Biophysical Techniques of Transcutaneous Drug Sampling …

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Biophysical Techniques of Transcutaneous Drug Sampling and Biophysical Techniques of Transcutaneous Drug Sampling and

Drug Delivery Drug Delivery

Srinivasa Murthy Sammeta

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BIOPHYSICAL TECHNIQUES OF TRANSCUTANEOUS DRUG SAMPLING AND DRUG

DELIVERY

A Dissertation

presented in partial fulfillment of requirements

for the degree of Doctor of philosophy

in the Department of Pharmaceutics

The University of Mississippi

By

SRINIVASA MURTHY SAMMETA

March 2011

Copyright Srinivasa Murthy Sammeta 2011

ALL RIGHTS RESERVED

ii

ABSTRACT

Monitoring the time course of drug in the skin is critical for determining the frequency

and dose of drug administration from safety and efficacy perspectives. Intermittent blood

sampling is used as a surrogate for approximating concentration of drugs in the tissues, which

leads to blood loss and discomfort to patients. A novel noninvasive technique called

“Electroporation and transcutaneous sampling” (ETS) was developed for estimating the drug

concentration in the skin extracellular fluid. The application of ETS technique in studying

dermatokinetics of cephalexin, ciprofloxacin, and 8-methoxypsoralen was investigated. The

results demonstrated the ability of ETS technique in dermatokinetic studies of drugs with

different physicochemical properties. ETS technique was also found to be a promising method

for noninvasive estimation of blood glucose levels.

Two novel techniques were developed for enhancing the transdermal delivery of drugs.

“ChilDrive”, a technique of combining regional cutaneous hypothermia with iontophoresis was

used for enhancing the bioavailability of transdermally administered drug in the deeper

musculoskeletal tissue like synovial fluid. The bioavailability of drugs in the synovial fluid of

knee joint was enhanced by ~6-12-fold and ~2-4-fold by ChilDrive when compared to passive

and iontophoretic transdermal drug delivery.

Magnetophoresis, a technique of enhancing transdermal drug delivery by application of

magnetic field was developed. The mechanistic studies demonstrated that transdermal

magnetophoresis of drugs was due to contribution of multiple factors such as magnetorepulsion,

magnetohydrokinesis and magnetically enhanced partition coefficient. Magnetophoretic patch

iii

system was designed and pharmacokinetic studies were performed. Magnetophoresis resulted in

higher dermal bioavailability of drugs compared to passive transdermal drug delivery. It was also

found from the in vitro studies that combination of chemical enhancers would further enhance

the efficiency of magnetophoretically mediated drug delivery enhancement.

iv

DEDICATION

Dedicated to my Amma and Nanna

v

ACKNOWLEDGEMENTS

I owe my deepest gratitude to my advisor, Dr. S. Narasimha Murthy for his continual

support and encouragement throughout my dissertation. I thank him for believing in me and

giving me an opportunity to work on various novel research topics. My progress during PhD

program at The University of Mississippi is only because of the unrelenting approach of Dr.

Narasimha Murthy.

I would like to thank Dr. Michael A. Repka, Dr. Soumyajit Majumdar, Dr. Seongbong Jo,

and Dr. John S. Williamson for being in my dissertation committee and evaluating my

dissertation with helpful suggestions. My special thanks to Dr. Michael A. Repka, Chair of

Pharmaceutics department for his endearing support. I would also like to thank Ms. Debbie P.

King, Staff Assistant for her help with all the departmental procedures.

Thanks to all the graduate students in pharmaceutics department for making my stay in

oxford enjoyable. I would like to thank Siva Ram Kiran Vaka, Dr. Anroop and Dr. Shiva Kumar

for helping in my research projects. My thanks to Dr. Babu Tekwani, Dr. Franck Dayan, Dr.

Chris Bowers, and Mr. Thomas Jamerson for access to their laboratory facilities.

Above all, I would like to thank my parents (Satyavathi and Nageswara Rao) for making

my dreams come true and my brother (Sree Ram), sister (Sirisha), brother-in-law (Kondal Rao)

and sister-in-law (Ajitha) who played a key role in my career.

vi

TABLE OF CONTENTS

ABSTRACT …………………………………………………………………………………… ii

DEDICATION………………………………………………………………………………….. iv

ACKNOWLEDGEMENTS…………………………………………………………………….. v

LIST OF TABLES…………………………………………………………………………… viii

LIST OF FIGURES……………………………………………………………………………… x

INTRODUCTION……………………………………………………………………………… 1

DERMAL DRUG LEVELS OF ANTIBIOTIC (CEPHALEXIN) DETERMINED BY

ELECTROPORATION AND TRANSCUTANEOUS SAMPLING (ETS) TECHNIQUE

………………………………………………………………………………..................................9

TRANSCUTANEOUS SAMPLING OF CIPROFLOXACIN AND 8-

METHOXYPSORALEN BY ELECTROPORATION (ETS TECHNIQUE)

………………………………………………………………………………................................26

NONINVASIVE TRANSCUTANEOS SAMPLING OF GLUCOSE BY

ELECTROPORATION..…………………………………………………………………… .......42

“CHILDRIVE”: A TECHNIQUE OF COMBINING REGIONAL CUTANEOUS

HYPOTHERMIA WITH IONTOPHORESIS FOR THE DELIVERY OF DRUGS TO

SYNOVIAL FLUID……………………………………………………………………… ..........52

MAGNETOPHORESIS FOR ENHANCING TRANSDERMAL DRUG DELIVERY:

MECHANISTIC STUDIES AND PATCH DESIGN…………………… ...................................68

vii

MAGNETOPHORESIS IN COMBINATION WITH CHEMICAL ENHANCERS FOR

TRANSDERMAL DRUG DELIVERY…………………… ........................................................90

REFERENCES…………………… ............................................................................................109

APPENDIX…………………… ..................................................................................................132

VITA…………………… ............................................................................................................134

viii

LIST OF TABLES

2-1. Pharmacokinetic parameters derived from the plasma concentration-time data after i.v. bolus

administration of 20 mg/kg of cephalexin in hairless rats ....................................................20

2-2. Mean pharmacokinetic parameters of cephalexin determined by ETS, microdialysis

techniques following administration of 20 mg/kg by i.v. bolus in hairless rats ...................21

2-3. Mean pharmacokinetic parameters of cephalexin determined by intraarticular microdialysis

in the synovial fluid following administration of 20 mg/kg by i.v. bolus in hairless rats ....22

3-1. Mean pharmacokinetic parameters of ciprofloxacin (unbound) in plasma and dermal ECF

(determined by ETS and microdialysis) following administration of 15 mg/kg by i.v. bolus

in hairless rats .......................................................................................................................38

3-2. Mean pharmacokinetic parameters of 8-methoxypsoralen (unbound) in plasma and dermal

ECF (determined by ETS and microdialysis) following oral administration of 5 mg/kg in

hairless rats............................................................................................................................40

5-1. AUC0-t and Cmax of diclofenac sodium in synovial fluid following transdermal (Passive,

Chil-Passive, Iontophoresis, ChilDrive), intravenous and intraarticular administration ......63

5-2. AUC0-t and Cmax of prednisolone sodium phosphate in synovial fluid following transdermal

(Passive, Chil-Passive, Iontophoresis, ChilDrive), intravenous and intraarticular

administration .......................................................................................................................64

6-1. Permeation flux and flux enhancement factor of LH and LB across porcine epidermis .......78

6-2. Permeation flux of LH and LB across pretreated porcine epidermis .....................................80

ix

6-3. AUC0-6h and Cmax of LH and LB in the skin extracellular fluid following transdermal

application of passive and magnetophoretic patch systems in vivo in rats ...........................88

7-1. Transdermal permeation flux of lidocaine hydrochloride from non-magnetophoretic and

magnetophoretic patch system ..............................................................................................99

7-2. Steady state flux of lidocaine hydrochloride, estradiol, and ferric pyrophosphate following

passive permeation and iontophoretic delivery across sandwich and single layer epidermis

with corresponding flux ratios ............................................................................................107

x

LIST OF FIGURES

1-1. Structure of skin........................................................................................................................1

2-1. Diagrammatic representation showing an experimental setup of electroporation and

transcutaneous sampling in hairless rats ...............................................................................14

2-2. Correlation between cephalexin concentration (5-40 µg/ml) in the reservoir compartment

and cephalexin sampled by ETS across hairless rat skin in vitro .........................................18

2-3. Time course of cephalexin in rat plasma () determined by blood sampling and dermal ECF

determined by microdialysis () and ETS () techniques following i.v. bolus

administration of 20 mg/kg of cephalexin ............................................................................19

2-4. Concentration-time profile of cephalexin in synovial fluid obtained by intraarticular

microdialysis after administration of 20 mg/kg of drug by i.v. bolus...................................22

2-5. Correlation between cephalexin levels in dermal ECF obtained from ETS with that of

synovial fluid drug levels ......................................................................................................23

2-6.Correlation between cephalexin levels in dermal ECF obtained from microdialysis with that

of synovial fluid drug levels .................................................................................................24

3-1. Correlation between ciprofloxacin concentration (2.5-40 µg/ml) in the reservoir

compartment and the amount sampled by ETS across hairless rat skin in vitro ..................34

3-2. Correlation between 8-methoxypsoralen concentration (2.5-40 µg/ml) in the reservoir

compartment and the amount sampled by ETS across hairless rat skin in vitro ...................35

xi

3-3. Time course of ciprofloxacin in rat plasma (-total drug; ♦-unbound drug) determined by

blood sampling and dermal ECF determined by microdialysis () and ETS () techniques

following i.v. bolus administration of 15 mg/kg of ciprofloxacin ........................................38

3-4. Time course of 8-methoxypsoralen in rat plasma (-total drug; ♦-unbound drug) determined

by blood sampling and dermal ECF determined by microdialysis () and ETS ()

technique following oral administration of 5 mg/kg of 8-methoxypsoralen ........................39

4-1. Relationship between glucose concentration in the reservoir compartment and glucose

sampled by ETS across porcine epidermis in vitro ...............................................................48

4-2. Clarke error grid analysis of venous blood glucose (reference) and venous blood glucose

predicted by ETS in Sprague-Dawley rats ............................................................................50

5-1. Skin and subdermal tissues .....................................................................................................55

5-2. Microdialysis probe implanted in the knee-joint for carrying out synovial fluid microdialysis

in hind limb of rat .................................................................................................................57

5-3. Design of iontophoretic patch with 'A' displaying the dorsal view and 'B' displaying the

ventral view ...........................................................................................................................59

5-4. Time course of diclofenac sodium in synovial fluid upon intraarticular () and intravenous

() administration .................................................................................................................62

5-5. Time course of prednisolone sodium phosphate in synovial fluid upon intraarticular () and

intravenous () administration ..............................................................................................63

5-6. Time course of diclofenac sodium in the synovial fluid following different modes of

transdermal administration (♦-Passive, -Chil-Passive, -Iontophoresis and -ChilDrive).

...............................................................................................................................................66

xii

5-7. Time course of prednisolone sodium phosphate in the synovial fluid following different

modes of transdermal administration (♦-Passive, -Chil-Passive, -Iontophoresis and -

ChilDrive). ...........................................................................................................................67

6-1. Experimental setup for permeation studies ............................................................................72

6-2. Cumulative permeation of LH across porcine epidermis in case of passive (♦), magnetic

field strength of 30 mT (), 150 mT () and 300 mT (). ..................................................78

6-3. Representative FT-IR spectra of porcine epidermis in absence of magnetic field (A),

presence of magnetic field (B) and after exposure to magnetic field for 80 h (C) ...............81

6-4. Photographs of magnetophoretic patch system used for in vivo studies ................................86

6-5. Time course of LH in the skin extracellular fluid following application of passive

transdermal patch () and magnetophoretic transdermal patch () in rats ...........................87

6-6. Time course of LB in the skin extracellular fluid following application of passive

transdermal patch () and magnetophoretic transdermal patch () in rats ...........................88

7-1. Transdermal permeation flux of lidocaine hydrochloride from non-magnetophoretic patch

systems ..................................................................................................................................97

7-2. Sandwich epidermis model ...................................................................................................100

7-3. Passive permeation of lidocaine hydrochloride across single layer () and sandwich

epidermis () .......................................................................................................................102

7-4. Passive permeation of estradiol across single layer () and sandwich epidermis () ..........103

7-5. Iontophoretic delivery of lidocaine hydrochloride across single layer () and sandwich

epidermis () .......................................................................................................................105

7-6. Iontophoretic delivery of estradiol across single layer () and sandwich epidermis () .....105

xiii

7-7. Iontophoretic delivery of ferric pyrophosphate across single layer () and sandwich

Epidermis () .....................................................................................................................106

1

CHAPTER-1

INTRODUCTION

1.1. Skin

Skin is the outermost organ of the body which acts as a barrier protecting the body from

physical, chemical or microbial attacks, ultraviolet radiation, acts as thermostat in maintaining

body temperature and prevents water loss from the body. Skin comprises of two distinct regions;

the epidermis and dermis which rests on the subcutaneous tissue (Figure 1-1) (1, 2).

Figure 1-1. Structure of skin

1.1.1. Epidermis

Epidermis is the outermost avascular layer with a thickness of ~0.075 to 0.15 mm. The

epidermal layer consists of 95% keratinocytes and the remaining 5% is composed of

melanocytes, langerhans cells and merkel cells. The epidermal region is further subdivided into

various layers like stratum corneum, stratum granulosum, stratum

Stratum Corneum

Epidermis

Dermis

Subcutaneous tissue

Musculoskeletal tissue

2

spinosum, and stratum basale. The outermost layer of epidermis, stratum corneum (10-20 µm) is

the main barrier that prevents in and out entry of substances. The stratum corneum or horny layer

represents a brick and mortar model with the dead, keratinized cells embedded in a mortar of

lipid bilayers. It consists of 75-80% of protein which is mainly keratin and 5-15% of lipid. The

major contents of lipid domain include ceramides, fatty acids, cholesterol, and sterol/wax esters.

Water also plays an important role along with keratinocytes and lipid layers in maintaining the

barrier integrity of stratum corneum. The stratum granulosum, spinosum and basale constitute

the viable epidermis which is present below the stratum corneum. Keratinocytes, melanocytes,

langerhans cells and merkel cells constitute the viable epidermis (3-6).

1.1.2. Dermis

The layer of skin just below the viable epidermis is the dermis which constitutes the bulk

of skin and made of fibrous and elastic tissue. The dermis has a thickness of ~2 to 4 mm and

consists of numerous structures like blood vessels, lymphatic vessels, nerve endings, hair,

sebaceous glands, and sweat glands. The dermal region has extensive capillary network which

connects to systemic circulation with branching from the arterioles and venules. The lymphatic

vessels aid in the drainage of particulate and liquid matter from the dermis. The dermis also has

scattered fibroblasts, macrophages, leucocytes, and mast cells. One square centimeter of skin is

associated with about three blood vessels, 360 cm of nerves, 10 hair follicles, 15 sebaceous

glands, and sweat glands (7, 8).

3

1.2. Transcutaneous sampling of drugs

In general, when the infection is present in the central pharmacokinetic compartment, the

activity of the drug is determined by the unbound drug concentration in the plasma. But, if the

infection is in the peripheral tissue like skin, it is the time course of unbound drug concentration

in the skin extracellular fluid (ECF) that is critical for successful drug therapy. ECF can be

considered as a liquid compartment in the close vicinity of cells in which drugs enter. In such

cases, monitoring drug levels in the plasma may not represent the actual levels in the affected

peripheral tissue. Moreover, the phlebotomical procedures are associated with blood loss and

discomfort in patients. Therefore, the time course of drugs in the affected tissue should be

monitored for determining the frequency and dose of drug administration from the safety and

efficacy perspectives (9-11).

1.2.1. Different techniques of sampling drugs from the dermal ECF

Conventional techniques of drug sampling from the dermal ECF include skin blister fluid

sampling and skin biopsy sampling. However, these techniques are invasive in nature and also

have a limitation with the number of samples that can be collected (12-16).

Microdialysis is a minimally invasive technique which is widely used for sampling of unbound

drug from the tissue extracellular fluid. To carryout cutaneous microdialysis a small probe

constructed with semipermeable membrane is inserted in the dermis and the principle involved is

that the physiological solution pumped through the probe is in equilibrium with the molecules

diffusing in the immediate surroundings (17). Microdialysis has been successfully employed for

the estimation of drugs like diclofenac, 5-fluorouracil, ibuprofen, propranolol, and nicotine in the

skin extracellular fluid (18-22). Microdialysis technique has certain limitations with

implementation in therapeutic drug monitoring. The drug recovery by microdialysis is affected

4

by various physicochemical and physiological factors and the major concern is the possibility of

damage to the skin microvasculature due to probe insertion leading to local ischemia and

effecting tissue metabolism (17, 23).

Reverse iontophoresis had been studied for transcutaneous sampling of caffeine, theophylline,

valproate, and lithium levels for therapeutic drug monitoring. It is an active technique where-in

the subdermal drug is driven to the surface of the skin by application of low intensity electric

current through skin (10, 24-26).

Ultrasound technique has also been used to sample interstitial fluid. This technique involves

permeabilization of skin by ultrasound followed by collection of interstitial fluid by application

of vacuum (10, 27, 28).

1.2.2. Electroporation and transcutaneous sampling (ETS)

Electroporation is considered to be one of the promising electrically mediated techniques

to enhance drug delivery across the skin involving reversible permeabilization of skin brought

about by application of short electrical pulses. Transient aqueous pathways are created in the

stratum corneum due to the application of high voltage short electrical pulses, which reseal to

regain the barrier property. The recovery of stratum corneum barrier property depends on the

nature, number and pulse voltage applied (29-32).

Electroporation and transcutaneous sampling (ETS) is a noninvasive technique for estimating the

time course of drugs in the dermal extracellular fluid. ETS is a technique of reversible

permeabilization of stratum corneum and sampling of drugs and analytes from the dermal

extracellular fluid by facilitating reverse diffusion of drug in the direction of dermis to stratum

corneum. The principle of ETS is similar to that of microdialysis; in case of microdialysis, the

recovery of the unbound drug from the dermal ECF is determined by the permeability of

5

microdialysis probe membrane whereas in case of ETS, the recovery of unbound drug is

determined by the skin permeability. In ETS, the diffusion of free drug across stratum corneum

is affected only by the free drug concentration in dermal extracellular fluid as the drug bound to

plasma proteins does not diffuse across stratum corneum following electroporation. Previous

studies have reported that electroporation is efficient in transporting molecules up to 10 kDa.

Hence, the transcutaneous flux of the drugs is proportional to the unbound drug concentration in

the dermal extracellular fluid (33-35).

The main objectives of ETS project are:

I. To develop a simple, noninvasive technique of sampling drugs and analytes from the

dermal extracellular fluid.

II. To study the time course of drug in the skin following drug administration by different

routes of drug delivery.

6

1.3. Transdermal drug delivery

Transdermal drug delivery involves the transport of drugs due to concentration gradient

from the skin surface into the systemic circulation. Delivery of drugs across the skin provides an

alternative to the conventional oral drug delivery and intravenous administration. The major

advantages of transdermal drug delivery are (36, 37):

a) Readily accessible and large surface area of skin for absorption.

b) Avoidance of hepatic first pass effect and gastrointestinal incompatibility.

c) Minimized side effects due to optimized blood concentration-time profile.

d) Self administration is feasible and patient compliant.

e) Predictable and extended duration of therapeutic activity.

f) Reduced frequency of drug administration.

g) Ease of termination of therapy by simple removal of drug source.

However, transdermal drug delivery has certain limitations like:

a) Stratum corneum layer of skin is the major barrier for delivery of drugs.

b) Suitable for low molecular weight and slightly lipophilic drugs only.

1.3.1. Different approaches to enhance transdermal drug delivery

Passive transdermal drug delivery is limited due to the barrier property of stratum

corneum. The different approaches used to enhance transdermal drug delivery include:

I. Chemical enhancers/penetration enhancers

II. Iontophoresis

III. Electroporation

IV. Ultrasound/ Sonophoresis

V. Microneedles

7

Chemical enhancers are widely used for enhancing the permeation of poorly penetrating drug

molecules through the skin. Numerous compounds like sulphoxides, azones, pyrrolidones,

alcohols, glycols, surfactants, and terpenes have been found to be efficient in enhancing the

transdermal drug delivery. The main mechanisms for enhanced transdermal drug delivery by

chemical enhancers or penetration enhancers include increased solubility of drug in the donor

formulation, increased partitioning into the stratum corneum, fluidization of lipid bilayers, and

disruption of intracellular proteins (38, 39).

Iontophoresis is a physical technique used for enhancing the transdermal drug delivery. It

involves the application of mild electric current, which aids in the movement of charged drug

molecules across the skin. The amount of drug delivered depends on the electric current applied,

duration of application and the area of skin surface to which current is applied. The main

mechanisms contributing to iontophoresis are electrophoresis and electroosmosis. Iontophoresis

approach had been used for enhancing transdermal delivery of drugs like 5-fluorouracil, sodium

nonivamide, lidocaine hydrochloride, dexamethasone sodium phosphate, fentanyl, timolol, and

acyclovir. The maximum current density that can be applied clinically is limited to 0.5 mA/cm2

(40-46).

Electroporation is a technique that involves transient structural perturbation of lipid bilayer

membranes due to the application of high voltage (60-120 V) electrical pulses. Electroporation

leads to the formation of transient aqueous pathways within the stratum corneum and the main

mechanisms for enhanced transdermal drug delivery are electrophoresis and enhanced diffusion.

The factors affecting transdermal drug delivery by electroporation include the pulse voltage,

number of pulses, pulse length, physicochemical properties of drug and formulation of drug

8

reservoir. Electroporation was shown to enhance transport of drugs like timolol, metoprolol,

fentanyl, calcein, calcitonin, and heparin (29, 31, 34, 47-51).

Sonophoresis is a technique of enhancing transdermal drug delivery by application of low

frequency ultrasound (20-100 kHz) on the skin. The mechanisms leading to enhanced drug

transport by sonophoresis include cavitation, thermal effects, induction of convective transport,

and mechanical effects. Sonophoresis was shown to increase the transdermal delivery of drugs

like lidocaine, corticosterone, estradiol, cyclosporine, and insulin (52-58).

Microneedle system consists of an array of micron sized needles which will penetrate the

epidermal region of the skin to create microchannels Based on application, microneedles used

can be solid microneedles or hollow microneedles. The effect of microneedles depends on the

type of needles used, fabrication, needle length, width, shape, and force of insertion.

Microneedles have been used to enhance the transdermal transport of compounds like

naltrexone, calcein, insulin, and DNA vaccination (59-64).

1.3.2. Novel approaches to enhance transdermal drug delivery

The two techniques that we have developed in our laboratory for efficient transdermal

drug delivery are

1. ChilDrive Iontophoresis

2. Magnetophoresis

ChilDrive iontophoresis is a technique of combining regional cutaneous hypothermia with

iontophoresis for enhancing the transdermal delivery of drugs to deeper musculoskeletal tissues.

Magnetophoresis is a technique of enhancing transdermal drug delivery by the application of

magnetic field strength.

9

CHAPTER-2

DERMAL DRUG LEVELS OF ANTIBIOTIC (CEPHALEXIN) DETERMINED BY

ELECTROPORATION AND TRANSCUTANEOUS SAMPLING (ETS) TECHNIQUE

2.1. ABSTRACT

The purpose of this project was to assess the validity of a novel electroporation and

transcutaneous sampling (ETS) technique for sampling cephalexin from the dermal extracellular

fluid (ECF). This work also investigated the plausibility of using cephalexin levels in the dermal

ECF as a surrogate for the drug levels in the synovial fluid. In vitro and in vivo studies were

carried out using hairless rats to assess the workability of ETS. Cephalexin (20 mg/kg) was

administered (i.v.) through tail vein and the time course of drug concentration in the plasma was

determined. In the same rats, cephalexin concentration in the dermal ECF was determined by

ETS and microdialysis techniques. In a separate set of rats, only intraarticular microdialysis was

carried out to determine the time course of cephalexin concentration in the synovial fluid. The

drug concentration in the dermal ECF determined by ETS and microdialysis did not differ

significantly from each other and so as were the pharmacokinetic parameters. The results provide

validity to the ETS technique. Further, there was a good correlation (~0.9) between synovial

fluid and dermal ECF levels of cephalexin indicating that dermal ECF levels could be used as a

potential surrogate for cephalexin concentration in the synovial fluid.

Keywords: Electroporation; Transcutaneous sampling; Microdialysis; Pharmacokinetics.

10

2.2. OBJECTIVES AND SIGNIFICANCE

The objectives of this study are:

I. To determine if ETS could be utilized for sampling of cephalexin from dermal ECF.

II. To ascertain if the dermal ECF levels can serve as a surrogate for synovial fluid levels of

cephalexin.

The most commonly occurring infections in people are skin infections. Particularly, children and

elderly people are mostly affected with skin infections due to lack of potent immune system.

Cephalosporins are most widely used for treatment of skin infections because of their safety

profiles (65-67). Cephalexin, a first generation cephalosporin antibiotic is mostly used because of

its activity against both the gram-positive and gram-negative microorganisms (68). In addition to

treatment of skin infections, cephalexin is also commonly used to treat the articular infections

(69, 70). Achieving therapeutically active drug levels at the site of infection is vital for any

antibiotic therapy. In general, when the infection is situated in the central pharmacokinetic

compartment, the activity of the drug is determined by the unbound drug concentration in the

plasma. However, in case of infections in the peripheral tissues such as skin and articular region,

it is the time course of concentration of unbound antibiotic in the respective tissue fluids, which

is crucial for successful treatment. In such cases monitoring the drug levels in the plasma may

not reflect the actual levels in the affected tissue. Therefore, the time course of antibiotics in the

affected tissue needs to be monitored for determining the frequency and dose of drug

administration from the safety and efficacy perspectives of the antibiotic therapy (71-75).

11

2.3. MATERIALS AND METHODS

2.3.1. Chemicals

Cephalexin hydrate was purchased from Sigma-Aldrich Inc. (St. Louis, MO), Phosphate

buffered saline (PBS, pH 7.4) premixed powder was obtained from EMD Chemicals

(Gibbstown, NJ), and all other chemicals were obtained from Fischer Scientific (Fairway, NJ).

2.3.2. In vitro experimental setup

The in vitro diffusion studies were carried out in Franz diffusion cells (FDC) (Logan

Instruments Ltd., Somerset, NJ) using hairless rat skin excised from the abdomen region.

Hairless rat skin is known to be a good model for topical and transdermal drug delivery studies

due to the similarity between the rat and human skin with respect to lipid content and water

uptake properties (76). Moreover, a good correlation of permeation data between the hairless rat

model and human skin models has been reported by several research groups in the past (77). The

skin was mounted on the diffusion cell in such a way that the epidermis side of the skin was in

contact with upper sampling compartment and dermal side with the lower reservoir

compartment. The active diffusion area of FDC was 0.64 cm2. Ag/AgCl electrode wires of 0.5

mm diameter (Alfa Aesar, Ward Hill, MA) made in form of circular rings were placed 2 mm

away from skin in both sampling and reservoir compartments. The sampling and the reservoir

compartments were filled with 0.4 and 5 ml PBS respectively and the skin was allowed to

equilibrate for 1 h. The AC electrical resistance of the epidermis was measured by placing a load

resistor RL (100 kΩ) in series with the epidermis. The voltage drop across the whole circuit (VO)

and across the skin (VS) was measured using an electrical set up consisting of a waveform

generator and a digital multimeter (Agilent Technologies, Santa Clara, CA). For measuring

12

resistance, voltage of 100 mV was applied at 10 Hz and the skin resistance in kΩ was

approximated from the formula:

SO

LS

VV

RVRs

−

= (1)

where RS is the skin resistance and RL is the load resistor in kΩ. The piece of skin which had a

resistance greater than 20 kΩ.cm2 was used for the experiment.

2.3.3. In vitro transcutaneous sampling studies

The sampling compartment was replaced with 0.4 ml of PBS (pH 7.4) and the reservoir

compartment was filled with 5 ml of cephalexin solution prepared in PBS (5-40 µg/ml). Thirty

square electrical pulses each of 10 ms duration at 120 V/cm2, 1 Hz was applied using ECM 830

Electro Square Porator (BTX Harvard apparatus, Holliston, MA). The electrical resistance was

measured immediately after application of electrical pulses to ensure skin permeabilization. PBS

from the sampling compartment was withdrawn 15 min after application of electrical pulses and

the amount of cephalexin sampled was analyzed by HPLC.

2.3.4. Ex vivo plasma protein binding

The blood was collected by cardiac puncture in rats and the plasma was separated by

centrifugation at 2000g at 4 ºC. Rat plasma was spiked with drug to provide concentration

ranging from 1-20 µg/ml. The spiked plasma samples were thoroughly mixed by vortexing and

allowed to equilibrate for 12 h at 4 ºC. After equilibration, protein free plasma was obtained by

using ultra filtration (Millipore Centrifree®

filtration units) by centrifugation of 0.5 ml of plasma

at 2000g for 20 min (71, 76). The amount of unbound drug present in the filtrate was measured

by HPLC after suitable dilution with PBS.

13

2.3.5. In vivo studies

The in vivo experimental procedures were approved by the Institutional Animal Care and

Use Committee (IACUC) at the University of Mississippi (Protocol#07-004). The in vivo studies

were carried out in hairless rats (Taconic, Hudson, Newyork) (250-300 g) under ketamine (80

mg/kg) and xylazine (10 mg/kg) anesthesia administered intraperitoneally.

Plasma sampling, ETS and microdialysis sampling was carried out in the same group of rats

(n=6). The samples by all three procedures were obtained at the same time points in each rat

simultaneously. Cephalexin solution of 20 mg/kg prepared in sterile isotonic saline was

administered by i.v. into tail vein as a bolus injection.

2.3.5.1. Cutaneous microdialysis

A 20G needle was inserted intradermally through a distance of 1 cm and a linear

microdialysis probe of 5 mm membrane length and 30 kDa cutoff molecular weight (BASi, West

Lafayette IN) was inserted through this needle and the needle was withdrawn leaving the probe

implanted in the dermal tissue. The inlet tube was connected to an injection pump (BASi, West

Lafayette, IN) and PBS was perfused at 2 µl/min flow rate for 30 minutes for equilibration. Two

samples were collected before drug administration. The drug was injected by i.v through the tail

vein after equilibration of the probe. Subsequently, the microdialysis samples were collected

continuously at every 15 minutes interval including at time points corresponding to ETS and

plasma sampling at 30, 60, 120, 180, 240, 300 and 360 min.

2.3.5.2. Electroporation and transcutaneous sampling

Prior to the drug administration, a custom made sampling cell was fixed using an

adhesive (Krazy glue, Elmers products Inc, Ohio) on the back of the rats (Figure 2-1). The

sampling cell was fitted with an Ag/AgCl electrode and the counter electrode was secured just

14

adjacent to the cell on the surface of the skin using a micropore surgical tape (3M Healthcare,

MN).

Figure 2-1. Diagrammatic representation showing an experimental setup of electroporation and

transcutaneous sampling in hairless rats.

The skin was hydrated with 100 µl of saline for 5 min before each sampling and was replaced

with 100 µl of PBS (sampling buffer). One blank sample was collected before drug

administration, and subsequent samples were collected at (30, 60, 120, 180, 240, 300 and 360

min). For ETS procedure, 30 electrical pulses each of 10 ms duration at 120 V/cm2, 1 Hz was

applied and the sampling fluid remained in the chamber for 15 min after pulsing.

2.3.5.3. Plasma pharmacokinetics of drug

For plasma pharmacokinetic studies, 100 µl of blood was collected by retro-orbital

bleeding before injection of drug and before each episode of transcutaneous sampling and

15

cutaneous microdialysis. The blood samples were diluted with 200 µl of PBS and plasma was

separated followed by protein precipitation and the plasma drug content was analyzed by HPLC.

2.3.5.4. Intraarticular microdialysis

In another set of rats (n=6), intraarticular microdialysis was carried out to determine the

amount of cephalexin present in the synovial fluid. After anaesthetizing the rats, the hind limb

was held in a fixed position and a 20G needle was passed through the knee joint capsule lateral

to the patellar ligament and a linear microdialysis probe of 5 mm length and 30 kDa cut off

molecular weight (BASi, West Lafayette, IN) was inserted through the needle (78, 79). The

needle was withdrawn leaving the probe implanted in the synovial cavity. PBS was perfused for

30 min prior to drug administration for equilibration at flow rate of 2 µl/min. Cephalexin

solution (20 mg/kg) was administered through the tail vein and microdialysis perfusion was

continued for 6 h with samples collected for 15 min interval including at time points

corresponding to ETS and cutaneous microdialysis sampling.

In case of both cutaneous and intraarticular microdialysis, the probe recovery was determined in

vivo by using retrodialysis method (80-82). For this, the probe was first equilibrated by perfusing

PBS at 2 µl/min for 30 min; followed by drug solution of known concentration for 30 min. After

equilibration, dialysate was collected for 15 min interval at 15, 30 and 45 min and the average

recovery of three time points was considered. The in vivo recovery rate was calculated using the

formula:

Recovery %=100- Concentration of dialysate

Concentration of perfusate X 100 (2)

16

2.3.6. Analytical method

The amount of cephalexin present in plasma, ETS and microdialysis samples were

analyzed by HPLC using Symmetry®

C18 column (4.6 x 150 mm) with UV detection at 254 nm.

Mobile phase consisted of a mixture of methanol and 2.5 mM sodium phosphate buffer, pH 5.6

(20:80 v/v) and the flow rate was 1 ml/min. The sensitivity of the method was 10 ng/ml and

linearity was between 10-1000 ng/ml (R2=0.99). To the plasma samples, equal volume of

acetonitrile was added to precipitate proteins and then centrifuged at 2000g for 10 min at room

temperature and the supernatant was analyzed for drug content (83, 84). ETS and microdialysis

samples were centrifuged and directly injected into HPLC system.

2.3.7. Data Analysis

The plasma pharmacokinetic parameters were calculated based on two compartment

model represented by the equation:

C = Ae-αt

+ Be-βt

(3)

where A, B are the pre-exponential constants, α is the distribution rate constant, β is the

elimination rate constant. The values of α, β, A and B are derived from curve fitting of

experimental data. The elimination half life (t1/2) was calculated using the formula 0.693/β and

area under the curve (AUC0-6h) was calculated using the trapezoid rule. The pharmacokinetic

parameters in case of dermal ECF and synovial fluid were calculated using non compartmental

pharmacokinetic model.

The statistical analysis was carried out using GraphPad Instat 3 software and the unpaired t-test

was selected for comparing the parameters obtained from ETS and microdialysis techniques.

P<0.05 was considered as level of significance. From Pearsons correlation, R2 and P-value were

17

calculated using Pearson Correlation (v1.0.3) in Free Statistics Software (v1.1.23-r1). The data

points shown in graphs are an average of 6 trials with error bars representing standard deviation.

2.4. RESULTS AND DISCUSSION

Calibration of ETS was carried out in vitro using freshly excised hairless rat skin model.

Known concentrations of drug were placed in the reservoir compartment and the drug was

sampled following electroporation. The amount of drug diffused in 15 min following application

of electrical pulses was plotted against respective reservoir concentrations (5-40 µg/ml) as

represented in Figure 2-2.

The linear relationship (R2=0.96) between the amount of drug sampled and the reservoir drug

concentration implies that the ETS samples would potentially represent the subdermal drug

concentration. The percentage recovery by ETS can be obtained by (slope X 100) from Figure 2-

2 and was found to be 3.06±0.2%. In control (across the untreated skin) the amount of drug

sampled was less than detectable levels. In case of electroporation trials, the resistance of skin

dropped ~74±8% whereas in case of control set of experiments, electrical resistance of skin did

not change significantly. The recovery of electrical resistance of electroporated skin was

insignificant within the sampling duration of 15 min, which is in agreement with previous reports

in case of ETS across the rat skin and porcine epidermis (74, 85).

18

Figure 2-2. Correlation between cephalexin concentration (5-40 µg/ml) in the reservoir

compartment and cephalexin sampled by ETS across hairless rat skin in vitro (y=0.03x,

R2=0.96).

The plasma protein binding of cephalexin revealed that the fraction of cephalexin bound to

plasma was 10.2±2.6% at concentrations between 1-20 µg/ml. Low protein binding of

cephalexin is considered to be one of the major reasons for its extensive distribution into the

peripheral tissues. The protein binding values were in agreement with 12.4% that was reported

by Tsai et al (68).

The concentration time profile of cephalexin in plasma and dermal extracellular fluid

(determined by ETS and cutaneous microdialysis) samples following i.v. administration of

cephalexin (20 mg/kg) is shown in Figure 2-3.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 10 20 30 40

Am

ou

nt

of

cep

hale

xin

sam

ple

d (

µg/m

l)

Concentration of cephalexin in reservoir

compartment (µg/ml)

19

Figure 2-3. Time course of cephalexin in rat plasma () determined by blood sampling and

dermal ECF determined by microdialysis () and ETS () tecniques following i.v. bolus

administration of 20 mg/kg of cephalexin.

The plasma concentration versus time data of cephalexin could be described by a two

compartment model (86). The pharmacokinetic parameters calculated for plasma drug

concentration-time profile are given in Table 2-1. The plasma drug concentrations reported in

this project are comparable to that reported by Tsai et al in rats considering the difference in dose

between the two studies. The plasma elimination half life of cephalexin in the current study was

104.59±28.61 min (1.74±0.47 h) which agrees well with the elimination half life (1.4±0.81 h)

reported by Padoin et al (68, 86).

0

5

10

15

20

25

30

0 60 120 180 240 300 360

Cep

hale

xin

con

cen

trati

on

(µ

g/m

l)

Time (min)

20

Table 2-1. Pharmacokinetic parameters derived from the plasma concentration-time data after i.v

bolus administration of 20 mg/kg of cephalexin in hairless rats.

Parameter i.v. bolus

A (µg/ml) 52.35±1.41

B (µg/ml) 11.89±1.04

t1/2 (min) 104.59±28.61

α (1/min) 0.03355±0.004

β (1/min) 0.00688±0.001

AUC0-6h (µg.min/ml) 3160.93±250.35

The recovery of cephalexin by the microdialysis probe in the cutaneous tissue was found to be

21.14±5.26% whereas the recovery of cephalexin by ETS was only about 3.06±0.2% which is

about 7-fold less than that of microdialysis. Although ETS has the advantage of being

noninvasive as opposed to microdialysis, the later has the limitation with the amount of drug that

could be sampled from the dermal ECF. Nevertheless, recovery could likely be improved by

using more vigorous electrical protocol and/or by increasing the sampling duration. In both

microdialysis as well as ETS techniques, the amount of cephalexin present in the dermal ECF in

rats was calculated using the amount sampled and the corresponding recovery values as follows.

Dermal ECF concentration=sample concentration

percentage recovery (4)

In the current study, the point to point comparison of the drug concentration in the dermal

extracellular fluid and the pharmacokinetic parameters, i.e. Cmax, Tmax, AUC0-6h and t1/2,

21

determined by ETS and microdialysis techniques did not differ significantly (unpaired t-test,

P<0.05) (Table 2-2). This provides validity to the ETS technique of sampling cephalexin.

Table 2-2. Mean pharmacokinetic parameters of cephalexin determined by ETS, microdialysis

techniques following administration of 20 mg/kg by i.v bolus in hairless rats.

Parameter ETS Microdialysis P-value

Tmax (min) 120 120 --

Cmax (µg/ml) 13.09±1.92 12.64±1.90 0.39

AUC0-6h(µg.min/ml) 2512.35±250.14 2473.66±202.43 0.42

t1/2 (min) 106.61±17.81 96.37±12.33 0.22

The amount of drug present in the synovial fluid is shown in Figure 2-4 and the pharmacokinetic

parameters are given in Table 2-3. In this case, the in vivo microdialysis probe recovery was

found to be 10.64±3.44%. The Cmax in case of synovial fluid (3.23±0.58 µg/ml) was four fold

less than the dermal ECF (~13.09 µg/ml). This data suggests that relatively higher doses of

cephalexin would be required to achieve effective drug levels in the synovial fluid.

22

Figure 2-4. Concentration-time profile of cephalexin in synovial fluid obtained by intraarticular

microdialysis after administration of 20 mg/kg of drug by i.v. bolus.

Table 2-3. Mean pharmacokinetic parameters of cephalexin determined by intraarticular

microdialysis in the synovial fluid following administration of 20 mg/kg by i.v bolus in hairless

rats.

Parameter Synovial fluid

Tmax (min) 120

Cmax (µg/ml) 3.233±0.58

t1/2 (min) 96.215±8.08

AUC0-6h (µg.min/ml) 654.10±101.35

0.0

1.0

2.0

3.0

4.0

0 60 120 180 240 300 360

Cep

hale

xin

con

cen

trati

on

(µ

g/m

l) i

n t

he

syn

ovia

l fl

uid

Time (min)

23

The drug levels from synovial fluid were plotted against drug levels in dermal ECF obtained by

ETS and microdialysis techniques. A good correlation of 0.92 (P=0.00029) and 0.91

(P=0.00047) was observed between the drug levels in synovial fluid and dermal ECF obtained

from ETS (Figure 2-5) and microdialysis (Figure 2-6).

Figure 2-5. Correlation between cephalexin levels in dermal ECF obtained from ETS with that

of synovial fluid drug levels (y=3.78x, R2=0.92).

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

Con

cen

trati

on

of

cep

hale

xin

in

der

mal

EC

F

(µg/m

l )

(ET

S)

Concentration of cephalexin (µg/ml) in synovial

fluid

24

Figure 2-6. Correlation between cephalexin levels in dermal ECF obtained from microdialysis

with that of synovial fluid drug levels (y=3.72x, R2=0.91).

From this relationship, it could be said that in rats, the drug level in the skin represents ~3.7X of

that in the synovial fluid. Establishing such correlation between the dermal ECF and the

concentration of drug in internal tissues would help in monitoring the drug levels of peripheral

tissues which are extremely difficult to access. From the results of this experiment it appears that

dermal ECF levels of cephalexin could be used as potential surrogate for cephalexin levels in the

synovial fluid.

ETS is a noninvasive method of cutaneous drug sampling and is expected to be relatively safer

than microdialysis method. However, there are concerns about potential skin damage due to the

application of electrical pulses. Many research groups have evaluated the safety of skin

electroporation in animal models and human subjects. Vanbever et al. have reported that

0

2

4

6

8

10

12

14

16

0 1 2 3 4

Con

cen

trati

on

of

cep

hale

xin

in

der

mal

EC

F

(µg/m

l) (m

icro

dia

lysi

s)

Concentration of cephalexin (µg/ml) in synovial

fluid

25

reversible mild skin reactions occurred following the application of 15 electrical pulses of 250 V

and 200 ms in vivo in hairless rats (87). Wong et al. have shown that electroporation can be

carried out in humans without causing pain at 150 V, 1 ms, 60 pulses by using microelectrode

array (88). The protocol that was applied in current experiments was 120 V, 30 square pulses

each of 10 ms duration which is rather mild than the protocols applied on human subjects in

other studies. The extent of skin damage depends on the applied electrical protocol and the

electrode design. Therefore the optimum electrical protocols need to be evaluated in vivo for

tolerability, morphological, histological, and biochemical changes in the skin before

implementation in clinical practice.

2.5. CONCLUSIONS

ETS is a potential noninvasive technique that could be developed for sampling of drugs

from the skin tissue. However, the major limitation of the technique is low recovery, which

limits the application of the technique to drugs which are less protein bound and which are

present considerably in high amounts in the dermal ECF. One of the most interesting outcomes

of the present work was that the dermal ECF concentration of cephalexin correlated well with the

concentration in synovial fluid.

Acknowledgements

This project described was supported by Grant Number AR053097 from National Institute of

Arthritis and Musculoskeletal and Skin Diseases.

“Reprinted from Journal of Pharmaceutical Sciences, 98, S.M. Sammeta, Siva Ram K. Vaka, S.

Narasimha Murthy, Dermal drug levels of antibiotic (cephalexin) determined by electroporation

and transcutaneous sampling (ETS) technique, 2677-2685, Copyright (2009), with permission

from John Wiley and Sons.”

26

CHAPTER-3

TRANSCUTANEOUS SAMPLING OF CIPROFLOXACIN AND

8-METHOXYPSORALEN BY ELECTROPORATION (ETS TECHNIQUE)

3.1. ABSTRACT

The novel technique of transcutaneous sampling of drugs by electroporation was

developed to study the dermatokinetics of ciprofloxacin and 8-methoxypsoralen. The selected

drugs differ in their aqueous solubility and also with respect to the extent of protein binding.

Ciprofloxacin (15 mg/kg) was administered i.v. through tail vein whereas 8-methoxypsoralen (5

mg/kg) was given by oral administration in hairless rats and the time course of drug

concentration in the plasma was determined. Drug concentration in the dermal extracellular fluid

(ECF) was determined by ETS and microdialysis sampling techniques. The drug concentration in

the dermal ECF determined by ETS and microdialysis did not differ significantly from each

other and so as were the pharmacokinetic parameters. The results show that ETS can be utilized

as a potential technique for sampling of drugs from the dermal extracellular fluid.

Keywords: Electroporation; Transcutaneous sampling; Hairless rats; Microdialysis;

Pharmacokinetics; Extracellular fluid.

27


The objectives of the study are:

I. To assess the feasibility of ETS technique for drugs of different physicochemical

properties.

II. To assess the workability of ETS technique, when drugs are administered via different

routes.

Two drugs ciprofloxacin and 8-methoxypsoralen which possess different physicochemical

properties and also differ in their extents of protein binding are selected as model drugs. For the

purpose of assessing the validity of the technique, the two drugs were administered by different

routes in this project (Ciprofloxacin by i.v. bolus and 8-methoxypsoralen by oral route).

Ciprofloxacin, a broad spectrum antibacterial agent belonging to the group of fluoroquinolones is

effective against microbial infections localized in the skin. The time course of ciprofloxacin in

the skin needs to be monitored for determination of the frequency and dose from safety and

efficacy perspectives (71, 89-91).

8-methoxypsoralen, a furocoumarinic is used in conjunction with UV radiation (PUVA therapy)

for the treatment of dermatoses like vitilgo and psoriasis (92-94). The effectiveness of 8-

methoxypsoralen depends on ultraviolet-A irradiation and optimum response will be elicited

only when the drug concentration in skin is over the effective concentration. For this reason, it is

imperative to determine drug concentration in skin to achieve better PUVA therapy (95-97).


3.3.1. Chemicals

Ciprofloxacin Hydrochloride, 8-Methoxypsoralen were purchased from Sigma-Aldrich

Inc (St.Louis, MO), Phosphate buffered saline (PBS, pH 7.4) premixed powder was obtained

28

from EMD Chemicals (Gibbstown, NJ), and all other chemicals were obtained from Fischer

Scientific (Fairway, NJ).

3.3.2. In vitro transcutaneous sampling studies

The in vitro diffusion studies were carried out in Franz diffusion cells (FDC) (Logan

Instruments Ltd., Somerset, NJ) using hairless rat skin excised from the abdomen region.

Hairless rat skin is considered to be a good model for topical and transdermal drug delivery

studies due to the similarity between the rat and human skin with respect to lipid content and

water uptake properties (76). Moreover, a good correlation of permeation data between the

hairless rat model and human skin models have been reported by several research groups in the

past (77). The skin was mounted on the diffusion cell in such a way that the epidermis side of the

skin was in contact with upper sampling compartment and dermal side with the lower reservoir

compartment. The active diffusion area of FDC was 0.64 cm2. Ag/AgCl electrode wires of

0.5 mm diameter (Alfa Aesar, Ward Hill, MA) made in the form of circular rings were placed

2 mm away from skin in both sampling and reservoir compartments. The sampling compartment

and the reservoir compartment were filled with 0.4 and 5 ml PBS, respectively and the skin was

allowed to equilibrate for an hour. The AC electrical resistance of the epidermis was measured

by placing a load resistor RL (100 kΩ) in series with the epidermis. The voltage drop across the

whole circuit (VO) and across the skin (VS) was measured using an electrical set up consisting of

a waveform generator and a digital multimeter (Agilent Technologies, Santa Clara, CA). For

measuring resistance, voltage of 100 mv was applied at 10 Hz and the skin resistance in kΩ was

approximated. The piece of skin, which had a resistance greater than 20 kΩ.cm2 was used for the

experiment.

29

Later, the sampling compartment was replaced with fresh 0.4 ml of PBS and reservoir

compartment was filled with 5 ml of ciprofloxacin solution prepared in PBS (2.5-40 µg/ml). In

case of 8-methoxypsoralen, the sampling compartment was replaced with PBS:ethanol (50:50)

and the reservoir compartment was filled with 5 ml of 8-methoxypsoralen solution (2.5-

40 µg/ml) prepared in PBS:ethanol (50:50). Thirty square electrical pulses each of 10 ms

duration at 120 V/cm2, 1 Hz was applied using ECM 830 Electro Square Porator (BTX Harvard

apparatus, Holliston, USA). The electrical resistance of the skin was measured immediately after

application of the electrical pulses to ensure skin permeabilization. The solution from the

sampling compartment was withdrawn 15 min after application of electrical pulses and the

amount of drug was analyzed by HPLC.

3.3.3. Ex vivo plasma protein binding

The blood was collected by cardiac puncture in rats and the plasma was separated by

centrifugation at 2000g at 4 °C. Rat plasma was spiked with drug to prepare samples of

concentration ranging from 1 to 40 µg/ml. The spiked plasma samples were thoroughly mixed by

vortexing and allowed to equilibrate for 12 h at 4 °C. After equilibration, protein free plasma was

obtained by carrying out ultra filtration (Millipore Centrifree®

filtration units) by centrifugation

of 0.5 ml of plasma at 2000g for 20 min. The amount of unbound drug present in the filtrate was

measured by HPLC after suitable dilution with PBS (74, 98).

3.3.4. In vivo studies


Use Committee (IACUC) at the University of Mississippi (Protocol#07-004). The in vivo studies

were carried out in hairless rats (Taconic, Hudson, Newyork) (250-300 g) under ketamine (80

mg/kg) and xylazine (10 mg/kg) anesthesia administered intraperitoneally.

30

Blood sampling, ETS and microdialysis sampling was carried out in the same group of rats

(n=6).The samples by all three procedures were obtained at the same time points in each rat

simultaneously. Ciprofloxacin solution (150-180 µl) of 15 mg/kg prepared in sterile isotonic

saline was administered by i.v. into tail vein as a bolus injection, whereas in case of 8-

methoxypsoralen, solution (415-500 µl) of 5 mg/kg prepared in 50:50 of PBS:ethanol was given

by oral gavage using a ball ended feeding needle.

3.3.4.1. Cutaneous microdialysis

For cutaneous microdialysis, a 20G needle was inserted intradermally through a distance

of 1 cm and penetration depth of 2 mm. A linear microdialysis probe of 5 mm membrane length

and 30 kDa cutoff molecular weight (BASi, West Lafayette IN) was inserted through this needle

and the needle was withdrawn leaving the probe implanted in the dermal tissue. The proper

placement of probe in the dermis was confirmed by making an incision at the site of probe

implantation after completion of the study. Any experiment, in which the probe was not placed

horizontally at the intended depth was not considered.

The inlet tube was connected to an injection pump (BASi, West Lafayette, IN) and perfusion was

set to a flow rate of 2 µl/min. The probe was equilibrated for 30 min (99-101). The perfusate was

PBS for studies involving ciprofloxacin and PBS:ethanol (50:50) for 8-methoxypsoralen. Two

samples were collected before drug administration. The drug was administered after equilibration

of the probe. Subsequently the microdialysis samples were collected continuously at every 15

min interval including at time points corresponding to ETS and plasma sampling at 30, 60, 120,

180, 240, 300 and 360 min. The microdialysis probe recovery was determined in vitro by placing

microdialysis probe in PBS containing known drug concentration and perfusing the probe with

PBS (for ciprofloxacin) and 50:50 PBS:ethanol (for 8-methoxypsoralen) at flow rate of 2 µl/min.

31

The dialysate coming out of the probe is collected every 15 min and analyzed for drug content

(102, 103). The in vitro recovery rate was calculated using the formula:

% Relative recovery= concentration of dialysate

concentration of sampleX 100 (5)

3.3.4.2. Transcutaneous sampling by electroporation

A custom made sampling cell was fixed using an adhesive (Krazy glue, Elmers products

Inc., Ohio) on the back of the rats. The sampling cell was fitted with a Ag/AgCl electrode and

the counter electrode was secured just adjacent to the cell on the surface of the skin using a

micropore surgical tape (3 M Healthcare, MN). The skin was hydrated with 100 µl of saline for

5 min before each sampling and was replaced with 100 µl of sampling buffer (PBS for

ciprofloxacin, PBS:ethanol (50:50) for 8-methoxypsoralen). One blank sample was collected

before drug administration, and subsequent samples were collected at 30, 60, 120, 180, 240, 300

and 360 min. For ETS procedure, thirty square electrical pulses each of 10 ms duration at

120 V/cm2, 1 Hz was applied and the sampling fluid remained in the chamber for 15 min after

pulsing.

3.3.4.3. Plasma pharmacokinetics of drugs

For plasma pharmacokinetic studies, 100 µl of blood was collected by retro-orbital

bleeding before administration of drug and before each episode of transcutaneous sampling and

cutaneous microdialysis. The blood samples were diluted with 200 µl of PBS and plasma was

separated by centrifugation at 2000g. In case of ciprofloxacin, plasma samples were subjected to

protein precipitation with equal volume of 0.5 M perchloric acid, followed by centrifugation at

10000g for 10 min and drug content was analyzed by HPLC (90). 8-methoxypsoralen in the

plasma was extracted with methylene chloride for 30 min followed by centrifugation at 5000g at

4 C for 10 min, the organic phase was separated and evaporated to dryness at room temperature

32

under nitrogen and the residue was dissolved in PBS:ethanol (50:50) and analyzed by HPLC

(104).


The amounts of ciprofloxacin and 8-methoxypsoralen present in plasma, ETS and

microdialysis samples were analyzed by high performance liquid chromatography. The HPLC

system (Waters, MA) consisted of a chromatographic pump (Waters 1525) and an autosampler

(Waters 717 plus).

Analysis of ciprofloxacin samples were carried out using a fluorescence detector (Waters 2475)

at an excitation wavelength of 278 nm and emission wavelength of 445 nm. Symmetry®

C18

column (4.6 x 150 mm) was used and the mobile phase consisted of a mixture of acetonitrile and

0.1 M aqueous monopotassium phosphate solution adjusted to pH 2.5 with orthophosphoric acid

(15:85 v/v) with flow rate of 2 ml/min (90). The linearity range was between 1-1000 ng/ml

(R2=0.99).

Analysis of 8-methoxypsoralen samples were carried out using a UV detector (Waters 2487) at

248 nm using Symmetry®

C18 column (4.6 x 150mm). The mobile phase consisted of a mixture

of methanol, acetonitrile and water (2:30:68) and the flow rate was 1ml/min (104, 105).

3.3.6. Data analysis

The pharmacokinetic parameters were calculated using non compartmental

pharmacokinetic model. The terminal elimination rate constant (λz) was determined from the

slope of terminal exponential phase of the logarithmic plasma concentration-time curve. The

elimination half life (t1/2) was calculated using 0.693/λz .The area under the curve (AUC0-t) was

caclulated using the trapezoidal rule and AUC0-∞ was obtained by adding Clast/λz to AUC0-t. In

33

case of 8-methoxypsoralen, the maximum plasma concentration (Cmax ) and the time to reach

maximum plasma concentration (Tmax ) were determined from the concentration-time curves.

The statistical analysis was carried out using GraphPad Instat 3 software and the t-test was

selected for comparing the parameters obtained from ETS and microdialysis techniques. P<0.05

was considered as level of significance. The data points shown in graphs are an average of 6

trials with error bars representing standard deviation.


Solubility of drug in the sampling fluid is one of the key determinants of recovery of drug

in case of both ETS and microdialysis. PBS was found to be an appropriate solvent for sampling

ciprofloxacin. However, in the case of 8-methoxypsoralen, the recovery was poor with PBS

which was likely due to the low water solubility of 8-methoxypsoralen (water solubility of 8-

methoxypsoralen is ~55.8 µg/ml). The recovery of 8-methoxypsoralen was improved with 50:50

PBS:ethanol system in both ETS and microdialysis techniques.

Electroporation leading to permeabilization of the skin is indicated by the drastic drop in

electrical resistance of the skin. The drop in electrical resistance has been reported to be due to

formation of transient aqueous pathways in the lipid domain of the stratum corneum. The

aqueous pathways reseal and the skin will regain its barrier property (thus its original electrical

resistance). The extent of drop in electrical resistance and the duration required for the skin to

recover is determined by the applied electrical protocol. With the protocol of 120 V, 10 ms, 30

pulses applied in this project, the drop in skin resistance was ~70±5%. There was no significant

recovery in skin resistance during the sampling time of 15 min. However, the skin resistance

recovered within few hours reflecting the fact that the electrical protocol was not too aggressive

to bring about irreversible impairment of the skin barrier. This was in agreement with the

34

observations in previous studies carried out across the rat skin and porcine epidermis (35, 74,

85).

Transcutaneous sampling was also carried out across the untreated skin (did not apply electrical

pulses) which served as control set of experiment. In case of untreated skin (control samples),

detectable level of drugs could not be sampled within the sampling duration of 15 min. In case of

ETS, detectable amounts of drug was sampled even at low reservoir drug concentrations. The

amount of drug sampled in 15 min following application of electrical pulses was plotted against

the corresponding reservoir drug concentrations (Figure 3-1 and 3-2).

Figure 3-1. Correlation between ciprofloxacin concentration (2.5-40 µg/ml) in the reservoir

compartment and the amount sampled by ETS across hairless rat skin in vitro (y=0.048x,

R2=0.96).

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

0 10 20 30 40

Am

ou

nt

of

cip

rofl

oxaci

n s

am

ple

d (

µg/m

l)

Concentration of ciprofloxacin in reservoir


35

A linear relationship (R2=0.96 and 0.98 for ciprofloxacin and 8-methoxypsoralen respectively)

was observed between the amount of drug sampled and the reservoir drug concentration,

indicating that the subdermal drug concentration can be represented by ETS samples. The

percentage recovery by ETS (slope X 100) was 4.8±0.8% and 7.6±1.3% for ciprofloxacin and 8-

methoxypsoralen respectively. The recovery of 8-methoxypsoralen was less (~0.5%) when ETS

was carried out with PBS alone.

Figure 3-2. Correlation between 8-methoxypsoralen concentration (2.5-40 µg/ml) in the

reservoir compartment and the amount sampled by ETS across hairless rat skin in vitro

(y=0.076x, R2=0.98).

The free drug present in the blood plasma equilibrates rapidly with the skin ECF. In otherwords,

the extent of distribution of drug into peripheral tissues is known to be determined predominantly

0.0

0.6

1.2

1.8

2.4

3.0

3.6

0 10 20 30 40

Am

ou

nt

of

8-m

eth

oxyp

sora

len

sam

ple

d (

µg/m

l)

Concentration of 8-methoxypsoralen in reservoir


36

by the plasma protein binding. Generally, the drugs that show high protein binding penetrate less

into the peripheral compartment. Ex vivo plasma protein binding studies were carried out for

both ciprofloxacin and 8-methoxypsoralen to assess the relationship between the protein binding

and tissue penetration. The plasma protein binding of ciprofloxacin was found to be

20.65±3.93% at concentrations between 1-40 µg/ml which was in aggreement with 16-40%

reported by other research groups (106-108). The fraction of 8-methoxypsoralen bound to

plasma was 80.97±6.85%. This was comparable to 91.4% reported by Pibouin et al (109). The

two drugs chosen for this project differ significantly with respect to their protein binding

property. One of the major objectives of this project was to assess the workability of the ETS

technique in case of drugs which exhibit different extents of protein binding.

The in vitro recovery of ciprofloxacin and 8-methoxypsoralen by microdialysis probe were

found to 3.47±0.35% and 16.41±3.32% respectively. Generally microdialysis is regarded as less

suitable for sampling lipophilic drugs because, microdialysis method of drug sampling involves

perfusion of aqueous sampling fluid through the dialysis probe during which the exchange of

ingredients between the perfusion fluid and tissue fluid takes place. The perfusate liquids are

preferably aqueous and therefore when the drug to be sampled is highly lipophilic, the recovery

of the drug with the aqueous perfusion liquids would be relatively very less (101, 110, 111). It is

not recommended to use organic solvents as the perfusate could alter the dialysis membrane

properties and may also cause potential toxicity to the tissue. However, in this study, we found

that the permeability of the dialysis membrane did not change significantly even after immersing

the membrane for over 24 hours in the 50:50 PBS:ethanol system.

In case of ETS, as the technique is noninvasive and the sampling fluid is in contact with the

tissue for a short period of time during drug sampling, use of hydroalcoholic systems pose

37

relatively less severe risk as compared to microdialysis. This shows that ETS technique could be

used to sample even lipophilic drugs, by changing the the sampling fluid constitution

appropriately. This is considered as one of the the advantages of ETS over microdialysis

technique.

In the current experiments, as the animals were anesthetized, there were no notable irritation

symptoms due to electroporation or microdialysis. There was no inflammation due to application

of electrical pulses or insertion of probe. However, there was slight bleeding during the

implantation of the probe which stopped within 1 or 2 min completely.

From both microdialysis and ETS techniques, the amount of ciprofloxacin and 8-

methoxypsoralen present in the dermal ECF in rats was calculated using the amount sampled and

the corresponding recovery values as follows:

Dermal ECF concentration=sample concentration

percentage recovery (6)

The concentration time profile of ciprofloxacin (15 mg/kg) in plasma and dermal ECF

(determined by ETS and cutaneous microdialysis) following i.v. bolus administration was

represented in Figure 3-3. The pharmacokinetic parametes calculated from non-compartmental

analysis are given in Table 3-1.

The plasma elimination half life of ciprofloxacin in the present study was found to be

124.12±25.99 min. This was comparable to 102.3±26.2 min and 91±8.6 min reported by

Nouaille-Degorce et al. and Tsai et al. respectively (91, 106).

38

Figure 3-3. Time course of ciprofloxacin in rat plasma (-total drug; ♦-unbound drug)

determined by blood sampling and dermal ECF determined by microdialysis () and ETS ()

techniques following i.v. bolus administration of 15 mg/kg of ciprofloxacin.

Table 3-1. Mean pharmacokinetic parameters of ciprofloxacin (unbound) in plasma and dermal

ECF (determined by ETS and microdialysis) following administration of 15 mg/kg by i.v bolus

in hairless rats.

Parameter ETS Microdialysis

Plasma

Tmax (min) 30 30 30

Cmax (µg/ml) 0.906±0.046 0.729±0.196 1.695±0.131

AUC0-∞ (µg.min/ml) 88.29±20.41 89.71±25.18 240.33± 29.59

t1/2 (min) 55.84±13.44 82.36±22.54 124.12±25.99

0.0

0.5

1.0

1.5

2.0

2.5

0 60 120 180 240

Cip

rofl

oxaci

n c

on

cen

trati

on

(µ

g/m

l)

Time (min)

39

Figure 3-4 represents the concentration-time profile of 8-methoxypsoralen in plasma and dermal

ECF (determined by ETS and cutaneous microdialysis) following oral administration of 5 mg/kg

of 8-methoxypsoralen. The pharmacokinetic parametes calculated from non-compartmental

analysis are given in Table 3-2.

Figure 3-4. Time course of 8-methoxypsoralen in rat plasma (-total drug; ♦-unbound drug)

determined by blood sampling and dermal ECF determined by microdialysis () and ETS ()

technique following oral administration of 5 mg/kg of 8-methoxypsoralen.

The Cmax and Tmax following oral administration of 5 mg/kg of 8-methoxypsoralen to hairless

rats was found to be 0.64±0.14 µg/ml and 120 min respectively, which is comparable to that

reported by Roelandts et al. Roelandts et al. have reported that following oral adminstration of 10

0.0

0.1

1.0

10.0

0 60 120 180 240 300 360

8-m

eth

oxyp

sora

len

con

cen

trati

on

(µ

g/m

l)

Time (min)

40

mg/kg of 8-methoxypsoralen to rats, Cmax and Tmax were ~1.2 µg/ml and 150 min respectively

(112).

Table 3-2. Mean pharmacokinetic parameters of 8-methoxypsoralen (unbound) in plasma and

dermal ECF (determined by ETS and microdialysis) following oral administration of 5 mg/kg in

hairless rats.

Parameter ETS

Microdialysis

Plasma

Tmax (min) 120 120 120

Cmax (µg/ml) 0.338 ± 0.080 0.280 ± 0.051 0.644 ± 0.142

AUC0-∞ (µg.min/ml) 116.08 ± 29.22 116.10 ± 22.85 120.72 ± 36.25

t1/2 (min) 243.83 ± 18.43 237.76 ± 15.83 97.15 ± 6.72

The point to point comparison of the drug concentration in the dermal ECF and the

dermatokinetic parameters, i.e. Cmax, Tmax, AUC0-∞ and t1/2 determined by ETS and microdialysis

technique for ciprofloxacin and 8-methoxypsoralen did not differ significantly (t-test, P<0.05)

(Table 3-1 and Table 3-2). This provides validity to the ETS technique for sampling drugs like

ciprofloxacin and 8-methoxypsoralen. The study also demonstrates that ETS could be

implemented in dermatokinetic investigation regardless of route of administration of drugs.

Successful development of a sampling device based on the ETS concept would be very useful in

carrying out dermatokinetic investigations of drugs. The noninvasiveness of the technique

permits frequent sampling of drugs which helps in relatively more precise calculation of the

kinetic parameters. In addition, the technique could be used to sample drugs in routine

therapeutic drug monitoring. ETS is a potential technique for cutaneous sampling of drugs and

analytes and could replace the invasive blood sampling procedure in future.

41

3.5. CONCLUSIONS

The results of this project showed the workability of ETS in case of drugs with different

solubility and different degree of protein binding. In addition, the present work also demonstrates

the workability of the ETS technique in case of intravenous and oral routes of drug

administration.

Acknowledgements

This project described was supported by Grant Number AR053097 from National Institute of

Arthritis and Musculoskeletal and skin Diseases.

“Reprinted from International Journal of Pharmaceutics, 369, Srinivasa M. Sammeta, Siva Ram

K. Vaka, S. Narasimha Murthy, Transcutaneous sampling of ciprofloxacin and 8-

methoxypsoralen by electroporation (ETS technique), 24-29, 2009, with permission from

Elsevier.”

42

CHAPTER-4

NONINVASIVE TRANSCUTANEOUS SAMPLING OF GLUCOSE BY

ELECTROPORATION

4.1. ABSTRACT

Monitoring of blood glucose levels involves the invasive method of withdrawing blood

which causes inconvenience to patients. The objective of this study was to investigate the

efficiency of the noninvasive electroporation and transcutaneous sampling (ETS) technique for

predicting blood glucose levels. In vitro studies were carried out in Franz diffusion cells using

porcine epidermis to assess the feasibility of transcutaneous sampling of glucose. In vivo, the

ETS technique was assessed in the diabetes-induced Sprague-Dawley rat model. Glucose was

sampled following the application of 30 electrical pulses of 1 ms duration at 120 V/cm2, 1 Hz.

Clarke error grid analysis was carried out for the venous blood glucose levels that were

determined by the ETS with reference to those measured by a glucose meter. The amount of

glucose sampled by the ETS method both in vitro and in vivo was proportional to the dermal

glucose concentration. All data points from in vivo studies were in A and B zones of Clarke error

grid analysis, and the mean absolute relative error was 12.8%. Results of the present study

demonstrate that ETS technique could be developed as a noninvasive method of predicting

venous blood glucose levels in people with diabetes.

Keywords: Electroporation and transcutaneous sampling; Glucose; In vitro; In vivo

43


The objective of the study was to investigate the feasibility of utilizing ETS technique to

sample glucose from dermal ECF that correlates with blood glucose levels.

Diabetes mellitus is a major health concern. Diabetes can lead to chronic complications such as

heart disease, blindness, renal failure, peripheral vascular disease, and limb amputation (113,

114). For proper diabetes management, it is very important that blood glucose levels are checked

regularly. This involves the invasive method of collecting blood through a finger stick system

which has been a major inconvenience for patients (115). Some patients avoid glucose

measurements due to needle phobia. To overcome this problem, different noninvasive blood

glucose monitoring methods have been developed, including near-infrared spectroscopy, far-

infrared spectroscopy, radio wave impedance, reverse iontophoresis, ultrasound, blister

technique, and microneedles (116-120). ETS technique involves application of a few short

electrical pulses on the surface of the skin, which permeabilize the stratum corneum by creating

transient aqueous pathways. The dermal glucose diffuses into the sampling fluid, which is in

contact with the permeabilized region of the skin.


4.3.1. Chemicals

Glucose, alloxan monohydrate, and the glucose assay kit (GAHK-20) were purchased

from Sigma-Aldrich Inc. (St. Louis, MO); 10X phosphate-buffered saline (PBS) premixed

powder was obtained from EMD Chemicals (Gibbstown, NJ).

44

4.3.2. Skin

Porcine belly skin was obtained from a local abattoir. Pieces of skin wrapped in

aluminum foil were heated to 60 °C for 2 min, and the epidermis was gently peeled off the skin.

The fresh epidermis was used for in vitro glucose sampling by electroporation.

4.3.3. In vitro glucose sampling by electroporation

In vitro diffusion studies were carried out in Franz diffusion cells (Logan Instruments

Ltd., Somerset, NJ) using porcine epidermis. The stratum corneum side of the skin was in

contact with the upper sampling compartment and the ventral side with the reservoir

compartment. The active diffusion area was 0.64 cm2. Ag/AgCl electrode wires of 0.5 mm

diameter (Alfa Aesar, Ward Hill, MA) made in the form of circular ring were placed 2 mm away

from the skin in both the sampling and the reservoir compartments. The upper sampling

compartment and the reservoir compartment were filled with 0.4 and 5 ml PBS respectively. The

electrical resistance of the epidermis was measured by placing a load resistor RL (100 kΩ) in

series with the epidermis. Voltage drop across the whole circuit (VO) and across the epidermis

(VS) were measured using a multimeter (Agilent Technologies, Santa Clara, CA). The piece of

porcine epidermis with a resistance greater than 20 kΩ.cm2 was used for the experiment.

The upper sampling compartment was filled with 0.4 ml of PBS, pH 7.4, and the lower reservoir

compartment was filled with 5 ml of glucose solution in PBS with concentrations between 50

and 400 mg/dl. Electroporation was carried out using an ECM 830 Electro Square Porator (BTX

Harvard Apparatus, Holliston, MA). The electroporation protocol was 30 pulses each of 1 ms

duration at 120 V/cm2 of active diffusion area. PBS from the sampling compartment was

withdrawn 15 min after the application of electrical pulses, and the amount of glucose was

measured using the glucose assay kit by UV spectrophotometer at 340 nm. The in vitro

permeability coefficient, Pin vitro (cm/min) was calculated using the formula:

45

Pin vitro =Glucose flux in 15 min

Glucose concentration in reservoir compartment (7)

4.3.4. In vivo glucose sampling by electroporation


Use Committee at the University of Mississippi (Protocol #07-028). In vivo studies were carried

out in Sprague-Dawley rats (Harlan, Indianapolis, IN) (200–224 g) under ketamine (80 mg/kg)

and xylazine (10 mg/kg) anesthesia. The moisture content in the epidermal layers and in the

dermis of each rat was measured using the Delfin moisture meter-SC and the Delfin moisture

meter-D (Delfin Technologies Ltd., Kuopio, Finland) before sampling to ensure the absence of

edema or inflammation (121).

Glucose sampling by ETS was carried out using a custom made sampling cell. The back portion

of rats was shaved, and a custom-made in vivo electroporation cell was fixed using an adhesive

(Krazy Glue, Elmers Products Inc., Columbus, OH). The cell contains a sample collection

chamber in which one of the Ag/AgCl electrodes was placed and the other electrode, which acts

as a counter electrode, was fixed just adjacent to the cell on the surface of the skin using

micropore surgical tape (3M Healthcare, St. Paul, MN). The skin was hydrated with 100 µl of

saline for 5 minutes before each sampling and was replaced with 400 µl of sampling buffer.

Thirty square electrical pulses each of 1 ms duration at 120 V/cm2 were applied, and the

sampling fluid remained in the chamber for 15 min after pulsing. The sampling fluid was

withdrawn, and the amount of glucose present was measured using the glucose assay kit (the

limits of detection were 0.1-5 mg/dl).

4.3.4.1. In vivo transcutaneous permeability coefficient

The calibration used to convert sampling chamber glucose to venous equivalent values is

nothing but determination of the permeability coefficient in rats having steady normal glucose

46

levels. Constant venous glucose levels were confirmed by triplicate samples. Venous glucose

levels and corresponding ETS glucose levels of 18 data points in normal rats were used for

calibration. The normal venous glucose range in Sprague-Dawley rats is ~150-180 mg/dl (122).

The in vivo permeability coefficient, Pin vivo (cm/min), of rat skin was calculated using the

formula:

Pin vivo = Glucose flux in 15 min

Venous blood glucose concentration (8)

4.3.4.2. Sampling in diabetes-induced rats

Nine rats were used, of which three were in the control group and six were in the test

group. Transcutaneous sampling of glucose by ETS was carried out in the test group only. In the

control group, transcutaneous sampling was carried out without the application of electrical

pulses. All nine rats were injected with alloxan (200 mg/kg in saline) intraperitoneally. Generally

the duration required to induce irreversible diabetes with alloxan is ~24 h (123). Blood and

transcutaneous samples were obtained in all rats before injecting alloxan and also after 24 and 36

h after the injection of alloxan. The venous blood glucose was measured using a glucose meter

(True Track Smart System). From the ETS glucose levels, venous blood glucose concentrations

are predicted using the formula:

Venous blood glucose concentration= Glucose flux in 15 min

Pin vivo

(9)

4.3.5. Data Analysis

The clinical utility of the method was determined based on the plot of data on a Clarke

error grid and determining the percentage of points located in the clinically acceptable A and B

47

zones. Mean absolute relative error was also calculated to estimate the clinical accuracy using

the formula

% Mean absolute relative error= Venous glucose-Venous glucose predicted from ETSVenous glucose X 100 (10)


Blood glucose is in homoeostasis with the peripheral tissue extracellular fluid. Therefore,

any change in the blood glucose is reflected in glucose levels in the tissue extracellular fluid. The

transdermal permeability of a polar diffusant such as glucose is limited because of the barrier

properties of stratum corneum. Therefore, transcutaneous sampling of glucose requires a

technique that can reversibly permeabilize the stratum corneum and facilitate the rapid diffusion

of glucose from dermal ECF into the sampling fluid. In vitro diffusion studies were carried out

across the porcine epidermis to assess the feasibility of ETS for sampling glucose. Sampling

without the application of electrical pulses (control) was not possible, as the glucose transfer by

passive diffusion was below detectable levels, whereas a significant amount of glucose diffused

following electroporation of the porcine epidermis. The electrical resistance of the epidermis

dropped by an average of 70±6% by the application of electrical pulses, indicating

permeabilization of the skin. In vitro ETS data are represented in Figure 4-1. Along with other

factors, the rate of diffusion is governed predominantly by the concentration of glucose in the

dermal extracellular fluid (presuming that the concentration of glucose in the sampling

compartment is negligible compared to the total glucose concentration in the reservoir

compartment). Therefore, the amount of glucose sampled by ETS was proportional to the

concentration of glucose in the reservoir compartment fluid (R2=0.93). The in vitro permeability

coefficient (Pin vitro) of porcine epidermis permeabilized by electroporation was 1.9±0.3×10-4

cm/min.

48

Figure 4-1. Relationship between glucose concentration in the reservoir compartment and

glucose sampled by ETS across porcine epidermis in vitro.

Preclinical studies were carried out in a diabetes-induced Sprague-Dawley rat model to assess

the workability of the technique in vivo. The moisture content in the rat skin was measured to

ensure that there was no edema/inflammation in the region of glucose sampling. The

measurement also confirms the uniformity of degree of hydration of epidermal layers of skin.

The Delfin moisture meters measure the skin moisture contents in terms of dielectric constant of

the skin. The average dielectric constant values from moisture meters SC and D were 9.94±1.65

and 20.67±3.31, respectively. The in vivo permeability coefficient (Pin vivo) of rat skin for glucose

was found to be 1.95±0.15×10-5

cm/min. Again, a good linear correlation between the venous

glucose levels and the amount of glucose sampled by ETS was observed (R2=0.87).

0

10

20

30

40

50

0 100 200 300 400

Glu

cose

sam

ple

d b

y E

TS

(m

g/d

l)

Glucose concentration in the reservoir

compartment (mg/dl)

49

Generally, Clarke error grid analysis was performed to assess the clinical utility of glucose

monitoring devices. The analysis divides the reference and measured glucose into five zones-A,

B, C, D, and E. Values in zones A and B are considered clinically acceptable, whereas values in

zones C, D, and E lead to significant errors. At this stage we do not know if one can translate

results from rat model experiments to human application. However, assessing preclinical data

from a clinical perspective would provide some insight into the clinical applicability of the

technique. The venous blood glucose determined by blood sampling (x axis) (reference values)

and that predicted by the ETS techniques (y axis) are shown in Figure 4-2. All data points were

within Clarke error grid A and B zones. A mean absolute relative error of 12.8% was found for

all measurements (n=18) from in vivo data.

50

Figure 4-2. Clarke error grid analysis of venous blood glucose (reference) and venous blood

glucose predicted by ETS in Sprague-Dawley rats.

4.5. CONCLUSIONS

Results support our hypothesis that the concentration of glucose sampled by ETS would

be proportional to the concentration of glucose in the dermal extracellular fluid. ETS could be

developed as a noninvasive method of monitoring blood glucose in people with diabetes.

Acknowledgements

This work has been supported by a Graduate Student Council Research Grant awarded by the

Graduate Student Council, The University of Mississippi.

This work was also partially supported by the National Institute of Arthritis and Musculoskeletal

and Skin Diseases, Grant AR053097.

51

We acknowledge “The Epsilon Group,” Virginia for their support in carrying out Clarke error

grid analysis. We also acknowledge Delfin Technologies Ltd., Kuopio, Finland, for lending us

the moisture meters.

“With kind permission from Journal of Diabetes Science and Technology, Noninvasive

Transcutaneous Sampling of Glucose by Electroporation, 2, 2008, 250-254, S. Srinivasa Murthy,

V. Siva Ram Kiran, S.K. Mathur and S. Narasimha Murthy.”

52

CHAPTER-5

“CHILDRIVE”: A TECHNIQUE OF COMBINING REGIONAL CUTANEOUS

HYPOTHERMIA WITH IONTOPHORESIS FOR THE DELIVERY OF DRUGS TO

SYNOVIAL FLUID

5.1. ABSTRACT

Bioavailability of drugs in the synovial fluid when administered via transdermal route is

highly limited due to the dermal clearance. The purpose of this project was to assess the

efficiency of ChilDrive (CD) technique to improve the drug targeting to the synovial fluid. CD is

a technique of transdermal delivery of drugs combining regional hypothermia and iontophoresis.

Diclofenac sodium and prednisolone sodium phosphate were administered by transdermal route

(Passive, Iontophoresis, Chil-Passive and ChilDrive) at the knee-joint region of hind limb in

Sprague-Dawley rats for 6 h. Intraarticular microdialysis was carried out to determine the time

course of drug concentration in the synovial fluid. Drug levels in synovial fluid after intravenous

and intraarticular administration were also determined. Iontophoretic delivery increased the

AUC0-t (area under the curve) of drugs in the synovial fluid by 3-fold over passive delivery

(0.86±0.04 and 2.0±0.06 µg.h/ml for diclofenac sodium and prednisolone sodium phosphate

respectively). CD resulted in an AUC0-t of 5.2±0.69 and 24.6±1.97 µg.h/ml for diclofenac

sodium and prednisolone sodium phosphate which was ~6-12-fold

53

higher than passive and 2-4-fold higher than iontophoresis. The results support our hypothesis

that CD improves bioavailability of drugs to the synovial joints. CD could be developed as a

potential noninvasive technique for treatment of arthritis.

Keywords: Intraarticular microdialysis; Iontophoresis; Synovial fluid; Transdermal


The objective of the study was:

I. To assess the effect of iontophoresis combined with regional hypothermia (ChilDrive) for

enhancing the bioavailability of drugs in the synovial fluid.

The different types of joints present in the human body are classified into synovial, cartilaginous

and fibrous joints based on the degree and type of movement. Synovial joints are the most freely

moveable joints present in the knee, elbow, shoulder, hip, wrist and neck region of the human

body (124). The presence of lubricating synovial fluid differentiates synovial joints from

cartilaginous and fibrous joints. Arthritis is the most prevalent disorder affecting synovial joints

and a major cause of disability among people of all ages. During arthritis, the synovial fluid

secretion is affected due to the damage caused to the synovial membrane either due to body’s

own immune cells or due to break down of articular cartilage. This leads to thickening of the

synovial membrane, eventually resulting in swollen joints and decreased movement between

bones of the synovial joints. The major types of arthritis include rheumatoid arthritis,

osteoarthritis, juvenile rheumatoid arthritis and infectious arthritis. Pain and inflammation are

associated with arthritis (125). The most common treatment modalities for arthritic conditions

involve oral NSAIDs, including cyclooxygenase (COX) 2 inhibitors and intraarticular injection

of steroids. NSAIDs act by inhibiting the COX enzymes which are responsible for the

conversion of arachidonic acid into prostaglandins, which is a major cause of inflammation

54

(126). Corticosteroids suppress the inflammatory parameters, like erythrocyte sedimentation rate

and C-reactive protein, resulting in decreased disease activity (127). Unfortunately, most of the

drugs are known to cause severe gastric irritation and other gastrointestinal disturbances

following oral administration. In addition, the systemic side effects are unavoidable due to

distribution of drugs (128, 129). Moreover, the amount of drug reaching the synovial joint would

be only a small fraction of the total administered dose. Although intraarticular injections provide

maximum bioavailability in the synovial cavity, they are invasive in nature and therefore do not

allow frequent administration (130). Harvey and Hunter suggested that the number of injections

in a single joint should not be more than four in a single year (131).

The synovial fluid present in joints is an ultrafiltrate of plasma which is continuously absorbed

and replenished by synovial lining of joint cavity, with a rapid turnover time of ~2 h (132, 133).

This is one of the main constraints in the treatment of arthritis as it results in rapid clearance of

drug from the synovial cavity. This necessitates either frequent administration or continuous

infusion of drugs into the synovial fluid to maintain effective level of drugs through the course of

treatment. In an infusion-type drug delivery system, a steady state drug level could be maintained

over prolonged durations. However, intraarticular infusion is practically not feasible unless the

patient is hospitalized and bedridden.

Transdermal delivery of drugs is noninvasive and is associated with advantages such as steady

state input of drugs, prolonged therapeutic activity, and minimized gastrointestinal and systemic

side effects. However, passive delivery of drugs through skin is not capable of achieving

required drug levels in the subdermal musculoskeletal tissues mainly due to the fact that the

majority of the drug that penetrates into skin will be cleared by the dermal circulation (134, 135).

One of the approaches to enhance deeper penetration of drugs is likely to increase the rate of

55

drug delivery higher than the rate of dermal clearance (dQ/dt>Rcl) (Figure 5-1). The rate of drug

delivery is known to increase when iontophoresis is applied across the skin due to the repulsive

driving force on the drug ions. However, the rate of drug delivery by iontophoresis across skin is

limited by the maximum current density that can be applied. The maximum current that is

tolerable and safe is in the range of 0.4-0.5 mA/cm2

(136, 137).

Figure 5-1. Skin and subdermal tissues. dQ/dt represents rate of drug permeation across skin and

Rcl represents rate of dermal clearance of drug. The amount of drug reaching the subdermal

tissues could be increased by rendering Rcl<dQ/dt.

The second approach would be to reduce the rate of dermal clearance in order to accumulate

drug in the dermal tissue. Some research groups have attempted to enhance the delivery of drugs

to subdermal tissues by reducing the rate of dermal clearance with the help of vasoconstrictors

(138). Vasoconstrictors constrict the blood vessels and there-by reduce the blood perfusion rate

(thus the drug clearance) in the tissue. Higaki et al. have shown that there was ~3.3-9.6-fold

enhancement in the amount of antipyrine, salicylic acid, and diclofenac reaching muscle

followed by reducing blood flow upon topical administration of vasoconstrictor phenylephrine

56

(139). But the use of vasoconstrictors over long durations is likely to cause severe undesirable

systemic and cardiovascular effects. Therefore, use of a biophysical technique that reduces the

regional blood perfusion would be safer than the vasoactive drugs.

Vuksanovic and coworkers studied the effect of temperature on the regional blood flow in skin

and have shown that when the skin temperature is >31 °C, the blood flow in the skin remains

constant. However, a rapid decrease in blood flow can be seen when skin temperature drops <31

°C due to vasoconstriction (140). Unlike the vasoactive drugs, temperature induced

vasoconstriction is a regional effect and hence regarded as relatively safe.

Diclofenac sodium and prednisolone sodium phosphate were selected as model drugs to assess

our hypothesis that regional hypothermia during iontophoretic transdermal delivery at the joints

leads to increased bioavailability of drugs in the synovial fluid.


5.3.1. Chemicals

Diclofenac Sodium, Prednisolone Sodium Phosphate were procured from Sigma-Aldrich

Inc (St. Louis, MO), Phosphate buffered saline (PBS, pH 7.4) premixed powder was obtained

from EMD Chemicals (Gibbstown, NJ) and all other chemicals were obtained from Fischer

scientific (Fairway, NJ).

5.3.2. Animals

All the experimental studies were performed on 24 male Sprague-Dawley rats (200-250g)

under ketamine (80 mg/kg) and xylazine (10 mg/kg) anesthesia administered intraperitoneally.

The rats were procured from Harlan (Indianapolis, IN) and maintained on a 12-12 h light/dark

cycle in an animal facility with unlimited access to food and water. The animal studies were

57

approved by the Institutional Animal Care and Use Committee (IACUC) at the University of

Mississippi (Protocol#09-006).

5.3.3. Intraarticular microdialysis

Intraarticular microdialysis was carried out to determine the amount of drug present in

the synovial fluid. After anaesthetizing the rats, the hind limb was held in a fixed position and an

introducer needle with split tubing was passed through the knee joint capsule, the needle was

removed and a CMA 20 microdialysis probe of 4 mm length and 20 kDa molecular weight cut

off (CMA, North Chlemsford, MA) was inserted through the split tubing. The split tubing was

withdrawn leaving the probe implanted in the synovial cavity. The implantation of probe in the

synovial cavity of rat is shown in Figure 5-2. PBS was perfused for 30 min prior to drug

administration for equilibration at flow rate of 2 µl/min. The probe recovery was determined in

vivo by using retrodialysis method. The proper placement of the probe in the synovial cavity was

confirmed by making an incision at the site of probe implantation after completion of studies.

Figure 5-2. Microdialysis probe implanted in the knee-joint for carrying out synovial fluid

microdialysis in hind limb of rat.

58

5.3.4. Intravenous and intraarticular delivery of drugs

Diclofenac sodium and prednisolone sodium phosphate were given by intravenous route

through the tail vein (4 mg/kg) and intraarticular microdialysis was carried out for 6 h to

determine the time course of drug reaching the synovial fluid. Also, drug solutions were injected

directly into the synovial cavity (50 µl) in separate set of rats and intraarticular microdialysis was

carried out for 6 h.

5.3.5. Transdermal drug delivery

5.3.5.1. Design of iontophoretic patch

The iontophoretic patch (Figure 5-3) was fabricated using a woven adhesive backing

membrane fitted with a snap type Ag/AgCl electrode. One cm2 foam pad was attached onto the

backing membrane above the electrode. Drug solution (1%) made in PBS was filled in the foam

pad just before the application of patch. Similar patch was made which was filled with PBS and

acts as counter electrode.

The patch containing drug solution was placed on the lateral side of the rat knee and the counter

electrode was placed on the opposite side for all the transdermal drug delivery studies.

Intraarticular microdialysis was carried out for 6 h and the dialysate was collected every hour to

determine the amount of drug reaching the synovial fluid. In all the cases, drug containing patch

was applied only after the equilibration of microdialysis probe in the synovial joint.

59

Figure 5-3. Design of iontophoretic patch with ‘A’ displaying the dorsal view and ‘B’ displaying

the ventral view.

5.3.5.2 Passive drug delivery

Passive drug delivery studies were carried out for both the drugs by using the patches

fabricated with electrodes to maintain similar experimental conditions compared to

iontophoresis. In passive drug delivery, no current was applied.

5.3.5.3 Iontophoretic drug delivery

Iontophoresis was carried out using an IOMED Phoresor (Iomed Inc, Salt Lake city, UT)

by connecting the lead clips from the iontophoresis unit to the snap on the drug electrode and

counter electrode. Electrical current of 0.5 mA/cm2 was applied continuously for 6 h.

5.3.5.4. Chil-Passive drug delivery

Passive drug delivery carried out under regional hypothermia was termed as “chil-

passive” drug delivery. Regional hypothermia was induced by placing the hind limb of the rats

on a water pad circulated continuously with water at reduced temperature (15-20 C) for the

entire period of study.

60

5.3.5.5. ChilDrive (CD) drug delivery

Iontophoresis carried out under regional hypothermia was termed as “ChilDrive” (CD).

Regional hypothermia was induced according to method mentioned earlier. With exception of

regional hypothermia, all other experimental conditions were similar to iontophoretic drug

delivery.

5.3.6. Analytical methods

The amount of diclofenac sodium and prednisolone sodium phosphate present in

microdialysis samples was analyzed by high performance liquid chromatography. The HPLC

system (Waters, MA) consisted of a chromatographic pump (Waters 1525), an autosampler

(Waters 717 plus), a UV detector (Waters 2487) and a Symmetry®

C18 column (4.6 x 150 mm).

Analysis of diclofenac sodium samples was carried out at a wavelength of 278 nm and the

mobile phase consisted of a mixture of acetonitrile and 0.01 M potassium dihydrogen phosphate

(pH 6.3), (35:65 v/v) with flow rate of 1 ml/min (141). The linearity range was between 10-1000

ng/ml (R2=0.99).

Analysis of prednisolone sodium phosphate samples was carried out at a wavelength of 242 nm

and the mobile phase was prepared by mixing 250 ml of isopropanol with 2 ml H3PO4 and

diluting with water to 900 ml. The pH of the solvent was adjusted to 3.0 with 1.0 M NaOH and

then diluted to 1000 ml with water and the flow rate was set to 1 ml/min (142). The linearity

range was between 10-1000 ng/ml (R2=0.99).

5.3.7. Statistical analysis

The statistical analysis of AUC0-t data after transdermal, intravenous and intraarticular

administration were determined by performing Unpaired t-test (GraphPad, InStat 3.0) to

61

determine the level of significance between two groups. P<0.05 was considered as the level of

significance.


Microdialysis technique was employed as a tool to investigate the time course of drug

concentration in the synovial fluid (79, 143). The in vivo probe recovery was performed using

retrodialysis method and was found to be 19.28±4.99% for diclofenac sodium and 23.01±5.48%

for prednisolone sodium phosphate.

5.4.1. Intravenous and intraarticular delivery of drugs

Drugs were administered intravenously to determine the fraction of the total dose

reaching the synovial fluid. The time course of drug in the synovial fluid upon intravenous

administration is shown in Figure 5-4 and 5-5.

The AUC0-t in the synovial fluid after intravenous administration was found to be 0.609±0.02

µg.h/ml for diclofenac and 1.445±0.17 µg.h/ml for prednisolone. The total AUC0-t in the

synovial fluid by systemic administration was comparable with that of passive topical delivery of

drugs (Table 5-1 and 5-2) and was significantly less than all the other modes of drug

administration. From these results it appears that the systemic delivery of drug for treating

synovial disorders may not end in remarkable results unless the dose and frequency are high

enough to maintain effective drug levels over long duration.

62

Figure 5-4. Time course of diclofenac sodium in synovial fluid upon intraarticular () and

intravenous () administration.

The Cmax and Tmax in synovial fluid for diclofenac following intravenous administration were

0.174 µg/ml and 4 h. Similarly for prednisolone sodium phosphate they were found to be 0.339

µg/ml and 2 h. Obviously, delivery of drugs directly into the synovial cavity resulted in

significantly higher bioavailability than all the other modes of drug administration (Table 5-1

and 5-2). The concentration-time profile followed a mono-exponential disposition curve (Figure

5-4 and 5-5).

0.1

1

10

100

0 2 4 6

Log c

on

cen

trati

on

of

dic

lofe

nac

sod

ium

in

syn

ovia

l fl

uid

(µ

g/m

l)

Time (h)

63

Figure 5-5. Time course of prednisolone sodium phosphate in synovial fluid upon intraarticular

() and intravenous () administration.

Table 5-1. AUC0-t and Cmax of diclofenac sodium in synovial fluid following transdermal

(Passive, Chil-Passive, Iontophoresis, ChilDrive), intravenous and intraarticular administration.

AUC0-t (µg.h/ml) Cmax (µg/ml)

Passive 0.864 ± 0.042 0.188 ± 0.022

Chil-Passive 1.336 ± 0.105 0.287 ± 0.024

Iontophoresis 2.683 ± 0.152 0.644 ± 0.036

ChilDrive 5.208 ± 0.693 1.259 ± 0.098

Intravenous 0.609 ± 0.028 0.174 ± 0.009

Intraarticular 77.829 ± 5.422 25.974 ± 1.340

0.1

1.0

10.0

100.0

1000.0

0 2 4 6

Log c

on

cen

trati

on

of

pre

dn

isolo

ne

sod

ium

ph

osp

hate

in

syn

ovia

l fl

uid

(µ

g/m

l)

Time (h)

64

Table 5-2. AUC0-t and Cmax of prednisolone sodium phosphate in synovial fluid following

transdermal (Passive, Chil-Passive, Iontophoresis, ChilDrive), intravenous and intraarticular

administration.

AUC0-t (µg.h/ml) Cmax (µg/ml)

Passive 2.003 ± 0.067 0.468 ± 0.106

Chil-Passive 3.958 ± 0.303 0.863 ± 0.055

Iontophoresis 6.364 ± 0.486 1.724 ± 0.423

ChilDrive 24.605 ± 1.975 7.360 ± 1.032

Intravenous 1.445 ± 0.174 0.339 ± 0.032

Intraarticular 479.946 ± 45.040 141.890 ± 20.179

The AUC0-t in synovial fluid after intraarticular administration was 77.82±5.42 and

479.94±45.04 µg.h/ml for diclofenac sodium and prednisolone sodium phosphate respectively.

However, one should note that the drugs administered into the synovial cavity were eliminated

rapidly as reflected by the elimination half-lives of drugs in the synovial fluid (t1/2 was 1.55±0.02

h for diclofenac and 1.19±0.01 h for prednisolone respectively). The rapid elimination of drug

could be attributed to the rapid turnover of synovial fluid and/or to the metabolism of drug in the

synovial fluid. Relatively longer half-life of diclofenac in the synovial fluid is likely due to

slightly higher protein binding (>98%) compared to prednisolone (65-91%) (144, 145).

5.4.2. Passive drug delivery

Vuksanovic et al. have clearly demonstrated using the laser doppler studies that the blood

perfusion rate in the skin tissue is decreased under hypothermic conditions (140). The decreased

blood flow is likely to result in decreased rate of dermal clearance of drugs. Passive drug

delivery studies were carried out at normal body temperature and at regional hypothermia

65

condition (Chil-Passive) for both the drugs. The drug concentration-time profile in the synovial

fluid was used to calculate the AUC0-t. The total AUC0-t in synovial fluid after passive

transdermal administration of drugs was almost doubled due to induction of regional

hypothermia (Table 5-1 and 5-2).

5.4.3. Iontophoretic drug delivery

Application of iontophoresis (0.5 mA/cm2) resulted in enhanced bioavailability of drugs

into the synovial cavity (Figure 5-6 and 5-7). Iontophoresis mediated delivery of drugs resulted

in 3-fold (P<0.01) higher AUC0-t in synovial fluid compared to passive drug delivery (Table 5-1

and 5-2). These results are in agreement with previous reports in which other authors showed

that iontophoresis aids in better treatment of joint disorders. Aiyejusunle et al. have reported that

cathodal iontophoresis (0.1-0.3 mA/cm2) of sodium salicylate resulted in reduced pain and

functional disability. Bender et al. have shown that the concentration of etofenamate in the

synovial fluid following iontophoresis was twice that of etofenamate levels in serum and hence

topical iontophoresis of etofenamate was suitable for alleviation of arthritis in superficially

located joints (146, 147).

In case of CD, iontophoresis was carried out by placing hind limb portion of rat on a water pad

through which water at 20 ºC was circulated, so that temperature of skin was maintained in the

range of ~20-25 ºC throughout the study. Reducing skin temperature has been widely used in

treatment techniques like cryotherapy where-in temperatures in the range of 7-20 C have been

used. As hypothermia is induced regionally in the present studies, it is less likely to lead to any

physiological changes in the whole body. Chesterton et al. reported that physiological changes

like local analgesia, reduced nerve conduction velocity, and reduced metabolic enzyme activity

are seen only at temperatures below 14 C (148).

66

Figure 5-6. Time course of diclofenac sodium in the synovial fluid following different modes of

transdermal administration (♦-Passive, -Chil-Passive, -Iontophoresis and -ChilDrive).

The AUC0-t of drug in the synovial fluid due to CD was ~2-4-fold (P<0.01) higher than

iontophoresis in case of both the drugs. This is exactly the fold of enhancement observed

between passive transdermal and chil-passive as well. Compared to passive drug delivery, the

AUC0-t in the synovial fluid of drugs when administered by CD was ~6-12-fold higher (P<0.01).

Interestingly, all the synovial concentration-time profiles following transdermal drug

administration showed a typical initial increase followed by decrease in drug concentration

regardless of the mode of drug delivery. It is likely that the change in the synovial fluid turnover

rate due to insertion of the probe might have caused this kind of concentration-time profile.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0 1 2 3 4 5 6

Syn

ovia

l fl

uid

con

cen

trati

on

of

dic

lofe

nac

sod

ium

(µg/m

l)

Time (h)

67

Figure 5-7. Time course of prednisolone sodium phosphate in the synovial fluid following

different modes of transdermal administration (♦-Passive, -Chil-Passive, -Iontophoresis and

-ChilDrive).

5.5. CONCLUSION

Regional hypothermia is one the most promising approaches to reduce the rate of clearance of

drug from the skin. ChilDrive can be developed as a potential way of treating musculoskeletal

disorders. The technology is likely to be more patient compliant than oral or parenteral delivery

of drugs.

“With kind permission from Springer Science+Business Media: Pharmaceutical Research,

“ChilDrive”: A technique of combining regional cutaneous hypothermia with iontophoresis for

delivery of drugs to synovial fluid, 26, 2009, 2535-2540, Srinivasa M. Sammeta and S.

Narasimha Murthy.”

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

0 2 4 6

Syn

ovia

l fl

uid

con

cen

trati

on

of

pre

dn

isolo

ne

sod

ium

ph

osp

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(µ

g/m

l)

Time (h)

68

CHAPTER-6

MAGNETOPHORESIS FOR ENHANCING TRANSDERMAL DRUG DELIVERY:

MECHANISTIC STUDIES AND PATCH DESIGN

6.1. ABSTRACT

Magnetophoresis is a method of enhancement of drug permeation across biological

barriers by the application of magnetic field. The present study investigated the mechanistic

aspects of magnetophoretic transdermal drug delivery and also assessed the feasibility of

designing a magnetophoretic transdermal patch system for the delivery of lidocaine. In vitro drug

permeation studies were carried out across the porcine epidermis at different magnetic field

strengths. The magnetophoretic drug permeation “flux enhancement factor” was found to

increase with the applied magnetic field strength. The mechanistic studies revealed that the

magnetic field applied in this study did not modulate permeability of the stratum corneum

barrier. The predominant mechanism responsible for magnetically mediated drug permeation

enhancement was found to be “magnetokinesis”. The octanol/water partition coefficient of drugs

was also found to increase when exposed to the magnetic field. A reservoir type transdermal

patch system with a magnetic backing was designed for in vivo studies. The dermal

bioavailability (AUC0-6h) from the magnetophoretic patch system in vivo in rats was significantly

higher than the similarly designed non-magnetophoretic control patch.

Keywords: Magnetophoresis; Transdermal; Lidocaine; Partition coefficient; Enhancer

69



I. To investigate the technique of magnetophoresis for enhancing transdermal drug

permeation.

II. To resolve the mechanisms leading to enhanced transdermal drug permeation by

magnetophoresis.

III. To assess the feasibility of designing a simple magnetophoretic transdermal patch

system.

Transdermal permeation of polar and hydrophilic drugs is limited by the barrier property of

stratum corneum. Therefore, several chemical and physical permeation enhancement techniques

have been explored to improve the transdermal delivery of poorly permeable drugs (29, 149-

151). In this direction, Murthy et al. investigated the plausibility of using magnetic field to

enhance the transdermal permeation of drugs. The authors reported for the first time that the

static magnetic field could facilitate the transdermal permeation of drugs such as benzoic acid,

salbutamol sulfate and terbutaline sulfate (152, 153). The authors speculated that the enhanced

transdermal drug permeation by the applied magnetic field could be a combination of multiple

mechanisms such as magnetokinesis and alteration of barrier property of skin. A recent study by

Krishnan et al. utilized an electromagnetic field to enhance the drug permeation across the skin.

Krishnan et al. reported that the potential mechanism for enhanced transdermal drug permeation

by the electromagnetic field of 5 mT was due to modulation of the permeability of stratum

corneum (154). There are a number of reports regarding the use of magnetite incorporated

particulate systems in drug delivery and imaging. An externally applied magnetic field has been

shown to direct the systemically administered magnetoliposomes or nanoparticles to the target

70

tissue (155, 156). The mechanism associated with this approach is attraction of the ferromagnetic

substance, magnetite, towards the magnetic field. The present project involved delivery of a drug

across the skin by application of magnetic field, where drug molecules which are diamagnetic in

nature are repelled away from the magnetic field. The base and salt form of a local anesthetic,

lidocaine which is a diamagnetic molecule was selected as the candidate for the present study.


6.3.1. Materials

Lidocaine Hydrochloride (LH) was purchased from Spectrum Chemicals (New

Brunswick, NJ), lidocaine base (LB), D-mannitol and sodium chloride were procured from

Sigma-Aldrich Inc. (St. Louis, MO). [3H] Water and D-[1-

14C] Mannitol were procured from

American Radiolabeled Chemicals, Inc. (St. Louis, MO) and all other chemicals were obtained

from Fischer Scientific (Fairway, NJ). Neodymium magnets were purchased from United

Nuclear Scientific (Laingsburg, MI) and K&J Magnetics, Inc. (Jamison, PA).

6.3.2. Porcine epidermis

Porcine belly skin was obtained from a local abattoir. Pieces of skin wrapped in

aluminum foil were heated to 60 ºC for 2 min and the epidermis was gently peeled off the skin.

The fresh epidermis was placed on glass microscopic slides and kept dry at 4 ºC and was used

within three days. Prior to use, the epidermis was hydrated with normal saline for an hour.

6.3.3. General in vitro experimental setup

A vertical Franz-type diffusion apparatus (PermeGear, Inc. Hellertown, PA) was used for

all resistance measurements and drug permeation studies across the porcine epidermis. A piece

of porcine epidermis was placed between the two compartments of the diffusion apparatus, one

serving as the donor and other as the receiver compartment. The active diffusion area of the

71

epidermis was 0.2 cm2. The receiver compartment (5 ml) and donor compartment (0.2 ml) were

filled with isotonic saline solution. Ag/AgCl electrode wires of 0.5 mm diameter (Alfa Aesar,

Ward Hill, MA) made in the form of concentric rings were placed 2 mm away from the porcine

epidermis in both the donor and the receiver compartments. Before carrying out permeation

studies, the electrodes were connected to the electrical set up for resistance measurement. The

electrical resistance of the epidermis was measured and only pieces of epidermis, which had

resistances greater than 20 kΩ.cm2 were used for the experiments. During the in vitro drug

permeation studies the temperature of receiver compartment was maintained at 37±1 C by water

circulation.

6.3.4. Passive drug permeation studies

After the electrical resistance measurement, the isotonic saline in the donor compartment

was replaced with 0.2 ml of LH solution (10 mg/ml) or LB solution (1.66 mg/ml) prepared in

isotonic saline and the permeation studies were carried out for 8 h. During the course of

permeation studies, at each sampling time point (2, 3, 4, 5, 6, 7 and 8 h) 0.2 ml of the receiver

compartment solution was sampled out, and was subsequently replaced with fresh isotonic saline

solution. The amount of drug in the samples was analyzed by HPLC.

6.3.5. Magnetophoretic drug permeation studies

The experimental set up was similar to passive drug permeation studies, and the

magnetic field was applied by placing two neodymium magnets (1.2 cm length x 0.6 cm width x

0.6 cm thickness) on either side of the donor compartment and at a distance of 2 mm above the

porcine epidermis (Figure 6-1). Permeation studies were carried out for 8 h at different magnetic

field strengths of 30, 150 and 300 mT. The amount of drug permeated across porcine epidermis

72

into the receiver compartment solution was determined by analyzing the samples withdrawn at

different time points as discussed in section 6.3.4.

Figure 6-1. Experimental set up for permeation studies. Porcine epidermis ‘A’ was sandwiched

between the donor compartment ‘B’ filled with drug solution and receiver compartment ‘C’

filled with isotonic saline. Two neodymium magnets ‘D’ were placed on either side of the donor

compartment. The donor and receiver compartments were fitted with Ag/AgCl electrode wires

for measurement of electrical resistance.

6.3.6. Drug permeation across the dialysis membrane

The drug permeation studies were carried out across Spectra/Por®

dialysis membrane

(1000 Da molecular weight cut-off) in the absence and presence of magnetic field (300 mT)

following exactly the same procedure described in the passive and magnetophoretic drug

permeation studies (Section 6.3.4 and 6.3.5).

6.3.7. Drug permeation across the pretreated epidermis (epidermis exposed to magnetic

field)

The porcine epidermis was pretreated (for 24 h) by exposure to the magnetic field by

using a similar set up as shown in Figure 6-1, without placing any medium in the donor

compartment. After the pretreatment, the donor chamber was filled with drug solution and the

73

passive drug permeation studies were carried out across the pretreated epidermis similar to that

discussed in section 6.3.4.

6.3.8. [3H] Water and D-[1-

14C] Mannitol transport studies

The experimental set up was similar to that used for the passive and magnetophoretic

drug permeation studies (Section 6.3.4 and 6.3.5). The donor compartment was filled with 0.2 ml

of [3H] water (20 µCi/ml) in isotonic saline for water transport studies. Mannitol (1 mg/ml) and

[1-14

C] mannitol (5 µCi/ml) in isotonic saline was placed in the donor compartment for mannitol

transport studies. The receiver compartment was filled with isotonic saline in both the studies. In

the case of magnetophoretic permeation studies, a magnetic field of 300 mT was applied. After

the passive and magnetophoretic permeation studies, tritiated water and mannitol transport across

porcine epidermis was measured by withdrawing samples at regular intervals for a period of 8 h.

The amount of [3H] water and [

14C] mannitol was determined by scintillation counting

(Scientisafe Econo 2 cocktail, Fischer Scientific, Fairway, NJ; liquid scintillation analyzer, Tri-

Carb 1600 TR, Packard Bioscience, Meriden, CT).

6.3.9. Transepidermal Water Loss (TEWL) studies

The TEWL was measured using a Vapometer (Delfin Technologies, Kuopio, Finland).

Porcine epidermis was mounted between the receiver and donor compartments of the diffusion

cell (Figure 6-1). Isotonic saline solution was placed only in the receiver compartment and the

donor compartment was kept empty. The whole set up was placed in a temperature (37 °C) and

humidity (RH 55%) controlled chamber and the TEWL was measured by fixing the Vapometer

directly on the donor compartment. TEWL was measured prior to the start of the experiment.

The test set was exposed to magnetic field (300 mT). The TEWL was measured at different time

points up to 8 h.

74

6.3.10. Fourier Transform Infrared (FT-IR) spectroscopic studies

The porcine epidermis was subjected to FT-IR spectroscopic studies in absence and

presence of magnetic field. Infrared spectra were obtained in the transmission mode using a

Nicolet 6700 FT-IR spectrometer in the frequency range 4000-1000 cm-1

. Sixty four scans were

collected at 4 cm-1

resolution. The same piece of porcine epidermis was used for recording the

spectra in absence and presence of magnets. Two magnets were placed on the surface of porcine

epidermis at a distance of 6 mm with unlike poles facing each other (magnetic field of 300 mT)

for recording the spectra. The magnets used, distance between magnets and polarity were same

as those used for in vitro permeation studies.

6.3.11. Effect of magnetic field on octanol-water and stratum corneum (SC)-aqueous

vehicle partitioning of drug

To determine the octanol-water partition coefficient, deionized water equilibrated with an

equal volume of octanol was used to prepare the drug solution. 0.2 ml of drug solution

(concentrations of 50, 100, 250 and 500 µg/ml) was placed in contact with 0.2 ml of octanol in a

glass vial. The two phases were mixed thoroughly by vigorous shaking for 6 h and allowed to

equilibrate for next 18 h. After equilibration, 20 µl of aqueous and octanol phases were

withdrawn for measurement and calculation of “initial partition coefficient”. The vials from the

test group were placed in presence of magnetic field (300 mT) and the control set were not. Only

the aqueous phase was placed in presence of magnetic field. After 24 h, the “final partition

coefficient” of the drug in the control and test set of vials was determined. The average

difference in the partition coefficient (final partition coefficient value-initial partition coefficient

value) was compared between control and the test sets.

75

To determine SC-vehicle partition coefficient, preweighed samples of SC (isolated by trypsin

digestion method as shown by Essa et al.) were placed in 0.2 ml of drug solution in isotonic

saline (100 µg/ml) in a glass vial for 48 h (157). A magnetic field of 300 mT was applied on the

bulk of the vehicle in the vial, only to the test group. At the end of 48 h, the amount of drug

present in the vehicle and SC were measured to estimate the partition coefficient of drug.

6.3.12. Fabrication of magnetophoretic transdermal patches

Magnetophoretic transdermal patches were designed by affixing a series of neodymium

magnets (10 mm length x 1.5 mm width x 1.5 mm thickness) on a nonmagnetic backing

membrane which is placed over an adhesive 3M™ foam tape (3M Drug Delivery Systems, St.

Paul, MN). About 200 mg of LH and LB gel (2% w/w) prepared in HPMC base (4% w/v) was

spread over the magnets (1.5 cm2) as a thin layer, which acts as drug reservoir. The magnetic

field strength at the skin surface of the magnetophoretic patch system was 450 mT. In the case of

passive transdermal patches, the neodymium magnets were replaced with similar nonmagnetic

metal pieces.

LH gel was prepared using HPMC in deionized water; whereas, LB gel was prepared using

HPMC in hydroalcoholic solution (water:alcohol, 70:30) due to the limited solubility of LB in

aqueous medium.

6.3.13. In vivo dermal bioavailability studies of magnetophoretic patch system

The animal studies were approved by the Institutional Animal Care and Use committee

(IACUC) at the University of Mississippi (Protocol#09-031). The in vivo studies were performed

on 12 male Sprague-Dawley rats (200-250 g) under ketamine (80 mg/kg) and xylazine (10

mg/kg) anesthesia administered intraperitoneally. The rats were procured from Harlan

(Indianapolis, IN).

76

Cutaneous microdialysis was carried out in rats by inserting a 20G needle intradermally through

a distance of 2 cm and a linear microdialysis probe of 5 mm membrane length and 30 kDa cut-

off molecular weight (BASi, West Lafayette, IN) was inserted through this needle and the needle

was withdrawn leaving the probe implanted in the dermal tissue. The inlet tube was connected to

an injection pump (BASi, West Lafayette) and isotonic saline solution was perfused at a flow

rate of 2 µl/min. After equilibration of the probe for 30 min, a transdermal patch system was

placed above the region where microdialysis probe was inserted and studies were carried out for

a period of 6 h. For both the salt (LH) and base (LB) forms of the drug, animal studies were

performed using passive and magnetophoretic patch systems respectively. The probe recovery

was determined in vivo by retrodialysis method.


The amount of lidocaine hydrochloride and lidocaine were analyzed by high performance

liquid chromatography. The HPLC system (Waters, MA) consisted of a chromatographic pump

(Waters 1525), autosampler (Waters 717 plus) and an UV absorbance detector (Waters 2487).

Symmetry®

C18 column (4.6 x 150 mm) was used and the mobile phase consisted of a mixture

(14/86 v/v) of acetonitrile and potassium dihydrogen phosphate 0.05 M (pH adjusted to 4.0) 1.3

ml/min at 216 nm (158).


Statistical analysis was carried out using GraphPad InStat 3 software. Unpaired t-test was

performed and P<0.05 was considered as the level of significance.

77


Porcine epidermis has been accepted as one of the most appropriate models for

transdermal drug delivery studies due to its similarities in structure and lipid composition of

stratum corneum with the human (159-161). Therefore, porcine epidermis was employed in all

the in vitro experiments in the present study. The magnetic field of different strengths (30-

300 mT) was generated by varying the distance between the poles of bar magnets or by using

magnets of different strengths. The magnetic field strengths were measured at the surface of the

epidermis using a hand-held Gauss/Tesla meter (F.W. Bell, Model # 4048).

6.4.1. Magnetophoresis of LH across the porcine epidermis

The data from Figure 6-2 and Table 6-1 clearly demonstrate the ability of magnetic field

to enhance the permeation of LH across the porcine epidermis. The permeation flux

enhancement factor (EF) (Permeation flux in presence of magnetic field / flux in absence of

magnetic field) was found to increase linearly with increasing applied magnetic field strength as

shown in the insert graph in Figure 6-2. The relationship between the permeation flux

enhancement factor for LH and magnetic field strength could be presented as EF = 0.027S+1,

(R2=0.92) where ‘EF’ is the flux enhancement factor and ‘S’ is the strength of magnetic field at

the surface of the epidermis.

78

Figure 6-2. Cumulative permation of LH across porcine epidermis in case of passive (♦),

magnetic field strength of 30 mT (), 150 mT () and 300 mT (). Insert graph shows plot of

magnetic field strength vs. flux enhancement factor.

Table 6-1. Permeation flux and flux enhancement factor of LH and LB across porcine

epidermis.

Magnetic

field (mT)

LH LB

Permeation flux

(µg/cm2/h)

Flux

enhancement

factor (EF)

Permeation

flux (µg/cm2/h)

Flux

enhancement

factor (EF)

0 0.18 ± 0.06 1.0 0.57 ± 0.12 1.0

30 0.53 ± 0.09 2.9 0.75 ± 0.18 1.3

150 1.01 ± 0.17 5.6 1.14 ± 0.29 2.0

300 1.61 ± 0.12 8.9 2.23 ± 0.22 3.9

79

The concept of utilizing magnetic field for enhancement of transdermal drug delivery emanated

from the hypothesis that diamagnetic substances, which are generally repelled away from the

external magnetic field would be driven across the biological barrier along the direction of the

magnetic field gradient. However, the diamagnetic susceptibility is considered to be a very weak

property and the driving force generated under mT range magnetic field is regarded as rather

mild. Despite this, a significant transdermal permeation enhancement of drug was observed in

the presence of magnetic field suggesting that the magnetophoresis phenomenon could involve

multiple mechanisms.

Generally, transdermal physical permeation enhancement techniques are known to cause

enhanced drug permeation across the biological barriers by mechanisms such as kinesis of drug

molecules and/or by alteration of the biological barrier. Iontophoresis is one of the techniques

which is known to enhance the transdermal drug permeation predominantly by driving the drug

ions due to electrorepulsion. On the other hand, techniques such as electroporation, sonophoresis

and thermoporation enhance the transdermal delivery of drugs by disruption of the stratum

corneum barrier. To resolve the role of different mechanisms in case of magnetophoresis,

systematic mechanistic studies were carried out as discussed further in this study.

6.4.2. Permeation across the pretreated epidermis (epidermis exposed to magnetic field)

To assess the plausible effect of an applied magnetic field on the barrier property, the

epidermis was pretreated by exposure to magnetic field (30, 150 and 300 mT) for 24 h. Passive

permeation studies (in absence of magnetic field) were carried out across the pretreated

epidermis. Permeation experiments across the untreated epidermis served as control. The

permeation flux across the epidermis did not differ significantly between pretreated and

untreated epidermis suggesting that prior exposure of the epidermis to magnetic field did not

80

bring about any long lasting change in the epidermal permeability (Table 6-2). However, this

experiment did not rule out the possibility of any potential real time microstructural changes in

the epidermis during the magnetophoretic drug permeation studies.

Table 6-2. Permeation flux of LH and LB across pretreated porcine epidermis (Epidermis

exposed to magnetic field for 24 h). The epidermis was exposed to different magnetic field

strengths and the drug permeation studies were carried out across the pretreated epidermis.

Pretreatment

Magnetic field (mT)

Permeation flux after pretreatment (µg/cm2/h)

LH LB

0 0.17 ± 0.05 0.59 ± 0.09

30 0.18 ± 0.03 0.56 ± 0.13

150 0.16 ± 0.06 0.60 ± 0.19

300 0.17 ± 0.03 0.57 ± 0.16

TEWL and electrical resistance have been considered as two promising parameters used to

assess the barrier integrity of the stratum corneum (162, 163). However, TEWL is known to be

relatively less sensitive than electrical resistance as it can only reflect mechanical disruption of

the barrier. On the other hand, electrical resistance is relatively more sensitive and could change

significantly even due to any microstructural changes in the stratum corneum. TEWL and

electrical resistance of the porcine epidermis were measured at different time points while the

epidermis was being exposed to magnetic field. The initial TEWL and electrical resistance of the

porcine epidermis was 6.15±0.71 g/m2/h and 38.1±1.2 kΩ.cm

2 respectively. The TEWL value

across the intact epidermis that has been reported by other groups agrees well with that found in

this case (164, 165). The TEWL and electrical resistance remained constant during study period

of 8 h. Therefore, it is most likely that, at the applied magnetic field strengths in these

experiments, no significant real time transient structural alterations occurred in the epidermis.

81

To further confirm the non-interaction of the magnetic field with the stratum corneum lipids, the

epidermis were subjected to FT-IR studies. The FT-IR spectra of the stratum corneum side were

recorded before and after exposure to the magnetic field. A custom designed skin holder was

used to hold the magnets in place even while the spectra were recorded. There was no significant

shift in the peaks in the lipid region (2920 and 2850 cm-1

) or in the amide regions (1650 and

1550 cm-1

) as clearly seen in the representative spectra shown in Figure 6-3, even after exposure

to magnetic field for over 80 h. This data also supplements the hypothesis that the magnetic field

strength used in this project has negligible or no effect on the skin structure and permeability.

Figure 6-3. Representative FT-IR spectra of porcine epidermis in absence of magnetic field (A),

presence of magnetic field (B) and after exposure to magnetic field for 80 h (C).

6.4.3. Magnetokinesis of drug

Magnetokinesis is a phenomenon of forced propagation of drug molecules under the

applied magnetic field. Magnetokinesis of molecules and particles under a gradient magnetic

field has been reported in the past by several research groups (166, 167). There are at least two

82

potential mechanisms that can contribute to magnetokinesis in the present experimental set up;

magnetorepulsion and magnetohydrokinesis. Magnetorepulsion could be described as driving of

the drug molecules (due to the repulsive force experienced by the induced diamagnet) in the

presence of external magnetic field. Magnetohydrokinesis mediated drug transport is due to the

movement of water across the membrane under the influence of an external magnetic field.

These mechanisms are comparable to electrorepulsion and electroosmosis in the case of

iontophoretic transdermal drug delivery. To reassess if magnetokinesis is one of the mechanisms

leading to transdermal drug permeation enhancement, permeability studies were also carried out

across the dialysis membrane with a cut-off size of 1000 Da. A nonbiological barrier like dialysis

membrane was chosen to eliminate any effect of magnetic field on the barrier. Interestingly,

application of magnetic field (300 mT) enhanced the permeation flux of LH across the dialysis

membrane also by ~ 4-fold over passive permeation (passive: 10.79±2.26 µg/cm2/h vs. magnet:

38.98±6.21 µg/cm2/h). These results suggest that magnetokinesis is one of the predominant

mechanisms responsible for drug permeation enhancement at these applied magnetic field

strengths. However, these experiments did not differentiate the relative contribution due to

magnetorepulsion and magnetohydrokinesis.

Magnetohydrokinesis across the epidermis under the influence of the applied magnetic field was

investigated by studying the transport of tritiated water across the epidermis. There was a

significant difference between the water transport flux across the epidermal membrane in the

absence (32.63±5.25 nl/cm2/h) and presence (57.21±8.63 nl/cm

2/h) of magnetic field (300 mT)

indicating that hydrokinesis could be one of the factors contributing significantly to the overall

drug transport due to magnetokinesis.

83

Enhanced transport of water would result in proportionately enhanced transport of dissolved

substances as well. Therefore to add supportive evidence to the magnetohydrokinesis

phenomenon, transport of 14

C-mannitol across the epidermis was investigated. The application of

magnetic field (300 mT) resulted in ~2.7-fold enhancement (5.18±1.88 ng/cm2/h) in the transport

flux of mannitol as compared to passive diffusion (1.89±0.49 ng/cm2/h). The contribution of

magnetohydrokinesis to the drug transport across biological membranes may become more

apparent when higher magnetic field strengths are applied and may become remarkably

significant when a more concentrated drug solution is placed in the donor chamber.

6.4.4. Effect of magnetic field on octanol-water and SC-aqueous vehicle partitioning of

drug

One of the predominant factors known to determine the permeation of drugs across the

skin is the partition coefficient. Generally, chemical permeation enhancers improve the drug

permeation by increasing the partitioning of drug into the skin lipids. Sun et al. have shown that

induction of magnetic field enhanced the extraction of acetone due to the increase in partition

coefficient (168). In the present work, the effect of exposure to magnetic field on the partition

coefficient of drug between octanol and water phases was investigated. The partition coefficient

of LH in the absence of magnetic field was 13.80±4.79 and in the presence of magnetic field

(300 mT) was found to be 28.94±2.11. In the present set up for determination of partition

coefficient, the aqueous phase was exposed to the magnetic field and the gradient exists along

the aqueous→organic phase direction. It is likely that the drug molecules are driven to the

interface along the field gradient which in turn modulates the thermodynamic equilibrium in a

way that favors more partitioning of drug into the octanol phase. The other potential reason for

enhanced octanol partitioning of the drug could be due to the changes in the solvent properties

84

due to exposure to the magnetic field. The SC-aqueous vehicle partition coefficient of LH was

also found to increase to 0.125±0.02 in presence of magnetic field compared to 0.037±0.012 in

absence of magnetic field.

6.4.5. Magnetophoresis of lidocaine base

LB has a better skin permeation property than its salt (LH) as it is relatively more

lipophilic than LH (log P of LB is 2.02 vs. 1.15 for LH under the present experimental

conditions). Even in case of LB, an increasing trend in the enhancement factor was observed

with increasing magnetic field strengths (Table 6-1) (EF= 0.009S+1). However, in general, at any

given magnetic field strength, the enhancement factor was higher in case of LH than LB. This

appears to be just because the passive permeation flux of LB was ~3-fold higher than that of LH

(Table 6-1).

Similar to LH, even in the case of LB, there was no enhancement in permeation flux across the

porcine epidermis pretreated by exposure to the magnetic field (Table 6-2). When permeation

studies of LB were performed across the dialysis membrane, an enhancement of ~3-fold was

observed in the presence of magnetic field (300mT) (passive: 4.95±1.99 µg/cm2/h vs. magnet:

14.47±3.06 µg/cm2/h).

The octanol-water partition coefficient of LB was 103.61±4.06 and it was increased to

128.55±3.91 in presence of magnetic field (300 mT). The SC-aqueous vehicle partition

coefficient of LB in the presence of the magnetic field (0.171±0.03) was increased when

compared to that in the absence of magnetic field (0.092±0.01). As compared to the ~110%

increase in the partition coefficient of LH, the increase in the case of LB was significantly less

(~24%). Comparable to this, the increase in partition coefficient between SC-vehicle was

enhanced by ~237% in the case of LH as compared to ~85% in case of LB. This is likely due to

85

the greater inherent ability of LB to partition into octanol or SC lipids as compared to LH. From

all the above studies it is again evident that the predominant mechanism of drug permeation

enhancement is likely to be magnetokinesis. Additionally, the enhanced drug partitioning into the

lipid domain of the stratum corneum could be one of the potential mechanisms contributing,

which is more apparent in the case of the hydrophilic molecule than the lipophilic molecule.

6.4.6. Magnetophoretic transdermal patch system

One of the major tasks that need to be addressed when any physical permeation

enhancement technique is developed is its applicability in vivo. Techniques such as sonophoresis

and electroporation have been developed as device based techniques for short duration treatment

or pretreatment. The drug is delivered during the treatment or post treatment from a formulation.

Approaches such as iontophoresis and microneedles could be conveniently incorporated into a

transdermal patch system for prolonged drug delivery (169, 170). Magnetophoresis is a

phenomenon which occurs only in presence of the magnetic field or it is rather an induced effect.

Therefore it is more appropriate to incorporate this mechanism in the transdermal patch system

intended for prolonged application. As a continuation of the transdermal magnetophoresis

project, a simple transdermal patch system incorporated with magnetic field as the backing layer

was designed for in vivo drug delivery applications. A patch system with better aesthetic

appearance and workability could be made by improving the proposed system. LH and LB gels

were prepared using HPMC (4% w/v) for use as a drug reservoir in the patch system. The

backing membrane consists of a sequence of magnets arranged in parallel. The control patch was

developed by using similar nonmagnetic metal pieces in the backing layer. The design of patch

system is shown in Figure 6-4.

86

Figure 6-4. Photographs of magnetophoretic patch system used for in vivo studies. In Figure a,

the magnetic backing could be clearly seen as it is not filled with the gel. In Figure b, the gel is

filled in the cavity ‘B’ (1.5 cm length, 1 cm width and 0.1 cm thickness) above the magnets. ‘A’

is the adhesive membrane to secure the patch onto the skin.

6.4.7. Transdermal drug delivery in vivo in rats

In vivo studies were carried out in rats whereas porcine epidermis was used for all the in

vitro studies. Though in vitro and in vivo data cannot be correlated due to the use of different

skin models, the in vivo data with rat model would supplement the findings from in vitro studies

and thus demonstrate the workability of the technique. Cutaneous microdialysis was employed as

a tool to investigate the time course of drug concentration in the skin. The in vivo microdialysis

probe recovery was found to 28.3±5.2 and 12.9±3.8% for LH and LB respectively. The patch

systems were applied on the abdomen region of rats. The time course of drug in the skin

extracellular fluid upon application of control and magnetophoretic transdermal patch systems is

shown in Figure 6-5 and 6-6 for LH and LB respectively.

a b

A

B

87

Figure 6-5. Time course of LH in the skin extracellular fluid following application of passive

transdermal patch () and magnetophoretic transdermal patch () in rats.

The area under the curve (AUC0-6h) which represents the dermal bioavailability and the

maximum concentration (Cmax) for both the salt and base forms of drug are given in Table 6-3.

LB being more lipophilic tends to permeate in higher amounts into the skin compared to LH.

Moreover, the use of ethanol for incorporating the lipophilic LB in the aqueous gel systems

might have also lead to enhanced passive drug permeation. The AUC0-6h was ~2-3-fold higher in

case of magnetophoretic patch system as compared to nonmagnetic control patch system (Table

6-3).

0

1

2

3

4

5

0 1 2 3 4 5 6

Con

cen

trati

on

of

LH

in

th

e sk

in e

xtr

ace

llu

lar

flu

id (

µg/m

l)

Time (h)

88

Figure 6-6. Time course of LB in the skin extracellular fluid following application of passive

transdermal patch () and magnetophoretic transdermal patch () in rats.

Table 6-3. AUC0-6h and Cmax of LH and LB in the skin extracellular fluid following transdermal

application of passive and magnetophoretic patch systems in vivo in rats.

Parameter

LH LB

Passive

patch

Magnetophoretic

patch

Passive

patch

Magnetophoretic

Patch

AUC0-6h

(µg.h/ml) 10.57 ± 2.05 23.02 ± 2.96 25.21 ± 4.05 53.96 ± 2.63

Cmax (µg/ml) 2.03 ± 0.26 4.56 ± 0.16 4.87 ± 0.48 10.39 ± 0.72

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Con

cen

trati

on

of

LB

in

th

e sk

in e

xtr

ace

llu

lar

flu

id (

µg/m

l)

Time (h)

89

6.5. CONCLUSIONS

Transdermal magnetophoresis is a phenomenon of the application of magnetic field to

enhance the drug delivery across the skin. This study demonstrates that the magnetic field could

be utilized to enhance the drug delivery across the skin. The magnetic field is believed to be a

relatively safer technique for use on skin as it was found to have no effect on the skin structure at

the field strengths utilized in this project. The in vivo studies demonstrated the feasibility of

developing a magnetophoretic transdermal patch system. The magnetophoretic patch systems

deliver drugs at a higher rate than the nonmagnetophoretic patch systems.

Acknowledgements

The project described was partially supported by Grant Number 5P20RR021929 from the

National Center for Research Resources. The content is solely the responsibility of the authors

and does not necessarily represent the official views of the National Center for Research

Resources or the National Institutes of Health.

The authors would like to thank Dr. Babu Tekwani (Principal Scientist, NCNPR, The University

of Mississippi) for the radioactive lab facility and Dr. Franck E. Dayan (Plant

physiologist/Biochemist, USDA-ARS, NCNPR, The University of Mississippi) for the liquid

scintillation counter. The authors would also like to thank Mr. Thomas Jamerson (Laboratory

Physicist, Department of Physics and Astronomy, The University of Mississippi) for helping

with the magnetic field strength measurements. The authors would also like to thank 3M Drug

Delivery Systems (St. Paul, MN) for providing gift samples of 3MTM

9773 Foam Tape.

“Reprinted from Journal of Controlled Release, 148(2), S. Narasimha Murthy, Srinivasa M.

Sammeta, C. Bowers, Magnetophoresis for enhancing transdermal drug delivery: Mechanistic

studies and patch design, 197-203, Copyright (2010), with permission from Elsevier.”

90

CHAPTER-7

MAGNETOPHORESIS IN COMBINATION WITH CHEMICAL ENHANCERS FOR

TRANSDERMAL DRUG DELIVERY

7.1. ABSTRACT

The objective of the present project was to investigate the effect of combination of

magnetophoresis, a novel physical permeation enhancement technique and chemical permeation

enhancers on the transdermal delivery of drugs. The in vitro drug permeation studies were

carried out across freshly excised abdominal skin of Sprague-Dawley rats using transdermal

patch systems (magnetophoretic and non-magnetophoretic) of lidocaine hydrochloride.

Lidocaine hydrochloride gel prepared using HPMC was spread over the magnets as a thin layer.

To investigate the effect of chemical permeation enhancers, menthol, DMSO, SLS and urea (5%

w/v) were incorporated in the gels prior to loading on the patch system. The amount of drug

permeated across the skin was estimated by withdrawing samples from receiver compartment at

different time points. The permeation flux of lidocaine from magnetophoretic patch was about 3-

fold higher (3.07±0.6 µg/cm2/h) than that of the control (non magnetic patch) (0.94±0.3

µg/cm2/h). Incorporation of chemical permeation enhancers in the gel enhanced the

magnetophoretic permeation flux by ~5-7-fold. The enhancement factor due to combination of

chemical permeation enhancer and magnetic field was found to be additive of the individual

effects. From the mechanistic studies, magnetic systems were found to enhance the drug

91

permeation via follicular pathway whereas the chemical enhancers increase the permeability of

bulk of the epidermis.

Keywords: Transdermal patch system, Follicular pathway, Chemical permeation enhancers,

Magnetophoresis.



I. To investigate the effect of chemical permeation enhancers on the magnetophoretically

mediated transdermal drug delivery.

II. To investigate the potential mechanisms contributing to the drug delivery enhancement

across skin, when chemical enhancers and magnetophoresis are used in combination.

Various chemical enhancers and physical permeation enhancement techniques have been

investigated for overcoming the stratum corneum barrier and enhancing the transdermal delivery

of drugs (171-173). In general, physical permeation enhancement techniques are more efficient

than chemical enhancers. Several research groups have studied the effect of combination of

chemical enhancers and physical permeation enhancement techniques like iontophoresis,

electroporation and ultrasound to assess the plausibility of deriving synergistic benefits. Use of a

combination of enhancers is reported to mutually enhance the efficacy and also increase the

safety of enhancers. Oh et al. have reported that the transdermal transport of zidovudine was

enhanced synergistically when iontophoresis was used in combination with chemical enhancers

like propylene glycol and oleic acid (174). Ganga et al. showed that the combination of azone

and iontophoresis enhanced transdermal permeation of metoprolol synergistically (175).

Combination of ultrasound and sodium lauryl sulfate was reported to result in synergistic

enhancement in permeation of mannitol by Mitragotri et al. (176). Magnetophoresis is a

92

phenomenon of enhancing drug permeation across the biological barriers by application of

magnetic field. Previous work by Murthy et al. has shown that magnetophoresis leads to

enhanced transdermal drug delivery in in vitro and in vivo studies (177). The predominant

mechanism for drug permeation enhancement was found to be magnetokinesis and enhanced

partitioning of drug into stratum corneum. In this study, the experimental data regarding

permeation enhancement of lidocaine hydrochloride using chemical enhancers and magnetic

field will be provided first followed by mechanistic studies (157, 178).


7.3.1. Materials

Lidocaine Hydrochloride (LH) was obtained from Spectrum Chemicals (New Brunswick,

NJ), Estradiol, Ferric pyrophosphate, Menthol, Dimethyl sulfoxide (DMSO) and Sodium lauryl

sulfate (SLS) were purchased from Sigma-Aldrich Inc (St. Louis, MO). PBS 10x liquid

concentrate and Urea were procured from EMD Chemicals Inc (Cincinnati, OH) and all other

chemicals and reagents were obtained from Fischer Scientific (Fairway, NJ). Neodymium

magnets were purchased from K&J Magnetics Inc (Jamison, PA).

7.3.2. Drug transport from transdermal patch systems

The in vitro transport of lidocaine hydrochloride from transdermal patch systems across

the freshly excised rat abdominal skin was studied using vertical Franz diffusion cells

(diffusional area of 0.64 cm2 and receiver compartment volume of 5 ml). The barrier integrity of

rat skin was confirmed by measuring the electrical resistance according to previously published

methods and pieces of skin which had a resistance greater than 20 kΩ.cm2 were only used for the

drug permeation studies

93

The design of transdermal patch system was similar to that described in section 6.3.12 and

Figure 6-4. Lidocaine hydrochloride (2% w/w) containing gel was prepared in HPMC base (4%

w/v) using deionized water and was spread over the magnets as a thin layer, which acts as drug

reservoir. DMSO, urea and SLS were incorporated in the gels prepared in deionized water

whereas menthol was incorporated in gel prepared in hydroalcoholic solution (water:alcohol,

70:30). The receiver compartment of diffusion cell was filled with PBS and drug permeation

studies were carried out by placing gel filled transdermal patches over the skin with samples

withdrawn at regular intervals of time over a period of 8 h.

7.3.3. Drug permeation studies across sandwich epidermis model

The mechanistic studies were carried out across the sandwich epidermis model prepared

from porcine epidermis. Sandwich epidermis model includes a layer of stratum corneum sample

placed over porcine epidermis. Stratum corneum samples are prepared from porcine epidermis

floated overnight on a aqueous solution containing 0.0001% w/v of trypsin and 0.5% w/v sodium

bicarbonate at 37 C according to method followed by Essa et al. and El Maghraby et al. (157,

178).

Passive and iontophoretic delivery of lidocaine hydrochloride, estradiol, and ferric

pyrophosphate were carried across sandwich epidermis and single layer epidermis and the steady

state flux was calculated. In case of lidocaine hydrochloride transport studies, the receiver

compartment of diffusion cell was filled with 5 ml of PBS, pH 7.4 and donor compartment was

filled with 0.5 ml of lidocaine hydrochloride (10 mg/ml) in PBS, pH 7.4. In case of estradiol

transport studies, the receiver compartment was filled with 5 ml of PBS, pH 7.4 containing 10%

HPßCD and the donor compartment was filled with 0.5 ml of estradiol (0.25 mg/ml) in PBS, pH

7.4 containing 40% alcohol. In case of ferric pyrophosphate transport studies, the receiver

94

compartment was filled with 5 ml of PBS, pH 5 and the donor compartment was filled with 0.5

ml of ferric pyrophosphate (50 mg/ml) in PBS, pH 5. Anodal iontophoresis was carried out for

lidocaine hydrochloride and estradiol, whereas cathodal iontophoresis was used for ferric

pyrophosphate. In all the cases, 0.3 mA/cm2 of electric current was applied using an Iomed

Phoresor.


The amount of lidocaine hydrochloride, estradiol were analyzed by high performance

liquid chromatography and ferric pyrophosphate was analyzed by UV spectrophotometer. The

HPLC system (Waters, MA) consisted of a chromatographic pump (Waters 1525), autosampler

(Waters 717 plus) and an UV absorbance detector (Waters 2487). Symmetry®

C18 column (4.6 x

150 mm) was used for analysis of lidocaine hydrochloride and the mobile phase consisted of a

mixture (14/86 v/v) of acetonitrile and potassium dihydrogen phosphate 0.05 M (pH adjusted to

4.0) with a flow rate of 1.3 ml/min at 216 nm (158). Phenomenox®

Luna C18 column (4.6 x 150

mm) was used for analysis of estradiol and the mobile phase consisted of mixture of acetonitrile

and 0.1% ortho-phosphoric acid (6:4) with a flow rate of 1 ml/min at 212 nm (179). Ferric

pyrophosphate samples were analyzed at 510 nm using UV spectrophotometer after the addition

of Ferrover®

reagent (180).


Statistical analysis was carried out using GraphPad InStat 3 software. Unpaired t-test was

performed and P<0.05 was considered as the level of significance. All the data are represented as

average of 3-5 trials with standard deviation.

95


7.4.1. Development of transdermal patch system incorporated with chemical enhancer and

magnetic backing

The magnetophoretic patch system consists of magnets arranged in parallel on the

adhesive backing membrane. A row of magnetic blocks were used instead of a single magnet

mainly to impart flexibility to the patch system. Magnets in the backing membrane were replaced

with nonmagnetic pieces in case of non-magnetophoretic patches. The patches containing

nonmagnetic backing were used to study the effect of chemical permeation enhancers on the

delivery of drug from the patch system. The drug along with chemical enhancers incorporated in

the gel was filled into the cavity of adhesive backing membrane. About 200 mg of gel was

loaded onto the patch system which contained lidocaine hydrochloride equivalent to 2 mg.

Different chemical enhancers were incorporated into the gel to enhance the drug permeation

across the skin. DMSO is known to be an effective permeation enhancer for both hydrophilic and

lipophilic drugs. On application to skin, DMSO is known to denature the proteins and change the

confirmation of intercellular keratin from α helical to ß sheet and also interacts with the

intercellular lipid regions of the stratum corneum (181-183). Terpenes have been reported to be

safe and effective permeation enhancers for both hydrophilic and lipophilic drugs (184). Menthol

has been studied as permeation enhancer for hydrophilic drugs like 5-fluorouracil and

propranolol hydrochloride (185, 186). The main reason for enhancement of permeation of

hydrophilic drugs by menthol is due to the disruption of the stratum corneum lipids by breakage

of hydrogen bonds between the ceramide groups (187). Urea has been known to act as a skin

permeation enhancer by increasing the stratum corneum water content and thus leading to

opening of hydrophilic diffusion channels within the stratum corneum. SLS is known to

96

solubilize the stratum corneum lipids and also interact with keratin to enhance the overall skin

permeability (39, 149).

7.4.2. Magnetophoresis mediated drug delivery enhancement

The passive permeation flux of lidocaine hydrochloride across rat skin was found to be

0.94±0.13 µg/cm2/h. The permeation flux of lidocaine hydrochloride was enhanced by ~3-fold

(3.07±0.43 µg/cm2/h) in case of magnetophoretic patch system compared to that of passive.

7.4.3. Effect of chemical enhancers on the drug delivery from transdermal patch systems

Incorporation of chemical enhancers has resulted in an increase in the permeation flux of

lidocaine hydrochloride compared to control (gel without any chemical enhancer). The

concentration of all the four enhancers used in the present study was at 5% w/v. The drug

permeation enhancement factor of different enhancers could be represented in the following

decreasing rank order: menthol > SLS > urea > DMSO. Incorporation of menthol in the gel

formulation resulted in ~3.7-fold enhancement (3.50±0.69 µg/cm2/h) in the flux of lidocaine

hydrochloride. Due to limited solubility of menthol in the aqueous vehicle, 30% v/v of alcohol

was used in the gel formulation to incorporate menthol (the flux enhancement by 30% v/v

alcohol alone in absence of menthol was found to be ~1.4-fold over control). SLS resulted in

~2.6-fold enhancement (2.44±0.39 µg/cm2/h) in the permeation flux over the control patch. On

the other hand, urea and DMSO lead to ~1.7-fold (1.62±0.27 µg/cm2/h) and ~1.1-fold (1.07±0.24

µg/cm2/h) enhancement in the flux over the control patch. Among the enhancers, menthol and

SLS resulted in greater enhancement compared to urea and DMSO. Except DMSO, all the other

chemical enhancers showed a significant enhancement in the flux of lidocaine hydrochloride

(Figure 7-1).

97

Figure 7-1. Transdermal permeation flux of lidocaine hydrochloride from non-magnetophoretic

patch systems. ‘Control’ indicates patch system without any chemical enhancer. ** indicates

statistically insignificant and * indicates statistically significant compared to control.

The effect of pretreatment was not considered because the effective pretreatment varies with

each chemical enhancer and would not clearly reflect the relative efficiency. Moreover, in real

life situation, the patch containing combination of chemical enhancers and magnetophoresis will

be applied by the patients. Therefore, the studies included simultaneous application of both the

enhancers (chemical enhancers and magnetophoresis) in the patch system to simulate the in vivo

practice.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Control DMSO Urea SLS Menthol

Per

mea

tion

flu

x o

f li

doca

ine

hyd

roch

lori

de

(µg/c

m2/h

)

**

*

*

*

98

7.4.4. Effect of combination of chemical enhancers and magnetophoresis on the drug

delivery

Combination of chemical enhancers and magnetophoresis resulted in enhanced

permeation flux compared to both individual chemical enhancer effect and magnetophoretic

effect and was additive in most of the cases (Table 7-1). Definitely, there was a significant

increase in the enhancement efficiency with this combination approach. Moreover, the results

clearly demonstrated the feasibility of incorporating chemical permeation enhancers and

magnetophoresis technique in a simple transdermal patch system, which could be of potential

benefit in enhancing the transdermal delivery of poorly permeable drugs. The plausible reason

for an additive effect is likely due to the difference in the predominant mechanism of drug

delivery enhancement between the chemical enhancers and magnetophoresis. Among the

enhancers used, menthol and SLS are known to act predominantly on the lipoidal regions

whereas DMSO and urea act on the keratinocytes. Our earlier studies have shown that the

magnetically mediated transdermal drug delivery is more likely due to interaction of the

magnetic field with the drug molecule than with the stratum corneum barrier. In the mechanistic

studies sandwich epidermis model was used to elucidate the predominant mechanism of

enhancement of drug delivery by the application of magnetic field.

99

Table 7-1. Transdermal permeation flux of lidocaine hydrochloride from non-magnetophoretic

and magnetophoretic patch system. ‘Control’ in the table indicates patch system without any

chemical enhancer. Enhancement factor is the ratio of permeation flux obtained with respect to

flux from control (non-magnetophoretic patch system).

Enhancer

Non-magnetophoretic patch Magnetophoretic patch

Permeation flux

(µg/cm2/h)

Enhancement

Factor

Permeation flux

(µg/cm2/h)

Enhancement

Factor

Control 0.94 ± 0.13 1.00 3.07 ± 0.43 3.26

DMSO 1.07 ± 0.24 1.13 3.82 ± 0.48 4.06

Urea 1.62 ± 0.27 1.72 4.85 ± 0.24 5.15

SLS 2.44 ± 0.39 2.59 4.93 ± 0.37 5.24

Menthol 3.50 ± 0.69 3.72 6.06 ± 0.43 6.44

7.4.5. Mechanistic studies

In a recent report, the results of the mechanistic studies clearly demonstrated that

magnetokinesis was the predominant mechanism responsible for enhanced transdermal

permeation (177). However, the relative contribution of appendageal pathway in magnetokinesis

has not been investigated. Mechanistic studies were carried out using sandwich epidermis model

developed by El Maghraby et al. to elucidate the role of appendageal pathway in drug

permeation across the skin (178). In the sandwich epidermis model, the stratum corneum layer

overlaid on the epidermal membrane forms the top layer and blocks most of the appendageal

pathways present in underlying epidermal membrane (Figure 7-2).

100

Figure 7-2. Sandwich epidermis model. 2a represents single layer epidermis with unblocked

appendageal pathway and 2b represents sandwich epidermis with a stratum corneum layer

overlaid on the epidermal membrane. Appendageal pathway of underlying epidermal membrane

is blocked by the overlaid stratum corneum layer and appendageal pathway of the overlaid

stratum corneum layer is blocked by the underlying epidermal membrane. In case of sandwich

epidermis, the permeation flux of hydrophilic drugs which permeate mostly through appendageal

pathway would be reduced by less than half and be close to zero due to blockage of appendageal

pathway. However, the permeation flux of lipophilic drugs which permeate mostly through the

bulk of epidermis (non-appendageal pathway) would be reduced by half due to increase in

thickness of the barrier, in the case of sandwich epidermis.

In this study porcine epidermis was used to carry out mechanistic studies using sandwich model

due to ease of separation of epidermis when compared to rat skin. El Maghraby et al. have

carried out permeation studies across the sandwich epidermis and compared it with that across

the epidermis alone (178). The authors reported that the permeation through sandwich epidermis

model would be much reduced in comparison to epidermis if appendageal pathway played an

active role in the permeation of drug. It was reported that appendageal pathway played only a

101

minor role in the penetration of liposomal formulations containing estradiol. Essa et al. used the

same model to examine the role of skin appendages in the passive, iontophoresis and liposomal

penetration of drugs and reported that the appendageal pathway played a major role in the

passive permeation of hydrophilic mannitol, where as it was negligible in case of lipophilic

estradiol (157). The authors also reported that the appendageal pathway had a significant role in

the flux enhancement by iontophoresis (157, 178).

Initially, a few set of experiments were dedicated to validate the sandwich epidermis model

reported by El Maghraby et al. Permeation studies were carried out using lidocaine

hydrochloride, estradiol and ferric pyrophosphate. Lidocaine hydrochloride is a cationic

hydrophilic drug and estradiol is lipophilic in nature. Ferric pyrophosphate represents a high

molecular weight anionic hydrophilic compound. Therefore, the three marker drugs differ in

their extent and pathways of transdermal permeation.

7.4.5.1. Passive permeation studies across sandwich epidermis and single layer epidermis

The passive permeation flux ratio of sandwich model to single layer epidermis (FSW/SL)

would be close to 0.5, if appendageal pathway had no role in drug permeation and the FSW/SL

would be <0.5 and close to zero if the drug permeation was via appendageal pathway. In general,

highly hydrophilic drugs permeate through skin mainly via appendageal pathway whereas

lipophilic drugs permeate mainly through the bulk of the skin (188, 189). Based on this one

would expect FSW/SL would be <0.5 for lidocaine hydrochloride and ferric pyrophosphate which

are hydrophilic and 0.5 for estradiol which is lipophilic in nature.

The steady state flux of lidocaine hydrochloride was found to be 1.45±0.02 µg/cm2/h across the

single layer porcine epidermis. The flux decreased significantly to 0.24±0.01 µg/cm2/h across the

sandwich epidermis (Figure 7-3). The FSW/SL was ~0.16 indicating that the appendageal pathway

102

had a significant role in the passive permeation of lidocaine hydrochloride (Table 7-2). However,

the diffusion across the bulk of the epidermis is not completely negligible in this case. This is

likely because of the presence of unionized fraction of the drug, which tends to selectively

diffuse via bulk of the epidermis pathway.

Figure 7-3. Passive permeation of lidocaine hydrochloride across single layer () and sandwich

epidermis ().

The passive permeation flux of estradiol was found to be 0.042±0.004 µg/cm2/h and 0.023±0.003

µg/cm2/h across single layer epidermis and sandwich epidermis respectively with FSW/SL of 0.54

(Figure 7-4, Table 7-2). The results indicate that the appendageal pathway did not play a

significant role in the permeation of lipophilic estradiol and hence the flux was reduced by

almost half across the sandwich epidermis. This was in agreement with the results reported by

Essa et al. for estradiol permeation across the human epidermis (157).

0

3

6

9

12

15

0 2 4 6 8

Am

ou

nt

of

lid

oca

ine

hyd

roch

lori

de

per

mea

ted

(µ

g/c

m2)

Time (h)

103

Ferric pyrophosphate did not result in any passive permeation across the porcine epidermis

which could be due its high molecular size and extremely hydrophilic nature.

Figure 7-4. Passive permeation of estradiol across single layer () and sandwich epidermis ().

7.4.5.2. Iontophoretic drug delivery across sandwich and single layer epidermis

Anodal iontophoresis would favor the transdermal delivery of lidocaine hydrochloride

and at pH over 5, both electroosmosis and electrophoresis contribute to the enhanced transport of

drug. In case of estradiol, permeation could be enhanced only by electroosmosis mechanism due

to lack of any charge on the molecule (157). Ferric pyrophosphate was found to be poorly

permeable across the skin by passive diffusion and the permeation could be enhanced

significantly by cathodal iontophoresis at pH over 5. In case of ferric pyrophosphate, recent

studies by Murthy and Vaka have shown that electroosmosis does not play any role in the

enhanced permeation and the main pathway for enhanced permeation by iontophoresis is through

appendageal pathway (180). So, in the present study, anodal iontophoresis was carried out for

lidocaine hydrochloride and estradiol whereas cathodal iontophoresis was carried out for ferric

0.0

0.6

1.2

1.8

2.4

0 12 24 36 48 60 72

Am

ou

nt

of

estr

ad

iol

per

mea

ted

(µg/c

m2)

Time (h)

104

pyrophosphate. The flux of lidocaine hydrochloride across sandwich epidermis decreased to

2.86±0.63 µg/cm2/h in comparison to 12.54±1.98 µg/cm

2/h across single layer epidermis and

IFSW/SL (ratio of iontophoretic flux across sandwich model to single layer epidermis) was found

to be 0.22 (Figure 7-5, Table 7-2). In case of estradiol IFSW/SL was found to be 0.19 with a flux of

0.022±0.002 µg/cm2/h across the sandwich epidermis model and 0.111±0.007 µg/cm

2/h in case

of single layer epidermis (Figure 7-6, Table 7-2). Assuming complete blockade of appendageal

pathway, IFSW/SL should be close to zero. But permeation of both lidocaine and estradiol was

observed which might be due to the electroosmosis mediated transport of drug through the

additional pores created in the bulk of the skin by application of iontophoresis. These results are

in agreement with that reported by others (157, 178). Murthy and Vaka demonstrated that the

iontophoretic delivery of ferric pyrophosphate occurs only through appendageal pathway across

the rat skin (180). In this study using porcine epidermis, IFSW/SL of ferric pyrophosphate was only

0.04, with flux of 1.58±0.10 µg/cm2/h and 37.69±4.68 µg/cm

2/h across sandwich and single layer

epidermis respectively (Figure 7-7, Table 7-2). These results of the validation studies confirm

that the appendageal pathway is almost completely blocked in the sandwich epidermis model and

it could serve as an excellent model to investigate the contribution of appendageal pathway in the

drug transport. Barry has also proposed that sandwich epidermis model can be applied

effectively to analyze appendageal pathway in case of ultrasound and magnetophoresis studies in

addition to passive diffusion and electrical methods (190). Therefore, the sandwich epidermis

model was used in case of magnetophoresis mediated studies to elucidate the mechanism of

appendageal pathway.

105

Figure 7-5. Iontophoretic delivery of lidocaine hydrochloride across single layer () and

sandwich epidermis ().

Figure 7-6. Iontophoretic delivery of estradiol across single layer () and sandwich epidermis

().

0

15

30

45

60

75

90

0 2 4 6

Am

ou

nt

of

lid

oca

ine

hyd

roch

lori

de

del

iver

ed (

µg/c

m2)

Time (h)

0.0

0.4

0.8

1.2

1.6

2.0

0 3 6 9 12 15 18

Am

ou

nt

of

estr

ad

iol

del

iver

ed

(µg/c

m2)

Time (h)

106

Figure 7-7. Iontophoretic delivery of ferric pyrophosphate across single layer () and sandwich

epidermis ().

0

150

300

450

600

750

0 2 4 6 8 10 12 14

Am

ou

nt

of

ferr

ic p

yro

ph

osp

hate

dee

liver

ed (µ

g/c

m2)

Time (h)

107

Table 7-2. Steady state flux of lidocaine hydrochloride, estradiol, and ferric pyrophosphate

following passive permeation and iontophoretic delivery across sandwich and single layer

epidermis with corresponding flux ratios.

Drug

Flux (µg/cm2/h) Flux ratio

(sandwich

/single

layer)

Role of

appendageal

pathway Sandwich

epidermis

Single layer

epidermis

Passive permeation

Lidocaine

Hydrochloride 0.24 ± 0.01 1.45 ± 0.02 0.16 Significant

Estradiol 0.023 ± 0.003 0.042 ± 0.004 0.54 Insignificant

Ferric

pyrophosphate - - - -

Iontophoretic delivery

Lidocaine

Hydrochloride a 2.86 ± 0.63 12.54 ± 1.98 0.22 Significant

Estradiol b 0.022 ± 0.002 0.111 ± 0.007 0.19 Significant

Ferric

pyrophosphate c 1.58 ± 0.10 37.69 ± 4.68 0.04 Significant

aAnodal iontophoresis

bAnodal iontophoresis

cCathodal iontophoresis

The potential mechanisms are electroosmosis and electrorepulsion in case of b and c

respectively. In case of a both electrorepulsion and electroosmosis contribute together.

7.4.5.3. Magnetophoretic delivery of lidocaine hydrochloride across sandwich and single

layer epidermis

These studies were carried out to determine the extent of contribution of appendageal

pathway in magnetically enhanced drug permeation. Drug permeation studies were carried out

across porcine epidermis using transdermal patch system. The flux of lidocaine hydrochloride

from magnetophoretic patch system across single layer epidermis and sandwich epidermis was

found to be 3.87±0.30 µg/cm2/h and 0.99±0.26 µg/cm

2/h respectively. The

MFSW/SL (ratio of

magnetophoresis flux across the sandwich model to single layer epidermis) would have been

close to 0.5 if appendageal pathway had no significant role in the drug permeation by

108

magnetophoresis. But, M

FSW/SL was found to be 0.25 indicating that appendageal pathway played

a significant role in the magnetophoretic delivery of lidocaine hydrochloride.

From the mechanistic studies reported earlier and in the present paper, it is more evident that the

magnetic field enhances the drug delivery across the skin predominantly by magnetokinesis

across the appendageal pathway without significantly affecting the stratum corneum barrier. In

addition, the present study indicates that the use of chemical permeation enhancers which

interact with the bulk of the epidermis (non appendageal region) would improve the overall drug

delivery efficiency.

7.5. CONCLUSIONS

The results indicate that the magnetically mediated drug permeation enhancement could

be further enhanced by incorporation of suitable chemical permeation enhancers. The magnetic

systems were found to enhance the drug permeation via appendageal pathway.

Acknowledgements

The project described was supported by Grant Number 5P20RR021929 from the National Center

for Research Resources. The content is solely the responsibility of the authors and does not

necessarily represent the official views of the National Center for Research Resources or the

National Institutes of Health. This project was also partially supported by Grant Number

HD061531A from Eunice Kennedy Shriver National Institute of Child Health & Human

Development (NICHD).

109

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APPENDIX

LIST OF PUBLICATIONS

1. S.M. Sammeta, S.R.K. Vaka, S.K. Mathur S.K, and S.N. Murthy. Non invasive

transcutaneous sampling of glucose by electroporation. J Diabetes Sci Technol. 2(2):250-254

(2008).

2. S.M. Sammeta, S.R.K. Vaka, and S.N. Murthy. Transdermal drug delivery enhanced by low

voltage electropulsation (LVE). Pharm Dev Technol. 14(2):159-164 (2009).

3. S.M. Sammeta, S.R.K. Vaka, and S.N. Murthy. Transcutaneous sampling of ciprofloxacin

and 8-methoxypsoralen by electroporation (ETS) technique. Int J Pharm. 369(1-2):24-29

(2009).

4. S.M. Sammeta, S.R.K. Vaka, and S.N. Murthy. Dermal drug levels of antibiotic (cephalexin)

determined by electroporation and transcutaneous sampling (ETS) technique. J Pharm Sci. 98

(8):2677-2685 (2009).

5. S.M. Sammeta, and S.N. Murthy. "ChilDrive": a technique of combining regional cutaneous

hypothermia with iontophoresis for the delivery of drugs to synovial fluid. Pharm Res.

26(11):2535-2540 (2009).

6. S.M. Sammeta, S.R.K. Vaka, and S.N. Murthy. Transcutaneous electroporation mediated

delivery of doxepin-HPCD complex: a sustained release approach for treatment of

postherpetic neuralgia. J Control Release. 142(3):361-367 (2010).

133

7. S.N. Murthy, S.M. Sammeta, and C. Bowers. Magnetophoresis for enhancing transdermal

drug delivery: mechanistic studies and patch design. J Control Release. 148(2):197-203

(2010).

8. S.M. Sammeta, M.A. Repka, and S.N. Murthy. Magnetophoresis in combination with

chemical enhancers for transdermal drug delivery. Drug Dev Ind Pharm. 2011 (In press).

134

VITA

Srinivasa Murthy Sammeta was born on July 23, 1981 in Hyderbad, AP, India. He

completed his B.Pharmacy in 2003 from Sri Venkateswara College of Pharmacy, Osmania

University, Hyderabad, India and M.Pharmacy in Pharmaceutics in 2006 from PES College of

Pharmacy, RGUHS, Bangalore, India. Sammeta joined Department of Pharmaceutics, The

University of Mississippi as a graduate student in Spring 2007. Sammeta has published over 8

research papers as primary author and 7 research papers as secondary author in peer reviewed

international journals. He received Graduate Student Council Research Grant and CORE-NPN

Fellowship in 2007. Sammeta was inducted into Rho Chi Honor society in 2009. He also

received AstraZeneca Travelship award for AAPS, best poster presentation award at SRDG-

Pharm Forum, and Annual Poster Session, NCNPR and CORE-NPN Fellowship during 2009.

Sammeta was awarded outstanding oral presentation award at SRDG-Pharm Forum in 2010. He

was Vice-Chair of AAPS-UM Student chapter during 2009 and played a key role in the

organizing of SRDG-Pharm Forum. Sammeta is also a Student member of AAPS-

Dermatopharmaceutics Focus Group Steering Committee.

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