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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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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101. Extensive gastrointestinal metabolic conversion limits the oral
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102. A controlled trial of rotigotine monotherapy in early Parkinson's disease.
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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
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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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114. A randomized, double-blind study of a skin patch of a dopaminergic
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115. Transdermal lisuride delivery in the treatment of Parkinson's disease.
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119. Transdermal ion migration. Phipps, B and Gyory, R. 1992, Advanced
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121. Theoretical models of iontophoretic delivery. Kastings, G B. 1992,
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124. Quantitative structure-permeation relationship for iontophoretic transport
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125. Iontophoretic delivery of ropinirole hydrochloride: effect of current density
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Introduction
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138. Iontophoretic delivery of apomorphine II: an in vivo study in patients with
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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
Chapter I Selegiline
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
Chapter I Selegiline
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
Chapter I Selegiline
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
Chapter I Selegiline
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.
Chapter I Selegiline
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.
Chapter I Selegiline
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).
Chapter I Selegiline
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
Chapter I Selegiline
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.
Chapter I Selegiline
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).
Chapter I Selegiline
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
Chapter I Selegiline
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
Chapter I Selegiline
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
Chapter I Selegiline
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
Chapter I Selegiline
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
Chapter I Selegiline
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.
Chapter I Selegiline
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
Chapter I Selegiline
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.
Chapter I Selegiline
61
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5. SELEDO: a 5-year long-term trial on the effect of selegiline in early
parkinsonian patients treated with levodopa. Przuntek, H, et al. 1999, European
Journal of Neurology, Vol. 6, pp. 141-150.
6. Investigation by Parkinson's Disease Research Group of United Kingdom
into excess mortality seen with combined levodopa and selegiline treatment in
patients with early, mild Parkinson's disease: further results of randomised trial and
confidential inquiry. Ben-Shlomo, Y, et al. BMJ, Vol. 316, pp. 1191-1196.
7. Fourteen-year final report of the randomized PDRG-UK trial comparing
three initial treatments in PD. Katzenschlager, R, et al. 2008, Neurology, Vol. 71,
pp. 474-480.
8. Selegiline in de novo parkinsonian patients: the French selegiline
multicenter trial (FSMT). Allain, H, Cougnard, J and Neukirch, H C. Suppl. 136,
1991, Acta Neurologica Scandinavica, Vol. 84, pp. 73-78.
Chapter I Selegiline
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.
13. Pharmacokinetics and absolute bioavailability of selegiline following
treatment of healthy subjects with the selegiline transdermal system (6 mg/24 h): a
comparison with oral selegiline capsules. Azarro, A J, et al. 2007, Journal of
Clinical Pharmacology, Vol. 47, pp. 1256-1267.
14. Multiple dose pharmacokinetics of selegiline and desmethylselegiline
suggest saturable tissue binding. Laine, K, et al. 2000, Clinical Neurology, Vol. 23,
pp. 23-27.
15. A new low-dose formulation of selegiline: clinical efficacy, patient
preference and selectivity for MAO-B inhibition. Clarke, A, et al. 2003, Journal of
Neural Transmission, Vol. 110, pp. 1257-1271.
16. Iontophoretic drug delivery. Kalia, Y N, et al. 2004, Advanced Drug
Delivery Reviews, Vol. 56, pp. 619-658.
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.
Chapter I Selegiline
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.
25. Theoretical models of iontophoretic delivery. Kastings, G B. 1992,
Advanced drug delivery rewievs, Vol. 9, pp. 177-199.
26. 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.
27. Characterisation of the iontophoretic permselectivity properties of human
and pig skin. Marro, D and Delgado-Charro, M B. 2001, Journal of Controlled
Release, Vol. 70, pp. 213-217.
28. 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.
Chapter I Selegiline
64
29. In vitro and in vivo evaluation of transdermal iontophoretic delivery of
hydromorphone. Padmanabhan, R V, Phipps, J B and Lattin, G A. 1990, Journal
of Controlled Release, Vol. 11, pp. 123-135.
30. Quantitative structure-permeation relationship for iontophoretic transport
across the skin. Mudry, B, et al. 2007, Journal of Controlled Release, Vol. 122, pp.
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,
Pharmaceutical Research, Vol. 21, pp. 844-850.
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.
38. The role of electroosmotic flow in transdermal iontophoresis. Pikal, M J. 2001, Advanced Drug Delivery Reviews, Vol. 46, pp. 281-305.
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
Chapter II Pramipexole
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.
Chapter II Pramipexole
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
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
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
Chapter II Pramipexole
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
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
Chapter II Pramipexole
69
Effect of competing ions
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
Chapter II Pramipexole
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
Chapter II Pramipexole
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
Chapter II Pramipexole
72
Equation 4 −−Λ
=∞
ClPPX Fµµ
Equation 5 −+
=ClPPX
PPXPPXt µµ
µ
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
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
Chapter II Pramipexole
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
Chapter II Pramipexole
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 ⋅=
Chapter II Pramipexole
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
Chapter II Pramipexole
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.
Chapter II Pramipexole
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
Chapter II Pramipexole
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#
Chapter II Pramipexole
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
Chapter II Pramipexole
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
#
Chapter II Pramipexole
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.
Chapter II Pramipexole
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
Current intensity [mA]
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.
Chapter II Pramipexole
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
Chapter II Pramipexole
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
Chapter II Pramipexole
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.
Chapter II Pramipexole
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.
Chapter II Pramipexole
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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1710-1713.
24. 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.
25. Iontophoretic permselectivity of mammalian skin: characterisation of hairless
mouse and porcine membrane models. Luzardo-Alvarez, A, et al. 1998,
Pharmaceutical Research, Vol. 15, pp. 984-987.
26. In vivo iontophoretic administration of ropinirole hydrochloride. Luzardo-Alvarez, A, Delgado-Charro, M B and Blanco-Méndez, J. 2003, Journal of
Pharmaceutical Sciences, Vol. 92, pp. 2441-2448.
27. Contributions of electromigration and electroosmosis to iontophoretic drug
delivery. Marro, D, et al. 2001, Pharmaceutical Research, Vol. 18, pp. 1701-1708.
28. The role of electroosmotic flow in transdermal iontophoresis. Pikal, M J. 2001,
Advanced Drug Delivery Reviews, Vol. 46, pp. 281-305.
29. Transport mechanisms in iontophoresis. III. An experimental study of the
contributions of electroosmotic flow and permeability change in transport of low and
high molecular weight solutes. Pikal, M J and Shah, S. 1990, Pharmaceutical
Research, Vol. 7, pp. 222-229.
30. 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.
31. Effect of charge and molecular weight on transdermal peptide delivery by
iontophoresis. Abla, N, et al. 2005, Pharmaceutical Reserach, Vol. 22, pp. 2069-
2078.
32. Electrorepultion versus electroosmosis: effect of pH on the iontophoretic flux of
5-fluorouracil. Merino, V, et al. 1999, Pharmaceutical Research, Vol. 16, pp. 758-
761.
Chapter II Pramipexole
91
33. 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.
34. Characterisation of the iontophoretic permselectivity properties of human and
pig skin. Marro, D and Delgado-Charro, M B. 2001, Journal of Controlled Release,
Vol. 70, pp. 213-217.
35. 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.
36. In vivo studies concerning a pH gradient in human stratum corneum and upper
epidermis. Ohman, H and Vahlquist, A. 1994, Acta Dermato-Venerologica, Vol.
74, pp. 375-379.
37. Dopamine agonists in Parkinson’s disease. Yamamoto, M and Anthony, S. 2008, Expert Reviews, Vol. 8, pp. 671-677.
38. Clinical pharmacokinetic and pharmacodynamic properties of drugs used in
Parkinson's disease. Deleu, D, Northway, M G and Hanssens, Y. 2002, Clinical
Pharmacokinetics, Vol. 41, pp. 261-309.
39. Tetko, I V, et al. ALOGPS 2.1 home page. Virtual Computational Chemistry
Laboratory. [Online] [Cited: 10 May 2006.] http://www.vcclab.org.
40. Transdermal ion migration. Phipps, B and Gyory, R. 1992, Advanced Drug
Delivery Reviews, Vol. 9, pp. 137-176.
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
Chapter III Piribedil
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
Chapter III Piribedil
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
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 were then
wrapped in Parafilm® and stored at -20°C for no longer than four months. Both,
dorsal and abdominal skin, from two different pigs was used in the experiments.
Iontophoresis 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.
Chapter III Piribedil
95
Single ion experiments
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]
Mannitol Conc. [mM] pH
Current Intensity
[mA] n*
Concentration effect
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
Effect of competing ions
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
Chapter III Piribedil
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
Chapter III Piribedil
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):
Chapter III Piribedil
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
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 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
Chapter III Piribedil
99
replicateds with standard deviation. All regressions were performed using the least
square method, and regression significance was tested with the runs test.
Results and discussion
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
]
Chapter III Piribedil
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
Chapter III Piribedil
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).
Chapter III Piribedil
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.
Chapter III Piribedil
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]
Chapter III Piribedil
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.
Chapter III Piribedil
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
Current intensity [mA]
Flux
[µm
ol/h
]
Chapter III Piribedil
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:
Chapter III Piribedil
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.
Chapter III Piribedil
108
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Chapter III Piribedil
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28. The role of electroosmotic flow in transdermal iontophoresis. Pikal, M J. 2001,
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32. Effect of amino acid sequence on transdermal iontophoretic peptide delivery.
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33. Contributions of electromigration and electroosmosis to iontophoretic drug
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34. Transdermal ion migration. Phipps, B and Gyory, R. 1992, Advanced drug
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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.
Chapter IV Pergolide
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
Chapter IV Pergolide
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 and methods
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
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
wrapped in Parafilm® and stored at -20°C for no longer than four months. Both,
dorsal and abdominal skin, from two different pigs was used in the experiments.
Iontophoresis 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
Chapter IV Pergolide
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.
Chapter IV Pergolide
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]
Total Cation 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
10:90 to 95:5 in 10min) was performed using a Phenomenex Luna™ HILIC, HPLC
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
Chapter IV Pergolide
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):
Equation 1 )101log(loglog pHpKaPD −+−=
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.
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 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 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.
Chapter IV Pergolide
118
Results and discussion
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
Chapter IV Pergolide
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
Chapter IV Pergolide
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
Current intensity [mA]
Perg
olid
e Fl
ux [n
mol
/h]
Chapter IV Pergolide
121
Single-ion experiments
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
Chapter IV Pergolide
122
presence of background electrolyte does not have a major negative impact on the
magnitude of the drug transdermal flux.
Bibliography
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Chapter IV Pergolide
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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
Chapter V Trihexyphenidyl
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
Chapter V Trihexyphenidyl
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
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
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.
Iontophoresis 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
Chapter V Trihexyphenidyl
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
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]
Mannitol Conc. [mM] pH
Current Intensity
[mA] n*
Concentration effect
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
Effect of competing ions
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
Chapter V Trihexyphenidyl
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]
Total Cation 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
Chapter V Trihexyphenidyl
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
10:90 to 95:5 in 10min) was performed using a Phenomenex Luna™ HILIC, HPLC
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
Chapter V Trihexyphenidyl
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).
Equation 2 )101log(loglog pHpKaPD −+−=
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
Chapter V Trihexyphenidyl
132
Equation 5 −+
=ClTHP
THPTHPt µµ
µ
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 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 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.
Results and discussion
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.
Chapter V Trihexyphenidyl
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
]
Chapter V Trihexyphenidyl
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]
Chapter V Trihexyphenidyl
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
Chapter V Trihexyphenidyl
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.
Concentration effect
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).
Chapter V Trihexyphenidyl
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
Current intensity [mA]
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
Chapter V Trihexyphenidyl
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.
Chapter V Trihexyphenidyl
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
Chapter V Trihexyphenidyl
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
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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.
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Chapter V Trihexyphenidyl
141
5. Clinical pharmacokinetic and pharmacodynamic properties of drugs used in
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7. Anticholinergic drugs: response of parkinsonism not responsive to
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8. Anticholinergic drugs used in Parkinson's disease: An overlooked class of
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9. Pharmacokinetics of trihexyphenidyl after long-term and short-term
administration to dystonic patients. Burke, R E and Fahn, S. 1985, Annals of
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10. Development and application of a specific and sensitive
radioimmunoassay for trihexyphenidyl to a pharmacokinetic study in humans. He, H, et al. 1995, Journal of Pharmaceutical Sciences, Vol. 84, pp. 561-567.
11. Metabolism and urinary excretion of benzhexol in humans. Nation, R L; Triggs, E J; Vine, J;. 1978, Xenobiotica, Vol. 8, pp. 165-169.
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14. Moffat, A C, Osselton, M D and Widdop, B. Clarke's Analysis of Drugs
and Poisons 2004. London : Pharmaceutical Press, 2004.
Chapter V Trihexyphenidyl
142
15. 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.
16. Partition coefficients and their uses. Albert, L, Corwin, H and David, E. 1971, Chemistry Reviews, Vol. 71, pp. 525-616.
17. Physicochemical properties and transport behaviour of piribedil:
Considerations on its membrane-crossing potential. Tsai, R, et al. 1992,
International Journal of Pharmaceutics, Vol. 80, pp. 39-49.
18. Atkins, P W. Molecules in motion: ion transport and molecular diffusion.
Physical Chemistry. 6th ed. Oxford : Oxford University Press, 1978, pp. 723-759.
19. Young, T F. Electrochemical Information. [book auth.] Bruce H Billings, et
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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-
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21. Lidocaine Iontophoresis Versus Eutectic Mixture of Local Anesthetics
(EMLA®) for IV Placement in Children. Galinkin, J, et al. 2002, Anesthesia and
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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
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24. Reverse iontophoresis - Parameters determining electroosmotic flow: I.
pH and ionic strength. Santi, P and Guy, R H. 1995, Journal of Controlled Release,
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Chapter V Trihexyphenidyl
143
25. Characterisation of the iontophoretic permselectivity properties of human
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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.
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31. Transdermal ion migration. Phipps, B and Gyory, R. 1992, Advanced
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33. In vitro and in vivo evaluation of transdermal iontophoretic delivery of
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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
Chapter VI Entacapone
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
Chapter VI Entacapone
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 and methods
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
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
wrapped in Parafilm® and stored at -20°C for no longer than four months. Both,
dorsal and abdominal skin from two different pigs was used in the experiments.
Iontophoresis 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
the epidermal side facing the donor chamber. The volume of both cells was 3.3ml
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
Chapter VI Entacapone
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
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
Effect of competing ions
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
Chapter VI Entacapone
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]
Total Cation Conc. [mM]
pH Current Intensity
[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).
Equation 1 )101log(loglog pHpKaPD −+−=
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
Chapter VI Entacapone
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
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 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 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.
Chapter VI Entacapone
150
Results and discussion
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
]
Chapter VI Entacapone
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
Current intensity [mA]
Enta
capo
ne F
lux
[nm
ol/h
]
Chapter VI Entacapone
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
Chapter VI Entacapone
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),
Chapter VI Entacapone
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.
Chapter VI Entacapone
155
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14. Physicochemical properties and transport behaviour of piribedil:
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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,
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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. 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.
18. Characterisation of the iontophoretic permselectivity properties of human
and pig skin. Marro, D, Guy, R H and Delgado-Charro, M B. 2001, Journal of
Controlled Release, Vol. 70, pp. 213-217.
Chapter VI Entacapone
157
19. Effect of charge and molecular weight on transdermal peptide delivery by
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2078.
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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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24. In vitro and in vivo evaluation of transdermal iontophoretic delivery of
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Controlled Release, Vol. 11, pp. 123-135.
25. Iontophoretic delivery of ropinirole hydrochloride: effect of current density
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26. Contributions of electromigration and electroosmosis to iontophoretic drug
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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
Chapter VII Human skin
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 and methods
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.
Chapter VII Human skin
160
Iontophoresis experiments
The skin 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 (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]
Total Cation Conc. [mM]
pH Current Intensity
[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
NCHTB ID 2004B/203 NCHTB ID 2004B/230 NCHTB ID 2004B/239 NCHTB ID 2005B/137 NCHTB ID 2004B/35
PBD 100% 30 0 30 5.0 0.4 30
NCHTB ID 2004B/240 NCHTB ID 2004B/229 NCHTB ID 2005B/39 NCHTB ID 2005B/48 NCHTB ID 2005B/228
THP 100% 30 0 30 5.4 0.4 30
NCHTB ID 2005B/46 NCHTB ID 2005B/136 NCHTB ID 2004B/204 NCHTB ID 2005B/164 NCHTB ID 2004B/211
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
Chapter VII Human skin
161
Analytical Methods
Analytical methods, used to quantify each drug were described in previous
chapters referring to each drug or electroosmotic marker.
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 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. 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.
Results and discussion
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
Chapter VII Human skin
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
Chapter VII Human skin
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.
Mannitol, pH5.0, 30mM, 100%
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
Mannitol, pH5.4, 30mM, 100%
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.
Mannitol, pH4.0, 50mM, 5%
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.
Chapter VII Human skin
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
Chapter VII Human skin
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
Chapter VII Human skin
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
Chapter VII Human skin
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.
Chapter VII Human skin
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.
Chapter VII Human skin
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.
Chapter VII Human skin
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
Chapter VII Human skin
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
Chapter VII Human skin
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.
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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
General discussion and conclusions
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).
General discussion and conclusions
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.
General discussion and conclusions
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
General discussion and conclusions
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 ‐‐
General discussion and conclusions
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
General discussion and conclusions
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
General discussion and conclusions
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.
General discussion and conclusions
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
General discussion and conclusions
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
General discussion and conclusions
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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General discussion and conclusions
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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 AV SD N 1 0.1 0.0 0.0 0.1 0.0 0.1 0.05 0.03 6 2 0.4 0.3 0.2 0.3 0.3 0.4 0.32 0.06 6 3 0.6 0.5 0.4 0.4 0.5 0.6 0.50 0.09 6 4 0.5 0.4 0.7 0.6 0.8 0.6 0.61 0.11 6 5 0.5 0.5 0.7 0.8 0.9 0.7 0.69 0.14 6 6 0.7 0.8 0.7 0.7 0.5 0.7 0.70 0.10 6 SEL‐05 30mM pH=4.5 100% 0.4mA
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
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 0.5 0.5 1.5 1.9 0.4 0.5 1.0 1.1 1.1 1.0 0.9 0.8 0.92 0.46 12 2 1.4 1.5 1.6 2.0 1.3 1.2 1.6 1.8 1.5 1.6 1.6 1.4 1.55 0.21 12 3 1.3 1.4 1.3 1.5 1.4 1.2 1.6 1.9 1.5 1.6 1.5 1.6 1.47 0.18 12 4 1.4 1.4 1.2 2.1 1.5 1.2 1.6 1.9 1.4 1.6 1.5 1.6 1.52 0.26 12 5 1.3 1.5 1.2 1.9 1.5 1.3 1.5 1.9 1.3 1.5 1.5 1.6 1.50 0.21 12 6 1.3 1.5 1.3 1.6 1.5 1.4 1.5 2.0 1.3 1.5 1.5 1.6 1.50 0.19 12
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
t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 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
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 N 1 0.0 0.4 0.2 0.2 0.3 0.3 0.2 0.1 6 2 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.0 6 3 0.4 0.3 0.3 0.4 0.3 0.2 0.3 0.1 6 4 0.3 0.2 0.3 0.4 0.3 0.3 0.3 0.1 6 5 0.4 0.2 0.3 0.4 0.3 0.3 0.3 0.1 6 6 0.3 0.2 0.4 0.4 0.5 0.4 0.4 0.1 6 SEL‐42 30mM pH=3.5 100% 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
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 6.82 8.00 6 2 20.6 22.8 11.1 9.2 8.2 9.9 13.63 6.37 6 3 21.4 12.7 14.5 13.3 12.5 12.3 14.44 3.52 6 4 17.5 17.6 13.0 9.8 10.3 9.2 12.91 3.80 6 5 11.2 13.3 9.7 8.3 9.3 6.6 9.73 2.31 6 6 8.4 10.9 8.6 7.1 8.5 5.9 8.23 1.68 6
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
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
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
t[h] Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Mean SD N1 5.0 2.6 3.7 2.8 3.7 3.9 3.61 0.85 6 2 14.5 13.7 14.7 11.7 9.1 12.1 12.62 2.13 6 3 17.2 19.1 16.8 17.0 11.6 15.1 16.14 2.54 6 4 17.3 22.8 19.3 19.9 10.0 15.7 17.51 4.38 6 5 15.6 17.1 14.1 15.2 7.9 13.6 13.90 3.19 6 6 17.8 19.3 16.2 16.3 8.8 14.3 15.46 3.68 6
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
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‐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
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‐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
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 4.2 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
PPX‐27 100% 90mM pH=8 0.4mA R1 R2 R3 R4 R5 AV SD N
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
PPX‐22 100% 30mM pH=8 0.1mA 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 0.01 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
R1 R2 R3 R4 R5 R6 AV SD N
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
PPX‐23 100% 30mM pH=8 0.2mA R1 R2 R3 R4 R5 R6 AV SD N
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
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 4.2 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
R1 R2 R3 R4 R5 AV SD N h1 0.29 0.39 0.31 0.42 0.34 0.35 0.05 5 h2 0.51 0.64 0.53 0.67 0.60 0.59 0.07 5 h3 0.53 0.70 0.62 0.62 0.67 0.63 0.06 5 h4 0.58 0.66 0.64 0.62 0.68 0.63 0.04 5 h5 0.56 0.67 0.54 0.63 0.69 0.62 0.07 5 h6 0.56 0.68 0.60 0.62 0.68 0.63 0.05 5
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
PPX‐30 75% 30mM pH=8 R1 R2 R3 R4 R5 R6 AV SD N
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
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 4.2 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]
PPX‐24 10% 30mM pH=5 R1 R2 R3 R4 R5 R6 AV SD N
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
PPX‐31 50% 30mM pH=5 R1 R2 R3 R4 R5 R6 AV SD N
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
PPX‐32 75% 30mM pH=5 R1 R2 R3 R4 R5 R6 AV SD N
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
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 0.8 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
R1 R2 R3 R4 R5 AV SD N h1 9.1 5.7 6.1 2.8 4.7 5.7 2.3 5 h2 7.9 4.3 7.5 4.9 11.7 7.3 2.9 5 h3 11.8 6.3 7.6 6.4 12.8 9.0 3.1 5 h4 16.6 12.3 9.2 11.2 12.6 12.4 2.7 5 h5 15.4 14.1 9.1 11.1 12.0 12.3 2.5 5 h6 10.6 11.9 8.3 11.2 12.7 11.0 1.7 5
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 0.0 0.0 5 h2 2.5 1.9 1.6 2.3 0.7 1.8 0.7 5 h3 2.3 2.1 2.0 3.2 0.6 2.0 0.9 5 h4 3.4 3.8 2.8 3.0 2.7 3.1 0.5 5 h5 3.5 3.1 2.9 3.3 2.1 3.0 0.5 5 h6 4.2 3.5 3.7 3.7 3.3 3.7 0.3 5
Concentration Effect, co‐ion Pramipexole Flux [µmol/h]
PPX‐4 50% 30mM pH=8 0.4mA R1 R2 R3 R4 R5 R6 AV SD N
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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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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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
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
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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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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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
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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
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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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Current effect Mannitol flux [nmol/h]
PER‐7 5% 50mM pH4.0 0.1mA
R1 R2 R3 R4 R5 R6 AV SD N
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
R1 R2 R3 R4 R5 R6 AV SD N
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
R1 R2 R3 R4 R5 R6 AV SD N
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
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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
PER‐10 25% 29mM pH4.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N
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
PER‐1 50% 29mM pH4.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N
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
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 00.00* 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
PER‐2 10% 50mM pH4.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N
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
R1 R2 R3 R4 R5 R6 AV SD N
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
R1 R2 R3 R4 R5 R6 AV SD N
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
R1 R2 R3 R4 R5 R6 AV SD N
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
R1 R2 R3 R4 R5 R6 AV SD N
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]
PER‐5 100% 1mM pH5.0 0.4mA R1 R2 R3 R4 R5 R6 AV SD N
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
R1 R2 R3 R4 R5 R6 AV SD N
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
R1 R2 R3 R4 R5 R6 AV SD N
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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