hour delivery, the required amount of chlorides can be estimated as 0.45mol/cm2 at

current density of 0.5mA/cm2. This is an order of magnitude higher than the solubility

of piribedil in water. Hence, 24 hour long delivery would require the use of an

additional source of chlorides, i.e. a hydrochloric salt, which limits the effectiveness of

the transport, due to ionic competition. Nonetheless, the number 0.45mol/cm2 does

not take into account the Cl- ions migrating from beneath the skin during the

iontophoresis. This can be a significant amount, as chlorides constitute the main

buffer of the human body, and as a counter-ion, carry anywhere from 20% (34) to as

much as 70% (24) of the total current. Also, as shown in Figure 6, piribedil transport

is unaffected by ionic competition within 0.5 to 1.0 range of the molar fraction. This

indicates that addition of an equal amount of external chlorides, should not have a

significant impact on the transdermal flux.

Moreover, the 11.6µmol/h target flux would assure the plasma levels obtained

after 30 days of administration of Trivastal Retard (50mg of piribedil). These fluxes

are relatively high in comparison to others, reported as effective (from 50 to 100 nM

(10)). Further study, with longer delivery time and in vivo conditions, is required to

more precisely determine the feasibility of iontophoretic delivery of piribedil in vivo.


105

Figure 5 The dependence of piribedil flux on applied current. The regression line is represented by the equation y=0.98±0.04·x+0.002±0.009. The dotted line limits the 95% confidence interval of the regression. The transport number calculated from the slope, after taking into account the units, is 0.0263±0.0011.

Co-ion delivery

In the next step, the effect of ionic competition was studied. Sodium ions were

chosen as the competing ions, for they are often included in drug formulations as

stabilising or buffering agents. In two series of experiments, piribedil was

iontophoretically delivered from donor solutions containing various PBD+/Na+ mole

fractions (Table 2).The flux of piribedil and mannitol as a marker of electroosmosis

was measured. The results are presented in Figure 6. The graph illustrates, that after

the initial flux increase, from 10% to 50% mole fraction, piribedil transport becomes

insensitive on decreasing content of competing ions in the donor. This can be

observed for both values of pH. Because of ionic competition and the lower cation

permselectivity of the skin, the flux values for pH 3.5 are lower, than these measured

at pH 5.0. The profile of a co-ion piribedil delivery can hardly be approximated by a

straight line (Figure 6a). This is different to small inorganic ions (Na+, K+, NH4+ and

Li+), larger organic molecules (lidocaine (24)), or the previously discussed selegiline

and pramipexole, In fact, drugs flux profile is more similar to previously investigated

lipophilic compounds: quinine and propranolol (33). The latter two are believed to

strongly interact with the skin, changing its permselective properties.

The impact of piribedil mole-fraction in the donor on mannitol transport seems

to underline the analogous transport profile of the piribedil, quinine and propranolol,

when delivered solutions containing competing ions. In Figure 6b, a sharp decline in

mannitol transport is observed after the addition of even relatively small amounts of

piribedil. 10% piribedil mole fraction caused a five-fold reduction in mannitol

transport, as compared to the control experiment. The further increase of the piribedil

0.0 0.1 0.2 0.3 0.4 0.50.0

0.1

0.2

0.3

0.4

0.5


Flux

[µm

ol/h

]


106

content, to 25%, continued to decrease the mannitol flux. There was however no

difference in mannitol transport for 25 and 100% piribedil mole fraction. Similar

electroosmosis reduction was reported for quinine and propranolol (33). These

observations might further support the concept of piribedil accumulating in the skin

and attenuating its negative charge, hence the electroosmotic effect. Confirmation of

this concept would, however, require further studies.

0% 25% 50% 75% 100%0.0

0.1

0.2

0.3

0.4

0.5

pH 3.5pH 5.0

a Mole Fraction

Flux

[µm

ol/h

]

0% 25% 50% 75% 100%0

25

50

75

100

Piribedil Mole FractionM

anni

tol F

lux

[nm

ol/h

]

Figure 6 a) Piribedil fluxes as a function of its mole fraction in donor solution for two pH values, 3.5 and 5.0. b) Transdermal transport of mannitol as a function of piribedil mole fraction in donor at pH 5.0.

In an attempt to model the transdermal drug transport, Phipps and Gyory (34)

derived an expression that links a transport number of a drug (td), with its mole

fraction in the donor solution, during movement across uncharged and homogenous

membrane:

Equation 7

XZBtt

ttt

t

ed

d

d

d

d

⋅⋅−

++−

−=

00

0

0

0

111

1

1

where td0 and te0 are the transport numbers of the drug and the competing cation, in

the single-ion situation; Z is the ratio of cation valences; X is the mole fraction of the

drug. The parameter B was described as the proportionality constant that relates the

ratio of cations concentrations in donor solution, to these in the membrane:


107

Equation 8 ds

es

d

e

CCB

CC

=

where Ce and Cd are the concentrations of inorganic cation and the drug in the donor

solution, respectively; and Ces and Cds are concentrations of inorganic cation and

drug in the skin. After rearrangement of Equation 8, factor B can be expressed as:

Equation 9 d

e

dsd

ese

PP

CCCCB ==

The Pe and Pd are the partition coefficients of competing cation and drug between

donor solution and the skin. The data from the experiments for both pH values (5.0

and 3.5) was fitted with the Equation 7, assuming the te0=0.6 (35). The curves

obtained are presented in Figure 6a. The fitting rendered two identical B values for

both data sets, BpH5.0=BpH3.5=0.384. The equal values of factor B for both plots

indicate, that at both pH values piribedil penetrates similarly into stratum corneum.

Furthermore, the low value of B factor indicates the possibility, that during

iontophoresis the partitioning of piribedil into the skin occurs. This stays in

accordance with the high octanol water partition coefficient measured for piribedil.

Conclusions

Piribedil is an intermediate charge carrier and does not carry more than 3% of

charge during iontophoresis across pig skin. The iontophoretic delivery rate of single-

ion piribedil seems sufficient to assure the therapeutic plasma levels. However,

formulating the patch appropriate for a 24 hour delivery can be very hard, due to

limited water solubility of the drug. The main factors controlling piribedil transport are

pH of the donor solution and the current intensity. To lesser extent piribedil

transdermal flux is dependent on competition of the background electrolyte present in

the donor; particularly at mole fractions higher than 50% flux seems unaffected.

Piribedil limited the electroosmotic flux in fashion similar to other lipophilic

compounds. No effect of piribedil concentration, when delivered as a singe-ion, was

observed. The transport number of piribedil measured in water is significantly higher

than the transdermal one, suggesting slower transdermal transport, and possibly,

accumulation of the drug in the stratum corneum.


108

Bibliography

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its metabolite S 584. Creese, I. 1974, European Journal of Pharmacology, Vol. 28,

pp. 55-58.

2. Alpha-2-Adrenoceptor antagonists: a new approach to Parkinson's disease?

Berfel-Courbon, C, et al. 1998, CNS Drugs, Vol. 10, pp. 189-207.

3. Antiparkinsonian agent piribedil displays antagonist properties at native, rat, and

cloned, human alpha(2)-adrenoceptors: cellular and functional characterization.

Millan, M J, et al. 2001, Journal of Pharmacology and Experimental Therapeutics,

Vol. 297, pp. 876-887.

4. Early piribedil monotherapy of Parkinson's disease: A planned seven-month report

of the REGAIN study. Rascol, O, et al. 2006, Movement Disorders, Vol. 21, pp.

2110-2115.

5. Efficacy of piribedil as early combination to levodopa in patients with stable

Parkinson's disease: A 6-month, randomized, placebo-controlled study. Zigler, M, et al. 2003, Movements Disorders, Vol. 18, pp. 418-425.

6. The Parkinson-Control study: A 1-year randomized, double-blind trial comparing

piribedil (150 mg/day) with bromocriptine (25 mg/day) in early combination with

levodopa in Parkinson's disease. Castro Caldas, A, et al. 2006, Movement


7. End-of-dose akinesia after a single intravenous infusion of dopaminergic agonist

piribedil in Parkinson's disease patients: a pharmacokinetic/pharmacodynamic,

randomized. double-blind study. Simons, N, et al. 2005, Movement Disorders, Vol.

7, pp. 803-809.

8. Pharmacodynamic and pharmacokinetic properties of piribedil: rationale for use in

Parkinson's disease. Allain, H. 2001, Disease Managemet and Health Outcomes,

Vol. 12, pp. 41-48.


109

9. Piribedil plasma levels after chronic oral administration of Trivastal Retard 50 (150

mg/day) in parkinsonian patients. Allain, H, et al. 2001, Parkinsonism & Related

Disorders, Vol. 7, p. S51.

10. Cinétique plasmatique du piribedil par voie intraveineuse et corrélation avec le

tremblement parkinsonien. Ziegler, M, Spampinato, U and Rondot, P. 1991, JAMA,

Vol. (French ed; special issue), pp. 26-30.

11. Physicochemical properties and transport behaviour of piribedil: Considerations

on its membrane-crossing potential. Tsai, R, et al. 1992, Internationa Journal of

Pharmaceutics, Vol. 80, pp. 39-49.

12. Intermittent vs continuous levodopa administration in patients with advanced

Parkinson disease: a clinical and pharmacokinetic study. Stocchi, F, et al. 2005,

Archives of Neurology, Vol. 62, pp. 905-910.

13. A randomized, double-blind study of a skin patch of a dopaminergic agonist,

piribedil, in Parkinson’s disease. Monastruc, J L, et al. 1999, Movement Disorders,

Vol. 14, pp. 336-341.

14. Iontophoretic delivery of amino acids and amino acid derivatives across the skin

in vitro. Green, P G, et al. 1991, Pharmaceutical Research, Vol. 8, pp. 1113-1120.

15. Partition coefficients and their uses. Albert, L, Corwin, H and David, E. 1971,

Chemistry Reviews, Vol. 71, pp. 525-616.

16. Atkins, P W. Molecules in motion: ion transport and molecular diffusion. Physical

Chemistry. 6th ed. Oxford : Oxford University Press, 1978, pp. 723-759.

17. Young, T F. Electrochemical Information. Bruce H Billings, et al. American

Institute of Physics Handbook. 2nd. New York : McGraw-Hill, Inc, 1963, 5, pp. 5-263.

18. Iontophoretic delivery of model inorganic and drug ions. Phipps, J B, Padmanabhan, R V and Lattin, G A. 1989, Journal of Pharmaceutical Sciences,

Vol. 78, pp. 365-369.


110

19. In vitro optimisation of dexamethasone phosphate delivery by iontiophoresis.

Sylvestre, J P, Guy, R H and Delgado-Charro, M B. 2008, Physical Therapy, Vol.

88, pp. 1177-1185.

20. Iontophoretic delivery of ropinirole hydrochloride: effect of current density and

vehicle formulation. Luzardo-Alvarez, A, Delgado-Charro, M B and Blanco-Mendez, J. 2001, Pharmaceutical Research, Vol. 18, pp. 1714-1720.


hydromorphone. Padmanabhan, R V, Phipps, J B and Lattin, G A. 1990, Journal of


22. Transport mechanisms in iontophoresis. II. Electroosmotic flow and transference

number measurements for hairless mouse skin. Pikal, M and Shah, S. 1990,






24. Optimizing iontophoretic drug delivery: identification and distribution of charge

carrying species. Marro, D, et al. 2001, Pharmaceutical Research, Vol. 18, pp. 1709-

1713.

25. Iontophoretic delivery of apomorphine in vitro: physiochemic considerations. Li, G L, Danhof, M and Bouwstra, J A. 2001, Pharmaceutical Research, Vol. 18, pp.

1509-1513.

26. Iontophoresis-faciliated transdermal delivery of verapamil. I. In vitro evalutaion

and mechanistic studies. Wearley, L, Liu, J C and Chien, Y W. 1989, Journal of


27. Electrorepulsion versus electroosmosis: effect of pH on the iontophoretic flux of

5-fluorouracil. Merino, V, et al. 1999, Pharmaceutical Research, Vol. 16, pp. 758-

761.


111

28. The role of electroosmotic flow in transdermal iontophoresis. Pikal, M J. 2001,

Advanced Drug Delivery Reviews, Vol. 46, pp. 281-305.

29. Iontophoretic delivery across the skin: electroosmosis and its modulation by drug

substances. Hirvonen, J and Guy, R H. 1997, Pharmaceutical Research, Vol. 14,

pp. 1258-1263.

30. Effect of lipophilicity on in vivo iontophoretic delivery. II. B-blockers. Tashiro, Y, et al. 2001, Biological and Pharmaceutical Bulletin, Vol. 24, pp. 671-677.

31. Iontophoresis of dexamethasone phosphate: competition with chloride ions.

Sylvestre, J P, et al. 2008, Journal of Controlled Release, Vol. 131, pp. 41-46.

32. Effect of amino acid sequence on transdermal iontophoretic peptide delivery.

Schuetz, Y B, et al. 2005, European Journal of Pharmaceutical Sciences, Vol. 26.



34. Transdermal ion migration. Phipps, B and Gyory, R. 1992, Advanced drug

delivery reviews, Vol. 9, pp. 137-176.

35. Electromigration of ions across the skin: Determination and prediction of transport

numbers. Mudry, B, Guy, R H and Delgado-Charro, M B. 2006, Journal of


Chapter IV Pergolide

112

Chapter IV – Pergolide Introduction

Pergolide (Permax®), (PER) is very potent,

semisynthetic, ergoline-derived dopamine

agonist, which has the affinity to the whole

spectrum of dopamine receptors. Pergolide binds

the strongest to D2–D4 receptors, while the affinity

to D1 and D5 subtypes is rather mild (1). It also

possesses certain α1-adrenergic and 5-HT

agonist activity (2). It is used in treatment of

Parkinson’s disease and restless leg

syndrome (3; 4).

The efficacy of pergolide in treatment of

Parkinson’s disease has been proven in several clinical trials. The three years

PELMOPET study compared pergolide with levodopa/carbidopa therapy, in 167

previously untreated patients (5). Similarly to other dopamine agonists, levodopa

appeared significantly more effective in terms of controlling the parkinsonian

symptoms, and also had a better side effects profile (6). However, after three years,

pergolide treatment resulted in substantial relief of symptoms, and delayed the time

of onset of dyskinesias. Pergolide has also ability of alleviating the severe levodopa-

induced dyskinesias, when applied in high doses (7). High-dose pergolide therapy

managed to improve dyskinesia, on average by 43%, measured by unified

Parkinson’s disease rating scale (UPRDS), in 81% of patients. In another study,

pergolide was assessed for efficiency as an adjuvant to levodopa/carbidopa therapy

(8). In this randomized, placebo-controlled, multicentre trial, it was demonstrated that

pergolide is an effective supplement to levodopa/carbidopa treatment. Co-

administration of pergolide allowed for reduction of levodopa dose (by on average

24.7%), significantly improved the motor scores, eased the daily activities, and

reduced the off-time. In addition, the study of Koller and co-workers (9) compared the

safety, tolerability and efficacy of pergolide and tolcapone, when co-administrated

with levodopa, in 204 patients. The efficacy of both drugs was similar; however,

NH

H S

CH3

CH3

H

NH

Figure 1 Pergolide chemical structure.Mw=314.5.


113

patients better tolerated tolcapone. Other study compares two ergoline dopamine-

agonists, pergolide and bromocriptine (10). This double-blind study, conducted on 24

patients, found no difference in efficacy for these drugs. Finally, it should be

mentioned, that pergolide is associated with valvular heart disease (11; 12) and

pleural fibrosis (13), thus it is recommended only as second-line therapy of

Parkinson’s disease.

Pergolide is used in Parkinson’s disease treatment at 2-2.5mg daily, in three

divided doses, with maximum dose of 5mg/day (14). It is one of the longest acting

dopamine agonist, with half-life of 21h ranging from 6 to 64h in parkinsonian patients

(15; 16). The Cmax and area under the time against the plasma-concentration curve

(AUC) were found to be linearly dependent on the pergolide dose, in range of 0.25 to

1.0mg. The average measured Cmax in long-term therapy was 127±49, 236±125 and

472±264ng/L for the dose of 0.25, 0.5 and 1.0mg respectively. The mean apparent

clearance was reported as 457±247L/h. Bioavailability of pergolide varies from 0.2 to

0.6 (17). Also, pergolide is known to bind to serum proteins at 90-95% (2).

Pergolide physicochemical properties are reported, as follows: Pergolide

molecular mass is 314.5Da; pKa and Log P were reported as 7.8 and 4.0,

respectively (18). Very poor water solubility has also been noted (18). All these

factors suggest, that pergolide is an average candidate for transdermal

iontophoresis; however, as it is a very potent drug and its daily dosage does not

surpass 5mg (14), transdermal delivery should require relatively low fluxes. To

calculate the target-flux required for clinically effective pergolide delivery, two

approaches could be applied: 1) The maximal daily dose is 5mg (14); maximal

reported bioavailability was F=0.6 (17); the desired flux can be calculated as

Jss=F·Dose/24h=125µg/h=0.40µmol/h; 2) On the basis of the pharmacokinetic data

from the literature, the steady state flux Jss can be calculated as a product of:

apparent clearance (Cl’), bioavailability (F), and desired plasma concentration (Cpl),

Jss=Cl·F·Cpl. With the bioavailability of 0.6, the apparent clearance 457L/h, and the

Cmax=472ng/L, for 1mg dose after long-term administration, the Jss=0.41µmol/h. This

flux of pergolide seems easily achievable, even for weak charge carrier.

Transdermal formulation seemed a reasonable choice for pergolide delivery,

as it assures steady and controllable drug influx to the organism, what is of great


114

importance in case of Parkinson’s disease treatment (19). Previous attempts with

passive delivery of pergolide, from surfactant-based elastic vesicles, did not reach

desired fluxes (20). Iontophoresis enhances transdermal fluxes of drugs, and was

proven an effective mean of drug delivery for large number of drugs (21). In this

study, transdermal iontophoresis of pergolide on the pig skin model, in vitro was

examined. In particularly, the feasibility of delivering therapeutic doses and the

effects of pergolide donor concentration, its mole fraction, pH of donor solution and

current intensity were examined.


Materials

Pergolide (PER) mesylate was purchased from Tocris Bioscience, sodium

chloride, mannitol, tris hydrochloride, agarose, silver wire (99.99% pure), silver

chloride (99.999% pure) were obtained from Sigma Aldrich UK; hydrochloric acid,

sodium hydroxide, methanol HPLC grade (MeOH), tetrahydrofuran HPLC grade, and

n-octanol were supplied by Fisher Scientific UK; triethylamine and perchloric acid

was supplied by Acros Organics UK. Deionised water (resistivity≥18.2MΩ/cm) was

used to prepare all solutions.

Skin preparation


cold running water. The skin slices were taken from the epidermal side, with an



dorsal and abdominal skin, from two different pigs was used in the experiments.


On the day of the experiment, the necessary skin was thawed at them room

temperature. Subsequently, it was clamped between two standard, side-by-side

diffusion cells, with the epidermal side facing the donor chamber. The volume of both

cells was 3.3ml, and the transport area 0.78cm2. Homemade silver-silver chloride

electrodes (22) were placed in both chambers. A constant current of 0.4mA was


115

applied for six hours, using a Kepco APH 1000DM (Flushing, NY) power supply, such

that the donor chamber had higher potential. Both chambers were magnetically

stirred throughout the permeation experiment. The receptor solution contained

unbuffered 0.9% NaCl solution, of pH≈5.8, to provide the main counter-ion, Cl-. The

whole receptor solution was removed and sampled hourly. The donor solution was

replaced with the fresh one every hour to provide enough Cl- ions for correct

electrode electrochemistry.

On one occasion, to separate the electrodes from pergolide mesylate, both

electrodes were placed in separate electrode compartments containing 154mM NaCl

solution, to avoid electrode deterioration. Anode and cathode compartments were

then connected to the donor and receptor compartments, respectively, by salt

bridges. The salt bridge was composed of 100mM tris hydrochloride, in 2% agarose

gel.

Single-ion experiments

Two single-ion experiments were carried out in the pergolide study. In the first

one, saturated, 0.3mM pergolide solution at pH 5.0 adjusted with 0.1M HCl, was

used as donor solution to deliver pergolide. In the second one, salt bridges were

used, as described above and the donor formulation consisted of saturated, 14.5mM

pergolide mesylate solution at pH 4.0. No chloride ions were present in donor, thus

the anode decomposition was expected, due to the silver mesylate formation.

Co-ion pergolide delivery

Iontophoresis was carried out using the solutions containing pergolide and

sodium ions. Total concentration of positive ions was equal to either 29 or 50mM, pH

was adjusted with 0.1M HCl to 4.0. 30mM mannitol was present in the solution. Table

1 summarizes the formulations used in these experiments.

Passive control

Passive flux of pergolide was assessed using a 2.5mM pergolide in 47.5mM

NaCl solution with 30mM mannitol added at pH4.0. No current was applied and the

donor solution was never replaced.


116

Table 1 Iontophoretic formulations used in co-ion pergolide delivery. *The first number represents the pergolide number of replicates and the second one, the number of replicates for mannitol.

PER Mole

Fraction

PER Conc. [mM]

NaCl Conc. [mM]


Mannitol Conc. [mM]

pH Current Intensity

[mA] n*

Current effect 5% 2.5 47.5 50 30 4.0

0.0 5/00.1 6/60.2 6/60.4 6/6

Mole Fraction effect

5% 1.45 27.45 29 30 4.0 0.4

6/625% 7.25 21.75 6/650% 14.50 14.50 6/65% 2.5 47.5 50 30 4.0 0.4 6/0

10% 5.0 45.0 6/0

Analytical Methods

Pergolide

Pergolide was quantified using a Jasco HPLC system (composed of a PU-980

pump, an autosampler AS-1595, and a UV-VIS detector UV-975), by means of the

pergolide external standard calibration curve, in corresponding media. At least seven

standards, ranging from 1µM to 50µM, were used for quantification. Isocratic

separation was performed using Phenomenex Gemini™5, C18 HPLC column

(50 mm x 4.6 mm, 5µm), with a flow rate of 1 ml/min, and an injection volume of 10µl,

with column temperature of 25C. The mobile phase consisted of 70%H2O and

30%AcN, spiked with 200µl/l of triethylamine, and acidified to pH=2.5 with perchloric

acid. Detection was performed at wavelength of 280nm. The limit of detection was

0.1µM and the limit of quantification was accepted as 0.5µM. The precision and

accuracy of the method was assessed as 2% and 7% respectively.

Mannitol

Mannitol was quantified using a Shimadzu LCMS 2010 EV system with an

electrospray ionisation, in the following conditions: a gradient separation (H2O:AcN


column (150 mm x 4.6 mm, 5µm), with a flow rate of 0.5 ml/min, and an injection

volume of 10µl, with column temperature of 25C. Detection was performed at

m/z=218.85 in negative mode with detector voltage of 2.0kV. Data collection rate was

0.2s. Limit of detection was 20nM and limit of quantification was 100nM. The


117

quantification was made by means of external standards, with use of at least nine

standards (from 0.2µM to 40.0µM). The accuracy and precision of the applied

method was assessed as 6% and 7%, respectively.

Determination of distribution coefficient of pergolide

Log D of pergolide at pH4.0 was calculated based on the log P and pKa taken

from the literature as in Equation 1 (23):


Conductivity measurements and pergolide water mobility estimation

Because of a very poor water solubility of pergolide mesylate the estimation of

its water mobility by means of conductivity measurements was impossible.

Solubility study

The excess of pergolide mesylate added to 50ml of water, and the pH was

adjusted to one of the following values: 3.0, 3.6, 4.0, 4.3, 5.0, in three replicates for

each pH value. Mixtures were subsequently placed in water bath at 40°C for two

hours, and were vigorously shaken every 20min. Subsequently the solutions were

allowed to cool to room temperature. After centrifugation, supernatants were

separated and analysed for pergolide content.







average value of the three final hours of experiment. Transport numbers for each

replicate were calculated as in t#=z·F·J/I, from the average of fluxes for last three

hours of experiment. The data on the graphs is presented as mean value of all

replicates with standard deviation. All regressions were performed using the least

square method, and regression significance was tested with ANOVA.


118


Solubility study

The water solubility of pergolide mesylate was assessed for a series of pH

values. The results are presented on Figure 2. It is clear that with rising pH the

solubility rapidly drops. The negligible solubility at pH 5.0 limits the practical

possibility of delivery from the donor solutions of this acidity. At pH 4.0, solubility is

roughly 14.5mM, what seems reasonable concentration for iontophoretic delivery;

however, as pergolide was used as a mesylate salt, the presence of additional

chloride ions would be necessary in donor, to avoid the decomposition of Ag/AgCl

electrodes.

pH 3.0 pH 3.6 pH 4.0 pH 4.3 pH5.00

10

20

30

Perg

olid

e co

ncen

trat

ion

[mM

]

Figure 2 Solubility of pergolide mesylate in water, for different values of pH (n=3) in the absence of NaCl.

Small pergolide solubility at pH 5.0 made the conductimetric assessment of

water mobility of the drug impossible. On the other hand, at pH 4.0, the concentration

of hydronium ions was too high to allow correct measurements.

Co-ion experiments

Pergolide used in all the experiments was in form of mesylate, it was then

necessary to add a source of chlorides (NaCl), for the Ag/AgCl electrodes to keep the

intrinsic electrode voltage low. The effect of mole fraction of pergolide ions, and also

the effect of total ionic concentration was investigated in several experiments, using

sodium as competing ions (Table 1). The results are presented at Figure 3 and


119

Figure 4. It can be observed at Figure 3, that with rising mole fraction there is certain,

however not particularly marked increase in pergolide flux. Also, a concomitant sharp

drop in mannitol flux was detected. Both the phenomena: the increase in flux with

rising mole fraction, and limitation of the electroosmosis, were observed for other

drugs investigated in previous chapters. The increase of the drug flux is explained by

rising drug concentration and therefore competition for charge carrying. At the same

time, lipophilic, positively charged drug-molecules penetrating the skin (at pH 4.0 with

the stratum corneum iP of 4.3), could enhance the positive charge in the skin, thus

limiting the electroosmosis in the direction from anode to cathode. This in turn,

reduces the bulk volume flow. Similar fluxes for 0.05 and 0.1 pergolide mole fraction

can be explained by these two phenomena cancelling each other out.

0.05 0.10 0.500

5

10

15

20

25

Pergolide Mannitol

Pergolide Mole Fraction

Flux

[nm

ol/h

]

0.05 0.100

5

10

15

20

25

29mM 50mM

Pergolide Mole Fraction

Perg

olid

e Fl

ux [n

mol

/h]

Figure 3 The dependence of pergolide () and mannitol () flux on pergolide mole fraction in donor solution. The pH of solutions was 4.0 and the total molarity was 29mM.

Figure 4 The comparison of pergolide flux for the same values of mole fraction (0.05 and 0.1) and for different total molarities, 29mM () and 50mM (). The pH of the donor solutions was 4.0.

Interestingly, pergolide fluxes obtained for higher, 50mM, total ionic

concentration are statistically significantly higher, than the fluxes for 29mM donor

solutions. Similarly to 29mM solutions, for 50mM total molarity, the shift in mole

fraction from 0.05 to 0.1 did not affect the flux (Figure 4).

In order to verify, whether the delivery of pergolide is controllable by

adjustment of current, the four experiments were carried out, each one for different


120

current intensity (Table 1). The donor solution chosen for the experiments, was

0.05 pergolide mole fraction and 50mM total molarity at pH 4.0. This formulation was

found equally effective, as the one having pergolide at 0.1 mole fraction, or 0.5 mole

fraction, at total molarity 29mM. It also contained the least of the drug. The results of

current experiments are presented at Figure 5. The transport number can be

obtained from the slope of the regression line, rendered by the plot, as in equation

t#=J·F/z·I. The slope was estimated as 42.0±7.5, and the corresponding transport

number was t#=0.0011±0.0002. This transport number is very small in comparison to

previously investigated drugs. However, the related flux of 16.5±3.0nmol/h at current

intensity of 0.4mA, seem to be high enough to match the pergolide target flux

Jss=0.41µmol/h. With 0.5mA/cm2 being an acceptable current density for human

applications, the required area A of iontophoretic patch is given by:

A=2·(0.41µmol/h·0.78cm2 / 16.5nmol/h)=38.8cm2.

These estimations nonetheless, do not take into account several practical

issues. First, the intophoretic dosage form is meant to be applied once daily. This

long delivery requires a significant amount of chloride ions for the electrochemical

reaction at the anode. This amount was estimated in the discussion of Chapter III, to

be around 0.45M for the current density of 0.5mA/cm2. This high concentration of

could reduce the in vivo fluxes making the delivery ineffective.

Figure 5 Pergolide as a function of current intensity. The drug was delivered from donor solution of 50mM total cationic concentration, 0.05 pergolide mole fraction at pH4.0. The points render a straight line (p<0.05, r2=0.6) of equation y=42.0±7.5·y+0.1±1.7. The dotted line limits the 95% confidence interval.

0.0 0.1 0.2 0.3 0.40

10

20

30


Perg

olid

e Fl

ux [n

mol

/h]


121


Two single-ion experiments were carried out. In the first one, the saturated

solution of 14.5mM pergolide mesylate at pH 4.0 was used as a donor. The solution

was separated from the electrode by a salt bridge. The average flux obtained in this

experiment was 16.8±8.2nmol/h. The corresponding transport number was

0.0011±0.0005. This value is very similar to the transport number, obtained form the

slope of the curve on Figure 5. However, the formulations used in current

experiments (Figure 5) had pergolide only at 0.05 mole fraction. The fact, that the

delivery from formulations of so remote mole fractions (0.05 vs. 1) resulted in very

similar flux suggests, that the role of mole fraction in pergolide delivery is secondary.

In the work of Nugroho et al. it was suggested, that the iontophoretic transport could

be composed of two phases. The first is loading the skin with delivered compound

with the use of electric driving force, and then passive diffusion of the compound to

the subdermal compartment. The small effect of molar fraction in the iontophoretic

transport of pergolide can be explained by a much smaller partition rate from the ski

to the subdermal compartment in comparison to the skin loading rate. This

In the second experiment, where the saturated solution of 0.3mM pergolide

mesylate at pH 5.0 was used, the drug flux was much smaller, 2.1±1.1nmol/h. Also,

high voltages were building between the electrodes and, throughout the experiment,

the deterioration of electrodes was observed. This was most probably caused by low

saturation concentration of pergolide at applied pH and the absence of chlorides ions

in donor solution. This limits the practical use of this setup.

Conclusions

Pergolide is a very poor charge carrier, nonetheless, it can be successfully

delivered with the iontophoretic patch of reasonable size. The current applied during

transdermal iontophoresis affects the transport in linear way. However, because of

the small transport number, the regression line is rather flat. This might make the

controllable delivery of the drug difficult. Pergolide mesylate is likely to be delivered

from the solutions containing background electrolyte, to avoid mesylate reacting with

Ag/AgCl electrodes, and also because of its poor water solubility. However, the


122

presence of background electrolyte does not have a major negative impact on the

magnitude of the drug transdermal flux.

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5. Pergolide vesus levodopa monotherapy in early Parkinson's disease

patients: the PELMOPET study. Oertel, W H, et al. 2006, Movement Disorders, Vol.

21, pp. 343-353.



7. High-dose pergolide monotherapy in the treatment of severe levodopa-

induced dyskinesias. Facca, A and Sanchez-Ramos, J. 1996, Movement Disorders,

Vol. 11, pp. 327-341.

8. A multicenter double-blind placebo-controlled trial of pergolide as an adjunct

to Sinemet in Parkinson's disease. Olanow, W C, et al. 1994, Movement Disorders,

Vol. 9, pp. 40-47.


123

9. Randomized trial of tolcapone versus pergolide as add-on to levodopa

therapy in Parkinson's disease patients with motor fluctuations. Koller, W, et al. 2001, Movement Disorders, Vol. 16, pp. 858-866.

10. Comparison of pergolide and bromocriptine therapy in parkinsonism.

LeWitt, P A, et al. 1983, Neurology, Vol. 33, pp. 1009-1014.

11. Evidence-based medical review update: pharmacological and surgical

treatments of Parkinson's disease: 2001 to 2004. Goetz, C G, et al. 2005, Movement


12. Valvular heart disease associated with taking low-dose pergolide for

restless legs syndrome. Worthington, A and Thomas, L. 2008, European Journal of

Echocardiography, Vol. 9, pp. 828-830.

13. Pleuropulmonary Fibrosis after long-term treatment with the dopamine

agonist pergolide for Parkinson's disease. Tintner, R, et al. 2005, Archives of




15. Pergolide pharmacokinetics in patients with Parkinson's disease.

Thalamas, C, et al. Supplement 1, 2001, Parkinsonism & Related Disorders, Vol. 7,

pp. S71-S72.

16. Pergolide: Multiple dose pharmacokinetics in patients with mild to

moderate Parkinson's disease. Thalamas, C, et al. 2005, Clinical


17. Clinical pharmacokinetic and pharmacodynamic properties of drugs used

in Parkinson's disease. Deleu, D, Northway, M G and Hanssens, Y. 2002, Clinical





124

19. Intermittent vs continuous levodopa administration in patients with

advanced Parkinson disease: a clinical and pharmacokinetic study. Stocchi, F, et al. 2005, Archives of Neurology, Vol. 62, pp. 905-910.

20. Transdermal delivery of pergolide from surfactant-based elastic and rigid

vesicles: characterisation and in vitro transport studies. Honywell-Nguyen, P L, et al. 2002, Pharmaceutical Research, Vol. 19, pp. 991-997.




the skin in vitro. Green, P G, et al. 1991, Pharmaceutical Research, Vol. 8, pp. 1113-

1120.

23. Physicochemical properties and transport behaviour of piribedil:

Considerations on its membrane-crossing potential. Tsai, R, et al. 1992, Internationa

Journal of Pharmaceutics, Vol. 80, pp. 39-49.



Chapter V Trihexyphenidyl

125

Chapter V Trihexyphenidyl Trihexyphenidyl (THP) (Artane®),

known also as benzhexol, have been the first

synthetic anticholinergic drug introduced to

the market. It is a non-selective antimuscarinic

agent (1), with some activity of blocking N-

methyl-D-aspartate (NMDA) receptors (2).

Trihexyphenidyl has been long used in

Parkinson’s disease (PD) therapy, particularly

in young patients. After the introduction of L-

dopa, the importance of anticholinergic drugs

decreased; nevertheless they still play a significant role in PD treatment, as they

provide an alternative mechanism of action, and are able to alleviate some serious

symptoms of the disease. In fact, the mechanism of action of trihexyphenidyl and

other anticholinergics still remains unclear. Several possible ways, anticholinergics

could act on central nervous system, have been postulated. One hypothesis

postulates, that acetylcholine antagonists restore the balance between dopamine and

acetylcholine neurotransmission in basal ganglia, which is disrupted by

neurodegeneration of substantia nigra and decreased dopamine transmission in PD

(3). Another study (4) has shown that some anticholinergics function as strong

inhibitors of the presynaptic, carrier-mediated, dopamine-reuptake mechanism, in

which dopamine molecules, that enter the nerve terminal by means of the carrier, are

accompanied by two or more sodium ions and one chloride ion. Finally, some of

anticholinergics possess NMDA-receptor antagonist properties, which have a

beneficial effect on akinesia (5).

Although trihexyphenidyl has been present on the market for a long time, only

very few clinical studies investigated the benefit of its employment in PD therapy.

Takahashi et al. investigated the effect of 6mg/day of trihexyphenidyl, on

parkinsonian symptoms and cognitive abilities, in six de novo PD patients (6). All

patients showed clinical improvement in PD symptoms after seven weeks, while no

impairment in cognitive function was noted. As reviewed by Deleu et al. (5),

Figure 1 Chemical structure oftrihexyphenidyl. Mw=301.2Da

OHN


126

trihexyphenidyl therapy provides improvement of 24% of parkinsonian symptoms in

as much as 76% of patients. Another study (7), showed that trihexyphenidyl can be

beneficial in patients non-responsive to L-dopa.

There is relatively limited data on trihexyphenidyl pharmacokinetics. In fact, no

intravenous studies are available, thus some pharmacokinetic parameters, as

absolute bioavailability, are unavailable (8). Couple of studies explored

trihexyphenidyl pharmacokinetic parameters after oral administration. Burke and

Fahn (9) examined three de novo patients, and 14 patients on long-term

trihexyphenidyl therapy, after administration of five to ten milligrams of the drug. In

long-term patients the absorption was relatively fast, tmax=1.3±0.2h, and the half-life

was t½=3.2±0.7h. For the three patients who did not have the anticholinergic therapy

before, a marked distribution phase was observed, followed by the elimination phase,

after ingestion of the single oral dose of 10mg. The half-lives for both phases were,

t½α=1.3±0.5h and t½β=5.6±2.7h, respectively. The absorption was also fast,

tmax=1.5±0.5h. The assay employed in the study was, however, questioned for lack of

specificity (10). Another study (10) reported following parameters, that after a single

oral dose of 4mg trihexyphenidyl: tmax=1.3±0.6, Cmax=21.2±7.6nM, area under the

curve from 0 to 72nd hour AUC0-72=594.9±210.1nM·h, and biphasic half-life was

t½α=5.3±3.2h and t½β=32.7±6.3h, measured in eight patients. A different study looked

at the metabolism of trihexyphenidyl, and reported that the drug is mainly excreted in

urine, as hydroxy-metabolite, with the alicyclic group being hydroxylated (11).

Trihexyphenidyl is normally administered in dose starting from 1mg daily, increased

gradually to the usual maintenance dose of 5 to 15mg in three to four doses daily

(12). The maximal daily dose is 20mg.

Physicochemical parameters of THP are not very well investigated.

Dissociation constant is pKa=8.7 (13); logP=5.3 (14). These values along with small

molecular mass of 301.2Da suggested, that trihexyphenidyl might be delivered

transdermally. Transdermal iontophoresis in particular, offers steady drug delivery,

limits the number of daily applications, allows adjusting the drug influx and could help

with more accurate dosage of the drug to the patient.

In this study, transdermal iontophoresis of trihexyphenidyl across the pig skin

model in vitro was examined. In particularly, the feasibility of delivering therapeutic


127

doses, the effects of THP donor concentration, its mole fraction, pH of donor solution

and current intensity were examined. To investigate the magnitude of the

electroosmotic effect, contributing to total trihexyphenidyl transport, transdermal

mannitol transport was studied.

With the limited pharmacokinetic data, it is impossible to accurately assess the

transdermal flux, required to achieve upper range of therapeutic plasma

concentrations, hence the feasibility of iontophoretic delivery. Nonetheless, using the

data of He and co-workers (10), the least sufficient flux, that is the flux above which

the desired plasma concentration would certainly be achieved, can be evaluated as

follows: Assuming the total absorption of the drug in gastrointestinal tract, the

maximal value of clearance is equal to Cl=D/AUC=19.9L/h. To achieve the plasma

concentrations at the level of Cmax, the required flux has a value of

J=Cl·Cmax=0.42µmol/h.

Trihexyphenidyl Materials and methods

Materials

Trihexyphenidyl (THP) hydrochloride, sodium chloride, mannitol, silver wire

(99.99% pure), silver chloride (99.999% pure) were obtained from Sigma Aldrich UK;

hydrochloric acid, sodium hydroxide, acetonitrile HPLC grade (AcN) and n-octanol

were supplied by Fisher Scientific UK; triethylamine was supplied by Acros Organics

UK. Deionised water (resistivity≥18.2MΩ/cm) was used to prepare all solutions.

Skin preparation

Full thickness porcine skin, obtained from a local abattoir, was rinsed under


electric dermatome (Zimmer, Dover, OH), set to 750µm thickness, and then were

wrapped in Parafilm® and stored at -20°C, for no longer than four months. Both,

dorsal and abdominal skin from four different pigs was used in the experiments.


Skin was thawed at room temperature on the day of the experiment.

Subsequently, it was clamped between two standard, side-by-side diffusion cells,

with the epidermal side facing the donor chamber. The volume of both half-cells was


128

3.3ml, and the transport area 0.78cm2. Silver-silver chloride electrodes were placed

in both chambers (15). A constant current of 0.4mA was applied for six hours using a

Kepco APH 1000DM (Flushing, NY) power supply, such that the donor chamber had

higher potential. Both chambers were magnetically stirred throughout the permeation

experiment. The receptor solution contained 0.9% NaCl solution of pH≈5.8, to

provide the main counter-ion, Cl-. The whole receptor solution was removed and

sampled hourly. The unbuffered donor solution was replaced with fresh one every

two hours (unless otherwise stated), to provide enough Cl- ions for correct electrode

electrochemistry.


Single-ion experiments had trihexyphenidyl as the only cation in the donor

solution. This study was set up to investigate the effect of a) trihexyphenidyl

concentration; b) the current intensity; and c) the pH, on trihexyphenidyl transdermal

transport. 30mM mannitol was present in all solutions as a marker of electroosmosis.

The detailed donor solution formulations are presented in Table 1.

Table 1 Trihexyphenidyl single-ion formulations. *The second number refers to the number of replicates for mannitol flux measurements.

THP Conc. [mM]


Current Intensity

[mA] n*


20 30 5.4 0.4

6/6 30 12/6

pH effect 30 30

2.0

0.4

5/0 3.0 6/6 4.0 5/5 5.0 6/0 5.4 12/6 5.8 8/0

Current intensity

effect 30 30 5.4

0.0 5/0 0.1 6/0 0.2 6/0 0.4 12/6


Two series of experiments including competing ions in the donor solution were

performed to establish a) the role of competing ions, and b) the role of total ionic

concentration in transdermal delivery of THP. In the first series of experiments the


129

total ion concentration in donor formulation was kept constant at two values: 20mM

and 30mM, while the THP content was varied (from 12.5 to 100% mole fraction).

Subsequently, THP was delivered from formulations with a 12.5% THP mole fraction

with total cation concentration varying form 30 to 80mM; and 25.0% THP mole

fraction with total cation concentration varying from 20 to 40mM. 30mM mannitol was

present in all formulations as a marker of electroosmosis. The detailed composition of

the donor formulations is specified in Table 2.

Passive control

Passive flux of trihexyphenidyl was assessed using a similar experimental

setup, except that no current was applied and the donor solution was never replaced

(Table 1).

EO control

The reference electroosmotic flow of mannitol, in the absence of THP, was

measured using a donor solution containing 30mM NaCl and 30mM of mannitol at pH

of 5.0 and 5.8 adjusted with 0.01 HCl.

Table 2 Composition of iontophoretic formulations used in trihexyphenidyl co-ion experiments. *The second number refers to the number of replicates for mannitol flux measurements.

Mole Fraction

PBD Conc. [mM]

NaCl Conc. [mM]


pH THP Valence

Current Intensity

[mA] n*

12.5% 3.75 26.25

30 5.8 1.0 0.4

6/5 25% 7.5 22.5 6/6 50% 15.0 15.0 6/0 75% 22.5 7.5 5/0

100% 30.0 0 12/625% 5.0 25.0

20 5.7 1.0 0.4

6/0 50% 10.0 10.0 5/0 75% 15.0 5.0 6/0

100% 20 0 6/6

12.5% 3.75 26.25 30

5.8 1.0 0.4 6/5

5.0 35.0 40 12/010.0 70.0 80 6/0

25% 5.0 15 20

5.9 1.0 0.4 6/0

7.5 22.5 30 6/010.0 30.0 40 11/0


130

The blank mannitol flow, originating from the skin, was measured using donor

solution containing 30mM NaCl and no mannitol, at pH of 5.8 adjusted with

0.01M HCl.

Analytical Methods

Trihexyphenidyl

Trihexyphenidyl was quantified using a Jasco HPLC system (composed of a

PU-980 pump, an autosampler AS-1595, and a UV-VIS detector UV-975) by means

of trihexyphenidyl external standard calibration curve in corresponding media of at

least seven standards. Isocratic separation was performed using Dionex Acclaim™

120, C18 HPLC column (150 mm x 4.6 mm, 5µm) with flow rate of 1 ml/min and

injection volume 10µl with column temperature of 25C. The mobile phase consisted

of 60%H2O and 40%AcN spiked with 200µl/l of triethylamine and acidified to pH=2.5

with perchloric acid. Detection was performed at wavelength of 195nm. Limit of

detection was 0.1µM and limit of quantification was accepted as 1µM. The

concentrations of standards ranged form 5µM to 150µM. The precision and accuracy

of the method was 3% and 5%, respectively

Mannitol

Mannitol was quantified using a Shimadzu LCMS 2010 EV system, with

electrospray ionisation, in the following conditions: a gradient separation (H2O:AcN


column (150 mm x 4.6 mm, 5µm) with a flow rate of 0.5 ml/min and an injection

volume of 10µl with column temperature of 25C. Detection was performed at

m/z=218.85 in negative mode with detector voltage of 2.0kV. Data collection rate was

0.2s. Limit of detection was 20nM and limit of quantification was 100nM. The

quantification was made by means of external standards with use of at least nine

standards (from 0.2µM to 40.0µM). The accuracy and precision of the applied

method was assessed as 6% and 7%, respectively.

Solubility of trihexyphenidyl

The excess of trihexyphenidyl hydrochloride was added to: 50ml of water,

50ml of 60mM NaCl, and to 50ml of 100mM NaCl solution. The mixtures were


131

shaken in a water-bath, at the temperature of 50°C, for three hours. Subsequently

the solutions were let to cool down to room temperature. The pH of solutions was

checked. After centrifugation, supernatants were collected and analysed by HPLC for

THP content.

Determination of distribution coefficient of trihexyphenidyl

The volume of 4ml of n-octanol was added to 4ml of 1mM trihexyphenidyl HCl

solution of pH 5.0 and 4.0. Mixtures were shaken for 48 hours to achieve equilibrium

in 25°C. After that time, water and octanol phases were collected manually to

separate vials and were analysed by HPLC. Distribution coefficient was calculated

from Equation 1 (16).

Equation 1 water

oloc

CCD tanloglog =

LogP and pKa was estimated using Equation 2 (17).


Conductivity measurements and trihexyphenidyl water mobility estimation

The specific conductivity was measured for a range of trihexyphenidyl water

solutions (15, 10, 5, 2.5, 1.25mM), using a conductimeter Metrohm T-120, at 22C,

with a probe of cell constant K=0.93. The pH of solutions was measured. Then, the

molar conductivities (specific conductivity normalized by concentration) were

calculated, and plotted against the square root of concentration. Conductivity at the

infinite dilution was estimated by extrapolating the obtained curve to zero.

Subsequently, using Kohlraush law of independent ion migration (Equation 3, with

mobility of chloride ion, µCl-=7.9·10-8 m2/s/V, taken from literature) water mobility of

trihexyphenidyl ion was calculated (18; 19). Afterwards, water transport numbers

were estimated as in Equation 4 and Equation 5 (18).

Equation 3 −

∞−+

∞+∞ Λ+Λ=Λ CC

Equation 4 −−Λ

=∞

ClTHP Fµµ


132

Equation 5 −+

=ClTHP

THPTHPt µµ

µ







average value of the 4th-6th hour of iontophoresis. Transport numbers for each

replicate were calculated as t#=J·z·F/I, from the fluxes obtained. The data on the

graphs is presented as mean value of all replicates with standard deviation. All

regressions were performed using the least squares method.


Assessment of physicochemical characteristics

Solubility study

The concentrations of saturated THP·HCl solution in water, 60mM NaCl, and

100mM NaCl, was respectively: 31.5±0.2mM, 15.4±0.4mM, and 8.7±0.05mM, at

pH≈5.4. This allowed calculating the solubility product of THP·HCl KSP=1.0±0.1·10-3.

Conductivity study

Recently, it was found that water transport number can be a good predictive

factor for skin transport number (20). Indeed, a strong, positive correlation was found

between the two. Trihexyphenidyl molar conductivity for infinite dilution Λ∞, was

estimated by extrapolation of the plot of molar conductivity, as a function of square

root of concentration. The THP water transport-number was calculated as in

Equation 4 and Equation 5, and the value obtained was t#THP+=0.10±0.003.


133

Figure 2 Molar conductivity of THP HCl as a function of square root of concentration. The data points are the means of three replicates, with standard deviations (within the point). The regression was significant and rendered a straight line (r2=0.98, p<0.0001). The Y-intercept is the infinite dilution molar conductivity, Λ0

∞.

pH experiments

In this set of experiments the impact of donor pH on trihexyphenidyl

transdermal transport was studied. The range of pH values was restricted by the

solubility of trihexyphenidyl, which at pH 5.8 was 31.5±0.2mM. Although THP

solutions of higher pH can be achieved, small THP concentrations imposed by low

solubility, would make impossible the single ion delivery. At the pH 5.8, THP was

completely ionized, as its pKa=8.7. The lower threshold of pH was set as 2.1, what

was just below the 2.5 value considered as harmless in human use (21). The flux of

trihexyphenidyl was assessed for series of pH values (Table 1). The fluxes of both,

THP and mannitol are illustrated in Figure 3. It is visible that except for the lowest,

2.1, value of pH, the drug flux is roughly stable. In the range of pH, form 3.0 to 5.4,

no statistically significant differences were found (ANOVA, followed by Bonferroni

test). The flux for pH 5.8 was lower in comparison to fluxes at pH 4.0, 5.0 and 5.4, by

19% (p<0.01); the statistical analysis found no differences between fluxes at

the pH 3.0 and 5.8.

0 1 2 3 4 570

75

80

85

90

Λ∞

C1/2[mM]1/2

Mol

ar C

ondu

ctiv

ity [S

i ⋅cm

2 /mol

]


134

Figure 3 trihexyphenidyl () and mannitol () fluxes from a donor solution containing 30mM of drug as single-ion, and 30mM of electroosmotic marker, for various pH values. The intensity of current applied was 0.4mA in all cases.

Mannitol fluxes measured were all very small, compared to the 107±12nmol/h

reference mannitol flux (in absence of the drug), and are much closer to the blank

mannitol flux (originating from the skin). The small effect of pH on trihexyphenidyl

transport is quite surprising. The studies described in previous chapters, and other

investigating the effect of pH on drug transdermal transport (22; 23; 24), revealed

pH’s crucial role in the iontophoretic process. The pH of donor solution determines

ionisation of delivered drug and the direction and magnitude of electroosmotic flow

(25). Very low and high pH values will lead to substantial hydronium and hydroxyl ion

concentrations, which are very mobile and can impede iontophoretic transport.

Ionisation

In case of trihexyphenidyl, in range of pH investigated, no change in ionisation

was expected, as its pKa=8.7. This causes practically all the THP molecules to be

ionised at the studied pH values.

H+ competition

Because of different charge-passing mechanism, hydronium ions are very

mobile (µH+=35·10-8m2/sV). Due to high mobility they can effectively compete for

charge carrying even at small concentrations. However, in case of trihexyphenidyl,

except for the smallest pH value of 2.1, no competition can be expected from H+ ions.

2.1 3.0 4.0 5.0 5.4 5.80.0

0.1

0.2

0.3

0

1

2

3

4

pH

Trih

exyp

heni

dyl f

lux

[nm

ol/h

]

Mannitol Flux [nm

ol/h]


135

The drop of THP flux at pH2.1, probably due to H+ competition, can be observed in

Figure 3.

Electroosmosis

In Figure 3 it is visible that mannitol flux changes with the pH. This indicates

that the pH affects also the bulk volume flow. However, the magnitude of mannitol

transport in relation to the reference values, measured for 30mM NaCl and no drug in

donor (95.2±8.1nmol/h, 107.3±11.6nmol/h, for pH 5.0 and 5.8 respectively), shows

that the volume flow is highly restricted by THP. This suggests that electroosmotic

contribution to THP transport is likely to be small. This also might explain why the

changes in mannitol transport are not accompanied by changes in THP transport,

and in consequence, the little effect of pH on THP transdermal flux. In several

previous papers (26; 27; 28) it was suggested that iontophoresis of charged and

lipophilic molecules can cause the suppression or reversal of electroosmotic flow.

This would be caused by strong interaction of lipophilic moieties with negatively

charged proteins of skin, which could neutralize or reverse the net negative charge of

the skin.

Similar observations were made for drugs studied in previous chapters

(piribedil and pramipexole). The effect of pH was investigated in the similar

conditions, and comparable mannitol fluxes were obtained. It is particularly

interesting observing the mannitol and drug fluxes, for pH ranging from 4.0 to 5.0, for

all three drugs. It can be noted, that for steady mannitol flux pramipexole and piribedil

fluxes drop; while for THP, drug flux remains steady regardless of varying mannitol

flux. These data seems to show that 1) during transdermal iontophoresis of organic

molecules, in single-ion situation, the electroosmosis is severely restricted; 2)

changes in electroosmotic flux is often not followed by relevant changes in drug flux.

These observations raise a question, whether during single-ion drug iontophoresis,

the change of pH causes the relevant changes of drug flux because of changing the

electroosmotic effect, or rather some other factors might be involved.

On the other hand, the importance of electroosmosis on transdermal transport

was demonstrated previously, when delivering the drugs from buffered solutions.

Merino et al. (22) have shown that transdermal transport of 5-fluorouracil (5FU), can


136

be equally efficient when delivered by electroosmosis or electromigration, from

solutions with high background electrolyte content. Also, Abla and co-workers (23)

have shown the significant effect of electroosmosis in peptide delivery. Nonetheless,

these studies investigated the delivery of very hydrophilic drugs (in silico logP -0.9

and -1.7 for 5FU and kyotorphin (29)) from buffered solutions. During the

iontophoresis from solutions containing competing ions, the concentration of

delivered drug in the skin is smaller, than during single-ion delivery. They are then,

less prone to interact with the skin. Also, for hydrophilic drugs, higher concentration

might be required to interact with the lipophilic skin proteins. Also, the paper of Marro

et al. (28), and Hirvonen et al. (30), seem to confirm these ideas. The three drugs,

lidocaine, propranolol and quinine, delivered as single ions limit the electroosmosis

accordingly to their logP values. When delivered from solutions containing

background electrolyte, the inhibitory effect is less accentuated, and dependent on

drug concentration.


Similarly to the previously studied drugs, the effect of THP concentration on

transdermal transport was assessed in single-ion situation. The solubility of THP from

one side, and the minimal chlorides concentration from the other, restricted the

investigated concentration range to relatively narrow band, from 20mM to 30mM.

Unlike in case of piribedil or pramipexole, the decrease of THP hydrochloride

concentration to 15mM, resulted in very high voltage building up on the cells, and

subsequent rapid electrode decomposition. The behaviour of THP flux, as a function

of its concentration in donor, is no different than the drugs studied in previous

chapters (Figure 4). A t-test did not find any statistically significant differences

between the THP delivery from 20 or 30mM solution. The issue was discussed in

detail in previous chapters, and the explanation invoked was the dependence of the

flux in singe-ion situation only on relative mobilities of delivered ion and counter-ion,

taking part in iontophoretic process (31).


137

20mM 30mM 20mM 30mM0.00

0.05

0.10

0.15

0.20

0.25

0

1

2

3

4

5

Trih

exyp

heni

dyl f

lux

[µm

ol/h

]

Mannitol Flux [nm

ol/h]

0.0 0.1 0.2 0.3 0.4 0.50.00

0.05

0.10

0.15

0.20

0.25


Trih

exyp

heni

dyl f

lux

[µm

ol/h

]

Figure 4 Trihexyphenidyl () and mannitol () fluxes from solutions of different drug concentrations. The pH of donor solutions was 5.8 and the current was 0.4mA.

Figure 5 Linear regression betweentrihexyphenidyl flux and applied current (p<0.0001, r2=0.98). The dotted line limits the 95% confidence interval of the regression. The equation of the line is given by y=0.48±0.01·x+0.017±0.004. The transport number obtained from the slope after according the units is t#= 0.013±0.0003.

From practical point of view, this phenomenon is quite convenient, as it allows

for iontophoretic delivery of drugs with high efficiency, using the low concentrated

formulations.

Current effect

The following studied factor was the effect of applied current intensity on the

transport of THP. The drug flux was investigated for three different current

intensities. The control experiment consisted of the same experimental conditions,

except for no current was applied. The results are presented in Figure 5. Clearly, the

dependence is linear (p<0.0001, r2=0.98). The THP transport number can be

assessed from the slope of the regression, accordingly with equation t#=J·F/z·I. The

transport number calculated from the slope was t#=0.013±0.0003. This means that

only 1.3% of total charge passing through the skin during the experiment was carried

by trihexyphenidyl molecules. In comparison to drugs investigated previously in

similar conditions (pramipexole, selegiline, piribedil, lidocaine (32) or hydromorphone

(33)), this transport number is rather small. The estimated target flux, required to

deliver therapeutic doses of the drug was 0.42µmol/h. The highest flux measured at

0.4mA was roughly 0.2µmol/h. With the transport area of 0.78cm2, the current

density was 0.51mA/cm2, which is the top value regarded as safe. This shows, that


138

the effective delivery of the drug can be already achieved with the relatively small

patch, of total area A=2·(0.42µmol/h·0.78cm2 / 0.20µmol/h)=3.3cm2. However, to

achieve long lasting (24h) delivery, an additional source of chlorides is required. As

shown below trihexyphenidyl can suffer a presence of competing ions without a

significant decrease of a flux. Thus, it could be possible, that adding an external Cl- source and enlarging patch area could provide sufficient delivery rate. The additional

in vivo study would be required to confirm these predictions.

Co-ion delivery

In the subsequent set of experiments, the effect of competing sodium ions

was systematically studied. The study was set up to investigate the effect of total

ionic concentration and the molar fraction of THP to sodium ions, on THP

transdermal flux. The detailed compositions of investigated formulations are

summarized in Table 2. The fluxes were measured in two series of experiments, with

two different summary ionic concentrations, 20mM and 30mM. The results are

presented in Figure 6b. The two-way ANOVA found no statistical significance in

change of flux when total molarity was elevated from 20mM to 30mM. Mole fraction,

on contrary was found to have some impact on the flux. Unlike for small inorganic

cations, like sodium or potassium (34), or hydrophilic organic compounds like

lidocaine (32), the THP delivery was not a linear function of its molar fraction in the

donor. In fact, except for 0.125 mole fraction for 30mM series, the flux is relatively

stable and fluctuates slightly around 0.19µmol/h, with the overall variability CV=24%.

This degree of variability was previously attributed to the skin (33).

0.00 0.25 0.50 0.75 1.000.000

0.005

0.010

0.015

0.020

0.0

0.1

0.2

0.3

Mole Fraction

THP

Tran

spor

t Num

ber

THP Flux [µ m

ol/h]

0.25 0.50 0.75 1.000.0

0.1

0.2

0.320mM30mM

Mole Fraction

Trih

exyp

heni

dyl f

lux

[µm

ol/h

]

0.00 0.25 0.50 0.75 1.0005

10

152025

100150200

Mole Fraction

Man

nito

l Flu

x [n

mol

/h]

a b c

Figure 6 The effect of competing ions. A) THP transport number as a function of THP mole fraction in donor solution. Data is fitted to Phipps and Gyory model (31). Estimated B factor was 0.027±0.005. The total ionic concentration was 30mM, and the pH=5.6. B) The comparison of THP fluxes from solutions of 20mM and 30mM total ion concentration, and various drug mole fractions. C) Mannitol fluxes measured during THP iontophoresis for different mole THP mole fractions. The total molarity was 30mM, and the pH was 5.6.


139

In an attempt to model the transdermal drug transport, Phipps and Gyory (31)

derived an expression that links a transport number of a drug (td), with its mole

fraction in the donor solution, across an uncharged and homogenous membrane:

Equation 6

XZBtt

ttt

t

ed

d

d

d

d

⋅⋅−

++−

−=

00

0

0

0

111

1

1

where td0 and te0 are the transport numbers of drug and competing cation, in

the single-ion situation; Z is ratio of cation valences; X is the mole fraction of the

drug. The parameter B was described as the proportionality constant that relates the

ratio of cations concentrations in the donor solution, to these in the membrane:

Equation 7 ds

es

d

e

CCB

CC

=

where Ce and Cd are the concentrations of, respectively, inorganic cation and

drug in donor solution; and Ces and Cds are concentrations of inorganic cation and

drug in the skin. After rearrangement of Equation 7, factor B can be expressed as:

Equation 8 d

e

dsd

ese

PP

CCCCB ==

The Pe and Pd are the partition coefficients of competing cation and drug,

between donor solution and skin. The data from the experiments with 30mM total

ionic concentration was fitted with the Equation 6, assuming the te0=0.6 (34). Figure

6a depicts the measured transport numbers and the curve obtained form the fitting.

The B parameter obtained from the fit was 0.027±0.005. This small value of B

reflects the lipophilic character of the delivered drug in relation to the competing ion.

This observation stays in accordance with the measured LogP of trihexyphenidyl. Its

high value suggests the possibility of the drug partitioning into the skin. It is,

however, extremely hard to have a validated method of prediction of parameter B,

what limits the practical use of this approach (20).

To assess the electroosmotic effect in THP iontophoresis, the fluxes of

electroosmotic marker, mannitol, were measured. Results are presented in Figure

6c. A sharp drop of mannitol transport can be observed even for small THP mole


140

fraction. Similar phenomenon was observed previously for quinine and propranolol

(28), and also for piribedil, described earlier in this thesis. Strong limitation of

electroosmotic flow is characteristic to the highly hydrophobic, positively charged

species, as they can partition to the skin, and neutralize or reverse the negative

charge fixed on it.

Conclusions

Trihexyphenidyl is a relatively poor charge carrier, with the highest value of

transport number of only t#=0.014. However, due to the small doses required in

therapy, and moderate clearance, this is transport number is sufficient for effective

delivery of therapeutic doses. In comparison to the previously investigated drugs,

factors like pH or the presence of competing ions have relatively small effect on THP

transport. Among the studied factors the current intensity had the biggest effect on

THP transdermal delivery, and can be used to profile the drug level in plasma. The

water transport number of trihexyphenidyl is several times higher than its transport

number across the skin, and is not very good predictor of the drugs transdermal

charge-carrying properties.

Bibliography

1. Effect of trihexyphenidyl, a non-selective antimuscarinic drug, on

decarboxylation of L-dopa in hemi-Parkinson rats. Izurieta-Sanchez, P, Sarre, S and Michotte, Y. 1998, European Journal of Phamacology, Vol. 353, pp. 33-42.

2. A study of N-methyl-D-aspartate antagonistic properties of anticholinergic

drugs. McDonough, J H and Shih, T M. 1995, Pharmacology Biochemistry and

Behavior, Vol. 51, pp. 249-253.

3. An algorithm (decission tree) for the management of Parkinson's disease

(2001): treatment guidelines. Olanow, C W, Watts, R L and Koller, W C. Suppl 5,

2001, Neurology, Vol. 11 , pp. S1-S88.

4. Kinetics and block of dopamine uptake in synaptosomes from rat caudate

nucleus. Krueger, B K. 1990, Journal of Neurochemistry, Vol. 55, pp. 260-267.


141




6. The effect of trihexyphenidyl, an anticholinergic agent, on regional cerebral

blood flow and oxygen metabolism in patients with Parkinson's disease. Takahashi, S, et al. 1999, Journal of the Neurological Sciences, Vol. 167, pp. 56-61.

7. Anticholinergic drugs: response of parkinsonism not responsive to

levodopa. Yamada, H, et al. 2002, Journal of Neurology Neurosurgery and

Psychiatry, Vol. 72, pp. 111-113.




9. Pharmacokinetics of trihexyphenidyl after long-term and short-term

administration to dystonic patients. Burke, R E and Fahn, S. 1985, Annals of




11. Metabolism and urinary excretion of benzhexol in humans. Nation, R L; Triggs, E J; Vine, J;. 1978, Xenobiotica, Vol. 8, pp. 165-169.



13. Steroselective determination of trihexyphenidyl using carboxylmethyl-β-

cyclodextrin by capillary electrophoresis with field-amplified sample stacking. Li, H, et al. 2007, Microchemical Journal. doi:10.1016/j.microc.2007.11.002 .




142


the skin in vitro. Green, P G, et al. 1991, Pharmaceutical Research, Vol. 8, pp.

1113-1120.

16. Partition coefficients and their uses. Albert, L, Corwin, H and David, E. 1971, Chemistry Reviews, Vol. 71, pp. 525-616.


Considerations on its membrane-crossing potential. Tsai, R, et al. 1992,

International Journal of Pharmaceutics, Vol. 80, pp. 39-49.



19. Young, T F. Electrochemical Information. [book auth.] Bruce H Billings, et

al. American Istitute of Physics Handbook. 2nd. New York : McGraw-Hill, Inc, 1963,

5, pp. 5-263.

20. Prediction of iontophoretic transport across the skin. Mudry, B, Guy, R H and Delgado-Charro, M B. 2006, Journal of Controlled Release, Vol. 111, pp. 362-

367.

21. Lidocaine Iontophoresis Versus Eutectic Mixture of Local Anesthetics

(EMLA®) for IV Placement in Children. Galinkin, J, et al. 2002, Anesthesia and

Analgesia, Vol. 94, pp. 1484-1488.

22. Electrorepulsion versus electroosmosis: effect of the pH on the

iontophoretic flux of 5-fluorouracil. Merino, V, et al. 1999, Vol. 16, pp. 758-761.

23. Contributions of electromigration and electroosmosis to peptide

iontophoresis across intact and impaired skin. Abla, N, et al. 2005, Pharmaceutical

Research, Vol. 22, pp. 2069-2078.

24. Reverse iontophoresis - Parameters determining electroosmotic flow: I.

pH and ionic strength. Santi, P and Guy, R H. 1995, Journal of Controlled Release,

Vol. 38, pp. 159-165.


143




26. Iontophoretic delivery of nafarelin across the skin. Delgado-Charro, M B and Guy, R H. 1996, International Journal of Pharmaceutics, Vol. 117, pp. 165-172.

27. Iontophoresis of nafarelin across human skin in vitro. Rodriguez Bayón, A M and Guy, R H. 1996, Pharmaceutical Research, Vol. 13, pp. 798-800.



29. Tetko, I V, et al. Welcome to the ALOGPS 2.1 home page! Virtual

Computational Chemistry Laboratory. [Online] [Cited: 15 May 2006.]

http://www.vcclab.org/.



Vol. 14, pp. 1258-1263.

31. Transdermal ion migration. Phipps, B and Gyory, R. 1992, Advanced

Drug Delivery Reviews, Vol. 9, pp. 137-176.


charge carrying species. Marro, D, et al. 2001, Pharmaceutical Research, Vol. 18,

pp. 1709-1713.







Chapter VI Entacapone

144

Chapter VI Entacapone Entacapone (ETC) (Comtan®,

Stalevo®) is a selective, dose-dependent

catechol-O-methyl-transferase (COMT)

inhibitor, used in the treatment of

Parkinson’s disease as an adjunct to

levodopa treatment (1). It peripherally

inhibits the formation 3-O-methyldopa

and has no effect on central activity of

COMT (2).

Entacapone has been tested clinically in several phase IV trials. Keranen et al.

(3) studied the effect of single entacapone dose on the pharmacokinetics and

metabolism of levodopa, in 12 healthy men. It has been shown that entacapone

dose-dependently increased the area under the plasma concentration-time curve

(AUC) of levodopa (by 65% at 400mg ETC), and decreased the AUC of

3-O-methyldopa (by 58% at 400mg ETC), in comparison to levodopa alone. No

changes in Cmax or tmax of levodopa were observed. Other study investigated the

effectiveness of entacapone in 15 Parkinson’s disease patients. Chronic ETC

treatment led to decrease of levodopa dosage, on average by 27%, while mean

levodopa concentrations in plasma were increased by 23% (4). Another large

multicentre, controlled trial looked at the effect of entacapone on quality of life of

patients, with early Parkinson’s disease (5). The study revealed that in comparison to

levodopa/carbidopa, the combination of levodopa, carbidopa and entacapone

significantly increased the quality of life of patients, with no or minimal motor

fluctuations, as measured by Unified Parkinson’s Disease Rating Score (UPDRS)

scale,.

The pharmacokinetic parameters of entacapone were investigated in several

studies. Keranen and co-workers (6) investigated entacapone pharmacokinetics after

single oral (5-800mg) and intravenous (25mg) dose in 12 healthy men. Oral

absorption of the drug was fast, tmax ranging from 0.4 to 0.9 hours. The bioavailability

ranged from 0.30 to 0.46. After 5-50mg doses the elimination of the drug from the

Figure 1 Entacapone chemical structure. Mw=305.3Da

N

N

O

OH

OH

N+

O-

O

CH3

CH3


145

system was very fast, half-life was t½=0.3h. With higher doses, distribution phase was

observed, characterised by biphasic elimination half-life of t½α=0.32h and t½β ranging

from 1.6 to 3.4 hours. Total body clearance was 45L/h. Yet another study

investigated the pharmacokinetics of single-dose entacapone when co-administered

orally with levodopa, in 20 parkinsonian patients (7). Entacapone was shown to

reduce the activity of COMT by up to 48%, increased the AUC and t½ of L-dopa, and

decreased the AUC of homovanillic acid and 3-O-methyldopa. Furthermore, the use

of entacapone effectuated in longer motor response to L-dopa. The 200mg dose was

found to be the most effective. The plasma concentrations of entacapone after

frequent multiple dosing, ranged from 0.7 to 3.3µM (8).

In terms of physicochemical parameters, entacapone seems relatively good

candidate for transdermal iontophoresis. Entacapone is a weak acid (9) of pKa=4.5,

as it dissociates a proton from the hydroxyl group in meta position. The negative

charge is stabilised by the aromatic ring and a conjugated double bonds on the

lateral chain. It has a molecular mass of M=305.3Da. It is practically insoluble in

water buffers at low pH, while its aqueous solubility rises rapidly with pH, and

reaches the 5.7mM at pH 6.5 (9). The distribution coefficient decreases from around

2.0 at pH 4.0 to around 0.2 at pH 7.4 (10).

Transdermal iontophoresis possibly, could constitute an effective dosage form

for entacapone. Transdermal drug delivery assures circumventing the first pass

effect, what might lead to increased bioavailability and reduction of dose (11).

Another advantage of iontophoretic application can be the constant and steady

delivery, what might be particularly beneficial f drugs of short half-life, such as

entacapone. It could further result in stable reduction in COMT activity, and smaller

fluctuations in L-dopa plasma concentrations. Also, after ETC oral administration,

large inconsistency of drug plasma-levels has been observed (12). Iontophoresis

offers easy profiling of the drug input, what could help tuning the drug dosage to

patients’ requirements.

In this study, transdermal cathodal iontophoresis of entacapone on the pig

skin model in vitro was examined. In particularly, the feasibility of delivering

therapeutic doses and the effects of entacapone donor concentration, its mole

fraction, pH of donor solution and current intensity were examined. The transdermal


146

flux required to reach the upper range of concentrations obtained by prolonged

administration of 200mg of entacapone, eight times daily, can be calculated on the

basis of pharmacokinetic data (6) (7) (8). The reported clearance was Cl=45L/h and

the plasma concentrations C, varied from 0.7 to 3.3µM. The flux to maintain the

entacapone on the same levels, can be expressed as JETC=Cl·C. This renders the

range of fluxes, from 29.5 to 147.4µmol/h. Mainly due to relatively high therapeutic

plasma-concentrations required, the value of flux is also high, for the possibilities of

iontophoretic drug delivery. However, the study was still interesting to carry out, as

entacapone is an acidic compound, as opposed to all the other investigated

substances.


Materials

Entacapone (ETC) was purchased from Tocris Bioscience, sodium chloride,

mannitol, silver wire (99.99% pure), silver chloride (99.999% pure) were obtained

from Sigma Aldrich UK; hydrochloric acid, sodium hydroxide, methanol HPLC grade

(MeOH), tetrahydrofuran HPLC grade, and n-octanol were supplied by Fisher

Scientific UK; triethylamine and perchloric acid was supplied by Acros Organics UK.

Deionised water (resistivity≥18.2MΩ/cm) was used to prepare all solutions.

Skin preparation





dorsal and abdominal skin from two different pigs was used in the experiments.


On the day of the experiment skin was thawed at room temperature.

Subsequently, it was clamped between two standard, side-by-side diffusion cells with


and the transport area 0.78cm2. Silver-silver chloride electrodes (13) were placed in

both chambers. A constant current of 0.4mA was applied for six hours using a Kepco


147

APH 1000DM (Flushing, NY) power supply, such that the donor chamber had lower

potential. Both chambers were magnetically stirred throughout the permeation

experiment. The receptor solution contained unbuffered 0.9% NaCl solution at pH of

5.8 to provide the main counter-ion, Cl-. The whole receptor solution was removed

and sampled hourly. The unbuffered donor solution was replaced with fresh one

every hour, to limit the competition of Cl- ions released from cathode during the

iontophoretic process.


Single ion experiments had entacapone as the only negatively charged ion in

donor solution. This study was set up to investigate a) the effect of applied current on

entacapone transdermal transport b) effect of pH on entacapone transdermal

transport.

Table 1 Iontophoretic formulations used in single-ion entacapone experiments.

ETC Conc. [mM] pH

Current Intensity

[mA] n

pH effect 30

7.0

0.4

12

8.5 5

10.0 6

Current intensity

effect 30 7.0

0.0 6 0.1 6 0.2 6 0.4 12


Iontophoresis was carried out using the donor solutions, with entacapone and

chloride ions. Total concentration of negative ions was equal to 30mM, and the pH

was adjusted with 1M NaOH to 7.0.Table 2 summarizes the formulations used in

these experiments.

Passive control

The passive flux of entacapone was assessed using a similar experimental

setup, except that no current was applied, and the donor solution was never


148

replaced. The donor solution was composed of a) 30mM entacapone at pH 7.0

alkalised with 0.1M NaOH b) saturated entacapone solution at pH 3.0.

Table 2 Iontophoretic formulations used in co-ion entacapone experiments.

Mole Fraction

ETC Conc. [mM]

NaCl Conc. [mM]



[mA] n

25% 7.5 22.5

30 7.0 0.4

6 50% 15.0 15.0 6 75% 22.5 7.5 5

100% 30.0 0 12

Analytical Methods

Entacapone was quantified using a Jasco HPLC system (composed of a PU-

980 pump, an autosampler AS-1595, and a UV-VIS detector UV-975) by means of

the entacapone external standard calibration curve, in corresponding media of at

least seven standards. Isocratic separation was performed using Dionex Acclaim™

120, C18 HPLC column (150 mm x 4.6 mm, 5µm), with flow rate of 1 ml/min, and

injection volume of 10µl, with column temperature of 30C. The mobile phase

consisted of 50%MeOH, 40%H2O, and 10%THF, spiked with 200µl/l of triethylamine,

and acidified to pH=2.5 with perchloric acid. Detection was performed at wavelength

of 315nm. Limit of detection was 0.1µM and limit of quantification was accepted as

1µM. The concentrations of standards used ranged form 5µM to 150µM. The

precision and accuracy of the method were 4% and 4%, respectively.

Determination of distribution coefficient of entacapone at pH 7.0

The distribution coefficient was calculated as in Equation 1 (14) on the basis of

the data from the literature (10).


Conductivity measurements and entacapone water mobility estimation

The specific conductivity was measured for range of entacapone sodium water

solutions (10, 2, 1, 0.5, 0.2mM) using conductimeter Metrohm T-120 at 22C with the

probe of cell constant K=0.93. The pH of the solutions was measured. Then, values


149

of molar conductivities (specific conductivity normalized by concentration) were

calculated and plotted against square root of concentration. Conductivity at infinite

dilution was estimated by extrapolating the curve to zero. Subsequently, using

Kohlraush law of independent ion migration Equation 2, water mobility of entacapone

ion was calculated. The mobility of the sodium ion, µNa+=5.2·10-8 m2/s/V was taken

from literature (15). Afterwards, water transport numbers were estimated as in

Equation 3 and Equation 4 (16).

Equation 2 −

∞−+

∞+∞ Λ+Λ=Λ CC

Equation 3 +−

Λ=

∞

NaETC Fµµ

Equation 4 ++

=NaETC

ETCETCt µµ

µ







average value of the three final samplings. Transport numbers for each replicate

were calculated as t#=J·z·F/I, from the average of fluxes, for last three hours of

experiment. The data on the graphs are presented as mean value of all the

replicates, with standard deviations. All regressions were performed using the least

squares method.


150


Conductivity measurements.

Entacapone molar conductivity for infinite dilution Λ∞ was estimated by

extrapolation of the plot of molar conductivity as a function of square root of

concentration. Drug water transport number was calculated as in Equation 3 and

Equation 4, and was equal to t#ETC=0.16.

Figure 2 Linear regression of entacapone molar conductivity as a function of square root of concentration. The Y-intercept is the infinite dilution molar conductivity Λ∞=60.0. The data points are the means of three replicates with standard deviations.

Single ion experiments – current dependency

In this set of four experiments, the transport number was assessed as the

slope of flux-current intensity dependency, according to equation J=t#/z·F·I, where J is

the drug flux, z drug valence, t# is the transport number and I is the current intensity.

The current intensity was varied from 0.0 to 0.4mA (Table 1). The results are

presented on Figure 3. The transport number obtained from the slope was

0.012±0.002. This transport number allows for good control over the entacapone flux,

by adjusting the current; however it is not enough for efficient delivery of the drug

(see below). The regression-derived transport number does not differ significantly

from the average transport number, calculated for each point separately, what

reflects the minimal passive entacapone penetration at pH 7.0 (3.7±0.3nmol/h/cm2).

The passive penetration for pH 3.0 (3.6±0.1nmol/h/cm2) was not statistically different

from the value for pH 7.0. It is noteworthy, that the transdermal transport number is

again an order of magnitude lower than the one measured in water (Figure 2). It has

0 1 2 30

50

100

150

ETC NaNaCl

Λ0

C1/2 [mM]1/2

Mol

ar c

ondu

ctiv

ity [S

i*cm

2 /mol

]


151

been observed for other drugs studied in this work, like piribedil. The model

connecting the two transport numbers was, however, developed for the cationic

substances, and might not be accurate for the anions (17).

Figure 3 The effect of current intensity on entacapone transdermal flux. The obtained regression is significant (p<0.05). The goodness of fit is r2=0.8. The equation of regression is y=432.1±42·x – 7.7±11.7; The transport number corresponding to the value of the slope after taking into the account the applied units is t#=0.012. The dotted line limits the regression 95% confidence interval.

0.0 0.1 0.2 0.3 0.4 0.50

100

200

300


Enta

capo

ne F

lux

[nm

ol/h

]


152

Single ion experiments – effect of pH

In this set of experiments the impact of the pH on entacapone transport in

single-ion situation was investigated. The single-ion situation was used to limit the

ionic competition. Some degree of competition was expected, as during the

iontophoretic experiment chloride ions are released in the cathode. For this reason,

the donor solution was replaced hourly. The range of pH values was limited to pH 7.0

from one side by very low solubility of entacapone in acidic solutions; and to pH 10.0

from the other, by skin tolerance. The results of these experiments are presented on

Figure 4.

Figure 4 Entacapone flux and transport number as a function of the pH of donor solution, in single-ion situation. The total molarity of the donor solutions was 30mM at the beginning of each experiment.

It is clearly visible, that for pH values of 7.0 and 8.5 the flux is stable. In fact,

ANOVA test found no statistical difference between the two fluxes. The flux

measured for the pH 10.0 was significantly, roughly twice, higher. This is quite

surprising, as it is expected, that with increasing pH, the anion permselectivity of the

skin would drop (18). One of the possible explanations would be the double

ionization of the drug in this pH. The work of Abla et al. (19) has show previously that

bivalent molecules can have higher transdermal fluxes, than similar ones but

monovalent. Analogous effect was also obtained by Sylvestre and co-workers (20),

when delivering the dexamethasone phosphate. The formulations containing

monovalent drug, at pH 4.0, resulted in approximately halved transdermal flux in

comparison to the formulations at pH 7.5, where more than 85% of dexamethasone

phosphate was bivalent. It was previously reported, that the catechol derivatives can

bare a double negative charge (21); however the second ionization constant is very

high, and oscillates around 13. Nevertheless, it is possible for entacapone molecule,

pH 7.0 pH 8.5 pH 10.00.0

0.2

0.4

0.6

0.00

0.01

0.02

0.03

Enta

capo

ne F

lux

[ µm

ol/h

]

EntacaponeTransport N

umber


153

that strongly electron-withdrawing nitro group, and lateral chain containing double

bonds conjugated with aromatic ring, make the bivalent negative ion more stable.

This would cause the second ionisation constant to decrease. Further study on

entacapone ionization is needed to confirm this idea.

Even for the highest obtained flux, the 0.38±0.11µmol/h, achieving the

transdermal flux, able to maintain therapeutic concentration in plasma might be

difficult. The target fluxes range is from 29.5 to 147.4µmol/h. This allows calculating

the area of the patch as: A=2·Jt·Jmax/Atrans, where the Jt is the target flux; Jmax is the

maximal measured flux; and Atrans is the transport area used in the experiments.

Factor two reflects the need of having the same area at the cathode and the anode.

The area A, ranges from 121 to 605cm2. These high patch surfaces would make it

very expensive and impractical to use.

Co-ion delivery

During the cathodal iontophoresis the chloride ions are released at a constant

rate from the cathode as a result of passing current: AgCl + ē → Ag0+Cl-. This

process constantly increases the chloride ion concentration in donor solution. Even if

originally the entacapone formulation was just in water, after some time the

concentration of the will become significant. At the 0.4mA current, the rate of Cl-

release at the cathode is roughly 1.5·10-5mol/h. This means that with the volume of

the half cell 3.3ml, the concentration of chlorides in formulation would gradually rise

to 4.5mM after an hour. This is a comparable value to entacapone concentration.

This phenomenon was also experimentally observed (22). Sylvestre et al. measured

the concentrations of chlorides in donor, during the iontophoresis of dexamethasone

phosphate, and the value was very close to the one predicted. It is then possible, that

the in comparable concentrations, chloride ions will compete for charge carrying with

entacapone. Thus, the impact of competing ions was systematically studied with a

series of formulations listed in Table 2. The results are presented on Figure 5.

Primarily, the entacapone flux seemed to be linearly dependent on mole fraction in

donor solution in range from 0.25 to 0.75. Surprisingly, above the 0.75 the tendency

breaks. The t-test has found the flux for 0.75 mole fraction significantly higher than

the 1.0. It was quite unexpected, as for most drugs investigated before the single-ion

flux was higher (for selegiline, pramipexole, dexamethasone (22), lidocaine (23),


154

hydromorphone (24) or ropinirole (25)), or at least equal (for trihexyphenidyl, piribedil,

quinine, propranolol (26)) to the flux obtained during co-ion delivery. However, except

for dexamethasone, all abovementioned substances are delivered as cations, from

the anodal chamber. For anions the situation looks differently. In deed, lets consider

the following facts: a) In applied experimental setup, entacapone is delivered from

cathodal chamber, at pH 7.0, hence against the electroosmotic flux; b) The

magnitude of the latter is determined by the negative charge fixed on the skin; c)

During the single ion delivery, the concentration of the delivered drug in the skin is

higher, than for co-ion delivery. Possible explanation to the observed, unexpectedly

lower flux for single-ion might be, that the high concentration of ETC in the skin

enhances the bulk volume flow, occurring in the anode to cathode direction. That

impedes the transport of entacapone occurring from cathode to anode. Because the

concentration of the drug in the skin is the highest, when no competition occurs, this

effect is the most accentuated for single-ion situation.

0.00 0.25 0.50 0.75 1.000.0

0.1

0.2

0.3

0.00

0.01

0.02

Entacapone Mole Fraction

Enta

capo

ne F

lux

[ µm

ol/h

]

EntacaponeTransport N

umber

Figure 5 Co-ion delivery of entacapone at pH 7.0, from solutions containing diverse initial ETC mole fractions. The summary initial anion concentration in the formulation was 30mM.

Conclusions

Entacapone is a poor charge carrier in transdermal iontophoresis. The small

efficiency of the transdermal transport, together with the high target fluxes, make it

impossible to efficiently deliver entacapone by transdermal iontophoresis. Its delivery

is affected by the intensity of applied current in linear manner allowing adjustment of

transdermal drug flux. The pH also impacts the ETC transport, probably due to

double ionisation of entacapone molecule. The transport of the drug is also impaired

by the effect of competing ions, however in non-linear manner.


155

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catechol-O-methyltransferase inhibitor entacapone. Nissinen, E, et al. 1992,

Naunyn-Schmiedeberg's Archives of Pharmacology, Vol. 346, pp. 262-266.

3. The effect of catechol-O-methyltransferase inhibition by entacapone on the

pharmacokinetics and metabolism of levodopa in healthy volunteers. Keranen, T, et al. 1993, Clinical Neuropharmacology, Vol. 16.

4. Effect of peripheral catechol-O-methyltransferase inhibition on the

pharmacokinetics and pharmacodynamics of levodopa in parkinsonian patients. Nutt, J G, et al. 1994, Neurology, Vol. 44, pp. 913-919.

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6. Inhibition of soluble catechol-O-methyltransferase and single-dose

pharmacokinetics after oral and intravenous administration of entacapone. Keranen, T, et al. 1994, European Journal of Clinical Pharmacology, Vol. 46, pp. 151-157.

7. A double-blind pharmacokinetic and clinical dose-response study of

entacapone as an adjuvant to levodopa therapy in advanced Parkinson's disease.

Ruottinen, H M and Rinne, U K. 1996, Clinical Neuropharmacology, Vol. 19, pp.

283-296.

8. Pharmacokinetics of oral entacapone after frequent multiple dosing and

effects on levodopa disposition. Rouru, J, et al. 1999, Pharmacokinetics and

Disposition, Vol. 55, pp. 461-467.


156

9. Effects of aqueous solubility and dissolution characteristics on oral

bioavailability of entacapone. Savolainen, J, et al. 2000, Drug Development


10. The role of physicochemical properties of entacapone and tolcapone on

their efficacy during local intrastratial administration. Forsberg, M, et al. 2005,

European Journal of Pharmaceutical Sciences, Vol. 24, pp. 503-511.










Considerations on its membrane-crossing potential. Tsai, R, et al. 1992, International

Journal of Pharmaceutics, Vol. 80, pp. 39-49.

15. Young, T F. Electrochemical Information. In, Bruce H Billings, et al.

American Institute of Physics Handbook. 2nd. New York : McGraw-Hill, Inc, 1963, 5,

pp. 5-263.




transport numbers. Mudry, B, Guy, R H and Delgado-Charro, M B. 2006, Journal of






157



2078.

20. In vitro optimisation of dexamethasone phosphate delivery by

iontophoresis. Sylvestre, J P, Guy, R H and Delgado-Charro, M B. 2008, Physical

Therapy, Vol. 88, pp. 1177-1185.

21. Ionization constants of catechols and catecholamines. Schüsler-Van Hees, M T, Beijersbergen Van Henegouwen, G M and Driever, M F. 1983,

Pharmacy World & Science, Vol. 5, pp. 102-108.

22. Iontophoresis of dexamethasone phosphate: competition with chloride

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27. Prediction of iontophoretic transport across the skin. Mudry, B, Guy, R H and Delgado-Charro, M B. 2006, Journal of Controlled Release, Vol. 111, pp. 362-

367.

Chapter VII Human skin

158

Chapter VII – Human Skin Introduction

To study the permeation of compounds for human use in vitro, the excised

human skin seems the most reliable model. However, it is often expensive, and hard

to acquire in quantities required for laboratory purposes. Thus, in common practice,

many animal-skin models have been implemented and applied. Pig skin has been

one of the very frequently used models for transdermal drug-delivery experiments (1).

It has been shown, based on morphological and functional data, that the skin of

domestic pig is closer to human skin than other animal models (2). Nonetheless, pig

skin, in particular its the least permeable layer, stratum corneum (SC), differs from

human SC in composition (3). It was observed, that ceramide profile characteristic for

human SC was different from that of the pig skin. Also, the lipid spatial organisation

in stratum corneum was found different for both species (3; 4). Most probably, due to

these chemical and morphological differences, certain macroscopic characteristics of

pig stratum corneum, important from the point of view of drug delivery, differ form the

human SC. Marro et al. (5) compared the human and pig skin permselective

properties. The isoelectric points found for human and pig skin, were respectively 4.8

and 4.4. Also, for most of pH values suitable for cationic drug delivery (>4.0), pig skin

had similar degree of permselectivity (DP, defined as a quotient of electroosmotic

flow in predominant and opposite direction), except for pH7.4, where the DP of pig

skin was considerably higher. This suggests that at neutral pH, negative charge fixed

on the pig skin is greater than on the human skin in similar conditions. This in turn

result in different cation permeability of those membranes.

Several other authors compared the delivery of selected drugs through human

skin and various animal models, using both, passive and iontophoretic formulations.

Kanikkannan et al. (6) investigated the delivery of timolol across the skin of rat,

mouse, guinea pig, rabbit and human in vitro. Studied passive formulations resulted

in large interspecies variability of transdermal flux (two orders of magnitude), as

opposed to the iontophoretic formulations, for which there were very small, or no

differences in transport between the species. Similar observations were made by

Phipps et al. (7), who delivered hydromorphone through human and pig skin. Also


159

here, the differences in passive delivery rates between the two species were very

large, while being statistically insignificant for iontophoretic delivery.

In this work, the five drugs, the delivery of which appeared the most effective,

were re-examined for iontophoretic delivery, with full thickness human skin, in best

conditions elucidated in previous chapters. Subsequently, obtained fluxes and their

time-profiles were compared to corresponding ones, obtained with the pig skin

experiments. The objective, was to further confirm feasibility of delivering clinically

relevant doses of investigated drugs, and also to determine how fast required plasma

levels can be achieved via iontophoretic delivery.


Materials

Pramipexole (PPX) was obtained from Chemos GmbH, piribedil base (PBD)

and pergolide (PER) mesylate were purchased from Tocris Bioscience UK. Sodium

chloride, mannitol, paracetamol, silver wire (99.99% pure), silver chloride (99.999%

pure), selegiline (SEL) hydrochloride and trihexyphenidyl (THP) hydrochloride were

obtained from Sigma Aldrich UK; hydrochloric acid, sodium hydroxide, methanol

(MeOH) HPLC grade, tetrahydrofuran (THF) HPLC grade, acetonitrile HPLC grade

(AcN), perchloric acid and n-octanol were supplied by Fisher Scientific UK;

triethylamine was supplied by Acros Organics UK. Deionised water

(resistivity≥18.2MΩ/cm) was used to prepare all solutions.

Human skin samples

The use of human skin for drug transport studies was approved by the Bath

Research Ethics Committee (5th April 2007) REC reference number: 07/Q2001/57

Full thickness human skin samples of dimensions roughly 2cm by 2cm were obtained

from Nottingham City Human Tissue Bank. They were placed in separate plastic

bags and stored at -20°C for no longer than three months. On the day of experiment

the selected skin samples were thawed in room temperature. Subsequently, the

excess of fat on dermal side of the skin was removed. Skin was then rinsed under

deionised water and placed in between of two half-cells.


160


The skin was clamped between two standard, side-by-side diffusion cells with


and the transport area 0.78cm2. Homemade silver-silver chloride electrodes (6) were

placed in both chambers. A constant current of 0.4mA was applied for six hours

using a Kepco APH 1000DM (Flushing, NY) power supply, such that the donor

chamber had higher potential. Both chambers were magnetically stirred throughout

the permeation experiment. The receptor solution contained unbuffered 0.9% NaCl

solution of pH≈5.8, to provide the main counter-ion, Cl-. The whole receptor solution

was removed and sampled hourly. The donor solution was replaced with fresh one

every two hours (except for pergolide experiment, where it was replaced hourly) to

provide enough Cl- ions for correct electrode electrochemistry.

For each of studied drugs that could be efficiently delivered for the pig skin

experiments, an experiment with human skin was set up. The formulations used in

these experiments were the most efficient formulations previously investigated. The

specific formulations are listed in Table 1.

Table 1 Formulation used in experiments with human skin. *Mannitol was used as an electroosmotic marker in all cases except for selegiline, where paracetamol was employed.

Drug Mole

Fraction

Drug Conc. [mM]

NaCl Conc. [mM]



[mA]

Electro-osmotic Marker Conc.*

Skin Sample ID

PPX 100% 30 0 30 8.0 0.4 30

NCHTB ID 2004B/242 NCHTB ID 2004B/231 NCHTB ID 2004B/232 NCHTB ID 2004B/238 NCHTB ID 2005B/158

SEL 100% 30 0 30 4.5 0.4 15


PBD 100% 30 0 30 5.0 0.4 30


THP 100% 30 0 30 5.4 0.4 30


PER 5% 2.5 47.5 50 4.0 0.4 30

NCHTB ID 2005B/140/1 NCHTB ID 2005B/140/2 NCHTB ID 2005B/145 NCHTB ID 2005B/146


161

Analytical Methods

Analytical methods, used to quantify each drug were described in previous

chapters referring to each drug or electroosmotic marker.






was fixed at p < 0.05. Transport numbers for each replicate were calculated as in

t#=z·F·J/I. All data are reported as mean and standard deviation. Pearson test was

used to test the correlation between variables. The area under the curve (AUC) of the

obtained time-flux profiles were derived by integrating the 4th degree polynomial fits

to the acquired data.


The results of human skin experiments are presented in Figure 2. From the

graphs it is clear, that for all the drugs, fluxes reached in the human skin experiments

are smaller than in corresponding ones, with the pig skin. Also, the time to reach

steady state was essentially longer with human skin and, except for pramipexole,

none of the drugs reached steady state within six hours. The smaller fluxes were

expected, as full thickness human skin was employed in these experiments, while in

corresponding experiments, dermatomed pig skin was used. The full thickness

human skin was provided in small pieces (2cm by 2cm) and it was impossible to

dermatome. Thus, it was used intact in the iontophoretic experiments. As a

consequence it has to be noted, that the differences observed in the experiments

with different membrane models are a consequence of two changing factors: human

skin replacing the pig skin; and full thickness skin replacing the dermatomed one.

Mannitol/paracetamol fluxes through human and pig skin were very similar

(Figure 2). This might indicate that electroosmotic effect is similar for both

experimental setups. Also, along with inhibition factor close to 0.5, for the two drugs

with lowest values of log D (SEL and PPX), this suggests, that the dermis is rather

permeable barrier for hydrophilic substances. Previously, the permselective


162

properties of human and pig skin were compared by Marro et al. (5). This work

compared the mannitol fluxes through full thickness human skin, to those through

dermatomed pig skin. Mannitol was delivered from donor solutions containing

153mM of inorganic salt (NaCl + HEPES) of various pH. It was shown that mannitol

penetrates better across pig skin than human skin. From Figure 2 it is visible, that for

the experimental setup applied in this work, no clear difference between the fluxes

through both kinds of membrane can be observed. This difference could be

explained by the phenomenon observed before, for the examined drugs: that is the

increasing mole fraction of the drug in donor limits the electroosmotic flow through

the skin, probably by interacting with negatively charged moieties of the barrier and

neutralising its negative charge. It is possible that organic and lipophilic (relative to

NaCl and HEPES) molecules penetrating both membranes similarly decrease their

permselective properties to certain small level. Thus, during the iontophoresis of the

five substances, as in Figure 2, similar electroosmotic-marker fluxes are observed.

Comparing these two data sets might suggest, that during the single-ion

iontophoresis of organic drugs, the differences of the skin model are of less

importance, than when using the donor solutions with high content of salts.

-2 0 2 40.0

0.1

0.2

0.3

0.4

0.5

PPX SEL

PBD THP

PER

Log D

Inhi

bitio

n fa

ctor

Figure 1 Dependence of inhibition factor on the distribution coefficient for examined drugs at relevant pH. PPX – pramipexole; SEL – selegiline; PBD – piribedil; THP – trihexyphenidyl; PER – pergolide


163

Mannitol, pH8.0, 30mM, 100%

0 2 4 60

5

10

15

20

25

Time [h]

Man

nito

l Flu

x [n

mol

/h]

Pramipexole, pH8.0, 30mM, 100%

0 2 4 60

1

2

3

0.00

0.05

0.10

0.15

0.20

Time [h]

Pram

ipex

ole

Flux

[µm

ol/h

] Pramipexole Transport N

o.

Paracetamol, pH4.5, 30mM, 100%

0 2 4 6-5

0

5

10

15

20

25

Time [h]

Para

ceta

mol

Flu

x [n

mol

/h] Selegiline, pH4.6, 30mM, 100%

0 2 4 6

0.0

0.5

1.0

1.5

2.0

0.00

0.05

0.10

Time [h]

Sele

gilin

e Fl

ux [µ

mol

/h] Selegiline Transport N

o.


0 2 4 60

2

4

6

Time [h]

Man

nito

l Flu

x [n

mol

/h]

Piribedil, pH5.0, 30mM, 100%

0 2 4 60.0

0.2

0.4

0.6

0.00

0.01

0.02

0.03

0.04

Time [h]

Pirib

edil

Flux

[µm

ol/h

]

Piribedil Transport Num

ber


0 2 4 6

0

2

4

6

8

Time [h]

Man

nito

l Flu

x [µ

mol

/h]

Trihexyphenidyl, pH5.4, 30mM, 100%

0 2 4 6

0.0

0.1

0.2

0.3

0.000

0.005

0.010

0.015

0.020

Time [h]

Trih

exyp

heni

dyl F

lux

[µm

ol/h

]

Trihexyphyenidyl Transport No.


0 2 4 6

0

5

10

15

20

Time [h]

Man

nito

l Flu

x [n

mol

/h]

Pergolide, pH4.0, 50mM, 5%

0 2 4 60

10

20

30

0.0

0.5

1.0

1.5

2.0

Time [h]

Perg

olid

e Fl

ux [n

mol

/h]

10-3⋅ Pergolide Transport N

o.

Pig skinHuman skin

Figure 2 Fluxes of drug and electroosmotic marker through the dermatomed pig skin, and full thickness human skin. On the right side, the fitted lines represent the Nugroho et al. (9) model fitted in the acquired data. The best fit parameters are presented below, in Table 3.


164

Several factors contributed to the stronger barrier properties of the full

thickness human skin. First, the dermis part of human skin consisted an additional

barrier for the penetrating drugs, before they reached the donor compartment. In

total, the entire membrane thickness increased from roughly 0.5-1.0mm (for

dermatomed pig skin) to 0.5cm, what could slow down the drug transport. Secondly,

although dermis is relatively conductive in comparison to the SC, this fatty and

connective tissue could absorb some part of delivered drug, acting as a reservoir,

and slow down the permeation process.

This could happen particularly in case of the cations of lipophilic drugs, with

pKa close to 7.4 like pergolide (pKa=7.8) or piribedil (pKa=6.9). Reaching the dermis

at pH 7.4, large proportion of these drugs would dissociate the proton, become

neutral, and partition to the lipid-rich parts of dermis. In order to establish, what was

the effect of the dermis on the drug transport we defined the inhibition factor (IF) as

the quotient of AUC of the time-flux profile, for human and pig skin. Figure 1

represents the plot of inhibition factor as a function of distribution coefficient. The

correlation between the two factors has been found statistically insignificant (p=0.2).

On the other hand, it is interesting that the inhibition factor seem to change

accordingly with drug water solubility. The smallest value of IF was observed for the

drugs which HCl salts are freely soluble in water (SEL and PPX). The flux of drugs

with solubility of around 30mM at pH 5.0 (PBD and THP) was reduce by factor 0.4;

and the flux of minimally water soluble pergolide mesylate (0.3mM at pH5.0) was

reduced almost 10 fold. This observation is only made on five substances and needs

further confirmation

The importance of barrier effect of viable epidermis has been demonstrated

for the transdermal delivery of rotigotine (9). The drug was delivered through human

stratum corneum (HSC) and dermatomed human skin (DHS). It was observed, that

replacing HSC with DHS altered not only the maximal flux, but also the iontophoretic

profile in time. Equally to this study, the possible causes were described as 1) larger

thickness of the barrier; 2) epidermis acting like a drug depot; and 3) lack of vascular

drug-clearance in the in vitro study. The experiments in rotigotine study were

conducted for nine hours, as opposed to six hours presented above. The longer

experiment time allowed achieving and comparing the steady state fluxes for


165

rotigotine. Similar results were obtained for delivery of apomorphine across HSC and

DHS (8). The steady state flux and time to reach it were significantly longer in

experiments with full thickness human skin in comparison to stratum corneum

In terms of delivering the clinically relevant doses, human skin experiments

seem to suggest, that at least for some of the investigated drugs, it is feasible with

the experimental setup employed. The feasibility was previously assessed for each

drug, with pig skin model. Using the dermatomed pig skin as a membrane model

rendered the lag times (the time to reach steady state flux) within two to three hours.

With this short lag time, it was possible to base the feasibility estimations on the

steady state flux. With the experiments with the full thickness human skin this

approach is not valid as the lag times are relatively long. In fact only one drug,

pramipexole, has reached the steady state flux during the six hours of experiment. It

was published previously (10) that the steady state transdermal flux, might not be the

right parameter to base the feasibility estimations, and the entire profile should be

Table 2 Drug amounts delivered in six hours, compared to the required transport rate from hypothetical 50cm2 patch.

Drug Amount delivered in 6

hours using full thickness human skin

[µmol/cm2]

Amount delivered in 6 hours through

dermatomed pig skin [µmol/cm2]

Target iontophoretic

flux [µmol/6h/cm2]

Pramipexole 8.44±0.35 17.0±1.24 0.07

Selegiline 3.03±0.25 9.0±1.48 0.11

Piribedil 0.39±0.05 2.5±0.8 1.44

Trihexyphenidyl 0.20±0.04 1.4±0.23 0.06

Pergolide 0.006±0.002 0.087±0.044 0.05

taken into the account. In order to assess the feasibility of delivering therapeutical

doses we present several approaches First, comparing the total delivered amount


166

during the entire experiment and comparing it to the theoretical value of desired

iontophoretic flux (Table 2). The target iontophoretic flux was taken from Table 2 of

the Introduction chapter and it was normalized by 50cm2 of a hypothetical

iontophoretic patch.

From Table 2 it seems that three of the drugs, namely pramipexole selegiline

and trihexyphenidyl might be effectively delivered by iontophoresis. The values of

delivered dose across human skin, presented in Table 2 can be underestimated as

only the initial period of six hours is taken into account, when the flux values are

relatively low. It can be expected that during further hours, the drug flux will be higher

and so the delivery rate will be faster. This regards to the pig skin data in much

smaller extent as the time to reach the steady state was limited to two – three hours.

Also, the lack of vascular clearance is another factor that can impede the delivery

across the full thickness human skin. In the in vitro situation where no blood

perfusion takes place in the dermis can act as an additional barrier and a depot for a

drug diminishing its flux to the donor. In the in vivo studies, the dermis is irrigated

with blood vessels, so the practical significance of drug accumulation in dermis might

be smaller than observed, as the drug would be successively washed out from the

application site. During the in vitro experiment however, with the lack of vascular

clearance, this part of tissue acts as an additional barrier and drug reservoir. The

importance of vascular clearance was demonstrated in studies on iontophoretic

delivery of apomorphine (7; 8). The results of apomorphine delivery in vivo, were

more strongly correlated with in vitro dermatomed human skin model, than to the

human stratum corneum model. It was noted, that during the in vivo administration of

apomorphine no subcutaneous drug depot was formed.

This approach to evaluating the feasibility of sufficient delivery has however

some disadvantages. It does not take into the account the rate of delivery at given

time, thus it does not allow for estimating the plasma concentration. Nugroho et al.

(9) derived a pharmacokinetic model, describing the iontophoretic drug delivery in

time. It assumes, that the electric force drives drug molecules into the skin, which

consist a separate compartment, and from there drug diffuses passively to the

receptor compartment. The model based on Equation 1 allows predicting the values


167

of steady state flux (JSS); the diffusion constant K, from skin to receptor compartment;

and the lag time tL.

Equation 1 )1()( )( LttKSS eJtJ −−−=

The advantage of this model is that it takes into account all the non zero data

points, including those before reaching the steady state. As a consequence it can be

used to more accurately extrapolate the results from the in vitro to the in vivo studies

(Figure 3).

Figure 3 Nugroho et al. compartment model for iontophoretic drug delivery. The in vitro situation (A) is used to estimate the zero order delivery to the skin (Jss) and the passive diffusion constant (K), from the skin to donor compartment. These data, combined with plasma elimination constant (Kel) and volume of distribution are used to model plasma concentrations in vivo (B).

This model however, cannot predict accurately the steady state flux unless it is

actually achieved, what limits its utility. Table 3 summarizes the permeation

parameters obtained with the fitting. When applied to the data from Figure 2, the

model fits well most of the data obtained for the pig skin (except for pergolide where

large variability was observed), as for every drug steady state was achieved. On the

contrary, for the human skin data, where only pramipexole reached the steady state,

the fittings for other drugs are ambiguous or bare large errors. This is due to too few

non zero data points, a consequence of too short lasting experiment.


168

Table 3 Iontophoretic permeation parameters obtained with the fitting of the Nugroho et al. model.

Parameter Pig skin Human skin AV+SD RSD AV+SD RSD

PPX Jss [µmol/h/cm2] 2.66±0.08 3% 2.38±0.50 21%

K [h-1] 0.97±0.18 19% 0.39±0.19 49% tL[h] 0.54±0.11 20% 1.57±0.24 15%

SEL Jss [µmol/h/cm2] 1.34±0.06 4% 1.39±0.78 56%

K [h-1] 0.92±0.14 15% 0.30±0.29 97% tL[h] 0.05±0.06 121% 2.48±0.34 14%

PBD Jss [µmol/h/cm2] 0.41±0.02 5% ambiguous fitting due to

too few points K [h-1] 0.96±0.25 26%tL[h] 0.80±0.11 14%

THP Jss [µmol/h/cm2] 0.22±0.01 5%

ambiguous fitting due to too few points K [h-1] 0.77±0.15 20%

tL[h] 0.00±0.08 3376%

PER Jss [nmol/h/cm2] 72.79±236.30 325%

ambiguous fitting due to too few points K [h-1] 0.05±0.19 375%

tL[h] 0.10±0.63 601%

Table 4 Pharmacokinetic parameters selected drugs used to model the plasma concentrations during iontophoretic delivery.* The reported value is an apparent clearance, (clearance value divided by bioavailability)

Cl [L/h]

Therapeutic window

[nM] Vd [L] t½ [h] Kel [1/h] Reference

PPX 25 3-35 462±119 12.8±3.3 0.054±0.014 (13; 14; 15)

SEL 92 2-10 166±99 1.25±0.75 0.55±0.33 (13; 16; 17; 18)

PBD 77±38 30-150 1300±1100 12.1±10.4 0.057±0.049 (19; 20; 21; 22; 23)

THP 20* 200-600 162±46 5.6±1.6 0.124±0.035 (24; 25; 26; 13)

Combining the data form Table 3 with pharmacokinetic parameters of the

examined drug (Table 4), it is possible to predict plasma concentrations of a drug, for

in vivo iontophoretic delivery, at any given moment. Following Nugroho et al.

Equation 2 was used to predict the plasma profiles of the drugs.


169

Equation 2

++

+−⋅

=−−−

el

tKKt

el

tK

d

SS

KKee

Ke

VSJtC

elel1)(

where C(t) is the predicted plasma concentration at time t; Kel is the drug elimination

constant; K is the modelled diffusion constant; Vd is the volume of distribution; JSS is

the steady rate of delivery; and S is the patch area.

Figure 4 represents the predicted plots of plasma concentrations for four

drugs, delivered from patches of various concentrations. Pramipexole and selegiline

plot was carried out for delivery from only 1cm2, and piribedil and

Pramipexole

0 4 8 12 16 20 240

20

40

60

80

100

Time [h]

Plas

ma

conc

entr

atio

n [n

M]

Selegiline

0 4 8 12 16 20 240

10

20

30

40

50

Time [h]

Plas

ma

conc

entr

atio

n [n

M]

Piribedil

0 4 8 12 16 20 240

100

200

300

400

Time [h]

Plas

ma

conc

entr

atio

n [n

M]

Trihexyphenidyl

0 4 8 12 16 20 240

200

400

600

800

1000

Time [h]

Plas

ma

conc

entr

atio

n [n

M]

Human skinPig skin

Figure 4 Predicted plasma levels of selected drugs on the basis of the best fit parameters (Table 3) and Equation 2. Open circle plots represent the prediction carried out with parameters obtained for pig skin model, while the closed circles correspond to human skin model. The plots predict plasma levels for delivery from patch sized: 1, 5, 50 and 50cm2, respectively for pramipexole selegiline, piribedil and trihexyphenidyl. Dotted line represents the upper limit of reported therapeutic window.


170

trihexyphenidyl, both from 50cm2. Although the predictions presented above suggest,

that the iontophoretic delivery could be possible for all the four drugs, this would only

be true providing, that the JSS is kept at the level obtained for single-ion delivery. 24

hours long delivery at 0.5mA current intensity, requires an amount of chlorides in

donor that exceeds the solubility of piribedil and trihexyphenidyl HCl salts. Thus, in

these cases, a co-ion delivery might be unavoidable, which in turn, could reduce the

steady state flux (JSS). Also, in case of piribedil and trihexyphenidyl, the prediction

was only carried out for the data obtained form the pig skin model. This model might

overestimate the delivery parameters (JSS and K) and underestimates the lag time (tL)

as referred to in vivo situation, due to lack of the dermis. As a consequence, the

predictions for plasma concentrations of those drugs could also be too high, and the

estimated time to reach steady state, too short. Further in vivo study is needed to

verify the accuracy of predictions for THP and in particularly, PBD. The large errors

visible in Figure 4, are mainly due to the large discrepancies in pharmacokinetic

parameters of examined drugs (Cl and Vd)

Nevertheless, the plots for pramipexole and selegiline based on both, human

and pig skin models, indicate that the their transdermal delivery by iontophoresis is

feasible. Both of the hydrochloride salts of these drugs are very well soluble in water,

and can contain sufficient amount of chloride ions for long lasting delivery. Also, they

seem to be transported at high rate and in sufficient doses, even with small patch

areas, across both skin types (Table 2). The plasma-levels modelling, pictured at

Figure 4 seem to further confirm these observations. It is noteworthy, that for these

two drugs, the steady state flux predicted by Nugroho model, and the plasma levels

are similar for regardless of skin model employed.

One of the advantages of iontophoretic delivery is one-a-day application. It is

particularly important as the patients compliance to medical instructions decreases

with rising number of daily applications. However, in order to make an iontophoretic

patch work for 24 hours, it has to be supplied with enough chloride ions, for the

electrodes to work correctly. The amount of chlorides required per 1cm2 of a patch,

can be obtained as a product of current density (I=0.5mA/cm2) and time of the

iontophoresis (24h=86400s), normalized by the Faraday constant (F=96480C/mol).

Providing that 1ml of donor solution can be fitted over one square centimetre, a


171

concentration of 0.45M would be required to make chlorides available. This

concentration is certainly higher than the solubility of most of the examined drugs.

Selegiline HCl is miscible with water to very high concentration (>1M). The solubility

study of pramipexole showed that at pH 8.0 it can reach 0.2M concentration, and

possibly more (the saturation concentration was not reached). This drug however, is

a very good charge carrier, and from Table 2 it seems, that the achievable delivery

rate exceeds the target flux by an order of magnitude. Hence, lowering the current

intensity or adding another source of chloride ions (i.e. NaCl) can be effectively used

in order to assure correct electrochemistry at the electrodes, and in the same time,

delivering the drug at the sufficient rate. On the contrary, the solubility of more

lipophilic drugs, trihexyphenidyl and piribedil, at pH 5.0 is roughly ten times lower.

Previous estimations of “deliverability” of Trihexyphenidyl (Table 2) showed, that the

rate it can be delivered with, surpasses the required flux by a factor of three. Also the

study carried out in Chapter V suggests, that charge carrying properties of THP are

rather insensitive to the presence of competing ions, in range of 0.25 to 1.0 mole

fraction. This means that lowering the current and introducing external source of

chlorides could assure enough Cl- for 24 hours delivery at sufficient rate.

In the case of piribedil the results are ambiguous. The predictions of Nugroho

et al., based on the pig skin model suggest, that it could be feasible to deliver it

iontophoretically. However, as discussed before, the estimation based solely on the

data from the pig skin model may be too optimistic. In particular, that in the human-

skin experiment, long lag time and significantly lower fluxes were observed. Table 2

indicates, that the dose delivered during first six hours is lower than the required one.

Also, a very large variability (RSD reaching almost 100%), due significant

discrepancies in volume of distribution (responsible for 95% of an error), mean that in

some patients widely distributing the drug, reaching the plasma concentration close

to 150nM may be impossible. The usage of additional chloride source, required for a

24 hour long delivery, could reduce the steady state flux of PBD, making the

transport less effective. Nevertheless, pergolide transdermal flux, similarly to THP, is

up to 50% mole fraction insensitive to the presence of competing ions. Thus, a

moderate addition of external chloride source, could elongate the delivery without

compromising the maximal delivery rate. What is more, the threshold level for

piribedil, indicated in Figure 4, was the Cmax measured after 30-day administration of


172

50mg Trivastal Retard (13). It is significantly higher, in comparison to the other

plasma concentrations (50-100nM) reported as effective (14). In the in vivo study it

might then appear, that the required fluxes would be in fact lower, than estimated in

this work. It would be very interesting to test the in vivo delivery of piribedil, to verify

the validity of the skin membrane model and the plasma-level prediction

Finally, it is clear, that pergolide cannot be effectively delivered via

transdermal iontophoresis. With neither of the skin models was it able to cross the

membrane with the sufficient rate. Large lag time and big standard deviation of flux

made it very hard to control the flux by current adjustment.

Conclusions

The experiments with human skin further confirmed the feasibility of delivering

clinically relevant doses of the two antiparkinsonian drugs: pramipexole and

selegiline, by transdermal iontophoresis. The trihexyphenidyl seems also possible to

deliver, however requires a large patch area and an additional source of chlorides . It

was also shown that this would be impossible for pergolide. The results for piribedil

are unclear and require further study. The transport of the electroosmotic marker was

very similar for both types of membrane, suggesting the similar input of

electroosmotic effect.

Bibliography

1. Pig and guinea pig skin as surrogates for human in vitro penetration

studies: a quantitative review. Barbero, A M and Frasch, H F. 2008, Toxicology In

Vitro, Vol. 23, pp. 1-13.

2. Gastrointestinal and dermal absorption: interspecies differences.

Calabrese, E J. 1984, Drug Metabolism Reviews, Vol. 15, pp. 1013-1032.

3. Structure of the skin barrier and its modulation by vesicular formulations.

Bouwstra, J A, et al. 2003, Progress in Lipid Research, Vol. 42, pp. 1-36.

4. Lipid organization in pig stratum corneum. Bouwstra, J A, et al. 1995,

Journal of Lipid Research, Vol. 36, pp. 685-695.


173




6. Kanikkannan, N, Singh, J and Ramarao, P. In vitro transdermal transport

of timolol maleate: effect of age and species. Journal of Controlled Release. 2001,

Vol. 71, pp. 99-105.

7. Padmanabhan, R, Phipps, J and Lattin, G. In vitro and in vivo evaluation

od transdermal iontophoretic delivery of hydromorphone. Journal of Controlled

Release. 1990, Vol. 11, pp. 123-135.

8. Iontophoretic delivery of amino acids and amino acid derivatives across the

skin in vitro. Green, P G, et al. 1991, Pharmaceutical Research, Vol. 8, pp. 1113-

1120.

9. Compartamental modeling of transdermal iontophoretic transport: I. In vitro

modeli derivation and application. Nugroho, A K, et al. 2004, Pharmaceutical

Research, Vol. 21, pp. 1974-1984.

10. Transdermal iontophoresis of rotigotine: influence of concentration

temperature and current density in human skin in vitro. Nugroho, A K, et al. 2004,


11. Iontophoretic delivery of apomorphine I: in vitro optimization and

validation. van der Geest, R, Danhof, M and Bodde, H E. 1997, Pharmaceutical

Research, Vol. 14, pp. 1798-1803.

12. Iontophoretic delivery of apomorphine II: an in vivo study in patients with

Parkinson's disease. van der Geest, R, et al. 1997, Pharmaceutical Research, Vol.

14, pp. 1804-1810.




174

14. Steady-state pharmacokinetic properties of pramipexole in healthy

volunteers. Wright, C E, et al. 1997, Journal of Clinical Pharmacology, Vol. 37, pp.

520-525.









pp. 23-27.




19. Piribedil plasma levels after chronic oral administration of Trivastal Retard

50 (150 mg/day) in parkinsonian patients. Allain, H, et al. 2001, Parkinsonism &

Related Disorders, Vol. 7, p. S51.

20. Pharmacodynamic and pharmacokinetic properties of piribedil: rationale

for use in Parkinson's disease. Allain, H. 2001, Disease Managemet and Health

Outcomes, Vol. 12, pp. 41-48.

21. Cinétique plasmatique du piribedil par voie intraveineuse et corrélation

avec le tremblement parkinsonien. Ziegler, M, Spampinato, U and Rondot, P. 1991, JAMA, Vol. (French ed; special issue), pp. 26-30.

22. Efficacy of piribedil as early combination to levodopa in patients with stable

Parkinson's disease: A 6-month, randomized, placebo-controlled study. Zigler, M, et al. 2003, Movements Disorders, Vol. 18, pp. 418-425.


175

23. End-of-dose akinesia after a single dose intravenious infusion of the

dopaminergic agonist piribedil in Parkinson's disease patients:

pharmacokinetic/phamacodynamic, randomized, double-blind study. Simon, N, et al. 2005, Movement Disorders, Vol. 20, pp. 803-809.

24. Pharmacokinetics of trihexyphenidyl after long-term and short-term

administration to dystonic patients. Burke, R E and Fahn, S. 1985, Annals of







General discussion and conclusions

176

General discussion and conclusions The influence of formulation on transdermal iontophoresis of drugs

In this work, a variety of properties of iontophoretic formulation has been

studied, concerning their impact on transdermal iontophoresis of the six drugs used

in Parkinson’s disease. Factors, like the pH, the drug concentration, the effect of

competing ions, the drug mole fraction or the current intensity, have been studied.

Also, the physicochemical properties of the six drugs were measured and compared

with the acquired transport rates, in order to elucidate the effect of those properties

on the iontophoretic transport.

The pH

Generally, all the studied drugs were delivered more efficiently from the donor

solutions at the higher pH values. The magnitude of the pH effect was different,

depending on the drug. The better charge carriers, like pramipexole or selegiline,

were more affected by the pH than the mediocre ones, such as trihexyphenidyl. The

donor pH affected several parameters, crucial for iontophoretic deliver: 1) the

solubility of the drug; 2) the ionization of the drug; 3) the charge fixed on the skin,

hence the magnitude of electroosmosis; and 4) the competition of hydronium ions.

For some drugs, like pergolide, the concentration suitable for iontophoretic

delivery was only achievable at pH below 4.0. For others, like pramipexole, the

attaining of the appropriate concentrations was possible even for the pH as high as

11.0. The pH decided on the maximal concentration of the drug in solution.

The ionization of the drug was another important factor affected by the pH.

Previously, studies of Abla et al. (1) and Sylvestre et al. (2) showed positive effect of

charge fixed on the delivered molecule on their transport efficiency, for both cations

and anions. In presented work, pramipexole and possibly entacapone, were

examined as bivalent ions. For pramipexole, the effect of rising valence (caused by

decreasing pH) was hard to assess, as it coexisted with the changing permselectivity

of the skin. These two effects were not easy to separate. Possible double ionization


177

of entacapone was used as an explanation of rising drug flux with the pH. However, a

confirmation of a second ionization constant is needed to support this explanation.

The electroosmosis is one of the main phenomena governing transdermal

iontophoretic transport. It is dependent mainly on the charge fixed on the skin, during

the iontophoretic process. The latter is, in turn, dependent on the value of the pH in

the skin. The iP of the pig skin is of around 4.3 (3). Thus, for the values of the pH

above the iP the skin is negatively charged, and for those lower than the iP it is

positively charged. As described by Pikal et al. (4), the electroosmosis is directed

along the movement of counter-ion, as referred to the skin charge. Hence, the

delivery of the cations will be enhanced by the electroosmotic effect, for the skin pH

higher than the iP value, while the transport of anions will be enhanced, when the pH

of the skin is lower than the iP. The magnitude of the electroosmotic effect from

anode to cathode direction should then decrease with the rising donor pH. This was

observed for all the cationic drugs investigated. Namely, the flux of electroosmotic

marker decreased with pH during the iontophoresis of pramipexole, selegiline,

piribedil or trihexyphenidyl. It was then concluded, that the electroosmotic input to the

total drug flux also decreased. However, the magnitude of this input remained

unresolved.

At the low pH values (<3.0), the concentration of hydronium ions becomes

comparable to the molarity of solutions used in iontophoretic experiments. This

causes the competition of highly mobile H+ ions to hinder the drug delivery. For the

drugs studied, a sharp drop of flux was observed between pH 4.0 and 2.0.

The current intensity

One of the advantages of the iontophoretic drug delivery is the easily

controllable drug input. In accordance with the electrodiffusion theory (5), the drug

flux is proportional to the current intensity. The delivery of all the investigated drugs

depended linearly on the current applied. However, in case of pergolide, the slope of

the flux-current dependency was small, and it could be difficult to use this in practice,

as a means of dose adjustment. An analogous dependence of iontophoretic flux was

observed in many previous studies (6; 7; 8).


178

The concentration as single-ion

According with the Nernst-Planck theory (5), during the single-ion delivery, the

transport of the ion of interest should be independent of its concentration. This was

indeed observed for four drugs studied (PPX, SEL, PBD and THP). Due to the

solubility problems, pergolide and entacapone were not tested for the concentration

effect in single-ion delivery. Previously, similar observations were done for

hydromorphone, lidocaine and ropinirole (6; 8; 9)

Ionic competition

Generally, the presence of competing electrolyte limited the drug transport. It

was found that it is the molar fraction of the drug, rather than its nominal

concentration, on which the transdermal flux is dependent. The two profiles of

competition were observed for the investigated drugs. The fluxes of the most soluble

drugs, pramipexole and selegiline, were limited accordingly to their mole fraction in

the donor solution. The dependence of the flux on mole fraction was linear. The flux

of other drugs, like entacapone or trihexyphenidyl, was affected only for small mole

fractions of the drug. Then, after achieving the top level, the flux stabilized. The

difference was explained by the partitioning of the drug in the skin, and could be

modeled by the Phipps and Gyory model (6).

Feasibility of iontophoretic delivery of AP drugs

The issue of feasibility for all drugs was discussed in Chapter VII. In summary,

the primary objective of this work was to assess the feasibility of iontophoretic

delivery of the six drugs. As a result, it was shown that the fluxes of two of them

(pramipexole and selegiline) reach the target rate, required for effective drug delivery.

Across both, the pig and the human skin, the delivery rate and dose delivered were

high enough to assure effective drug concentrations. The modeling of plasma

concentration revealed that required plasma concentrations can be achieved, even

for a relatively small transport area. Furthermore, pramipexole experiments have

shown that it has good abilities of passively penetrating the skin. This makes it a

good candidate for passive transdermal application, which is much cheaper to

commercialize than an iontophoretic one. Similarly, selegiline is already formulated

as a passive patch.


179

Due to high therapeutical plasma concentrations and poor charge-carrying

properties entacapone and pergolide could not be effectively delivered via

iontophoretic application.

Trihexyphenidyl and pergolide have shown some promise in pig skin

experiments, but performed less well in experiments with full thickness human skin.

Modeling of plasma concentrations for iontophoretic delivery of those drugs seemed

to indicate that, with a large patch area, the achievement of efficient fluxes might be

possible. For these drugs additional in vivo tests are required, to establish the

feasibility of effective iontophoretic delivery. It Is particularly important in the case of

piribedil. It is now widely accepted that the constant plasma levels of dopamine

agonists are crucial in PD therapy (8). The attempts to deliver this drug

transdermally, using passive applications, were unsuccessful (7). The application of

iontophoresis could enable the transdermal route for this substance. As a

consequence, an increase in bioavailability could be expected. Also, in practice there

are other factors which are important in the applicability of iontophoretic dosage form.

Firstly, the delivery of pergolide might be difficult to control by the current adjustment,

due to its small transport number. The low value of transport number means that

even a large increase in the current results in only a small change of flux. This,

combined with the relatively large variability of pergolide transport, could make the

delivery hard to control, hence limiting its usage.

Electroosmotic input to the total transport

The results concerning the input of electroosmosis to the total iontophoretic

transport are puzzling. First, as discussed earlier, the fluxes of electroosmotic marker

might not be transported the same way. Hence, the calculation of the electroosmotic

contribution as DrugEO CVJ ⋅= might be not accurate. As mentioned earlier in this

work, the changes in mannitol flux are understood here only as an indicator of rising

or decreasing volume flow.

Furthermore, the experiments studying the effect of the pH in pramipexole

iontophoresis seem to indicate, that the input of electroosmosis can improve the

transport by half. This is however based on the assumptions, that there is negligible


180

electroosmotic effect at pH 5.0, and that the pH inside the skin is equal to the pH of

the donor.

On the other hand, gradually elevating mole fraction of the drug, results in 1)

increase in the drug flux; and 2) sharp limitation of mannitol flux, suggesting limitation

of electroosmotic flow. Furthermore, in the cases of selegiline and pramipexole,

limitation of mannitol transport by increasing drug concentration does not seem to

affect the slope of the drug-flux/mole fraction curve. This means that with mannitol

flux present, or not, the mole fraction has the same impact on drug flux. This, in turn,

suggests that the input of the electroosmotic flow to total drug delivery is rather small.

It is hard to distinguish effect of electroosmosis on the drug transport, as there

is no particular ion population that travels across the skin solely by electroosmosis or

electromigration. These two phenomena coexist, acting on delivered substances

parallely. It clearly requires further study to determine the importance of

electroosmosis in transdermal delivery of organic compounds.

Physicochemical properties of the drugs

One of the objectives of this study was to look at the physicochemical

parameters of investigated drugs on their impact on the drug transdermal transport.

The examined parameters were the ionic mobility in water, lipophilicity (measured as

log P and log D), molecular mass and water solubility. The investigated parameters

were measured or, if available, taken from the literature (Table 1).

Table 1 Physicochemical parameters of the six investigated drugs with transdermal fluxes obtained. *Data taken from the literature (9; 10; 11)

J6h Human

skin [μmol/h]

J6h Pig skin

[μmol/h]

J6hHuman

/ J6hPig

logD at pH 5.0 logP pK

Solubility at pH 5.0

[mM] Mw[Da]

Ionic water mobility [10‐4cm2/Vs]

SEL 0.91 1.22 0.75 0.7 3.6 7.4 >20% 187 3.0 PPX 1.91 2.45 0.78 ‐3.3 1.5 11.0 freely 211 2.7 PBD 0.14 0.35 0.40 0.9 2.8 6.9 34.1 298 3.9 THP 0.08 0.19 0.42 1.4 5.3* 9.3 31.5 301 1.9 ETC ‐‐ 0.17 ‐‐ 1.4 1.5* 4.5 0.32* 305 1.0 PER 0.002 0.02 0.11 0.2 4.0* 7.8 0.34 314 ‐‐


181

Lipophilicity

Previous research underlines the importance of the lipophilicity of a delivered

molecule for several reasons. Hirvonen et al. (12; 13) have shown that drugs,

depending on their lipophilicity, can have a major impact on the magnitude of the

electroosmotic effect, one of the main phenomena contributing to the

electrotransport. In the studied group of five β-blockers, those of the highest partition

coefficient inhibited the mannitol transport in the strongest manner. In contrast, those

hydrophilic ones had practically no effect on mannitol transport. It was suggested,

that such limitation of the electroosmosis could reduce the drug transport. The results

presented in this work seem to be consistent with the observations of Hirvonen and

co-workers. The most hydrophilic compound, pramipexole, at the concentration of

3mM (in 27mM NaCl solution) actually enhanced slightly the transport of

electroosmotic marker, while the most hydrophobic one, trihexyphenidyl, greatly

reduced it. Table 2 summarizes the fluxes of EO markers in the presence and in the

absence of a drug, in a co-ion situation. It also presents the inhibition factor (defined

as the reference EO flux divided by the flux in presence of a drug) and log P of a

drug. It is necessary to add, that both of the experimental setups, the one presented

in Table 2, and the one by Hirvonen et al. refer to the situation in which the drug is

delivered from the solutions containing background electrolyte. In the single-ion

situation all the examined drugs, even the most hydrophilic, decreased the

electroosmotic flux. However, the EO marker fluxes obtained for iontophoresis of the

hydrophilic drugs were less strongly inhibited in comparison to those lipophilic. This

seems to confirm the previous suggestions that only the combination of a lipophilic

“anchor” and a positive charge can substantially reduce the electroosmotic transport.

Table 2 The fluxes of electroosmotic marker compared to the reference flux (in absence of drug) across the pig skin, from donor solutions containing 0.1 and 1.0 mole mole fraction of a drug. MF – mole fraction.

EO Marker

Flux [nmol/h] Av±SD

Reference flux

[nmol/h] Av±SD

Inhibition factor at 0.1 MF Av±SD

Inhibition factor at

single-ion Av±SD

Log P

SEL 20.9±7.8 55.21±12.27 2.6±0.2 7.4±0.1 3.6

PPX 124.7±14.8 107.3±11.6 0.9±0.3 7.1±0.0 1.5

PBD 19.2±12 95.2±8.1 5.0±0.1 33.8±0.0 2.8

THP 16.5±2.6 95.2±8.1 5.8±0.0 50.6±0.0 5.3

PER 4.09±5.2 47.6±4.0 11.6±0.1 ‐‐ 4.0


182

In terms of the dependence of total iontophoretic flux, on the lipophilicity, there

seems to be a correlation between the single-ion steady state flux across the porcine

skin, and the partition coefficient (Pearson test, p<0.05). However, this is only visible

when the data presented in this study are combined with the data for hydromorphone

(14), and those of lidocaine, propranolol and quinine (15) (Figure 1).

0 2 4 60

1

2

3

4

5

HMPPPX

LIDSEL

THPPERQINPRP

Log P

Sing

le io

n io

ntop

hore

tic fl

ux[ µ

mol

/h/c

m2 ]

Figure 1 The plot of steady state flux during the single-ion transdermal iontophoresis across porcine skin as a function of log of partition coefficient. PRP – propranolol, QIN – quinine, LID – lidocaine, HMP – hydromorphone.

Among all other available drugs, the data of those four were used, as they

were acquired in similar iontophoretic conditions. The addition of these data widens

the spectrum of log P values of drugs tested. Nevertheless, there is a striking

difference of two orders of magnitude, between the maximal fluxes obtained for

selegiline (log P 3.6) and only slightly more lipophilic pergolide (log P 4.0). Also,

piribedil and trihexyphenidyl exhibited similar single-ion fluxes, despite the much

higher lipophilicity of the latter drug. The results similar to these were obtained for the

transdermal delivery of tripeptides (16). However, no direct correlation was found

between the iontophoretic flux and the lipophilicity nor the molecular weight for the

peptides studied. Also, in the β-blockers delivery in vivo (17), no clear correlation

between log P and transdermal flux was observed. In fact, the penetration into the

skin for pindolol (log P 0.08), was five times higher than that of atenolol (log P -0.11)

or metoprolol (log P 0.2). These results might suggest that a) some molecules are


183

particularly primed for crossing the skin, and b) that lipophilicity on its own, is not an

accurate predictive factor for transdermal delivery. Possibly, more elaborate methods

involving the 3D-spatial analysis of molecule shape and lipophilicity distribution in a

molecule is necessary to explain adequately the transport kinetics.

Figure 2 presents the plot of the delivered dose during the six hours of the

iontophoresis for the drugs examined, across the pig and the human skin, as a

function of distribution coefficient (at the pH applied for delivery). From the graph it is

clear that the delivered dose decreases with the increase of the log D of a drug. This

however, can be a summary effect of the two factors: the maximal flux achieved and

the time taken to achieve it. It can be speculated that the lipophilic substances can

deposit in lipid-rich skin layers and elongate the time to reach the steady state flux. In

fact, the Nugroho et al. kinetic model of iontophoretic drug delivery (18), which was

discussed in Chapter VII, considers the skin as a separate compartment, and

predicts the accumulation of delivered substances in it, and their further partitioning

to the subdermal compartments. In Figure 2 in Chapter VII, the time to reach steady

state can be read for each drug for the pig skin model. For selegiline it is 2h; for

pramipexole piribedil and trihexyphenidyl, 3h; and for pergolide 5h. For the

experiments with human skin, this is possible only in case of pramipexole, as the

experiment time was too short to achieve the steady state delivery. Thus, in case of

the time to reach steady state, no clear impact of drug lipophilicity can be observed.

-2 0 2 40

5

10

15

20

PPX SEL PBD THPPER

Human skinPig skin

Log D

Del

iver

ed d

ose

[ µm

ol]

Figure 2 The delivered dose of five investigated drugs during the six hours of iontophoresis across human and pig skin. The delivery was carried out in the single-ion situation described in the previous chapters.


184

Water mobility and molecular weight

Molecular weight and water ionic mobility are other factors, often mentioned as

deciding upon the transdermal transport. Mudry et al. (19) have shown that for small

inorganic molecules and several organic cations, there is a strong positive correlation

between transdermal transport number and ionic mobility. Also, it was found that

molecular weight had a negative effect on the transdermal transport number (19). In

this study, the water mobility of examined ions was measured and compared with the

data previously published, for similar size organic drugs (19), namely propranolol

quinine lidocaine and ropinirole. Figure 3a depicts the results obtained. As opposed

to the data of Mudry et al. (19), no correlation between the two values can be

detected. The observed difference to their findings can be explained by the fact that

different ions were employed in both studies. Mudry et al. used a wide span of ionic

substances: small, mobile inorganic ions like K+ or NH4+, organic ions like tetra-N-

methylammonium or larger organic ions like i.e. quinine. This allowed the whole

range of mobilities and transdermal transport numbers relevant to each ion to be

investigated. In this work however, the focus was more on drug substances, which

are usually organic molecules, larger than 150Da. Thus, the question to answer was:

“Is it possible to assess the transport number of an organic drug molecule, having its

water mobility?”

0.0 1.0 2.0 3.0 4.0 5.00.00

0.05

0.10

0.15

0.20

PBD

PPX

SEL

ROP

LID

QIN PRPTHP

104• Mobility (cm2 s-1 V-1)

Cat

ion

tran

spor

t num

ber

a

150 200 250 300 3500

1

2

3

4

SEL

PPX

LID

PRP PBD

THPQIN

PER

ROP

Molecular weight [Da]

Sing

le io

n io

ntop

hore

tic fl

ux[ µ

mol

/h]

b

Figure 3 The plot of transdermal transport number in single ion situation as a function of a) water mobility and b) molecular weight of a ionic species. SEL – selegiline, PPX – pramipexole, LID – lidocaine, ROP – ropinirole, PRP – propranolol, PBD – piribedil, THP – trihexyphenidyl, PER – pergolide, QIN – quinine.

Based on Figure 3a the answer to that question is no. This is probably because, with

the large organic molecules other effects such as partitioning to the skin or limiting


185

the electroosmotic effect, become more significant as compared to smaller inorganic

ions. These additional factors are not taken into account in Mudry’s model, thus it

does not accurately predict the transport number for organic molecules. On the

contrary, the correlation between molecular weight and iontophoretic flux was

detected (Pearson test, p<0.05), indicating that molecular size is a significant factor

to be taken under consideration when choosing the candidates for transdermal

iontophoresis (Figure 3b). Abla and co workers (1) have previously studied the effect

of molecular weight on a peptide model of iontophoretic deliver. From their study it

was apparent that there is a strong positive correlation between the transdermal flux

and charge/molecular weight ratio. The results obtained for the antiparkinsonian

drugs (Figure 4) seem to be in good accordance with their data for the peptides.

2 3 4 5 60

1

2

3

4

SEL

PPX

LID

PER

PBDTHP

ROP

QIN PRP

103 Charge/Mw

Iont

opho

retic

flux

[ µm

ol/h

/cm

2 ]

Figure 4 The single-ion flux of drugs as a function of the charge to molecular weight ratio. SEL – selegiline, PPX – pramipexole, LID – lidocaine, ROP – ropinirole, PRP – propranolol, PBD – piribedil, THP – trihexyphenidyl, PER – pergolide, QIN – quinine.

Solubility and pKa

Solubility characteristics are important parameters affecting transdermal drug

transport. They determine upon the maximal concentration of a drug in the donor

solution, which in turn has an influence on the applicable range of current and the

usage of external sources of chlorides. The ionization constant Ka determines the

range of pH of the donor from which the drug can be delivered.

The investigated antiparkinsonian agents were mainly weak bases, except for

entacapone which was a weak acid. The measured solubility and pKa data are

presented in Table 1. It is significant that the iontophoretic flux seem to follow

solubility, as the two best charge carriers, pramipexole and selegiline are readily

soluble in water. The intermediate ones, trihexyphenidyl and piribedil have solubility


186

around 30mM (at pH 5.0). Finally, the worst ones entacapone and pergolide have low

water solubility. More data are required in order to verify whether these two factors

are correlated, however, based on these results, it can be stipulated that water

soluble drugs remain in the skin water-channels in an ionised form where they are

subject to the driving electric force. Those sparingly soluble substances in contrast,

partition to the lipid-rich parts of skin. Few studies have reported investigations of the

effects of water solubility and this could be a productive area for future research.

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8. Avoidance of dyskinesia. Preclinical evidence for continuous dopaminergic

stimulation. Jenner, P. 1 Suppl, 2004, Vol. 62, pp. S47-S55.


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tetrahydrobenzothiazol and intermediate compounds. EP 1 884 514 A1 2008.

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their efficacy during local intrastratial administration. Forsberg, M, et al. 2005,

European Journal of Pharmaceutical Sciences, Vol. 24, pp. 503-511.



Vol. 14, pp. 1258-1263.

13. Transdermal delivery of peptides by iontophoresis. Hirvonen, J, Kalia, Y N and Guy, R H. 1996, Nature Biotechnology, Vol. 14, pp. 1710-1713.






16. Effect of amino acid sequence on transdermal iontophoretic peptide

delivery. Schuetz, Y B, et al. 2005, European Journal of Pharmaceutical Sciences,

Vol. 26.

17. Effect of lipophilicity on in vivo iontophoretic delivery. II. B-blockers.

Tashiro, Y, et al. 2001, Biological and Pharmaceutical Bulletin, Vol. 24, pp. 671-677.

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modeli derivation and application. Nugroho, A K, et al. 2004, Pharmaceutical

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165-172.

Appendix I

188

Appendix I. Selegiline Experiments Current Effect

Paracetamol Fluxes (100%, 30mM, pH4.7) [nmol/h] SEL‐38 30mM pH=4.8 100% 0.1mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 1.7 2.5 1.7 1.8 1.8 0.0 1.6 0.8 6 2 2.4 3.8 2.7 2.1 3.0 6.6 3.4 1.7 6 3 3.0 4.0 3.3 2.1 3.8 5.6 3.7 1.2 6 4 3.2 4.9 4.6 3.1 4.2 5.5 4.3 0.9 6 5 4.0 4.3 4.5 3.9 4.3 5.3 4.4 0.5 6 6 3.2 4.1 4.2 4.5 3.7 5.7 4.2 0.9 6 SEL‐37 30mM pH=4.8 100% 0.2mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 3.0 2.4 2.1 2.7 2.0 6.1 3.0 1.6 6 2 3.6 2.9 3.1 4.1 2.8 4.8 3.6 0.8 6 3 3.0 2.7 3.4 4.4 4.4 3.3 3.5 0.7 6 4 3.1 2.8 5.4 5.0 4.8 2.6 4.0 1.2 6 5 3.2 3.0 7.0 5.1 5.8 2.9 4.5 1.7 6 6 3.8 2.9 6.9 5.2 5.5 3.0 4.5 1.6 6 Sel‐36 30mM pH=4.8 pH=4.8 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 22.6 6.0 4.9 0.0 3.6 3.8 6.8 8.0 6 2 20.6 22.8 11.1 9.2 8.2 9.9 13.6 6.4 6 3 21.4 12.7 14.5 13.3 12.5 12.3 14.4 3.5 6 4 17.5 17.6 13.0 9.8 10.3 9.2 12.9 3.8 6 5 11.2 13.3 9.7 8.3 9.3 6.6 9.7 2.3 6 6 8.4 10.9 8.6 7.1 8.5 5.9 8.2 1.7 6

Selegiline Fluxes (100%, 30mM, pH4.7) [µmol/h] SEL‐38 30mM pH=4.8 100% 0.1mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N 1 0.04 0.03 0.05 0.07 0.05 0.22 0.08 0.07 6 2 0.22 0.19 0.23 0.24 0.22 0.36 0.25 0.06 6 3 0.32 0.32 0.35 0.36 0.37 0.38 0.35 0.02 6 4 0.33 0.37 0.38 0.37 0.41 0.37 0.37 0.03 6 5 0.36 0.39 0.39 0.43 0.43 0.38 0.40 0.03 6 6 0.41 0.40 0.41 0.41 0.44 0.34 0.40 0.03 6 SEL‐37 30mM pH=4.8 100% 0.2mA


t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 1 0.5 0.6 0.6 0.4 1.3 0.7 2 1.1 1.5 1.3 1.9 1.2 1.1 3 1.3 1.4 1.3 1.4 1.7 1.2 4 1.3 1.5 1.1 1.6 2.3 1.1 5 1.3 1.2 1.3 1.2 1.9 1.1 6 1.2 1.6 1.1 1.3 1.6 1.1 AV SD N 0.57 0.30 17 SEL‐02 30mM pH=4.6 100% 0.4mA SEL‐29 30mM pH=4.6 100% 0.4mA 1.32 0.31 17

t[h] Cell 1 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 1.35 0.18 17 1 0.0 0.7 0.7 0.4 0.5 0.0 0.9 0.8 0.5 0.5 0.5 1.43 0.33 17 2 2.0 1.4 1.4 1.3 1.3 0.6 1.3 1.3 1.2 1.3 1.3 1.18 0.27 17 3 1.6 1.4 1.3 1.1 1.5 1.2 1.2 1.1 1.2 1.5 1.5 1.21 0.22 15 4 1.6 1.3 1.8 1.0 1.8 1.3 1.2 1.1 1.2 1.5 1.4 5 1.2 0.9 1.0 0.6 1.2 1.2 1.1 1.1 1.0 1.5 1.3 6 1.2 0.9 0.8 0.4* 1.4 1.3 1.2 1.0 1.3 1.2 2.1*

Appendix I

189

Selegiline Conductivity measurements

Molar Conductivity [Si*cm2/mol]

Root of Concentration [mM]1/2 Metrohm conductimeter Oaklab conductimeter AV SD

2.24 95.6 95.7 95.7 93.9 94.1 94.5 94.2 0.3 Selegiline

1.41 99.3 99.7 99.9 97.0 96.7 97.3 97.0 0.3 1 101.6 101.4 101.5 100.0 100.0 100.2 100.1 0.1

0.71 102.3 102.2 102.3 101.6 102.8 101.6 102.0 0.7 0.45 105.3 106.3 106.3 105.9 107.8 106.4 106.7 1.0 0.47 125.3 130.0 127.2 127.5 2.4

NaCl

0.72 125.2 125.2 125.4 125.3 0.1 1 123.0 122.9 123.2 123.0 0.2

1.42 121.3 120.9 120.9 121.0 0.2 2.24 118.4 118.2 118.2 118.3 0.1 3.16 115.9 115.8 115.9 115.9 0.1

Mole Fraction EffectSelegiline Fluxes (pH=4.5(0.2), 120mM, 0.4mA) [µmol/h]

SEL‐31 10% pH=4.8 120mM 0.4mA SEL‐35 10% pH=4.8 120mM 0.4mA t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 0.1 0.0 0.1 0.1 0.1 0.0 0.1 0.5 0.1 0.0 0.1 0.0 0.09 0.14 12 2 0.3 0.4 0.3 0.3 0.3 0.2 0.3 0.3 0.2 0.2 0.2 0.1 0.26 0.05 12 3 0.3 0.4 0.3 0.6 0.5 0.4 0.3 0.3 0.3 0.5 0.4 0.3 0.40 0.08 12 4 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.2 0.30 0.02 12 5 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.29 0.02 12 6 0.3 0.4 0.3 0.3 0.3 0.3 0.3 0.4 0.3 0.3 0.3 0.3 0.30 0.03 12 SEL‐19 25% pH=4.6 120mM 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N 1 0.4 0.3 0.5 0.3 0.3 0.5 0.36 0.09 6 2 0.4 0.4 0.5 0.5 0.5 0.5 0.46 0.05 6 3 0.4 0.3 0.5 0.5 0.5 0.6 0.45 0.08 6 4 0.5 0.4 0.5 0.5 0.5 0.5 0.47 0.03 6 5 0.4 0.5 0.5 0.5 0.5 0.5 0.47 0.04 6 6 0.4 0.4 0.5 0.4 0.5 0.5 0.41 0.06 6 Sel‐30 50% pH=4.5 120mM 0.4mA Sel‐41 50% pH=4.5 120mM 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 0.4 0.6 0.6 0.7 0.3 0.4 0.4 0.3 0.4 0.3 0.5 0.5 0.43 0.12 12 2 1.3 0.9 1.2 1.2 1.2 1.1 1.0 1.2 1.1 1.0 1.0 1.1 1.11 0.11 12 3 1.1 1.2 0.9 0.9 1.1 0.9 1.1 1.3 1.2 1.3 1.1 1.2 1.11 0.15 12 4 1.0 1.1 0.9 0.8 1.1 0.9 1.1 1.4 1.3 1.3 1.0 1.2 1.11 0.19 12 5 1.0 1.1 0.9 0.9 1.0 0.8 1.0 1.0 1.0 1.0 0.8 1.0 0.98 0.09 12 6 0.9 1.1 0.9 0.9 1.0 0.9 1.1 1.1 1.1 1.1 0.8 1.0 0.99 0.10 12

Appendix I

190

Selegiline Fluxes (pH=4.5(0.2), 120mM, 0.4mA) [µmol/h] SEL‐20 75% pH=4.5 120mM 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 0.6 0.4 0.4 0.8 0.9 0.6 0.60 0.21 6 2 0.7 0.8 0.7 0.9 1.0 1.3 0.90 0.23 6 3 0.7 0.7 1.2 1.0 1.2 1.4 1.04 0.27 6 4 0.7 0.9 0.8 1.0 1.2 1.5 1.02 0.30 6 5 0.6 1.1 1.0 0.9 1.0 1.5 1.00 0.28 6 6 0.8 1.0 0.8 1.2 0.8 1.2 0.95 0.20 6

SEL‐32 100% pH=4.2 120mM 0.4mA SEL‐44 100% pH=4.1 120mM 0.4mA


Appendix I

195

Paracetamol fluxes (Selegiline, pH4.6(0.3), 120mM, 0.4mA) [nmol/h]

SEL‐31 10% pH=4.8 120mM 0.4mA SEL‐35 10% pH=4.9 120mM 0.4mA t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 11.4 27.3 25.5 23.3 4.7 9.6 5.6 5.9 6.2 6.4 6.0 6.6 11.5 8.6 12 2 18.4 48.8 23.1 12.8 14.8 11.3 12.7 10.6 11.7 11.2 11.5 14.2 16.8 10.7 12 3 17.7 52.0 16.1 23.2 23.6 21.4 16.9 15.2 17.0 15.3 14.8 18.0 20.9 10.2 12 4 22.2 55.3 20.5 19.5 22.2 19.7 20.1 19.1 21.8 19.9 18.3 23.5 23.5 10.1 12 5 24.7 36.8 23.1 24.8 24.1 23.9 17.2 15.4 16.3 15.0 15.0 16.9 21.1 6.4 12 6 27.8 34.5 23.8 22.0 22.4 25.7 13.0 9.4 10.0 9.2 9.3 10.2 18.1 8.9 12 Sel‐30 50% pH=4.5 120mM 0.4mA Sel‐41 50% pH=4.5 120mM 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 1.7 0.0 2.1 1.8 1.4 5.0 2.6 3.7 2.8 3.7 3.9 3.6 0.8 5 2 6.3 8.1 3.9 7.0 4.4 14.5 13.7 14.7 11.7 9.1 12.1 12.6 1.8 5 3 8.8 7.6 3.9 9.6 5.5 17.2 19.1 16.8 17.0 11.6 15.1 16.1 2.4 5 4 8.7 8.1 3.7 11.0 6.0 17.3 22.8 19.3 19.9 10.0 15.7 17.5 4.4 5 5 9.5 12.8 4.3 11.9 5.6 15.6 17.1 14.1 15.2 7.9 13.6 13.9 3.2 5 6 7.8 16.1 5.2 9.6 5.5 17.8 19.3 16.2 16.3 8.8 14.3 15.5 3.7 5 Sel‐44 100% pH=4.1 120mM 0.4mA


Appendix I

196

Concentration Effect – Single IonSelegiline Fluxes (100%, pH=4.3, 0.4mA) [µmol/h]

SEL‐02 30mM pH=4.6 100% 0.4mA SEL‐05 30mM

pH=4.5 100% 0.4mA

t[h] Cell 1 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 1 0.0 0.7 0.7 0.4 0.5 0.5 0.6 0.6 0.4 1.3 0.7 2 2.0 1.4 1.4 1.3 1.3 1.1 1.5 1.3 1.9 1.2 1.1 3 1.6 1.4 1.3 1.1 1.5 1.3 1.4 1.3 1.4 1.7 1.2 4 1.6 1.3 1.8 1.0 1.8 1.3 1.5 1.1 1.6 2.3 1.1 5 1.2 0.9 1.0 0.6 1.2 1.3 1.2 1.3 1.2 1.9 1.1 AV SD N 6 1.2 0.9 0.8 0.4* 1.4 1.2 1.6 1.1 1.3 1.6 1.1 0.6 0.3 17 1.3 0.3 17

SEL‐29 30mM

pH= 4.6 100%

0.4 mA 1.4 0.2 17

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 1.4 0.3 17 1 0 0.9 0.8 0.5 0.5 0.5 1.2 0.3 17 2 0.6 1.3 1.3 1.2 1.3 1.3 1.2 0.2 15 3 1.2 1.2 1.1 1.2 1.5 1.5 4 1.3 1.2 1.1 1.2 1.5 1.4 5 1.2 1.1 1.1 1.0 1.5 1.3 6 1.3 1.2 1.0 1.3 1.2 2.1*

SEL‐06 90mM pH=4.2 100% 0.4mA SEL‐34 90mM pH=4.2 100% 0.4mA

t[h] Cell 1 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 1 0.8 0.7 0.7 0.7 0.9 0.4 0.5 0.4 0.5 0.7 0.4 2 1.3 1.2 1.1 1.2 1.2 1.2 1.3 1.5 1.2 1.3 1.2 3 1.0 0.9 1.0 1.2 1.1 1.5 1.1 1.6 1.0 0.9 1.3 4 0.9 0.7 0.9 0.0* 1.0 1.1 0.8 1.1 0.7 0.4 1.0 5 1.2 1.0 1.1 1.6 1.2 1.0 0.7 1.1 0.8 0.7 0.8 Mean SD N 6 0.9 0.8 0.8 1.1 0.8 1.0 1.0 0.8 0.9 1.0 0.9 0.8 0.2 22 1.2 0.1 22 SEL‐18 90mM pH=4.2 100% 0.4mA SEL‐15 90mM pH=4.2 100% 0.4mA 1.1 0.2 21

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 1.0 0.2 21 1 0.9 0.9 0.8 0.9 0.9 1.0 1.1 0.8 0.7 1.0 1.0 1.0 0.2 22 2 1.2 1.3 1.2 1.3 1.3 1.5 1.0 1.2 1.3 1.1 1.1 0.9 0.2 22 3 1.3 1.2 1.2 2.6* 1.3 1.1 0.9 1.2 1.0 0.8 1.3 4 1.2 1.2 1.1 1.2 1.2 1.2 0.7 0.8 1.2 0.8 1.1 5 1.3 1.1 1.1 1.0 1.1 1.2 0.7 1.0 1.2 0.7 1.1 6 1.2 1.0 1.0 1.0 1.2 1.2 0.7 0.8 1.1 0.7 0.8

SEL‐44 100% pH=4.1 120mM 0.4mA SEL‐32 100% pH=4.2 120mM 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N 1 1.0 1.1 1.1 1.0 0.9 0.8 0.5 0.5 1.5 1.9 0.4 0.5 0.9 0.5 12 2 1.6 1.8 1.5 1.6 1.6 1.4 1.4 1.5 1.6 2.0 1.3 1.2 1.5 0.2 12 3 1.6 1.9 1.5 1.6 1.5 1.6 1.3 1.4 1.3 1.5 1.4 1.2 1.5 0.2 12 4 1.6 1.9 1.4 1.6 1.5 1.6 1.4 1.4 1.2 2.1 1.5 1.2 1.5 0.3 12 5 1.5 1.9 1.3 1.5 1.5 1.6 1.3 1.5 1.2 1.9 1.5 1.3 1.5 0.2 12 6 1.5 2.0 1.3 1.5 1.5 1.6 1.3 1.5 1.3 1.6 1.5 1.4 1.5 0.2 12

Appendix I

197

Paracetamol Fluxes (Selegiline 100%, pH=4.1, 0.4mA) [nmol/h] Sel‐33 pH=4.0 30mM 100% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N 1 6.8 5.2 5.5 4.8 4.8 12.9 6.6 3.1 6 2 8.1 7.7 6.9 5.6 6.8 12.5 7.9 2.4 6 3 8.6 11.2 9.0 9.7 10.6 11.1 10.0 1.1 6 4 7.4 14.0 9.9 12.2 13.1 10.6 11.2 2.4 6 5 5.5 13.9 9.0 11.6 16.9 8.5 10.9 4.1 6 6 6.0 14.0 7.3 10.6 13.4 5.4 9.5 3.8 6 7 4.9 13.0 6.5 11.5 11.9 5.0 8.8 3.7 6 Sel‐34 pH=4.2 90mM 100% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N 1 0.0 5.7 4.5 5.4 6.9 5.4 4.6 2.4 6 2 9.6 11.2 11.1 8.4 9.4 10.0 9.9 1.1 6 3 10.3 10.4 12.6 8.4 7.4 10.7 10.0 1.8 6 4 11.0 9.6 11.6 8.1 7.8 11.1 9.9 1.6 6 5 10.0 8.3 10.8 7.8 7.4 9.2 8.9 1.3 6 6 10.4 8.1 10.6 7.7 7.8 8.9 8.9 1.3 6 Sel‐44 pH=4.1 120mM 100% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N 1 6.6 7.4 6.3 6.9 7.2 10.0 7.4 1.3 6 2 6.7 7.8 7.3 8.5 7.1 7.5 7.5 0.6 6 3 5.3 8.7 6.3 7.3 5.8 7.7 6.9 1.3 6 4 5.2 9.4 4.6 6.3 5.6 6.7 6.3 1.7 6 5 4.8 9.9 5.2 5.8 5.2 6.6 6.3 1.9 6 6 4.7 10.2 4.4 5.1 5.2 6.7 6.0 2.2 6

pH EffectSelegiline Fluxes (Selegiline 100%, 30mM, 0.4mA) [µmol/h]

SEL‐26 30mM pH=2.0 100% 0.4mA t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 0.06 0.06 0.05 0.07 0.02 0.01 0.05 0.02 6 2 0.13 0.09 0.10 0.10 0.09 0.05 0.09 0.02 6 3 0.14 0.09 0.12 0.10 0.11 0.06 0.10 0.03 6 4 0.13 0.09 0.09 0.09 0.12 0.06 0.10 0.03 6 5 0.13 0.08 0.08 0.08 0.11 0.06 0.09 0.03 6 6 0.13 0.10 0.09 0.09 0.12 0.06 0.10 0.03 6 SEL‐28 30mM pH=3.0 100% 0.4mA


t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 0.6 0.5 0.5 0.5 0.6 0.5 0.5 0.1 6 2 1.2 0.9 1.0 0.5 0.9 0.9 0.9 0.2 6 3 1.2 0.8 0.9 0.4 0.6 0.8 0.8 0.3 6 4 0.9 0.6 0.8 0.4 0.4 0.6 0.6 0.2 6 5 0.6 0.4 0.5 0.3 0.3 0.4 0.4 0.1 6 6 0.6 0.4 0.4 0.2 0.3 0.4 0.4 0.1 6

Selegiline Fluxes (Selegiline 100%, 30mM, 0.4mA) [µmol/h] SEL‐33 30mM pH=4.0 100% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 0.3 0.1 0.3 0.1 0.1 0.5 0.2 0.2 6 2 1.2 0.9 1.1 0.7 0.9 1.3 1.0 0.2 6 3 1.2 1.3 1.3 1.2 1.3 1.2 1.3 0.0 6 4 0.9 1.5 1.3 1.4 1.5 1.0 1.3 0.2 6 5 0.7 1.4 1.1 1.3 1.5 0.7 1.1 0.3 6 6 0.5 1.4 0.9 1.1 1.3 0.6 1.0 0.4 6 7 0.5 1.3 0.8 1.0 1.2 0.4 0.9 0.4 6

Appendix I

198

SEL‐05 30mM pH=4.5 100% 0.4mA SEL‐29 30mM pH=4.6 100% 0.4mA t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 1 0.5 0.6 0.6 0.4 1.3 0.7 0.0 0.9 0.8 0.5 0.5 0.5 2 1.1 1.5 1.3 1.9 1.2 1.1 0.6 1.3 1.3 1.2 1.3 1.3 3 1.3 1.4 1.3 1.4 1.7 1.2 1.2 1.2 1.1 1.2 1.5 1.5 4 1.3 1.5 1.1 1.6 2.3 1.1 1.3 1.2 1.1 1.2 1.5 1.4 5 1.3 1.2 1.3 1.2 1.9 1.1 1.2 1.1 1.1 1.0 1.5 1.3 6 1.2 1.6 1.1 1.3 1.6 1.1 1.3 1.2 1.0 1.3 1.2 14.2* Mean SD N 0.6 0.3 17 SEL‐02 30mM pH=4.6 100% 0.4mA 1.3 0.3 17

t[h] Cell 1 Cell 3 Cell 4 Cell 5 Cell 6 1.4 0.2 17 1 0.0 0.7 0.7 0.4 0.5 1.4 0.3 17 2 2.0 1.4 1.4 1.3 1.3 1.2 0.3 17 3 1.6 1.4 1.3 1.1 1.5 1.2 0.2 15 4 1.6 1.3 1.8 1.0 1.8 5 1.2 0.9 1.0 0.6 1.2 6 1.2 0.9 0.8 0.4* 1.4

Paracetamol Fluxes (Selegiline 100%, 30mM, 0.4mA) [nmol/h] Sel‐42 30mM pH=3.5 100% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Mean SD N 1 0.0 2.7 2.1 2.8 4.8 2.47 1.73 5 2 3.2 4.1 1.5 3.5 5.2 3.48 1.36 5 3 2.3 3.1 1.0 2.1 3.9 2.49 1.09 5 4 1.6 2.5 0.9 1.4 2.9 1.88 0.81 5 5 1.1 1.6 0.7 1.0 2.1 1.31 0.55 5 6 1.5 1.6 0.7 1.1 2.3 1.42 0.59 5 Sel‐33 30mM pH=4.0 100% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 6.8 5.2 5.5 4.8 4.8 12.9 6.64 3.14 6 2 8.1 7.7 6.9 5.6 6.8 12.5 7.93 2.41 6 3 8.6 11.2 9.0 9.7 10.6 11.1 10.04 1.10 6 4 7.4 14.0 9.9 12.2 13.1 10.6 11.20 2.41 6 5 5.5 13.9 9.0 11.6 16.9 8.5 10.89 4.10 6 6 6.0 14.0 7.3 10.6 13.4 5.4 9.45 3.76 6 7 4.9 13.0 6.5 11.5 11.9 5.0 8.79 3.72 6 Sel‐36 30mM pH=4.8 1.00 0.4mA


Appendix I

199

Selegiline Fluxes (Selegiline 50%, 120mM, 0.4mA) [µmol/h] SEL‐40 120mM pH=3.0 50% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 0.5 0.2 0.2 0.3 0.2 0.3 0.3 0.1 6 2 0.3 0.3 0.2 0.2 0.3 0.4 0.3 0.1 6 3 0.2 0.2 0.1 0.2 0.3 0.2 0.2 0.1 6 4 0.2 0.2 0.2 0.2 0.4 0.2 0.2 0.1 6 5 0.2 0.2 0.2 0.2 0.4 0.2 0.2 0.1 6 6 0.2 0.2 0.2 0.2 0.4 0.2 0.2 0.1 6 SEL‐39 120mM pH=4 50% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 0.4 0.4 0.4 0.4 0.5 0.5 0.4 0.0 6 2 1.0 0.9 1.0 0.9 0.9 1.1 0.9 0.1 6 3 1.0 1.0 1.0 0.9 0.8 1.1 1.0 0.1 6 4 0.9 1.0 1.0 0.9 0.9 1.0 1.0 0.1 6 5 1.0 1.0 1.0 0.9 0.8 1.1 1.0 0.1 6 6 0.9 1.0 0.8 0.9 0.9 1.0 0.9 0.1 6 Sel‐30 120mM pH=4.5 50% 0.4mA Sel‐41 120mM pH=4.5 50% 0.4mA


Appendix I

200

Paracetamol Fluxes (Selegiline 50%, 120mM, 0.4mA) [nmol/h] Sel‐40 120mM pH=3.0 50% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 3.4 2.8 0.0 3.2 0.0 3.6 2.15 1.69 6 2 0.0* 2.8 3.5 4.4 5.8 2.6 3.81 1.31 5 3 1.7 2.0 2.7 2.7 2.0 1.5 2.13 0.51 6 4 0.6 0.7 1.9 1.6 0.7 0.7 1.02 0.55 6 5 0.4 0.5 2.7 0.9 0.7 0.8 1.01 0.84 6 6 0.4 0.6 3.3 0.7 1.6 3.2 1.64 1.32 6 Sel‐39 120mM pH=4.0 50% 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Mean SD N1 3.9 4.4 4.2 4.4 3.8 4.14 0.27 5 2 7.4 7.4 8.5 6.5 7.0 7.35 0.74 5 3 8.2 8.2 8.5 6.1 7.8 7.77 0.96 5 4 6.7 7.5 6.5 5.0 7.6 6.67 1.04 5 5 6.8 7.7 5.8 4.5 8.1 6.57 1.45 5 6 6.4 7.9 4.4 5.2 7.6 6.29 1.48 5 Sel‐41 120mM pH=4.5 50% 0.4mA


Concentration Effect – Co‐ion

Comparison of the Selegiline fluxes [µmol/h] for constant mole fractions (10%, 50%, 75%) and different total molarities (40, 50, 60, 120mM) (pH4.65, 0.4mA)

SEL‐31 10% pH=4.8 120mM 0.4mA SEL‐35 10% pH=4.8 120mM 0.4mA t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N 1 0.1 0.0 0.1 0.1 0.1 0.0 0.1 0.5 0.1 0.0 0.1 0.0 0.09 0.14 12 2 0.3 0.4 0.3 0.3 0.3 0.2 0.3 0.3 0.2 0.2 0.2 0.1 0.26 0.05 12 3 0.3 0.4 0.3 0.6 0.5 0.4 0.3 0.3 0.3 0.5 0.4 0.3 0.40 0.08 12 4 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.2 0.30 0.02 12 5 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.29 0.02 12 6 0.3 0.4 0.3 0.3 0.3 0.3 0.3 0.4 0.3 0.3 0.3 0.3 0.30 0.03 12 SEL‐14 10% pH=4.8 50mM 0.4mA

time Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N 1 0.2 0.2 0.1 0.6 0.1 0.2 0.24 0.19 6 2 0.3 0.3 0.3 0.4 0.2 0.3 0.29 0.05 6 3 0.3 0.3 0.3 0.3 0.2 0.3 0.29 0.02 6 4 0.3 0.3 0.3 0.3 0.2 0.3 0.28 0.03 6 5 0.1 0.4 0.4 0.3 0.3 0.3 0.29 0.08 6 6 0.1 0.4 0.4 0.3 0.4 0.3 0.32 0.10 6

Appendix I

201


Sel‐30 50% pH=4.5 120mM 0.4mA Sel‐41 50% pH=4.5 120mM 0.4mA t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 AV SD N1 0.4 0.6 0.6 0.7 0.3 0.4 0.4 0.3 0.4 0.3 0.5 0.5 0.4 0.1 12 2 1.3 0.9 1.2 1.2 1.2 1.1 1.0 1.2 1.1 1.0 1.0 1.1 1.1 0.1 12 3 1.1 1.2 0.9 0.9 1.1 0.9 1.1 1.3 1.2 1.3 1.1 1.2 1.1 0.1 12 4 1.0 1.1 0.9 0.8 1.1 0.9 1.1 1.4 1.3 1.3 1.0 1.2 1.1 0.2 12 5 1.0 1.1 0.9 0.9 1.0 0.8 1.0 1.0 1.0 1.0 0.8 1.0 1.0 0.1 12 6 0.9 1.1 0.9 0.9 1.0 0.9 1.1 1.1 1.1 1.1 0.8 1.0 1.0 0.1 12 SEL‐03 50% pH=4.5 60mM% 0.4mA SEL‐10 50% pH4.6 60mM 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 1 0.2 0.4 0.3 0.3 0.4 0.6 0.5 0.5 0.4 0.4 0.5 0.6 2 0.6 0.7 0.5 0.6 0.6 0.6 0.7 0.8 0.8 0.9 1.0 0.8 3 0.6 0.7 0.5 0.6 0.7 0.7 0.7 0.8 0.8 0.7 0.7 0.8 4 0.7 0.7 0.7 0.7 0.9 0.6 0.5 0.6 0.7 0.7 0.8 0.7 5 0.6 0.6 0.6 0.7 0.7 0.6 0.7 0.4 0.7 0.9 0.8 0.7 6 0.6 0.5 0.6 0.7 0.8 0.6 0.8 0.7 0.8 0.6 0.7 0.6 AV SD N 0.4 0.1 12 SEL‐43 50% pH=4.6 60mM 0.4mA 0.7 0.1 12

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 0.7 0.1 12 1 0.3 0.3 0.4 0.3 0.5 0.5 0.7 0.1 12 2 0.9 0.8 0.9 0.9 1.1 1.0 0.7 0.1 12 3 1.1 1.0 1.0 1.0 1.1 1.1 0.7 0.1 12 4 1.2 1.1 1.1 1.1 1.3 1.1 5 1.2 1.2 1.0 1.1 1.2 1.1 6 1.1 1.0 1.0 1.0 1.2 1.1


SEL‐20 75% pH=4.5 120mM 0.4mA t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 0.6 0.4 0.4 0.8 0.9 0.6 0.60 0.21 6 2 0.7 0.8 0.7 0.9 1.0 1.3 0.90 0.23 6 3 0.7 0.7 1.2 1.0 1.2 1.4 1.04 0.27 6 4 0.7 0.9 0.8 1.0 1.2 1.5 1.02 0.30 6 5 0.6 1.1 1.0 0.9 1.0 1.5 1.00 0.28 6 6 0.8 1.0 0.8 1.2 0.8 1.2 0.95 0.20 6 SEL‐12 75% pH=4.5 40mM 0.4mA

time Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 0.5 0.5 0.8 0.8 1.4 0.8 0.79 0.33 6 2 0.7 1.3 1.6 0.7 1.3 1.1 1.12 0.36 6 3 0.8 0.7 1.3 1.2 1.1 0.9 0.99 0.22 6 4 0.9 0.8 0.9 1.0 1.5 1.3 1.07 0.27 6 5 0.6 1.1 1.0 0.9 0.7 0.7 0.84 0.21 6 6 0.9 1.0 1.5 1.3 0.0** 1.1 1.18 0.24 5

Human Skin Selegiline Flux [µmol/h] SEL‐HS 100% 30mM pH4.5 0.4mA R1 R2 R3 R4 R5 AV SD N

h1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 h2 0.01 0.14 0.01 0.00 0.00 0.03 0.06 5 h3 0.18 0.49 0.21 0.12 0.04 0.21 0.17 5 h4 0.54 0.78 0.50 0.40 0.29 0.50 0.19 5 h5 0.84 0.92 0.69 0.69 0.55 0.73 0.14 5 h6 1.01 1.04 0.81 0.90 0.77 0.90 0.12 5

Paracetamol Flux [nmol/h] SEL 100% 30mM pH4.5 0.4mA R1 R2 R3 R4 R5 AV SD N

h1 0.8 0.0 0.0 0.6 1.5 0.6 0.6 5 h2 1.3 1.7 1.3 0.7 1.7 1.3 0.4 5 h3 3.8 5.1 4.3 2.2 2.4 3.6 1.2 5 h4 7.4 8.3 8.0 4.8 4.5 6.6 1.8 5 h5 10.9 10.2 10.3 7.5 6.3 9.0 2.0 5 h6 13.4 12.1 12.5 9.5 7.6 11.0 2.4 5

Appendix I

202

Paracetamol control (Paracetamol 15mM, NaCl 120mM, Selegiline 0%, 0.4mA) Par‐4 0% pH=6.8 120mM 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N 1 13.7 13.3 13.5 14.0 15.0 14.4 13.97 0.63 6 2 24.2 26.8 21.5 18.5 26.4 25.2 23.77 3.18 6 3 36.2 37.8 30.6 23.0 38.8 36.7 33.86 6.03 6 4 43.8 45.1 34.2 29.6 39.2 43.2 39.21 6.16 6 5 60.3 55.6 47.7 36.2 54.3 48.4 50.41 8.39 6 6 61.5 64.5 51.4 42.0 67.2 61.8 58.07 9.53 6 Par‐2 0% pH=4.6 120mM 0.4mA TM‐120 Par‐3 0% pH=4.6 120mM 0.4mA

t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N 1 8.4 9.8 6.7 8.5 7.1 6.6 3.3 3.6 2.7 1.6 3.1 3.3 5.40 2.75 12 2 18.5 28.1 23.6 24.4 22.6 23.4 16.3 13.1 15.0 10.3 16.2 16.3 18.98 5.34 12 3 34.3 36.5 34.5 28.3 37.0 39.8 28.2 21.9 24.1 26.8 26.9 28.9 30.59 5.64 12 4 50.6 49.0 66.9 58.9 60.2 54.4 37.5 39.6 35.8 33.5 41.5 40.0 47.32 10.93 12 5 57.0 54.9 66.5 73.8 60.8 59.4 59.9 49.6 41.0 66.9 46.8 60.6 58.10 9.12 12 6 55.8 47.8 76.3 70.0 59.4 58.6 85.9 43.6 68.6 52.1 49.2 60.66 13.11 11

Appendix II

203

Appendix II. Pramipexole Experiments Concentration Effect Single‐ion

Pramipexole flux [µmol/h] PPX‐39 100% 15mM pH=8 0.4mA

R1 R2 R3 R4 R5 AV SD N

h1 0.43 0.86 0.55 1.08 0.80 0.7 0.3 5h2 1.53 2.02 1.66 2.16 1.91 1.9 0.3 5 h3 2.00 2.27 2.05 2.30 2.17 2.2 0.1 5

h4 2.10 2.17 2.19 2.23 2.79 2.3 0.3 5h5 2.25 2.37 2.22 2.47 2.65 2.4 0.2 5h6 2.37 2.55 2.35 2.52 2.47 2.4 0.1 5

PPX‐8 100% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 R6 AV SD N

h1 0.6 1.0 1.1 1.5 1.8 1.1 1.0 0.63 18

h2 1.8 2.9 2.3 2.8 2.9 2.7 2.0 0.63 18h3 2.2 2.8 2.4 2.9 2.8 2.5 2.5 0.30 18

h4 2.5 2.6 2.4 3.1 2.8 3.1 2.5 0.26 18h5 2.6 2.5 2.5 2.9 2.9 3.2 2.7 0.23 18h6 2.7 0.0* 2.2 2.9 2.7 3.2 2.6 0.23 17

PPX‐15 100% 30mM pH=8 0.4mA PPX‐25 100% 30mM pH=8 0.4mA

R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6

h1 1.4 1.0 1.3 2.6 1.3 0.9 0.2 0.3 0.3 0.4 0.3 0.4 h2 2.1 2.0 2.3 1.3 2.3 2.0 1.2 1.4 1.2 1.1 1.1 1.7 h3 2.4 2.3 2.5 2.0 2.5 2.4 2.8 2.7 2.6 2.6 2.0 3.1 AV SD N h4 2.5 2.4 2.5 2.5 2.6 2.5 2.3 2.4 2.6 2.4 2.0 2.5 0.9 0.2 12 h5 2.6 2.5 2.6 2.6 2.6 2.7 2.3 2.6 2.8 3.0 2.2 2.7 1.9 0.3 12h6 2.5 2.6 2.6 2.7 2.7 2.7 2.3 2.6 2.3 2.5 2.5 2.5 2.2 0.2 12

2.3 0.2 11

PPX‐16 100% 60mM pH=8 0.4mA PPX‐28 100% 60mM pH=8 0.4mA 2.4 0.2 12 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 2.4 0.2 12

h1 1.0 0.9 0.9 1.4 0.8 0.7 1.0 0.6 0.9 0.7 1.0 0.8 h2 2.1 2.1 2.3 2.2 2.2 2.0 1.9 1.8 1.8 1.6 1.9 1.3 h3 2.4 2.3 2.5 2.5 2.1 2.2 2.4 2.4 2.1 1.9 2.2 1.8 h4 2.6 2.3 2.5 0.0* 2.3 2.3 2.5 2.2 2.2 1.9 2.3 2.2 h5 2.5 2.4 2.5 2.7 2.6 2.6 2.6 2.4 2.3 2.0 2.3 2.2 h6 2.5 2.4 2.4 2.5 2.5 2.4 2.3 1.8 2.3 2.5 2.2 2.7

Appendix II

204

Mannitol flux [nmol/h] PPX‐39 100% 15mM pH=8 0.4mA R1 R2 R3 R4 R5 AV SD N

h1 0.0 4.5 5.6 0.4 1.3 2.4 2.5 5 h2 1.6 1.6 4.6 5.9 5.3 3.8 2.1 5 h3 3.9 3.9 6.8 4.9 6.9 5.3 1.5 5 h4 5.6 4.5 9.5 6.4 9.2 7.1 2.2 5 h5 3.9 5.9 8.2 8.6 16.9 8.7 5.0 5 h6 8.6 9.8 10.1 10.8 22.2 12.3 5.6 5


h1 3.3 3.4 4.3 5.3 3.3 5.4 4.2 1.0 6 h2 6.6 5.6 10.5 6.9 6.7 11.2 7.9 2.3 6 h3 9.9 10.3 13.9 15.9 9.6 14.4 12.3 2.7 6 h4 11.8 13.1 16.1 14.3 12.8 16.2 14.1 1.8 6 h5 19.5 12.1 15.0 14.3 14.3 16.2 15.2 2.5 6 h6 17.0 12.1 17.4 76.6* 17.2 16.3 16.0 2.2 5

PPX‐28 100% 60mM pH=8 0.4mA R1 R2 R3 R4 R5 AV SD N

h1 8.2 27.1 43.8 15.7 9.4 20.8 14.9 5 h2 12.7 32.7 31.1 18.1 7.2 20.4 11.2 5 h3 11.3 15.4 20.4 13.9 7.0 13.6 5.0 5 h4 18.4 20.8 23.8 14.8 18.9 19.3 3.3 5 h5 15.1 17.4 18.5 11.8 19.5 16.5 3.1 5 h6 14.1 16.0 19.3 12.5 22.5 16.9 4.0 5


h1 0.1 0.2 0.1 7.7 0.1 1.6 3.4 5 h2 17.8 0.1 0.2 14.4 11.7 8.8 8.2 5 h3 17.9 0.0* 0.4 20.0 10.1 12.1 8.9 4 h4 25.5 11.9 9.1 23.5 16.2 17.2 7.2 5 h5 14.8 13.3 6.7 21.6 22.2 15.7 6.4 5 h6 9.7 11.4 14.1 22.6 34.9 18.5 10.4 5

Appendix II

205

Current Intensity Effect Pramipexole flux [µmol/h] (100%, 30mM, pH8.0)

PPX‐07 100% 30mM pH=8 0.0mA[µmol/h/cm2]

R1 R2 R3 R4 R5 R6 AV SD N

h0 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.000 6

h2 0.001 0.002 0.002 0.001 0.001 0.001 0.001 0.000 6

h4 0.002 0.003 0.001 0.004 0.001 0.002 0.002 0.001 6

h6 0.002 0.005 0.002 0.008 0.004 0.004 0.004 0.002 6


h1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6

h2 0.01 0.02 0.04 0.06 0.06 0.02 0.04 0.02 6

h3 0.16 0.25 0.29 0.31 0.30 0.20 0.25 0.06 6

h4 0.36 0.40 0.46 0.41 0.41 0.45 0.41 0.04 6

h5 0.35 0.43 0.51 0.57 0.49 0.48 0.47 0.08 6

h6 0.48 0.49 0.54 0.60 0.50 0.55 0.53 0.05 6

PPX‐23 100% 30mM pH=8 0.2mA


h1 0.1 0.1 0.2 0.1 0.4 0.2 0.18 0.11 6

h2 0.4 0.4 0.5 0.4 0.1 0.4 0.39 0.16 6

h3 0.9 0.7 1.0 0.8 1.1 0.6 0.84 0.18 6

h4 1.1 1.1 1.0 1.1 1.6 1.1 1.18 0.23 6

h5 1.2 1.2 1.3 1.2 1.0 1.6 1.24 0.19 6

h6 1.2 1.2 0.0* 1.0 1.7 1.2

1.25 0.28 5

PPX‐8 100% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 R6

h1 0.6 1.0 1.1 1.5 1.8 1.1 h2 1.8 2.9 2.3 2.8 2.9 2.7 h3 2.2 2.8 2.4 2.9 2.8 2.5

AV SD N

h4 2.5 2.6 2.4 3.1 2.8 3.1 1.0 0.63 18 h5 2.6 2.5 2.5 2.9 2.9 3.2 2.0 0.63 18h6 2.7 0.0* 2.2 2.9 2.7 3.2 2.5 0.30 18

2.5 0.26 18 2.7 0.23 18 2.6 0.23 17

PPX‐15 100% 30mM pH=80.4 mA PPX‐25 100% 30mM pH=8

0.4 mA

R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6

h1 1.4 1.0 1.3 2.6 1.3 0.9 0.2 0.3 0.3 0.4 0.3 0.4

h2 2.1 2.0 2.3 1.3 2.3 2.0 1.2 1.4 1.2 1.1 1.1 1.7

h3 2.4 2.3 2.5 2.0 2.5 2.4 2.8 2.7 2.6 2.6 2.0 3.1

h4 2.5 2.4 2.5 2.5 2.6 2.5 2.3 2.4 2.6 2.4 2.0 2.5

h5 2.6 2.5 2.6 2.6 2.6 2.7 2.3 2.6 2.8 3.0 2.2 2.7 h6 2.5 2.6 2.6 2.7 2.7 2.7 2.3 2.6 2.3 2.5 2.5 2.5

Appendix II

206

Mannitol flux [nmol/h] PPX‐22 100% 30mM pH=8 0.1mA R1 R2 R3 R4 R5 R6 AV SD N

h1 5.3 5.1 4.8 5.8 5.2 5.0 5.2 0.4 6 h2 4.3 3.2 4.1 5.4 3.3 3.2 3.9 0.9 6 h3 3.7 3.7 5.0 4.8 3.3 4.5 4.2 0.7 6 h4 3.8 3.2 5.0 4.4 3.5 4.4 4.0 0.7 6 h5 3.2 3.3 5.6 6.3 27.5* 4.6 4.6 1.4 5 h6 4.6 3.9 6.0 6.1 7.1 4.4 5.4 1.3 6


h1 4.4 9.8 9.8 7.8 9.4 11.8 8.8 2.5 6 h2 5.2 6.2 9.5 7.9 9.6 10.0 8.1 2.0 6 h3 7.8 7.2 11.5 10.6 14.9 9.0 10.2 2.9 6 h4 ** 6.9 11.5 10.5 16.3 10.1 11.1 3.4 5 h5 7.0 5.5 10.1 9.4 10.3 11.0 8.9 2.1 6 h6 7.3 6.7 10.1 7.5 15.0 8.5 9.2 3.1 6


h1 3.3 3.4 4.3 5.3 3.3 5.4 4.2 1.0 6 h2 6.6 5.6 10.5 6.9 6.7 11.2 7.9 2.3 6 h3 9.9 10.3 13.9 15.9 9.6 14.4 12.3 2.7 6 h4 11.8 13.1 16.1 14.3 12.8 16.2 14.1 1.8 6 h5 19.5 12.1 15.0 14.3 14.3 16.2 15.2 2.5 6 h6 17.0 12.1 17.4 76.6* 17.2 16.3 16.0 2.2 5

pH Effect Pramipexole flux [µmol/h]

PPX‐8 100% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 R6

h1 0.57 0.99 1.06 1.46 1.84 1.06 h2 1.82 2.89 2.34 2.84 2.87 2.66 h3 2.18 2.79 2.42 2.88 2.80 2.51 AV SD N h4 2.50 2.59 2.41 3.12 2.76 3.10 1.0 0.63 18 h5 2.58 2.47 2.52 2.86 2.87 3.15 2.0 0.63 18 h6 2.65 0.0* 2.22 2.91 2.74 3.22 2.5 0.30 18

2.5 0.26 18 PPX‐15 100% 30mM pH=8 0.4mA PPX‐25 100% 30mM pH=8 0.4mA l 2.7 0.23 18 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 2.6 0.23 17

h1 1.36 0.96 1.35 2.57 1.35 0.92 0.18 0.32 0.29 0.38 0.31 0.42 h2 2.14 2.04 2.29 1.29 2.26 2.03 1.20 1.40 1.25 1.08 1.13 1.70 total last 3h h3 2.39 2.33 2.51 2.02 2.45 2.35 2.79 2.65 2.62 2.65 2.01 3.12 AV SD N h4 2.52 2.38 2.48 2.52 2.57 2.51 2.31 2.35 2.61 2.38 1.98 2.46 2.6 0.24 53 h5 2.55 2.49 2.60 2.61 2.61 2.65 2.30 2.64 2.83 3.04 2.24 2.73 h6 2.53 2.58 2.61 2.69 2.66 2.66 2.35 2.62 2.27 2.52 2.48 2.50 PPX‐40 100% 30mM pH=7 0.4mA

R1 R2 R3 R4 R5 AV SD N h1 0.76 0.73 0.81 0.86 0.96 0.82 0.09 5 h2 1.85 1.75 1.94 1.87 2.00 1.88 0.09 5 h3 2.16 2.14 2.47 2.14 2.20 2.22 0.14 5 h4 2.27 2.21 2.33 2.27 2.39 2.29 0.07 5 h5 2.45 2.23 2.25 2.24 2.27 2.29 0.09 5 h6 2.37 2.29 2.54 2.38 2.38 2.39 0.09 5 PPX‐9 100% 30mM pH=5 0.4mA

R1 R2 R3 R4 R5 AV SD N h1 0.7 1.1 0.8 0.7 0.6 0.76 0.18 5 h2 1.4 1.5 1.4 1.3 1.4 1.40 0.07 5 h3 1.7 1.5 1.5 1.5 1.6 1.56 0.10 5 h4 1.7 1.3 1.3 1.5 1.7 1.48 0.21 5 h5 1.8 1.4 1.2 2.1 1.8 1.66 0.34 5 h6 2.3 1.8 1.4 1.8 2.2 1.90 0.35 5

Appendix II

207

PPX‐41 100% 30mM pH=4.0 0.4mA


PPX‐10 100% 30mM pH=2.5 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.1 0.1 0.3 0.2 0.2 0.1 0.18 0.09 6 h2 0.2 0.3 0.3 0.3 0.2 0.2 0.22 0.05 6 h3 0.2 0.3 0.3 0.2 0.2 0.2 0.24 0.05 6 h4 0.3 0.3 0.2 0.2 0.2 0.2 0.24 0.06 6 h5 0.3 0.3 0.2 0.2 0.2 0.2 0.22 0.04 6 h6 0.3 0.3 0.2 0.0* 0.2 0.2 0.25 0.03 5

Mannitol flux [nmol/h] PPX‐25 100% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 R6 AV SD N

h1 3.3 3.4 4.3 5.3 3.3 5.4 4.17 0.96 6 h2 6.6 5.6 10.5 6.9 6.7 11.2 7.94 2.31 6 h3 9.9 10.3 13.9 15.9 9.6 14.4 12.31 2.73 6 h4 11.8 13.1 16.1 14.3 12.8 16.2 14.05 1.80 6 h5 19.5 12.1 15.0 14.3 14.3 16.2 15.24 2.48 6 h6 17.0 12.1 17.4 76.6* 17.2 16.3 16.02 2.21 5

PPX‐40 100% 30mM pH=7 0.4mA R1 R2 R3 R4 R5 AV SD N h1 1.9 7.6 9.8 3.9 5.82 3.55 4 h2 3.5 9.0 11.5 3.6 6.87 4.01 4 h3 6.0 4.5 5.9 8.0 6.11 1.44 4 h4 7.3 6.6 15.2 5.2 8.58 4.50 4 h5 10.7 4.2 15.4 7.4 9.41 4.81 4 h6 12.0 7.5 15.5 8.2 10.81 3.68 4 PPX‐21 100% 30mM pH=5 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.0 0.0 0.0 0.0 0.0 0.00 0.00 5 h2 2.5 1.9 1.6 2.3 0.7 1.80 0.68 5 h3 2.3 2.1 2.0 3.2 0.6 2.03 0.94 5 h4 3.4 3.8 2.8 3.0 2.7 3.15 0.47 5 h5 3.5 3.1 2.9 3.3 2.1 2.98 0.53 5 h6 4.2 3.5 3.7 3.7 3.3 3.66 0.31 5

Mole Fraction Effect pH8.0 Pramipexole flux [µmol/h] (pH8.0, 30mM, 0.4mA)

PPX‐42 1% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.04 0.05 0.04 0.05 0.04 0.04 0.01 5 h2 0.06 0.05 0.06 0.06 0.05 0.06 0.00 5 h3 0.06 0.06 0.06 0.06 0.06 0.06 0.00 5 h4 0.06 0.07 0.06 0.06 0.06 0.06 0.00 5 h5 0.06 0.06 0.06 0.05 0.06 0.06 0.00 5 h6 0.06 0.06 0.06 0.06 0.06 0.06 0.00 5

Appendix II

208

PPX‐26 10% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.22 0.16 0.12 0.13 0.12 0.18 0.16 0.04 6 h2 0.44 0.32 0.48 0.44 0.51 0.54 0.46 0.08 6 h3 0.36 0.52 0.0* 0.53 0.60 0.57 0.51 0.09 5 h4 0.32 0.55 0.60 0.65 0.61 0.49 0.54 0.12 6 h5 0.0* 0.52 0.65 0.59 0.64 0.68 0.62 0.06 5 h6 0.47 0.75 0.51 0.75 0.64 0.66 0.63 0.12 6 PPX‐5 25% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.40 0.40 0.63 0.54 0.60 0.45 0.5 0.1 6 h2 0.79 0.75 0.88 0.85 0.74 0.68 0.8 0.1 6 h3 0.57 0.75 0.73 0.73 0.75 0.77 0.7 0.1 6 h4 0.63 0.78 0.84 0.87 0.88 0.79 0.8 0.1 6 h5 0.52 0.77 0.73 0.70 0.79 0.71 0.7 0.1 6 h6 0.53 0.70 0.80 0.76 0.83 0.76 0.7 0.1 6 PPX‐4 50% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.54 0.41 0.13 0.43 0.94 1.11 0.6 0.4 6 h2 1.04 1.15 0.67 1.12 1.16 1.22 1.1 0.2 6 h3 1.24 1.15 0.78 1.27 1.14 1.51 1.2 0.2 6 h4 1.44 1.17 0.87 1.30 1.29 0.92 1.2 0.2 6 h5 1.34 1.43 1.17 1.32 1.22 1.66 1.4 0.2 6 h6 1.36 1.45 1.04 1.31 1.19 1.58 1.3 0.2 6 PPX‐34 75% 30mM pH8.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.66 1.17 0.76 0.77 1.01 0.78 0.86 0.19 6 h2 1.50 1.83 1.68 0.38 1.84 1.66 1.48 0.55 6 h3 1.89 1.93 1.87 0.65 1.98 1.81 1.69 0.51 6 h4 1.88 1.75 1.92 1.96 2.03 1.82 1.89 0.10 6 h5 2.20 2.17 1.88 1.91 1.98 1.81 1.99 0.16 6 h6 2.00 1.76 1.93 2.00 1.97 1.99 1.94 0.09 6

PPX‐

8 100

% 30m

M pH=

8 0.4m

A R1 R2 R3 R4 R5 R6

h1 0.57 0.99 1.06 1.46 1.84 1.06 h2 1.82 2.89 2.34 2.84 2.87 2.66 h3 2.18 2.79 2.42 2.88 2.80 2.51 AV SD N h4 2.50 2.59 2.41 3.12 2.76 3.10 1.0 0.63 18 h5 2.58 2.47 2.52 2.86 2.87 3.15 2.0 0.63 18 h6 2.65 0.0* 2.22 2.91 2.74 3.22 2.5 0.30 18

2.5 0.26 18

PPX‐

15 100

% 30m

M pH=

8 0.4m

A PPX‐

25 100

% 30m

M pH=

8 0.4m

A 2.7 0.23 18 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 2.6 0.23 17

h1 1.36 0.96 1.35 2.57 1.35 0.92 0.18 0.32 0.29 0.38 0.31 0.42 h2 2.14 2.04 2.29 1.29 2.26 2.03 1.20 1.40 1.25 1.08 1.13 1.70 h3 2.39 2.33 2.51 2.02 2.45 2.35 2.79 2.65 2.62 2.65 2.01 3.12 h4 2.52 2.38 2.48 2.52 2.57 2.51 2.31 2.35 2.61 2.38 1.98 2.46 h5 2.55 2.49 2.60 2.61 2.61 2.65 2.30 2.64 2.83 3.04 2.24 2.73 h6 2.53 2.58 2.61 2.69 2.66 2.66 2.35 2.62 2.27 2.52 2.48 2.50

Appendix II

209

Mannitol flux [nmol/h] PPX‐26 10% 30mM pH=8 R1 R2 R3 R4 R5 R6 AV SD N h1 35.0 25.5 20.6 17.0 34.0 25.9 26.3 7.1 6 h2 80.9 69.5 61.7 67.2 103.9 73.3 76.1 15.1 6 h3 99.7 93.9 86.7 105.9 123.6 95.7 100.9 12.8 6 h4 116.7 112.0 103.6 133.5 138.7 120.3 120.8 13.2 6 h5 133.2 142.0 129.2 137.4 141.3 113.7 132.8 10.6 6 h6 89.0 119.6 111.0 130.9 137.9 134.5 120.5 18.4 6

PPX‐20 25% 30mM pH=8 R1 R2 R3 R4 R5 R6 AV SD N h1 7.6 5.1 6.6 0.0* 7.6 3.7 6.1 1.7 5 h2 31.8 15.9 15.8 16.5 28.2 13.7 20.3 7.7 6 h3 72.0 42.8 51.3 35.1 65.2 27.1 48.9 17.4 6 h4 95.7 72.7 76.0 49.5 80.7 35.5 68.3 22.0 6 h5 82.3 52.9 62.0 32.5 42.2 56.2 54.7 17.1 6 h6 31.5 106.8 64.6 88.4 69.2 83.0 73.9 25.6 6

PPX‐29 50% 30mM pH=8 R1 R2 R3 R4 R5 R6 AV SD N

h1 37.1 9.0 17.8 17.7 9.5 15.5 17.8 10.2 6 h2 64.4 17.1 42.5 46.5 28.2 35.3 39.0 16.3 6 h3 60.7 0.0* nd nd nd nd 60.7 nd 1 h4 85.1 55.9 61.6 97.3 0.0* 85.4 77.1 17.5 5 h5 113.3 69.9 78.6 106.5 85.2 90.1 90.6 16.5 6 h6 109.8 76.0 69.7 111.6 77.7 84.1 88.2 18.1 6


h1 4.2 5.8 7.1 11.5 7.7 4.9 6.9 2.6 6 h2 10.2 13.4 16.1 20.4 17.1 11.2 14.7 3.9 6 h3 16.9 18.0 25.8 27.5 31.4 14.8 22.4 6.7 6 h4 20.8 28.0 42.0 38.3 30.1 19.6 29.8 9.1 6 h5 27.5 26.3 36.1 41.0 29.1 16.7 29.5 8.4 6 h6 39.7 24.1 31.9 35.1 24.2 9.8* 31.0 6.8 5


h1 3.3 3.4 4.3 5.3 3.3 5.4 4.2 1.0 6 h2 6.6 5.6 10.5 6.9 6.7 11.2 7.9 2.3 6 h3 9.9 10.3 13.9 15.9 9.6 14.4 12.3 2.7 6 h4 11.8 13.1 16.1 14.3 12.8 16.2 14.1 1.8 6 h5 19.5 12.1 15.0 14.3 14.3 16.2 15.2 2.5 6 h6 17.0 12.1 17.4 76.6* 17.2 16.3 16.0 2.2 5

Appendix II

210

Mole Fraction Effect pH 5.0 Pramipexole flux [µmol/h]


h1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.0 6 h2 0.3 0.1 0.3 0.1 0.1 0.3 0.2 0.1 6 h3 0.4 0.2 0.5 0.2 0.2 0.5 0.4 0.1 6 h4 0.4 0.3 0.5 0.3 0.3 0.5 0.4 0.1 6 h5 0.5 0.2 0.4 0.3 0.3 0.5 0.4 0.1 6 h6 0.3 0.2 0.4 0.2 0.2 0.4 0.3 0.1 6

PPX‐37 25% 30mM pH5.0 0.4mA PPX‐

19 25% 30m

M pH=

5 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 AV SD N

h1 0.0 0.2 0.2 0.2 0.2 0.3 0.1 0.1 0.2 0.2 0.7 0.2 0.2 11 h2 0.5 0.6 0.5 0.5 0.6 0.6 0.3 0.3 0.4 0.3 0.4 0.5 0.1 11 h3 0.5 0.5 0.5 0.5 0.5 0.5 0.4 0.4 0.5 0.4 0.4 0.5 0.1 11 h4 0.5 0.5 0.5 0.5 0.5 0.5 0.4 0.4 0.5 0.4 0.4 0.5 0.1 11 h5 0.6 0.5 0.6 0.5 0.5 0.5 0.4 0.4 0.5 0.4 0.4 0.5 0.1 11 h6 0.5 0.5 0.5 0.5 0.5 0.4 0.4 0.4 0.5 0.4 0.4 0.5 0.0 11


h1 0.20 0.34 0.26 0.24 0.29 0.33 0.3 0.1 6 h2 0.55 0.63 0.41 0.53 0.66 0.58 0.6 0.1 6 h3 0.50 0.04* 0.65 0.50 0.77 0.63 0.6 0.1 5 h4 0.58 0.75 0.42 0.45 0.81 0.59 0.6 0.2 6 h5 0.73 0.67 0.60 0.53 0.79 0.57 0.6 0.1 6 h6 0.54 0.68 0.56 0.53 0.70 0.54 0.6 0.1 6


h1 0.33 0.54 0.69 0.52 0.32 0.40 0.5 0.1 6 h2 1.12 1.34 1.44 0.93 0.69 1.02 1.1 0.3 6 h3 1.57 1.32 1.47 1.36 0.61 0.94 1.2 0.4 6 h4 1.37 1.15 1.53 1.31 0.89 1.01 1.2 0.2 6 h5 1.28 1.22 1.47 1.28 1.17 1.00 1.2 0.2 6 h6 1.34 1.22 1.35 1.19 1.08 0.92 1.2 0.2 6


h1 0.7 1.1 0.8 0.7 0.6 0.8 0.2 5 h2 1.4 1.5 1.4 1.3 1.4 1.4 0.1 5 h3 1.7 1.5 1.5 1.5 1.6 1.6 0.1 5 h4 1.7 1.3 1.3 1.5 1.7 1.5 0.2 5 h5 1.8 1.4 1.2 2.1 1.8 1.7 0.3 5 h6 2.3 1.8 1.4 1.8 2.2 1.9 0.4 5

Appendix II

211

Mannitol flux [nmol/h] 0% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 15.5 25.6 12.8 15.0 21.4 18.1 5.3 5 h2 32.4 37.5 30.7 37.8 44.0 36.5 5.2 5 h3 58.6 62.6 44.9 58.9 63.9 57.8 7.6 5 h4 79.5 83.4 72.1 79.0 81.2 79.1 4.2 5 h5 93.4 97.5 81.7 92.3 87.1 90.4 6.1 5 h6 100.7 107.4 85.7 100.5 105.7 100.0 8.5 5

PPX‐37 25% 30mM pH5.0 0.4mA


PPX‐21 100% 30mM pH=5 0.4mA


Concentration Effect, co‐ion Pramipexole Flux [µmol/h]


h1 0.5 0.4 0.1 0.4 0.9 1.1 0.6 0.4 6 h2 1.0 1.2 0.7 1.1 1.2 1.2 1.1 0.2 6 h3 1.2 1.1 0.8 1.3 1.1 1.5 1.2 0.2 6 h4 1.4 1.2 0.9 1.3 1.3 0.9 1.2 0.2 6 h5 1.3 1.4 1.2 1.3 1.2 1.7 1.4 0.2 6 h6 1.4 1.5 1.0 1.3 1.2 1.6 1.3 0.2 6

PPX‐33 50% 60mM pH8.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N

h1 0.37 0.74 0.56 0.47 0.69 0.86 0.61 0.18 6 h2 0.81 1.25 1.14 1.03 1.27 1.35 1.14 0.20 6 h3 1.51 1.44 1.37 1.38 1.55 1.50 1.46 0.07 6 h4 1.51 1.46 1.39 1.33 1.65 1.38 1.45 0.12 6 h5 1.64 1.50 1.48 1.46 1.64 1.53 1.54 0.08 6 h6 1.45 1.51 1.55 1.40 1.36 1.63 1.48 0.10 6

Appendix II

212

Mannitol Flux [nmol/h] PPX‐29 50% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 R6 AV SD N

h1 37.1 9.0 17.8 17.7 9.5 15.5 17.8 10.2 6h2 64.4 17.1 42.5 46.5 28.2 35.3 39.0 16.3 6h3 60.7 0.0* nd nd nd nd 60.7 nd 1h4 85.1 55.9 61.6 97.3 0.0* 85.4 77.1 17.5 5h5 113.3 69.9 78.6 106.5 85.2 90.1 90.6 16.5 6h6 109.8 76.0 69.7 111.6 77.7 84.1 88.2 18.1 6

PPX‐33 50% 60mM pH8.0 0.4mA Mannitol Flux [µmol/h] R1 R2 R3 R4 R5 R6 AV SD N 5.4 5.4 5.4 5.4 5.4 13.2 6.7 3.2 6

h1 26.5 16.4 6.4 6.1 15.5 29.0 16.7 9.7 6 h2 31.9 23.3 6.3 5.9 6.2 45.8 19.9 16.7 6 h3 50.8 45.3 6.2 6.2 32.1 53.0 32.3 21.4 6 h4 68.8 55.2 8.3 8.5 60.9 68.3 45.0 28.8 6 h5 42.7 49.4 8.2 8.2 83.6 77.8 45.0 32.6 6 h6 66.1 81.9 7.3 6.9 85.7 82.8 55.1 37.8 6

Human Skin Experiments Mannitol Flux [nmol/h]

PPX‐36 100% 30mM pH8.0 0.4mA Human skin R1 R2 R3 R4 R5 AV SD N

h1 1.1 0.9 3.8 1.7 4.7 2.4 1.7 5 h2 7.5 5.8 6.4 10.9 4.2 7.0 2.5 5 h3 10.2 2.9 11.2 8.2 11.5 8.8 3.5 5 h4 12.3 6.1 16.8 11.3 14.7 12.2 4.1 5 h5 14.2 7.5 15.1 13.9 19.5 14.0 4.3 5 h6 16.2 9.1 22.6 16.3 17.8 16.4 4.8 5

Pramipexole Flux [µmol/h] PPX‐36 100% 30mM pH8.0 0.4mA Human skin R1 R2 R3 R4 R5 AV SD N h1 0.02 0.00 0.01 0.02 0.05 0.02 0.02 5 h2 0.37 0.18 0.27 0.43 0.70 0.39 0.20 5 h3 1.04 0.73 0.87 0.39 1.66 0.94 0.47 5 h4 1.46 1.25 1.24 1.59 2.02 1.51 0.32 5 h5 1.62 1.62 1.70 1.91 2.17 1.81 0.24 5 h6 1.90 1.78 1.85 1.90 2.16 1.92 0.14 5

Appendix II

213

50ml of 1mM pramipexole base (21.14mg/100ml) titrated with 2mM HCl ml HCl added

pH ml HCl added

pH ml HCl added

pH ml HCl added

pH

0.0 10.16 19.0 8.33 38.5 4.79 63.5 3.57 0.5 10.13 19.5 8.00 39.0 4.76 64.5 3.551.0 10.10 20.0 7.63 39.5 4.72 65.5 3.531.5 10.07 20.5 7.38 40.0 4.70 66.5 3.502.0 10.03 21.0 7.16 40.5 4.67 67.5 3.482.5 10.00 21.5 6.99 41.0 4.64 68.5 3.463.0 9.97 22.0 6.83 41.5 4.60 69.5 3.453.5 9.94 22.5 6.68 42.0 4.57 70.5 3.444.0 9.90 23.0 6.54 42.5 4.55 71.5 3.424.5 9.87 23.5 6.42 43.0 4.51 72.5 3.405.0 9.85 24.0 6.31 43.5 4.48 73.5 3.395.5 9.81 24.5 6.21 44.0 4.46 74.5 3.376.0 9.78 25.0 6.11 44.5 4.43 75.5 3.366.5 9.76 25.5 6.01 45.0 76.5 3.357.0 9.72 26.0 5.93 45.5 4.37 77.5 3.337.5 9.69 26.5 5.86 46.0 78.5 3.328.0 9.66 27.0 5.78 46.5 4.31 79.5 3.308.5 9.62 27.5 5.71 47.0 80.5 3.299.0 9.59 28.0 5.64 47.5 4.25 81.5 3.289.5 9.56 28.5 5.58 48.0 82.5 3.27

10.0 9.52 29.0 5.53 48.5 4.20 83.5 3.2610.5 9.50 29.5 5.47 49.0 84.5 3.2511.0 9.46 30.0 5.42 49.5 4.14 85.5 3.2411.5 9.41 30.5 5.37 50.0 86.5 3.2412.0 9.38 31.0 5.33 50.5 4.08 87.5 3.2312.5 9.34 31.5 5.28 51.0 88.5 3.2213.0 9.30 32.0 5.24 51.5 4.03 89.5 3.2113.5 9.25 32.5 5.20 52.5 3.97 90.5 3.2014.0 9.21 33.0 5.16 53.5 3.93 91.5 3.1914.5 9.16 33.5 5.11 54.5 3.87 92.5 3.1915.0 9.11 34.0 5.08 55.5 3.82 93.5 3.1815.5 9.05 34.5 5.04 56.5 3.78 94.5 3.1716.0 9.00 35.0 5.01 57.5 3.75 95.5 3.1616.5 8.93 35.5 4.97 58.5 3.71 96.5 3.1617.0 8.85 36.0 4.95 59.5 3.68 97.5 3.1517.5 8.74 36.5 4.91 60.5 3.65 98.5 3.1518.0 8.64 37.0 4.88 61.5 3.62 99.5 3.1418.5 8.51 37.5 4.85 62.5 3.60 100.5 3.14

38.0 4.82

Appendix II

214

Pramipexole Conductivity measurements Molar Conductivity [Si*cm2/mol]

Root of Concentration

[mM]1/2 AV SD

0.69 145.2 144.8 144.8 144.9 0.2

Pramipexole

1.01 283.8 283.9 284.0 283.9 0.1 1.42 530.2 530.4 530.3 530.3 0.1 2.24 1219.0 1219.0 1220.0 1219.3 0.6 3.16 2244.0 2245.0 2247.0 2245.3 1.5 0.47 125.3 130.0 127.2 127.5 2.4

NaC

l 0.72 125.2 125.2 125.4 125.3 0.1 1 123.0 122.9 123.2 123.0 0.2 1.42 121.3 120.9 120.9 121.0 0.2 2.24 118.4 118.2 118.2 118.3 0.1 3.16 115.9 115.8 115.9 115.9 0.1

Appendix III

215

Appendix III. Piribedil experiments Piribedil Fluxes [µmol/h] pH effect (100%, 30mM, 0.4mA)

PBD-01 100% 30mM pH2.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.03 0.05 0.02 0.07 0.06 0.12 0.06 0.04 6h2 0.08 0.12 0.07 0.11 0.12 0.12 0.10 0.02 6h3 0.08 0.12 0.06 0.06 0.07 0.05 0.07 0.02 6h4 0.07 0.08 0.10 0.07 0.08 0.08 0.08 0.01 6h5 0.10 0.20* 0.08 0.11 0.11 0.09 0.10 0.01 5h6 0.09 0.12 0.12 0.10 0.12 0.12 0.11 0.01 6 PBD-07 100% 30mM pH3.1 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.14 0.11 0.25 0.23 0.34 0.18 0.21 0.08 6h2 0.17 0.16 0.28 0.23 0.33 0.26 0.24 0.06 6h3 0.15 0.16 0.21 0.21 0.31 0.21 0.21 0.06 6h4 0.16 0.16 0.20 0.22 0.28 0.20 0.20 0.04 6h5 0.15 0.18 0.17 0.22 0.30 0.23 0.21 0.05 6h6 0.17 0.18 0.17 0.21 0.28 0.19 0.20 0.04 6 PBD-17 100% 30mM pH3.5 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.28 0.21 0.17 0.17 0.21 0.21 0.04 5h2 0.24 0.20 0.22 0.18 0.21 0.21 0.02 5h3 0.18 0.18 0.23 0.19 0.17 0.19 0.03 5h4 0.20 0.17 0.24 0.15 0.17 0.19 0.03 5h5 0.20 0.16 0.25 0.13 0.20 0.19 0.04 5h6 0.22 0.18 0.24 0.14 0.18 0.19 0.04 5 PBD-14 100% 30mM pH4.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.09 0.06 0.06 0.09 0.07 0.12 0.08 0.02 6h2 0.18 0.28 0.20 0.19 0.23 0.14 0.20 0.05 6h3 0.17 0.28 0.21 0.18 0.28 0.19 0.22 0.05 6h4 0.19 0.28 0.18 0.14 0.28 0.21 0.21 0.06 6h5 0.25 0.29 0.21 0.21 0.35 0.34 0.27 0.06 6h6 0.17 0.15 0.14 0.16 0.23 0.26 0.18 0.05 6 PBD-15 100% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.04 0.06 0.12 0.08 0.07 0.07 0.03 5h2 0.24 0.24 0.31 0.29 0.29 0.27 0.03 5h3 0.39 0.34 0.36 0.39 0.37 0.37 0.02 5h4 0.40 0.39 0.37 0.44 0.34 0.39 0.04 5h5 0.51 0.50 0.45 0.57 0.35 0.48 0.08 5h6 0.41 0.38 0.43 0.23 0.32 0.36 0.08 5

Appendix III

216

Mannitol Fluxes [nmol/h] pH effect (Piribedil 100%, 30mM, 0.4mA) PBD-14 100% 30mM pH4.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 5.0 0.3 0.3 0.0 0.3 1.05 1.92 6h2 5.0 0.0 2.0 1.3 6.6 2.48 2.72 6h3 3.3 0.0 2.3 0.7 6.9 2.20 2.67 6h4 3.6 0.0 2.0 1.3 6.3 2.20 2.41 6h5 2.3 0.0 0.7 0.7 4.6 1.38 1.80 6h6 24.0* 0.0 1.0 0.7 4.6 1.25 1.93 5 PBD-15 100% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 1.3 0.0 0.3 0.0 0.0 0.33 0.57 5h2 2.6 0.3 0.0 3.6 3.6 2.05 1.77 5h3 1.7 2.6 2.0 2.3 3.6 2.44 0.76 5h4 3.6 3.0 2.0 4.0 4.3 3.37 0.92 5h5 3.3 2.0 2.0 3.6 2.3 2.64 0.77 5h6 1.7 2.0 1.7 4.3 2.3 2.38 1.10 5

Piribedil Fluxes [µmol/h] Current effect (100%, 30mM, pH5.0)

PBD-10 100% 30mM pH5.0 0.0mA [µmol/h] R1 R2 R3 R4 R5 R6 AV SD N h2 0.009 0.005 0.005 0.006 0.007 0.011 0.007 0.003 6 h4 0.020 0.012 0.020 0.018 0.021 0.013 0.017 0.004 6 h6 0.019 0.008 0.011 0.022 0.014 0.007 0.014 0.006 6 PBD-18 100% 30mM pH5.0 0.1mA R1 R2 R3 R4 R5 AV SD N h1 0.03 0.03 0.05 0.03 0.04 0.04 0.01 5 h2 0.05 0.06 0.09 0.03 0.08 0.07 0.02 5 h3 0.08 0.09 0.11 0.11 0.11 0.10 0.02 5 h4 0.10 0.10 0.12 0.07 0.12 0.10 0.02 5 h5 0.10 0.11 0.11 0.07 0.11 0.10 0.02 5 h6 0.08 0.10 0.11 0.11 0.10 0.10 0.01 5 PBD-19 100% 30mM pH5.0 0.2mA R1 R2 R3 R4 R5 AV SD N h1 0.05 0.04 0.04 0.04 0.04 0.04 0.00 5 h2 0.07 0.07 0.08 0.09 0.11 0.08 0.02 5 h3 0.08 0.10 0.13 0.14 0.16 0.12 0.03 5 h4 0.08 0.17 0.15 0.18 0.24 0.16 0.06 5 h5 0.20 0.16 0.16 0.19 0.19 0.18 0.02 5 h6 0.15 0.16 0.15 0.15 0.17 0.15 0.01 5 PBD-15 100% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.04 0.06 0.12 0.08 0.07 0.07 0.03 5 h2 0.24 0.24 0.31 0.29 0.29 0.27 0.03 5 h3 0.39 0.34 0.36 0.39 0.37 0.37 0.02 5 h4 0.40 0.39 0.37 0.44 0.34 0.39 0.04 5 h5 0.51 0.50 0.45 0.57 0.35 0.48 0.08 5 h6 0.41 0.38 0.43 0.23 0.32 0.36 0.08 5

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217

Piribedil Fluxes [µmol/h] Concentration effect (100%, pH5.0, 0.4mA) PBD-13 100% 15mM pH5.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.04 0.09 0.09 0.19 0.11 0.12 0.11 0.05 6h2 0.28 0.30 0.25 0.33 0.30 0.35 0.30 0.03 6h3 0.36 0.38 0.33 0.42 0.06 0.40 0.33 0.13 6h4 0.39 0.46 0.25 0.44 0.41 0.42 0.39 0.08 6h5 0.38 0.43 0.38 0.42 0.41 0.43 0.41 0.03 6h6 0.39 0.44 0.40 0.42 0.39 0.17* 0.41 0.02 5 PBD-06 100% 22.5mM pH5.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.96 0.41 0.28 0.25 0.25 0.23 0.40 0.28 6h2 0.74 0.68 0.64 0.55 0.46 0.44 0.59 0.12 6h3 0.78 0.71 0.64 0.65 0.50 0.50 0.63 0.11 6h4 0.73 0.64 0.71 0.69 0.48 0.53 0.63 0.10 6h5 0.58 0.53 0.57 0.62 0.44 0.53 0.54 0.06 6h6 0.51 0.43 0.51 0.54 0.34 0.54 0.48 0.08 6 PBD-15 100% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.04 0.06 0.12 0.08 0.07 0.07 0.03 5h2 0.24 0.24 0.31 0.29 0.29 0.27 0.03 5h3 0.39 0.34 0.36 0.39 0.37 0.37 0.02 5h4 0.40 0.39 0.37 0.44 0.34 0.39 0.04 5h5 0.51 0.50 0.45 0.57 0.35 0.48 0.08 5h6 0.41 0.38 0.43 0.23 0.32 0.36 0.08 5

Mannitol Fluxes [nmol/h] Concentration effect (100%, pH5.0, 0.4mA)

PBD-

13 100% 15mM pH5.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.0 1.1 0.0 0.0 0.0 1.1 0.36 0.56 6h2 1.1 0.0 12.0 12.0 0.0 6.8 5.30 5.75 6h3 8.7 6.5 7.6 12.0 0.0 8.1 7.15 3.96 6h4 9.8 6.5 13.1 14.2 0.0 11.1 9.11 5.20 6h5 6.5 6.5 12.0 7.6 0.0 8.7 6.90 3.94 6h6 6.5 5.4 14.2 7.6 0.0 7.8 6.93 4.56 6

PBD-

15 100% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 1.3 0.0 0.3 0.0 0.0 0.33 0.57 5h2 2.6 0.3 0.0 3.6 3.6 2.05 1.77 5h3 1.7 2.6 2.0 2.3 3.6 2.44 0.76 5h4 3.6 3.0 2.0 4.0 4.3 3.37 0.92 5h5 3.3 2.0 2.0 3.6 2.3 2.64 0.77 5h6 1.7 2.0 1.7 4.3 2.3 2.38 1.10 5

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218

Piribedil Fluxes [µmol/h] Mole fraction Effect (pH5.0, 30mM, 0.4) PBD-16 10% 30mM pH5.0 0.4mA PBD-24 10% 30mM pH5.0 0.4mA

R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 AV SD N

h1 0.05 0.05 0.05 0.04 0.04 0.04 0.02 0.01 0.05 0.01 0.02 0.01 0.03 0.02 12 h2 0.13 0.13 0.08 0.12 0.11 0.10 0.08 0.05 0.11 0.05 0.10 0.06 0.09 0.03 12 h3 0.20 0.18 0.19 0.19 0.17 0.15 0.13 0.10 0.13 0.13 0.12 0.01* 0.15 0.03 11 h4 0.15 0.20 0.22 0.20 0.18 0.18 0.14 0.12 0.14 0.12 0.15 0.13 0.16 0.03 12 h5 0.11 0.18 0.25 0.25 0.19 0.15 0.11 0.15 0.09 0.16 0.16 0.16 0.17 0.05 12

h6 0.13 0.18 0.26 0.24 0.21 0.14 0.13 0.16 0.11 0.14 0.17 0.18 0.17 0.05 12

PBD-20 25% 30mM pH5.0 0.4mA


h1 0.03 0.03 0.02 0.02 0.02 0.04 0.02 0.01 6 h2 0.15 0.13 0.06 0.11 0.14 0.12 0.12 0.03 6 h3 0.21 0.20 0.16 0.19 0.21 0.19 0.19 0.02 6 h4 0.24 0.22 0.20 0.23 0.23 0.21 0.22 0.01 6 h5 0.23 0.23 0.21 0.26 0.26 0.24 0.24 0.02 6

h6 0.24 0.23 0.24 0.09* 0.28 0.25 0.25 0.02 5

PBD-04 50% 30mM pH5.0 0.4mA PBD-09 50% 28.2mM pH5.0 0.4mA PBD-21 50% 30mM pH5.0 0.4mA

R1 R2 R3 R4 R5 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 AV SD N

h1 0.23 0.10 0.10 0.19 0.63* 0.35 0.40 0.08 0.03 0.04 0.05 0.04 0.03 0.04 0.04 0.05 0.04 0.11 0.12 16 h2 0.26 0.23 0.20 0.22 0.31 0.34 0.29 0.19 0.12 0.12 0.13 0.18 0.12 0.21 0.19 0.21 0.17 0.20 0.07 17 h3 0.43 0.37 0.35 0.36 0.40 0.77 0.30 0.29 0.25 0.23 0.23 0.33 0.26 0.40 0.42 0.29 0.22 0.35 0.13 17 h4 0.33 0.21 0.35 0.42 0.34 1.06* 0.28 0.29 0.28 0.28 0.27 0.37 0.32 0.0* 0.48 0.33 0.22 0.32 0.07 15 h5 0.30 0.35 0.25 0.29 0.50 0.48 0.26 0.31 0.29 0.31 0.31 0.42 0.37 0.41 0.50 0.36 0.29 0.35 0.08 17

h6 0.41 0.29 0.32 0.34 0.51 0.45 0.28 0.31 0.29 0.29 0.31 0.43 0.34 0.39 0.49 0.36 0.27 0.36 0.08 17

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219

Piribedil Fluxes [µmol/h] Mole fraction Effect (pH5.0, 30mM, 0.4) PBD-08 75% 28.2mM pH5.0 0.4mA

R1 R2 R3 R4 R5 R6 AV SD N h1 0.15 0.23 0.26 0.23 0.04 0.06 0.16 0.09 6 h2 0.29 0.38 0.27 0.30 0.16 0.17 0.26 0.08 6 h3 0.39 0.45 0.36 0.31 0.29 0.40 0.37 0.06 6 h4 0.38 0.47 0.31 0.26 0.33 0.35 0.35 0.07 6 h5 0.39 0.46 0.31 0.25 0.35 0.33 0.35 0.07 6 h6 0.29 0.35 0.26 0.34 0.45 0.36 0.34 0.06 6 PBD-15 100% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.04 0.06 0.12 0.08 0.07 0.07 0.03 5 h2 0.24 0.24 0.31 0.29 0.29 0.27 0.03 5 h3 0.39 0.34 0.36 0.39 0.37 0.37 0.02 5 h4 0.40 0.39 0.37 0.44 0.34 0.39 0.04 5 h5 0.51 0.50 0.45 0.57 0.35 0.48 0.08 5 h6 0.41 0.38 0.43 0.23 0.32 0.36 0.08 5

Mannitol Fluxes [nmol/h] Mole fraction effect (Piribedil pH5.0, 30mM, 0.4mA)

Man Ref 0% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 15.5 25.6 12.8 15.0 21.4 18.07 5.26 5 h2 32.4 37.5 30.7 37.8 44.0 36.49 5.21 5 h3 58.6 62.6 44.9 58.9 63.9 57.79 7.56 5 h4 79.5 83.4 72.1 79.0 81.2 79.05 4.25 5 h5 93.4 97.5 81.7 92.3 87.1 90.39 6.10 5 h6 100.7 107.4 85.7 100.5 105.7 99.99 8.54 5 PBD-24 10% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD Nh1 2.7 2.2 2.5 1.8 2.3 2.3 2.31 0.30 6 h2 4.0 4.2 5.7 10.2 3.1 2.3 4.93 2.85 6 h3 4.6 3.9 10.5 12.0 3.2 2.9 6.17 3.99 6 h4 14.4 14.6 0.0* 32.8 7.8 9.2 15.77 9.98 5 h5 9.1 12.6 34.0 33.9 12.5 11.0 18.87 11.76 6 h6 10.1 5.3 35.8 32.5 24.3 9.1 19.51 13.07 6 PBD-20 25% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 1.1 1.6 5.8 0.0 0.0 2.0 1.73 2.13 6 h2 4.2 1.7 4.4 2.8 1.1 1.8 2.67 1.37 6 h3 5.2 1.1 4.5 8.5 3.5 3.9 4.44 2.43 6 h4 3.6 2.3 3.2 2.9 3.5 1.1 2.78 0.93 6 h5 3.4 1.8 2.9 2.8 3.9 1.4 2.68 0.94 6 h6 3.5 2.7 4.0 4.2 5.8 2.9 3.86 1.11 6 PBD-15 100% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 1.3 0.0 0.3 0.0 0.0 0.33 0.57 5 h2 2.6 0.3 0.0 3.6 3.6 2.05 1.77 5 h3 1.7 2.6 2.0 2.3 3.6 2.44 0.76 5 h4 3.6 3.0 2.0 4.0 4.3 3.37 0.92 5 h5 3.3 2.0 2.0 3.6 2.3 2.64 0.77 5 h6 1.7 2.0 1.7 4.3 2.3 2.38 1.10 5

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220

Piribedil Fluxes [µmol/h] Mole fraction Effect (pH3.5, 30mM, 0.4) PBD-22 10% 30mM pH3.5 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.02 0.04 0.04 0.05 0.04 0.04 0.01 5 h2 0.08 0.09 0.10 0.10 0.07 0.09 0.01 5 h3 0.10 0.08 0.08 0.07 0.06 0.08 0.01 5 h4 0.09 0.06 0.07 0.04 0.04 0.06 0.02 5 h5 0.09 0.04 0.07 0.04 0.04 0.06 0.02 5 h6 0.03 0.04 0.05 0.03 0.04 0.04 0.01 5 PBD-23 50% 30mM pH3.5 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.07 0.09 0.08 0.09 0.07 0.09 0.08 0.01 6 h2 0.21 0.20 0.18 0.21 0.18 0.23 0.20 0.02 6 h3 0.25 0.22 0.23 0.27 0.25 0.28 0.25 0.02 6 h4 0.30 0.25 0.25 0.26 0.26 0.25 0.26 0.02 6 h5 0.27 0.20 0.21 0.24 0.20 0.18 0.22 0.03 6 h6 0.22 0.16 0.15 0.19 0.18 0.15 0.18 0.03 6 PBD-17 100% 30mM pH3.5 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.28 0.21 0.17 0.17 0.21 0.21 0.04 5 h2 0.24 0.20 0.22 0.18 0.21 0.21 0.02 5 h3 0.18 0.18 0.23 0.19 0.17 0.19 0.03 5 h4 0.20 0.17 0.24 0.15 0.17 0.19 0.03 5 h5 0.20 0.16 0.25 0.13 0.20 0.19 0.04 5 h6 0.22 0.18 0.24 0.14 0.18 0.19 0.04 5

Piribedil Fluxes [µmol/h]Comparison of 25% mole fraction for different total molarity

PBD-05 25% 80mM pH5.1 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.13 0.07 0.00 0.22 0.11 0.09 0.10 0.07 6 h2 0.18 0.11 0.21 0.17 0.20 0.23 0.18 0.04 6 h3 0.32 0.20 0.37 0.41 0.34 0.37 0.34 0.07 6 h4 0.46 0.39 0.33 0.35 0.77 0.44 0.46 0.16 6 h5 0.30 0.21 0.31 0.43 0.33 0.32 0.32 0.07 6 h6 0.31 0.23 0.33 0.45 0.33 0.34 0.33 0.07 6 PBD-20 25% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.03 0.03 0.02 0.02 0.02 0.04 0.02 0.01 6 h2 0.15 0.13 0.06 0.11 0.14 0.12 0.12 0.03 6 h3 0.21 0.20 0.16 0.19 0.21 0.19 0.19 0.02 6 h4 0.24 0.22 0.20 0.23 0.23 0.21 0.22 0.01 6 h5 0.23 0.23 0.21 0.26 0.26 0.24 0.24 0.02 6 h6 0.24 0.23 0.24 0.09* 0.28 0.25 0.25 0.02 5

Appendix III

221

Human skin Piribedil Flux [µmol/h]

PBD 100% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.01 0.01 0.00 0.00 0.00 0.00 0.00 5h2 0.00 0.01 0.01 0.00 0.01 0.01 0.01 5h3 0.02 0.04 0.01 0.01 0.01 0.02 0.01 5h4 0.05 0.08 0.02 0.03 0.03 0.04 0.02 5h5 0.09 0.19 0.05 0.09 0.07 0.10 0.06 5h6 0.13 0.19 0.13 0.14 0.11 0.14 0.03 5 Mannitol Flux [nmol/h] PBD 100% 30mM pH5.0 0.4mA R1 R2 R3 R4 R5 AV SD N h1 1.4 1.1 3.2 1.4 1.2 1.7 0.9 5h2 1.1 1.5 3.1 0.9 1.3 1.6 0.9 5h3 1.2 3.1 3.3 1.6 1.9 2.2 0.9 5h4 1.7 3.4 4.1 3.0 3.1 3.1 0.9 5h5 2.0 4.5 4.6 4.1 3.8 3.8 1.0 5

h6 2.2 4.2 5.6 5.2 4.2 4.3 1.3 5

Appendix IV

222

Appendix IV. Pergolide Experiments Current effect

Pergolide flux [nmol/h] PER‐8 5% 50mM pH4.0 0.0mA R1 R2 R3 R4 R5 AV SD N

h0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 h2 0.00 0.00 0.27 1.54 0.00 0.36 0.67 5 h4 0.04 0.03 0.31 1.47 0.01 0.37 0.62 5 h6 0.12 0.08 0.27 1.49 0.05 0.40 0.62 5 PER‐7 5% 50mM pH4.0 0.1mA R1 R2 R3 R4 R5 R6 AV SD N

h1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6 h2 0.0 0.0 0.0 0.0 0.0 2.2 0.4 0.9 6 h3 1.7 5.4 4.3 0.5 1.2 5.8 3.2 2.3 6 h4 1.3 5.7 4.0 0.9 2.8 7.3 3.7 2.5 6 h5 2.8 4.5 4.1 1.4 4.7 7.1 4.1 1.9 6 h6 5.0 5.1 3.8 2.0 6.2 6.9 4.9 1.7 6 PER‐6 5% 50mM pH4.0 0.2mA R1 R2 R3 R4 R5 R6 AV SD N

h1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6 h2 0.6 1.9 1.3 2.5 1.7 2.0 1.7 0.7 6 h3 1.6 4.3 3.6 6.0 3.1 4.6 3.8 1.5 6 h4 3.1 7.5 5.9 9.7 5.2 7.9 6.5 2.3 6 h5 4.7 9.5 8.4 11.4 6.4 10.4 8.5 2.5 6 h6 6.2 10.7 11.1 1.34* 8.2 12.4 9.7 2.5 5 PER‐3 5% 50mM pH4.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N

h1 1.3 0.0 1.3 3.7 2.2 6.8 2.5 2.4 6 h2 0.9 1.2 5.8 9.0 10.2 13.5 6.8 5.1 6 h3 1.8 2.9 10.4 16.2 20.8 11.8 10.7 7.4 6 h4 3.4 5.4 17.8 20.6 00.0* 15.2 12.5 7.7 5 h5 5.6 8.0 18.5 20.3 27.1 21.7 16.9 8.4 6 h6 4.1 6.0 17.7 28.3 36.9 19.1 18.7 12.6 6

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223

Current effect Mannitol flux [nmol/h]

PER‐7 5% 50mM pH4.0 0.1mA


h1 0.2 0.2 0.1 0.2 0.4 0.1 0.2 0.1 6h2 0.2 0.3 0.1 0.1 0.2 0.1 0.2 0.1 6h3 0.7 0.0 0.7 3.0 0.2 0.0 0.8 1.1 6h4 6.2 3.8 9.1 3.9 3.8 3.6 5.1 2.2 6h5 6.1 3.7 14.8 5.4 6.1 3.6 6.6 4.2 6h6 8.5 3.6 14.2 4.8 6.3 3.7 6.9 4.1 6 PER‐6 5% 50mM pH4.0 0.2mA


h1 0.2 0.3 0.0 0.1 0.2 0.1 0.2 0.1 6h2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.0 6h3 1.4 0.1 0.0 0.0 0.1 0.1 0.3 0.5 6h4 6.8 0.0 0.2 0.1 0.1 0.2 1.2 2.7 6h5 7.0 9.2 0.0 0.0 0.1 0.1 2.7 4.2 6h6 12.9 12.6 0.1 0.1 0.3 0.0 4.3 6.5 6 PER‐3 5% 50mM pH4.0 0.4mA


h1 0.2 0.2 0.1 0.2 0.4 8.2 1.5 3.3 6h2 0.2 0.3 0.1 0.1 0.2 7.5 1.4 3.0 6h3 0.7 0.0 0.7 3.0 0.2 12.7 2.9 4.9 6h4 6.2 3.8 9.1 3.9 3.8 15.4 7.0 4.6 6h5 6.1 3.7 14.8 5.4 6.1 20.1 9.4 6.5 6h6 8.5 3.6 14.2 4.8 6.3 19.9 9.6 6.3 6

Appendix IV

224

Mole Fraction Effect Pergolide flux [nmol/h]

PER‐9 5% 29mM pH4.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N

h1 3.5 0.5 0.9 6.7 1.6 0.0 2.2 2.5 6 h2 19.5 3.0 4.2 12.3 14.0 1.0 9.0 7.4 6 h3 8.7 6.7 8.3 7.2 27.5 3.3 10.3 8.6 6 h4 9.1 8.2 9.4 9.2 23.2 5.0 10.7 6.3 6 h5 10.7 9.5 10.8 11.3 16.2 6.6 10.9 3.1 6 h6 11.2 9.5 10.8 11.4 10.8 8.9 10.4 1.0 6


h1 2.0 4.8 1.0 1.1 1.3 2.9 2.2 1.5 6 h2 5.5 3.3 2.6 2.8 3.8 4.6 3.8 1.1 6 h3 8.6 6.4 4.7 5.1 6.5 7.3 6.4 1.4 6 h4 12.3 9.7 7.5 7.6 9.6 10.5 9.5 1.8 6 h5 13.9 11.2 10.4 9.5 10.2 13.0 11.4 1.7 6 h6 14.2 13.6 9.9 9.9 8.6 12.0 11.3 2.3 6


h1 26.2 33.1 31.4 27.9 25.4 17.8 27.0 5.4 6 h2 18.1 18.3 21.9 19.0 15.9 15.0 18.0 2.4 6 h3 16.5 17.2 21.2 20.5 15.9 15.3 17.8 2.5 6 h4 18.8 17.8 24.8 19.6 17.5 16.4 19.2 3.0 6 h5 19.8 18.8 22.3 19.5 17.1 16.6 19.0 2.1 6 h6 18.5 18.2 25.7 18.8 17.9 17.0 19.4 3.1 6


h1 1.3 0.0 1.3 3.7 2.2 6.8 2.5 2.4 6 h2 0.9 1.2 5.8 9.0 10.2 13.5 6.8 5.1 6 h3 1.8 2.9 10.4 16.2 20.8 11.8 10.7 7.4 6 h4 3.4 5.4 17.8 20.6 00.00* 15.2 12.5 7.7 5 h5 5.6 8.0 18.5 20.3 27.1 21.7 16.9 8.4 6 h6 4.1 6.0 17.7 28.3 36.9 19.1 18.7 12.6 6


h1 11.5 12.6 2.4 2.7 2.5 1.0 5.4 5.1 6 h2 11.8 32.8 5.8 5.5 6.6 1.0 10.6 11.4 6 h3 10.7 26.1 7.7 21.4 10.2 2.2 13.1 9.0 6 h4 21.2 16.6 14.3 23.6 22.8 5.4 17.3 6.9 6

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225

h5 19.2 14.9 16.2 21.1 21.4 6.7 16.6 5.5 6h6 37.2 22.3 34.3 39.8 14.9 6.2 25.8 13.5 6

PER‐4 100% 14.5mM pH4.0 0.4mASALT BRIDGE


h1 8.6 1.7 2.0 7.8 0.9 0.8 3.6 3.6 6h2 20.3 11.6 7.7 10.2 4.7 1.5 9.3 6.5 6h3 22.1 23.2 8.4 10.6 7.0 4.5 12.6 8.0 6h4 31.6 26.8 8.2 16.7 8.5 6.6 16.4 10.6 6h5 23.7 27.9 15.3 15.1 11.1 6.3 16.6 8.0 6h6 23.9 26.3 10.4 17.6 18.5 8.4 17.5 7.1 6

Mannitol flux [nmol/h]

PER‐9 5% 29mM pH4.0 0.4mA


h1 5.7 0.4 0.9 2.1 0.6 8.2 3.0 3.2 6h2 9.8 6.1 0.1 3.1 8.1 7.5 5.8 3.6 6h3 10.6 13.6 17.1 13.6 9.0 12.7 12.8 2.8 6h4 16.2 13.3 17.5 6.7 12.1 15.4 13.5 3.9 6h5 23.2 13.0 19.8 16.2 4.3 20.1 16.1 6.8 6h6 25.1 13.0 16.6 14.8 2.9 19.9 15.4 7.5 6

PER‐

10 25% 29mM pH4.0 0.4mA


h1 0.6 5.1 0.3 1.4 2.5 0.3 1.7 1.9 6h2 3.1 2.3 3.5 2.8 2.5 3.5 2.9 0.5 6h3 3.8 3.5 5.1 2.5 3.4 4.6 3.8 0.9 6h4 4.2 3.4 6.6 5.3 3.7 10.9* 4.6 1.3 5h5 4.3 4.2 6.7 6.2 3.6 5.9 5.2 1.3 6h6 3.6 3.1 5.1 5.4 3.5 4.3 4.2 0.9 6 PER‐1 50% 29mM pH4.0 0.4mA


h1 0.0 0.3 0.3 1.0 0.0 1.6 1.7 1.9 6h2 0.6 0.0 2.4 0.7 2.9 5.9 2.9 0.5 6h3 0.8 0.3 7.9 0.3 3.0 1.7 3.8 0.9 6h4 8.3 1.7 2.3 0.0 1.3 1.0 4.6 1.3 5h5 3.0 0.3 1.0 0.0 0.7 0.7 5.2 1.3 6h6 1.7 0.0 0.3 0.0 0.3 0.3 4.2 0.9 6

Appendix IV

226

pH effect Pergolide flux [nmol/h]


h1 0.9 1.3 1.2 0.4 0.6 0.5 0.8 0.4 6 h2 1.8 2.5 1.8 1.2 2.5 2.2 2.0 0.5 6 h3 2.9 1.7 2.7 1.9 2.6 2.4 2.4 0.5 6 h4 1.9 5.2 3.3 1.2 1.3 1.6 2.4 1.6 6 h5 1.6 2.8 3.6 1.1 1.6 1.7 2.1 0.9 6 h6 1.0 2.9 2.9 1.4 1.7 1.5 1.9 0.8 6

PER‐4 100% 14.5mM pH4.0 0.4mASALT BRIDGE


h1 8.6 1.7 2.0 7.8 0.9 0.8 3.6 3.6 6 h2 20.3 11.6 7.7 10.2 4.7 1.5 9.3 6.5 6 h3 22.1 23.2 8.4 10.6 7.0 4.5 12.6 8.0 6 h4 31.6 26.8 8.2 16.7 8.5 6.6 16.4 10.6 6 h5 23.7 27.9 15.3 15.1 11.1 6.3 16.6 8.0 6 h6 23.9 26.3 10.4 17.6 18.5 8.4 17.5 7.1 6

Appendix V

227

Appendix V. Trihexyphenidyl Experiments Mole fraction effect

THP flux [µmol/h] (20mM, pH5.8(0.2), 0.4mA) THP‐21 25.0% 20mM pH6.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD Nh1 0.03 0.00 0.00 0.04 0.03 0.02 0.02 0.02 6h2 0.15 0.07 0.10 0.04 0.11 0.11 0.10 0.04 6h3 0.22 0.16 0.17 0.17 0.17 0.15 0.17 0.02 6h4 0.25 0.23 0.21 0.24 0.19 0.16 0.21 0.03 6h5 0.25 0.24 0.21 0.18 0.19 0.18 0.21 0.03 6h6 0.23 0.24 0.24 0.13 0.24 0.16 0.20 0.05 6

THP‐07 50.0% 20mM pH5.7 0.4mA R1 R2 R3 R4 R5 AV SD Nh1 0.08 0.07 0.04 0.31 0.02 0.10 0.12 5

h2 0.18 0.10 0.19 0.15 0.10 0.14 0.04 5 h3 0.22 0.14 0.26 0.17 0.13 0.19 0.06 5 h4 0.20 0.16 0.22 0.13 0.19 0.18 0.04 5h5 0.19 0.16 0.21 0.18 0.17 0.18 0.02 5h6 0.18 0.15 0.23 0.20 0.17 0.19 0.03 5

THP‐10 75.0% 20mM pH6.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N

h1 0.04 0.16 0.04 0.03 0.07 0.03 0.06 0.05 6h2 0.13 0.16 0.11 0.11 0.17 0.10 0.13 0.03 6h3 0.14 0.17 0.17 0.16 0.23 0.14 0.17 0.03 6h4 0.0* 0.16 0.15 0.16 0.18 0.11 0.15 0.03 5h5 0.16 0.15 0.16 0.16 0.15 0.10 0.15 0.03 6h6 0.19 0.15 0.12 0.16 0.12 0.12 0.14 0.03 6

THP‐28 100% 20mM pH5.5 0.4mA R1 R2 R3 R4 R5 R6 h1 0.04 0.05 0.04 0.05 0.04 0.04 h2 0.16 0.16 0.17 0.17 0.19 0.18

h3 0.21 0.19 0.19 0.20 0.22 0.21 AV SD N

h4 0.21 0.20 0.20 0.21 0.22 0.22 0.05 0.01 12h5 0.21 0.20 0.20 0.21 0.21 0.20 0.21 0.05 12h6 0.23 0.19 0.18 0.19 0.22 0.21 0.23 0.05 12 THP‐29 100% 20mM pH5.4 0.4mA 0.23 0.03 12 R1 R2 R3 R4 R5 R6 0.24 0.05 12h1 0.03 0.05 0.07 0.08 0.05 0.06 0.22 0.04 12h2 0.30 0.26 0.24 0.30 0.26 0.16 h3 0.27 0.33 0.25 0.27 0.13 0.26 h4 0.23 0.29 0.22 0.23 0.28 0.28 h5 0.26 0.35 0.19 0.23 0.29 0.29 h6 0.20 0.30 0.17 0.20 0.26 0.25

Appendix V

228

Mole fraction effect THP flux [µmol/h] (30mM, pH5.8(0.2), 0.4mA)

THP‐23 12.5% 30mM pH5.7 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.00 0.00 0.00 0.00 0.00 0.04 0.01 0.02 6 h2 0.05 0.05 0.08 0.07 0.06 0.06 0.06 0.01 6 h3 0.08 0.08 0.16 0.10 0.10 0.07 0.10 0.03 6 h4 0.13 0.12 0.18 0.14 0.12 0.10 0.13 0.03 6 h5 0.14 0.13 0.18 0.17 0.14 0.09 0.14 0.03 6 h6 0.13 0.14 0.20 0.18 0.14 0.08 0.15 0.04 6 THP‐26 25.0% 30mM pH5.7 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6 h2 0.05 0.05 0.00 0.08 0.00 0.00 0.03 0.03 6 h3 0.08 0.07 0.06 0.15 0.05 0.07 0.08 0.04 6 h4 0.09 0.10 0.07 0.17 0.06 0.10 0.10 0.04 6 h5 0.20 0.13 0.08 0.19 0.07 0.13 0.13 0.05 6 h6 0.20 0.13 0.08 0.15 0.08 0.12 0.13 0.04 6 THP‐11 50.0% 30mM pH5.9 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.12 0.10 0.11 0.10 0.00 0.00 0.07 0.05 6 h2 0.20 0.20 0.13 0.19 0.16 0.16 0.17 0.03 6 h3 0.24 0.24 0.19 0.24 0.24 0.18 0.22 0.03 6 h4 0.26 0.23 0.19 0.24 0.19 0.17 0.21 0.04 6 h5 0.21 0.23 0.17 0.21 0.17 0.15 0.19 0.03 6 h6 0.18 0.21 0.16 0.19 0.16 0.14 0.17 0.02 6 THP‐08 75.0% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.04 0.15 0.03 0.23 0.14 0.12 0.09 5 h2 0.18 0.16 0.11 0.11 0.21 0.15 0.05 5 h3 0.22 0.19 0.19 0.22 0.20 0.20 0.02 5 h4 0.21 0.23 0.23 0.22 0.23 0.22 0.01 5 h5 0.16 0.19 0.22 0.23 0.27 0.22 0.04 5 h6 0.19 0.23 0.24 0.24 0.28 0.23 0.03 5

Appendix V

229

THP‐12 100% 30mM pH5.4 0.4mA THP‐24 100% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 AV SD N

h1 0.08 0.24 0.29 0.13 0.33 0.21 0.00 0.02 0.04 0.02 0.00 0.10 0.12 0.12 12h2 0.15 0.21 0.20 0.18 0.19 0.18 0.07 0.15 0.14 0.11 0.08 0.17 0.15 0.05 12h3 0.21 0.21 0.20 0.22 0.22 0.21 0.16 0.21 0.19 0.19 0.19 0.22 0.20 0.02 12h4 0.21 0.21 0.20 0.22 0.21 0.22 0.21 0.23 0.22 0.22 0.24 0.23 0.22 0.01 12h5 0.20 0.21 0.17 0.21 0.20 0.22 0.22 0.24 0.20 0.23 0.25 0.24 0.22 0.02 12h6 0.19 0.20 0.20 0.20 0.20 0.20 0.20 0.22 0.18 0.19 0.21 0.18 0.20 0.01 12

Mole fraction effect THP flux [µmol/h] (40mM, pH5.8(0.2), 0.4mA)

THP‐22 12.5% 40mM pH5.9 0.4mA THP‐16 12.5% 40mM pH5.8 0.4mA R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 AV SD N

h1 0.00 0.00 0.02 0.01 0.03 0.03 0.01 0.01 0.01 0.01 0.05 0.06 0.02 0.02 12h2 0.03 0.04 0.09 0.10 0.09 0.09 0.09 0.15 0.09 0.08 0.09 0.10 0.09 0.03 12h3 0.08 0.07 0.13 0.12 0.16 0.16 0.08 0.15 0.07 0.08 0.07 0.09 0.11 0.03 12h4 0.17 0.11 0.16 0.15 0.18 0.18 0.07 0.16 0.06 0.07 0.05 0.07 0.12 0.05 12h5 0.22 0.13 0.20 0.17 0.24 0.22 0.06 0.11 0.05 0.06 0.05 0.06 0.13 0.08 12h6 0.26 0.17 0.24 0.19 0.21 0.19 0.05 0.09 0.05 0.05 0.04 0.05 0.13 0.08 12 THP‐09 25.0% 40mM pH6.1 0.4mA THP‐05 25.0% 40mM pH5.7 0.4mA R1 R2 R3 R4 R5 R1 R2 R3 R4 R5 R6 AV SD N

h1 0.00 0.03 0.01 0.50 0.02 0.00 0.00 0.07 0.00 0.05 0.07 0.07 0.14 11h2 0.04 0.09 0.09 0.16 0.04 0.10 0.07 0.12 0.09 0.14 0.19 0.10 0.05 11h3 0.05 0.13 0.12 0.27 0.08 0.13 0.27 0.11 0.17 0.20 0.16 0.15 0.07 11h4 0.14 0.15 0.14 0.19 0.10 0.18 0.22 0.18 0.12 0.18 0.14 0.16 0.04 11h5 0.13 0.16 0.15 0.21 0.11 0.16 0.21 0.18 0.10 0.22 0.14 0.16 0.04 11h6 0.15 0.18 0.15 0.26 0.13 0.20 0.24 0.21 0.14 0.22 0.14 0.18 0.04 11

Appendix V

230

THP‐06 12.5% 80mM pH5.6 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.07 0.02 0.04 0.00 0.01 0.04 0.03 0.02 6 h2 0.07 0.06 0.05 0.08 0.05 0.07 0.06 0.01 6 h3 0.09 0.09 0.08 0.11 0.07 0.13 0.10 0.02 6 h4 0.10 0.08 0.10 0.14 0.12 0.12 0.11 0.02 6 h5 0.10 0.12 0.08 0.11 0.09 0.11 0.10 0.02 6 h6 0.12 0.15 0.13 0.12 0.14 0.13 0.13 0.01 6

Mole fraction effect Mannitol fluxes [nmol/h] (THP 30mM, pH5.6(0.2), 0.4mA)

MAN‐01 0% 30mM pH5.8 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 68.3 29.9 31.2 51.4 33.0 29.2 40.5 16.0 6 h2 100.3 59.4 59.0 68.6 58.7 46.3 65.4 18.5 6 h3 85.1 61.5 66.7 76.9 73.7 62.4 71.0 9.2 6 h4 125.1 97.7 90.3 100.6 69.0 72.8 92.6 20.5 6 h5 110.7 97.3 100.2 103.4 103.0 83.3 99.6 9.2 6 h6 137.8 120.7 135.9 138.5 130.1 115.4 129.7 9.7 6

THP‐23 12.5% 30mM pH5.7 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.0 1.0 3.3 1.1 1.3 1.4 1.2 5 h2 6.8 5.3 8.7 5.1 4.6 6.1 1.7 5 h3 6.8 9.8 15.4 8.2 8.1 9.7 3.4 5 h4 12.9 17.5 15.7 16.7 13.7 15.3 1.9 5 h5 13.6 17.2 19.9 19.6 13.3 16.7 3.2 5 h6 13.9 18.8 20.1 19.6 14.4 17.4 3.0 5

THP‐26 25.0% 30mM pH5.7 0.4mA R1 R2 f R4 R5 R6 AV SD N h1 6.2 3.4 2.1 3.9 2.2 2.2 3.3 1.6 6 h2 8.3 5.2 3.8 6.0 4.0 4.2 5.2 1.7 6 h3 10.9 6.5 5.4 7.8 6.4 6.5 7.2 1.9 6 h4 8.5 6.5 4.0 7.4 5.2 5.2 6.1 1.6 6 h5 8.5 7.1 4.0 5.0 5.1 5.3 5.8 1.6 6 h6 72.0* 6.6 3.3 4.9 5.8 5.7 5.3 1.3 5

THP‐24 100% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 11.2 0.3 0.4 0.5 0.4 0.5 2.2 4.4 6 h2 9.0 0.3 3.1 2.8 5.2 0.2 3.4 3.3 6 h3 2.0 2.3 5.4 7.4 0.0* 0.8 3.6 2.7 5 h4 1.8 2.1 1.4 1.9 2.5 2.1 2.0 0.4 6 h5 3.9 1.7 1.9 2.2 2.1 1.6 2.2 0.9 6 h6 1.0 1.8 1.6 2.2 1.5 1.6 1.6 0.4 6

Appendix V

231

Concentration effect THP flux [µmol/h] (100%, pH5.4, 0.4mA)

THP‐12 100% 30mM pH5.4 0.4mA THP‐24 100% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 AV SD N h1 0.08 0.24 0.29 0.13 0.33 0.21 0.00 0.02 0.04 0.02 0.00 0.10 0.12 0.12 12h2 0.15 0.21 0.20 0.18 0.19 0.18 0.07 0.15 0.14 0.11 0.08 0.17 0.15 0.05 12h3 0.21 0.21 0.20 0.22 0.22 0.21 0.16 0.21 0.19 0.19 0.19 0.22 0.20 0.02 12h4 0.21 0.21 0.20 0.22 0.21 0.22 0.21 0.23 0.22 0.22 0.24 0.23 0.22 0.01 12h5 0.20 0.21 0.17 0.21 0.20 0.22 0.22 0.24 0.20 0.23 0.25 0.24 0.22 0.02 12h6 0.19 0.20 0.20 0.20 0.20 0.20 0.20 0.22 0.18 0.19 0.21 0.18 0.20 0.01 12

THP‐28 100% 20mM pH5.5 0.4mA

R1 R2 R3 R4 R5 R6 AV SD N h1 0.04 0.05 0.04 0.05 0.04 0.04 0.04 0.00 6h2 0.16 0.16 0.17 0.17 0.19 0.18 0.17 0.01 6h3 0.21 0.19 0.19 0.20 0.22 0.21 0.20 0.01 6h4 0.21 0.20 0.20 0.21 0.22 0.22 0.21 0.01 6h5 0.21 0.20 0.20 0.21 0.21 0.20 0.20 0.00 6

h6 0.23 0.19 0.18 0.19 0.22 0.21 0.20 0.02 6

Appendix V

232

Mannitol flux [nmol/h] (100%, pH5.4, 0.4mA)

THP‐24 100% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 11.2 0.3 0.4 0.5 0.4 0.5 2.19 4.39 6 h2 9.0 0.3 3.1 2.8 5.2 0.2 3.44 3.32 6 h3 2.0 2.3 5.4 7.4 0.0* 0.8 3.59 2.74 5 h4 1.8 2.1 1.4 1.9 2.5 2.1 1.97 0.36 6 h5 3.9 1.7 1.9 2.2 2.1 1.6 2.23 0.85 6 h6 1.0 1.8 1.6 2.2 1.5 1.6 1.63 0.38 6 THP‐29 100% 20mM pH5.4 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.8 0.2 1.1 0.1 0.3 0.0 0.42 0.44 6 h2 6.0 3.2 4.1 0.4 2.2 0.2 2.67 2.23 6 h3 2.8 8.3 2.4 4.3 3.6 0.5 3.66 2.64 6 h4 3.2 3.7 1.5 4.8 3.1 3.9 3.36 1.10 6 h5 4.5 6.6 2.1 3.5 4.5 4.9 4.36 1.49 6 h6 3.1 4.5 1.9 2.3 2.8 3.0 2.92 0.91 6

pH effect THP fluxes [µmol/h] (100%, 30mM,0.4mA)

THP‐19 100% 30mM pH2.1 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.04 0.04 0.04 0.05 0.05 0.04 0.00 5 h2 0.05 0.05 0.03 0.05 0.03 0.04 0.01 5 h3 0.07 0.08 0.04 0.08 0.05 0.06 0.02 5 h4 0.07 0.07 0.06 0.08 0.05 0.07 0.01 5 h5 0.07 0.10 0.07 0.10 0.06 0.08 0.02 5 h6 0.06 0.08 0.06 0.09 0.05 0.07 0.01 5 h7 0.08 0.09 0.09 0.11 0.07 0.09 0.01 5 THP‐15 100% 30mM pH3.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 0.06 0.04 0.05 0.06 0.03 0.05 0.05 0.01 6 h2 0.04 0.05 0.05 0.06 0.06 0.16 0.07 0.05 6 h3 0.16 0.09 0.11 0.07 0.11 0.17 0.12 0.04 6 h4 0.20 0.15 0.14 0.05 0.14 0.20 0.15 0.05 6 h5 0.21 0.18 0.16 0.05 0.17 0.22 0.16 0.06 6 h6 0.26 0.21 0.18 0.05 0.18 0.24 0.18 0.08 6

Appendix V

233

THP‐

17 100% 30mM pH4.0 0.4mA

R1 R2 R3 R4 R5 AV SD Nh1 0.07 0.05 0.14 0.04 0.08 0.08 0.04 5h2 0.22 0.17 0.18 0.18 0.24 0.20 0.03 5h3 0.26 0.23 0.22 0.25 0.27 0.25 0.02 5h4 0.24 0.22 0.19 0.22 0.25 0.22 0.02 5h5 0.24 0.22 0.23 0.22 0.24 0.23 0.01 5h6 0.20 0.19 0.22 0.21 0.20 0.21 0.01 5

THP‐

18 100% 30mM pH5.0 0.4mA


h1 0.03 0.04 0.08 0.03 0.05 0.03 0.04 0.02 6h2 0.08 0.17 0.26 0.14 0.22 0.09 0.16 0.07 6h3 0.14 0.24 0.31 0.19 0.26 0.14 0.21 0.07 6h4 0.15 0.24 0.26 0.21 0.26 0.15 0.21 0.05 6h5 0.17 0.25 0.25 0.23 0.26 0.18 0.22 0.04 6h6 0.16 0.20 0.21 0.20 0.24 0.17 0.20 0.03 6

THP‐

12 100% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 R6 h1 0.08 0.24 0.29 0.13 0.33 0.21 h2 0.15 0.21 0.20 0.18 0.19 0.18 h3 0.21 0.21 0.20 0.22 0.22 0.21 h4 0.21 0.21 0.20 0.22 0.21 0.22 h5 0.20 0.21 0.17 0.21 0.20 0.22 AV SD Nh6 0.19 0.20 0.20 0.20 0.20 0.20 0.12 0.12 12 0.15 0.05 12

THP‐

24 100% 30mM pH5.4 0.4mA 0.20 0.02 12 R1 R2 R3 R4 R5 R6 0.22 0.01 12h1 0.00 0.02 0.04 0.02 0.00 0.10 0.22 0.02 12h2 0.07 0.15 0.14 0.11 0.08 0.17 0.20 0.01 12h3 0.16 0.21 0.19 0.19 0.19 0.22 h4 0.21 0.23 0.22 0.22 0.24 0.23 h5 0.22 0.24 0.20 0.23 0.25 0.24 h6 0.20 0.22 0.18 0.19 0.21 0.18

THP‐

12 100% 30mM pH5.8 0.4mA

R1 R2 R3 R4 R5 R6 R7 R8 AV SD Nh1 0.06 0.02 0.06 0.09 0.10 0.09 0.07 0.05 0.07 0.03 6h2 0.16 0.13 0.12 0.18 0.16 0.20 0.17 0.18 0.16 0.03 6h3 0.16 0.15 0.16 0.44* 0.17 0.17 0.18 0.16 0.16 0.01 5h4 0.16 0.17 0.15 0.24 0.20 0.18 0.18 0.18 0.18 0.03 6h5 0.15 0.15 0.15 0.18 0.20 0.16 0.16 0.18 0.16 0.02 6h6 0.14 0.14 0.16 0.15 0.20 0.15 0.15 0.17 0.16 0.02 6

Appendix V

234

pH effect Mannitol fluxes [nmol/h] (100%, 30mM, 0.4mA)

THP‐15 100% 30mM pH3.1 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 1.1 0.0 0.0 2.0 1.6 1.1 0.98 0.83 6 h2 0.4 1.6 1.0 1.3 2.4 1.1 1.30 0.66 6 h3 3.1 1.5 1.2 2.3 2.0 1.7 1.94 0.68 6 h4 1.9 1.2 1.0 1.4 0.8 1.1 1.25 0.39 6 h5 1.8 1.5 1.1 1.7 1.1 1.0 1.36 0.35 6 h6 0.7 1.1 1.0 1.0 0.8 1.0 0.93 0.17 6 THP‐17 100% 30mM pH4.1 0.4mA R1 R2 R3 R4 R5 AV SD N h1 6.8 3.9 3.8 3.2 2.5 4.04 1.63 5 h2 2.8 2.5 2.8 2.9 4.0 2.99 0.60 5 h3 1.7 1.9 5.9 3.5 2.0 3.02 1.75 5 h4 3.1 3.0 3.4 2.9 2.5 2.97 0.30 5 h5 2.1 1.7 1.9 2.8 3.2 2.34 0.65 5 h6 5.0 4.8 1.9 4.0 2.9 3.71 1.33 5 THP‐24 100% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 R6 AV SD N h1 11.2 0.3 0.4 0.5 0.4 0.5 2.19 4.39 6 h2 9.0 0.3 3.1 2.8 5.2 0.2 3.44 3.32 6 h3 2.0 2.3 5.4 7.4 0.0* 0.8 3.59 2.74 5 h4 1.8 2.1 1.4 1.9 2.5 2.1 1.97 0.36 6 h5 3.9 1.7 1.9 2.2 2.1 1.6 2.23 0.85 6 h6 1.0 1.8 1.6 2.2 1.5 1.6 1.63 0.38 6

Appendix V

235

Current effect THP fluxes [µmol/h] (100%, 30mM, pH5.4)

THP‐14 100% 30mM pH5.4 0.0mA R1 R2 R3 R4 R5 AV SD N h2 0.03 0.04 0.04 0.04 0.09 0.05 0.02 5h4 0.04 0.04 0.02 0.02 0.04 0.03 0.01 5h6 0.03 0.03 0.02 0.02 0.03 0.03 0.01 5

THP‐25 100% 30mM pH5.4 0.1mA

R1 R2 R3 R4 R5 R6 AV SD N h1 0.05 0.00 0.04 0.00 0.03 0.03 0.02 0.02 6h2 0.05 0.04 0.04 0.04 0.03 0.04 0.04 0.01 6h3 0.06 0.04 0.04 0.04 0.03 0.04 0.04 0.01 6h4 0.06 0.05 0.05 0.05 0.04 0.05 0.05 0.01 6h5 0.08 0.06 0.06 0.05 0.04 0.07 0.06 0.01 6h6 0.06 0.07 0.06 0.06 0.04 0.07 0.06 0.01 6 THP‐27 100% 30mM pH5.4 0.2mA R1 R2 R3 R4 R5 R6 AV SD Nh1 0.03 0.03 0.03 0.04 0.02 0.04 0.03 0.00 6 h2 0.07 0.07 0.07 0.07 0.05 0.07 0.07 0.01 6h3 0.11 0.09 0.10 0.10 0.08 0.11 0.10 0.01 6h4 0.11 0.11 0.12 0.11 0.11 0.12 0.11 0.00 6h5 0.14 0.11 0.13 0.12 0.11 0.12 0.12 0.01 6h6 0.10 0.11 0.09 0.11 0.11 0.11 0.10 0.01 6 THP‐12 100% 30mM pH5.4 0.4mA THP‐24 100% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 AV SD Nh1 0.08 0.24 0.29 0.13 0.33 0.21 0.00 0.02 0.04 0.02 0.00 0.10 0.12 0.12 12h2 0.15 0.21 0.20 0.18 0.19 0.18 0.07 0.15 0.14 0.11 0.08 0.17 0.15 0.05 12h3 0.21 0.21 0.20 0.22 0.22 0.21 0.16 0.21 0.19 0.19 0.19 0.22 0.20 0.02 12h4 0.21 0.21 0.20 0.22 0.21 0.22 0.21 0.23 0.22 0.22 0.24 0.23 0.22 0.01 12h5 0.20 0.21 0.17 0.21 0.20 0.22 0.22 0.24 0.20 0.23 0.25 0.24 0.22 0.02 12

h6 0.19 0.20 0.20 0.20 0.20 0.20 0.20 0.22 0.18 0.19 0.21 0.18 0.20 0.01 12

Appendix V

236

Human skin THP flux [µmol/h]

THP 100% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 AV SD N h1 0.000 0.000 0.000 0.000 0.000 0.000 0.000 5 h2 0.000 0.000 0.000 0.000 0.000 0.000 0.000 5 h3 0.001 0.003 0.002 0.004 0.008 0.004 0.003 5 h4 0.007 0.038 0.011 0.022 0.037 0.023 0.014 5 h5 0.027 0.042 0.032 0.054 0.079 0.047 0.021 5 h6 0.068 0.069 0.055 0.071 0.144 0.082 0.036 5

Mannitol Flux [nmol/h]

THP 100% 30mM pH5.4 0.4mA R1 R2 R3 R4 R5 AV SD N h1 1.9 2.2 0.8 3.0 3.5 2.3 1.0 5 h2 1.3 1.7 1.6 3.4 2.4 2.1 0.8 5 h3 1.7 2.3 1.5 4.5 1.3 2.3 1.3 5 h4 0.0 0.9 2.0 2.3 0.1 1.1 1.1 5 h5 0.1 0.8 1.9 0.9 0.1 0.7 0.7 5 h6 0.0 2.9 0.1 2.8 0.9 1.3 1.4 5

THP conductivity C [mol/m3] σ [Si/m] C1/2

20.00 0.149 4.4715.00 0.112 3.8710.00 0.076 3.165.00 0.039 2.242.50 0.020 1.581.25 0.010 1.120.63 0.005 0.79

Appendix VI

237

Appendix VI. Entacapone Experiments Entacapone Fluxes [nmol/h] Current Effect

100% 30mM pH=7 0.1mA ETC-8 C1 C2 C3 C4 C5 C6 AV SD N h1 12.3 11.2 18.7 12.6 17.7 10.1 13.8 3.6 6 h2 8.4 8.3 9.4 7.9 8.2 8.5 8.4 0.5 6 h3 13.5 13.7 14.8 8.4 7.9 12.6 11.8 2.9 6 h4 19.9 22.6 22.4 12.0 9.7 19.7 17.7 5.5 6 h5 25.7 26.8 30.2 14.1 11.2 23.0 21.8 7.5 6 h6 29.1 33.0 36.2 20.2 15.6 27.8 27.0 7.8 6 100% 30mM pH=7 0.2mA ETC-9 C1 C2 C3 C4 C5 C6 AV SD N h1 21.5 20.4 21.0 0.8 2 h2 26.8 22.9 23.2 22.0 29.0 24.7 24.8 2.7 6 h3 47.8 34.4 37.4 34.2 53.8 40.5 41.3 7.9 6 h4 64.4 51.6 54.9 59.2 86.1 59.6 62.6 12.3 6 h5 66.7 65.7 68.1 86.3 104.4 70.8 77.0 15.4 6 h6 107.9 91.8 87.4 114.4 124.9 91.7 103.0 15.0 6 100% 30mM pH=7 0.4mA ETC-1 100% 30mM pH=7 0.4mA ETC-10 C1 C2 C3 C4 C5 C6 C1 C2 C3 C4 C5 C6 AV SD Nh1 17.7 16.2 23.5 0.0 58.1 21.7 13.7 14.4 13.1 19.8 15.8 9 h2 24.4 43.6 20.1 72.7 32.9 106.8 14.0 128.3 70.1 53.1 42.3 32.2 53.4 35.3 12 h3 46.7 77.6 39.9 134.0 75.3 148.6 23.1 165.1 126.0 121.8 92.2 56.3 92.2 46.3 12 h4 89.9 122.8 68.9 176.7 102.9 174.8 38.0 186.6 177.1 160.1 120.8 131.6 129.2 47.8 12 h5 127.0 123.5 99.8 204.7 151.6 201.7 63.7 217.2 221.7 193.9 157.9 117.8 156.7 51.4 12 h6 137.5 198.4 147.1 252.2 194.1 234.0 97.7 302.6 306.0 282.4 221.4 180.2 212.8 66.4 12

Appendix VI

238

Entacapone Fluxes [nmol/h] Moler Fraction Effect 25% 30mM pH=7 0.4mA ETC-4 C1 C2 C3 C4 C5 C6 AV SD N h1 31.8 29.6 23.1 23.1 21.3 25.8 4.6 5h2 59.4 25.4 52.3 49.3 55.9 49.3 48.6 12.0 6h3 83.6 37.0 70.9 77.4 80.9 74.4 70.7 17.1 6h4 100.4 55.1 89.0 88.3 100.5 93.5 87.8 16.9 6h5 109.0 76.1 93.2 93.2 107.4 108.8 98.0 13.0 6h6 69.3 94.0 86.4 79.9 67.5 103.9 83.5 14.2 6 50% 30mM pH=7 0.4mA ETC-3 C1 C2 C3 C4 C5 C6 AV SD N h1 10.6 12.1 9.1 6.2 15.6 3.1 9.5 4.4 6h2 93.2 73.5 63.3 44.7 83.9 62.0 70.1 17.3 6h3 111.1 102.8 99.3 71.4 120.1 96.6 100.2 16.5 6h4 152.4 130.4 109.8 96.6 146.6 111.7 124.6 22.2 6h5 156.8 138.3 132.6 124.6 159.0 137.9 141.5 13.6 6h6 162.0 168.3 140.1 174.0 147.3 180.4 162.0 15.6 6 75% 30mM pH=7 0.4mA ETC-2 C1 C2 C3 C4 C5 C6 AV SD N h1 40.8 31.8 40.7 47.0 37.3 47.8 40.9 6.0 6h2 124.3 116.6 132.6 119.8 119.6 128.4 123.5 6.1 6h3 160.3 203.8 178.6 169.6 197.1 167.4 179.5 17.4 6h4 148.8 231.2 215.2 223.3 228.7 199.3 207.8 31.1 6h5 252.8 237.2 261.5 215.9 284.4 233.2 247.5 24.0 6h6 233.0 277.4 284.8 257.0 267.8 240.7 260.1 20.5 6 100% 30mM pH=7 0.4mA ETC-1 C1 C2 C3 C4 C5 C6 h1 17.7 16.2 23.5 h2 24.4 43.6 20.1 72.7 32.9 106.8 h3 46.7 77.6 39.9 134.0 75.3 148.6 h4 89.9 122.8 68.9 176.7 102.9 174.8 AV SD N h5 127.0 123.5 99.8 204.7 151.6 201.7 19.8 15.8 9h6 137.5 198.4 147.1 252.2 194.1 234.0 53.4 35.3 12 100% 30mM pH=7 0.4mA ETC-10 92.2 46.3 12 C1 C2 C3 C4 C5 C6 129.2 47.8 12h1 0.0 58.1 21.7 13.7 14.4 13.1 156.7 51.4 12h2 14.0 128.3 70.1 53.1 42.3 32.2 212.8 66.4 12h3 23.1 165.1 126.0 121.8 92.2 56.3 h4 38.0 186.6 177.1 160.1 120.8 131.6 h5 63.7 217.2 221.7 193.9 157.9 117.8 h6 97.7 302.6 306.0 282.4 221.4 180.2

Appendix VI

239

Entacapone Fluxes [nmol/h] pH Effect

100% 30mM pH=7 0.4mA ETC-1 100% 30mM pH=7 0.4mA

ETC-10

C1 C2 C3 C4 C5 C6 C1 C2 C3 C4 C5 C6 AV SD N h1 17.7 16.2 23.5 0.0 58.1 21.7 13.7 14.4 13.1 19.8 15.8 9 h2 24.4 43.6 20.1 72.7 32.9 106.8 14.0 128.3 70.1 53.1 42.3 32.2 53.4 35.3 12 h3 46.7 77.6 39.9 134.0 75.3 148.6 23.1 165.1 126.0 121.8 92.2 56.3 92.2 46.3 12 h4 89.9 122.8 68.9 176.7 102.9 174.8 38.0 186.6 177.1 160.1 120.8 131.6 129.2 47.8 12 h5 127.0 123.5 99.8 204.7 151.6 201.7 63.7 217.2 221.7 193.9 157.9 117.8 156.7 51.4 12 h6 137.5 198.4 147.1 252.2 194.1 234.0 97.7 302.6 306.0 282.4 221.4 180.2 212.8 66.4 12

100% 30mM pH=8.5 0.4mA ETC-11

C1 C2 C3 C4 C5 AV SD N h1 0.0 18.7 14.4 15.8 0.0 9.8 9.0 5 h2 20.6 53.9 19.2 42.3 26.8 32.6 15.0 5 h3 63.8 109.4 45.7 80.4 76.8 75.2 23.4 5 h4 90.2 139.2 93.9 108.5 121.2 110.6 20.2 5 h5 108.9 189.3 122.1 148.0 142.1 35.4 4 h6 132.1 253.9 166.3 160.9 199.2 182.5 46.5 5

100% 30mM pH=10 0.4mA ETC-7

C1 C2 C3 C4 C5 C6 AV SD N h1 92.7 3.4 77.0 50.8 97.6 194.9 86.1 63.5 6 h2 264.7 30.0 360.4 185.7 241.2 234.0 219.3 109.2 6 h3 294.2 102.7 456.0 181.6 257.3 331.2 270.5 122.4 6 h4 307.3 125.3 465.6 231.6 301.1 630.6 343.6 179.2 6 h5 481.1 217.6 391.0 238.0 318.7 618.8 377.5 153.4 6 h6 569.2 356.6 377.4 274.5 337.1 505.9 403.4 111.2 6 h7 562.0 382.3 365.0 345.6 330.3 404.6 398.3 84.4 6

Appendix VI

240

Entacapone Conductivity CETC

[mM] C½ 1 [μSi/cm] 2 [μSi/cm] 3 [μSi/cm] AV [μSi/cm] AV [Si/m] Λ [Si·m2/mol]

9.9672 3.157 473.60 466.30 466.50 468.80 0.04688 0.00472.0139 1.419 109.30 110.00 109.80 109.70 0.01097 0.00541.023 1.011 55.73 56.10 56.98 56.27 0.00563 0.0055

0.5074 0.712 29.11 28.79 28.73 28.88 0.00289 0.00570.2015 0.449 11.72 11.86 11.91 11.83 0.00118 0.0059

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