Physical techniques to overcome the cutaneous barrier: how iontophoresis and fractional laser ablation change the delivery kinetics of drugs

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Physical techniques to overcome the cutaneous barrier: how iontophoresis and fractional laser ablation change the delivery

kinetics of drugs



The aim of this thesis was to investigate how different physical techniques, in particular iontophoresis and fractional laser ablation, could be used to enhance transdermal and cutaneous drug delivery. First part of the thesis describe how iontophoresis can be used to control transdermal delivery kinetics of drugs for neurodegenerative disorders. Second part of the thesis was focused on to investigate the transdermal iontophoretic delivery of degarelix.

Overall, this research highlights the complicated interaction between peptide structure, skin membranes and plasma proteins that must be judiciously considered in order to achieve significant delivery. Last part of the thesis presents a novel approach for creating intraepidermal drug reservoirs using fractional laser ablation to deposit microparticles loaded with either a low molecular weight drug (triamcinolone acetonide) or an antibody (cetuximab) so as to provide localized sustained delivery. The results justify that this combination approach is of particular interest for dermatological conditions.

SINGHAL, Mayank. Physical techniques to overcome the cutaneous barrier: how iontophoresis and fractional laser ablation change the delivery kinetics of drugs. Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5203

DOI : 10.13097/archive-ouverte/unige:104616 URN : urn:nbn:ch:unige-1046161

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Laboratoire de Biochimie Pharmaceutique

Physical techniques to overcome the cutaneous barrier:

How iontophoresis and fractional laser ablation change the delivery kinetics of drugs


Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention sciences pharmaceutiques


Mayank Singhal de

Delhi (Inde)

Thèse N° 5203


Atelier de reproduction ReproMail 2018


“If I have seen further it is by standing on the shoulders of giants.”

Sir Isaac Newton

“Either write something worth reading or do something worth writing.”

Benjamin Franklin

“The good thing about science is that it’s true whether or not you believe in it.”

Neil deGrasse Tyson


This thesis is dedicated to my parents, Mr. Murari L. Singhal and Mrs. Susheela Singhal, for the love and support they have given me my whole life to get me to where I am today.







Effect of continuous and multi-phasic current profiles on the iontophoretic transport of pramipexole, rasagiline and huperzine A: Depicting temporal variation and biodistribution in the skin

Abstract 33

1. Introduction 34

2. Materials and methods 36

2.1. Materials 36

2.2. Skin preparation 36

2.3. Iontophoresis set-up 36

2.4. Preparation of drug solutions 37

2.5. Continuous current delivery profiles 37

2.6. Multi-phasic current delivery profiles 37

2.7. Skin biodistribution 37

2.8. HPLC analysis 39

2.9. Statistical Analysis 39

3. Results and discussion 39

3.1. Continuous current delivery 39

3.2. Multi-phasic current delivery 42

3.3. Skin biodistribution 45

4. Conclusion 50

Acknowledgements 51

References 52


Controlled delivery of pramipexole from an iontophoretic transdermal patch system in vitro and in vivo



1. Introduction 58

2. Materials and methods 59

2.1. Materials 59

2.2. Skin preparation 59

2.3. Development of PRA patch 60

2.4. Development of PRA iontophoretic system 60

2.5. Iontophoresis set-up 60

2.6. Effect of current density 61

2.7. In vivo studies 61

2.8. Analytical methods 62

2.9. Statistical Analysis 64

3. Results and discussion 64

4. Conclusion 72

Acknowledgements 73

References 74


Controlled iontophoretic delivery in vitro and in vivo of ARN14140, a multi target compound for Alzheimer’s disease

Abstract 79

1. Introduction 80

2. Materials and methods 81

2.1. Materials 81

2.2. Skin preparation 82

2.3. Stability in the presence of skin and electrical current 82

2.4. Iontophoresis set-up 83

2.5. Effect of ARN14140 concentration on electrotransport 83

2.6. Effect of current density on electrotransport 83

2.7. Comparison of iontophoretic delivery across porcine and human skin 83

2.8. ARN14140 skin biodistribution 84

2.9. Iontophoretic delivery of ARN14140 from a gel formulation 84

2.10. In vivo experimental protocol 84

2.11. Statistical Analysis 85



3.1. Stability studies 85

3.2. Effect of increasing ARN14140 concentration on transport 86 3.3. Effect of increasing current density on ARN14140 permeation 87

3.4. ARN14140 skin biodistribution 89

3.5. Mechanism of ARN14140 transport 89

3.6. Delivery and transport efficiency 90

3.7. Validation with human skin 91

3.8. Permeation studies with hydroxyethyl cellulose gel 91 3.9. Iontophoretic delivery kinetics of ARN14140 in vivo 92

4. Conclusion 94

5. Supplementary information 95

5.1. Validation of HPLC-UV method for the quantification of ARN and ACE 95 5.2. Validation of UHPLC-MS/MS method for the in vivo samples 96

5.3. Extraction validation of porcine skin 97

Acknowledgements 98

References 99



An insight into electrically assisted delivery of degarelix: What happens outside and inside the skin?

Abstract 103

1. Introduction 104

2. Materials and methods 106

2.1. Materials 106

2.2. HPLC-UV analysis 106

2.3. Skin preparation 107

2.4. Stability studies 107

2.5. Iontophoretic delivery of degarelix from solution 108

2.6. Electrotransport from degarelix depot 110

2.7. Confocal laser scanning microscopy 112



3. Results 113

3.1. Demonstrating degarelix stability 113

3.2. Iontophoretic delivery of degarelix from solution 114 3.3. Electroactive release from degarelix s.c. depot 117

4. Discussion 119

5. Conclusion 124

Acknowledgements 125

References 126



Fractional laser ablation for the cutaneous delivery of triamcinolone acetonide from cryomilled polymeric microparticles: Creating intra-epidermal drug depots

Abstract 131

1. Introduction 132

2. Materials and methods 134

2.1. Materials 134

2.2. Preparation of TA loaded MP 134

2.3. Preparation of fluorescein/Nile Red loaded MP 136

2.4. Analytical methods 136

2.5. Characterization of MP 137

2.6. In vitro drug release study 138

2.7. Preparation of skin and P.L.E.A.S.E.® microporation 138 2.8. MP deposition in P.L.E.A.S.E.® porated skin and CLSM 138 2.9. Biodistribution of TA in the P.L.E.A.S.E.® porated skin 139

2.10. Statistical analysis 139

3. Results and discussion 140

3.1. Characterization of MP 140

3.2. In vitro drug release 144

3.3. CLSM to visualize MP deposition and fluorescein release in laser porated skin 146 3.4. Biodistribution of TA in the epidermis and upper dermis 148



4. Conclusion 152

5. Supplementary information 152

5.1. Validation of HPLC-UV method for the quantification of TA 152 5.2. Validation of UHPLC-MS/MS method for the quantification of TA 154

5.3. Selection of dissolution media 155

5.4. Optical microscopy images of laser-porated human skin 156

Acknowledgements 157

References 158


Enabling targeted cutaneous delivery of cetuximab to treat psoriasis using intraepidermal depots: Alternative approach to parenteral administration

Abstract 165

1. Introduction 166

2. Materials and methods 167

2.1. Materials 167

2.2. Preparation of cetuximab Loaded MP 168

2.3. Quantitative analysis of cetuximab by ELISA 168

2.4. Characterization of MP 168

2.5. Preparation of Skin and P.L.E.A.S.E. Microporation 170

2.6. MP delivery to the P.L.E.A.S.E. porated skin 170

2.7. Statistical analysis 171

3. Results and discussion 171

4. Conclusion 177

Acknowledgements 177

References 178






1 L'administration transdermique est une excellente alternative non-invasive pour l'administration de médicaments à action systémique. En plus de protéger le médicament de l'environnement gastro-intestinal agressif, elle fournit une cinétique d’administration du médicament plus contrôlée avec une compliance et une commodité supérieures pour le patient. Cependant, la fonction protectrice de la couche supérieure de la peau limite son application comme voie d'administration de médicament. Par conséquent, plusieurs techniques telles que l'iontophorèse et l'ablation factionnée au laser ont été développées pour élargir les horizons des médicaments candidats pour la voie transdermique afin de rendre le

“non-administrable” administrable.

Le but de cette thèse était d'étudier comment différentes techniques physiques pourraient être utilisées pour améliorer l’administration de médicaments cutanés et transdermiques.

La première partie de la thèse étudie spécifiquement l'application de l'iontophorèse pour contrôler la cinétique d’administration transdermique de petites molécules, en particulier pour des applications dans le traitement de maladies neurodégénératives. L'iontophorèse transdermique est une alternative intéressante pour l’administration contrôlée d'agents thérapeutiques impliqués dans le traitement des maladies neurodégénératives. L'une de ses propriétés clés est la capacité à utiliser le profil du courant pour contrôler la cinétique d’administration des médicaments et pour obtenir des profils complexes d'administration du médicament qui simulent les profils physiologiques souhaités. Le Chapitre 1 étudie la variation temporelle du transport du pramipexole (PRA), de la rasagiline (RAS) et de l'huperzine A (HUP) sous des profils de courants continus et multiphasiques et étudie les effets sur la perméation cumulative, le flux transdermique et la rétention médicamenteuse.

Des expériences initiales avec un courant continu ont permis d'établir une corrélation entre l’administration totale de PRA, RAS et HUP (c'est-à-dire la somme de la permeation cumulée et de la déposition cutané) et la quantité de charge transférée. Ensuite, des expériences avec des profils de courant multiphasiques ont validé la relation établie entre les quantités de charge transférées et l'apport total pour prédire la distribution totale de chaque médicament.

Les valeurs expérimentales étaient à ± 15% des valeurs prédites. L'effet de la densité du courant et de la durée d’application du courant sur la biodistribution cutanée de PRA en fonction de la profondeur de la peau a également été étudié et s'est avéré avoir un impact


2 de réservoirs de médicaments dans la peau.

Le Chapitre 2 évalue l’administration transdermique in vitro et in vivo de l'agoniste de la dopamine, PRA, à partir d'un système de patch ionophorétique. Des expériences préliminaires in vitro ont été menées pour optimiser la conception et le développement du patch iontophorétique. Il a été constaté qu'avec un modèle de patch à un compartiment, le compartiment du médicament en contact avec le compartiment des électrodes entraînait une décoloration de la peau due à l'absence de quantités suffisantes d'ions chlorure requis pour l'électrochimie à l'interface anodique. L'augmentation de la quantité de Cl- conduit à une diminution de l'efficacité d’administration du patch (c'est-à-dire qu’une fraction de la quantité appliquée est administrée). Un modèle à deux compartiments où le compartiment de médicament a été séparé du compartiment des électrodes a amélioré l’efficacité de l’administration du patch sans précipitation d'ions d'argent dans le compartiment de la formulation. Les études in vivo visaient à étudier les changements des profils plasmatiques lors de la modulation des densités de courant appliquées au cours d’une même expérience. La concentration plasmatique du médicament s'est avérée varier sous l'influence des différents profils de courant. La durée et la densité du courant appliqué ont également influencé l'administration de pramipexole dans le cerveau et le liquide cérébro-rachidien. Les profils de concentration plasmatique obtenus en utilisant des profils de courant continu et multiphasique ont mis en évidence le contrôle fourni par l'iontophorèse et sa capacité unique à changer rapidement la vitesse d'administration de médicament.

Après une étude approfondie de l’administration iontophorétique de PRA, RAS et HUP in vitro et aussi in vivo pour PRA, l'administration iontophorétique transdermique d'une nouvelle entité chimique, ARN14140, pour la maladie d'Alzheimer (MA) a été étudiée au Chapitre 3.

ARN14140 est un conjugué de la galantamine et la mémantine et module simultanément les voies cholinergiques et glutamatergiques pour traiter la MA. Il a une faible biodisponibilité orale et une pharmacocinétique sous-optimale ce qui a induit la necessité d’utilier une injection intracérébroventriculaire pour administrer l’ARN14140 directement au cerveau dans les études antérieures. Des expériences préliminaires ont été réalisées en utilisant la peau porcine in vitro et validées avec la peau humaine. L'ARN14140 était stable en présence de peau et lors de l'application du courant. La perméation cumulative de l'ARN14140 à travers la peau a augmenté avec la densité et la concentration du principe actif. L'efficacité


3 équivalente a été obtenue à travers la peau humaine et porcine. Des études in vivo chez des rats Wistar mâles ont révélé que 426,7 ± 42 nmol/cm2 et 1118,3 ± 73 nmol/cm2 d'ARN14140 ont été administrés respectivement à 0,15 et 0,5 mA/cm2 après 6 h d’ionthophorèse. Plus important encore, ARN14140 a également été détecté dans le cerveau. En conclusion, l'iontophorèse transdermique permet l'administration non invasive d'ARN14140 et il a été démontré que le médicament était capable d'atteindre le cerveau.

L'ionophorèse peut également être utile pour administer des peptides pour lesquels le besoin de stabilité et la sensibilité à la dégradation chimique et enzymatique limitent leur administration uniquement par voie parentérale uniquement. La deuxième partie se concentre sur l'étude de l’aptitude du courant électrique à contrôler l'administration d'un décapeptide

“degarelix” à travers la peau porcine et également à partir d'un dépôt sous-cutané. La capacité du courant électrique à améliorer l'administration de l'antagoniste de l'hormone de libération des gonadotrophines, degarelix, a été étudiée in vitro au Chapitre 4. Initialement, l'électrotransport de degarelix à densité de courant fixe (0,5 mA/cm2, 6 h) a été mesuré en fonction de la concentration (0,5 mM et 2 mM) à travers la peau porcine. Les résultats ont indiqué que l'augmentation de la concentration n'a pas augmenté l'administration de degarelix.

Cela est due à sa surface hydrophobe - en tant que cation lipophile, degarelix est capable de se lier à des sites de charge négative dans la peau et inhiber l'électro-osmose - déterminé par co- iontophorèse du paracétamol et en utilisant le derme séparé du reste de la peau par chaleur. La microscopie électronique a révélé l’agrégation du degarelix, dépendante du temps et de la concentration, altérant ainsi son électro-transport. Dans le but de provoquer la libération du degarelix à partir d'un dépôt sous-cutané, un nouveau modèle a été développé. Le degarelix s'est avéré former des agrégats en présence de protéines plasmatiques chargées négativement, neutralisant ainsi sa charge positive et empêchant ainsi sa réponse aux stimuli de courant électrique externes. Dans l'ensemble, cette recherche met en évidence l'interaction compliquée entre la structure peptidique, les membranes cutanées et les protéines plasmatiques qui doivent être judicieusement considérées afin d'améliorer l'administration.

La troisième partie de la thèse étudie une nouvelle approche de l'utilisation de l'ablation fractionnée au laser comme moyen de créer des dépôts intraépidermiques et de fournir ainsi une administration localisée soutenue et d'éviter les effets systémiques. Dans le Chapitre 5 un laser ablatif erbium: YAG a été utilisé pour permettre le dépôt cutané “sans aiguille” de


4 certaines thérapies dermatologiques pourrait être améliorée par l'utilisation de systèmes de réservoir intraépidermique de médicament "à dose élevé" qui permettent une administration topique prolongée et ciblée de médicament, par exemple, dans le traitement des chéloïdes et des cicatrices hypertrophiques. Les microparticules ont été préparées en utilisant une technique de cryobroyage qui a entraîné des efficacités de chargement de médicament d'environ 100%. Ils ont été caractérisés par plusieurs techniques différentes, y compris la microscopie électronique à balayage, la diffraction de rayon X et la calorimétrie différentielle à balayage. La TA a été quantifiée par des méthodes d'analyse HPLC-UV et UHPLC-MS / MS validées. Les études de libération in vitro ont démontré l'effet des propriétés du polymère sur la cinétique de libération de TA. La microscopie confocale à balayage laser a permis la visualisation de microparticules crybroyée contenant de la fluorescéine et du Nile Red dans les micropores cutanés, la libération subséquente de fluorescéine dans les micropores et sa diffusion à travers l'épiderme et le derme supérieur. La biodistribution de TA, c'est-à-dire la quantité de médicament en fonction de la profondeur dans la peau, après application de microparticules était beaucoup plus uniforme qu'avec une suspension de TA. De plus, l'administration était plus sélective pour le dépôt avec moins de perméation transdermique.

Ces résultats suggèrent que cette approche peut fournir une alternative efficace, ciblée et minimalement invasive aux injections intralésionnelle douloureuses pour le traitement des cicatrices chéloïdes.

Le dernier chapitre de cette thèse, le Chapitre 6, étudie la création d'un dépôt intra- épidermique de cetuximab en utilisant l'ablation fractionée par laser pour le traitement du psoriasis. Les microparticules ont été préparées en utilisant une technique de cryobroyage qui a abouti à des efficacités de chargement de presque 100%. Les microparticules ont été caractérisées pour confirmer l'incorporation et l'intégrité de l'anticorps en utilisant une coloration de protéine non-spécifique, un Western Blot et une chromatographie d'exclusion de taille et elles ont été visualisées en utilisant la microscopie électronique à balayage. Le cetuximab a été quantifié en utilisant la méthode ELISA développée en interne. Les études in vitro de dissolution ont démontré l'effet de la viscosité du polymère sur la cinétique de libération des anticorps. Des expériences de biodistribution ont été réalisées en utilisant la peau de porc. Des microparticules de différentes formes ont été développées à partir de deux polymères différents. L'immunocoloration non spécifique et Western Blot ont démontré la


5 biodistribution du cetuximab, c'est-à-dire la quantité d'anticorps en fonction de la profondeur de la peau, suite à l'application de microparticules a entraîné moins de perméation transdermique avec de plus grandes quantités présentes dans la peau que la solution contrôle.

Ces résultats suggèrent que cette approche peut fournir une alternative ciblée, efficace et minimalement invasive à l'administration par voie parentérale pour le traitement du psoriasis.


7 Transdermal delivery is an excellent non-invasive alternative for the delivery of systemically acting drugs. In addition to protecting the drug from the aggressive gastrointestinal environment, it provides controlled drug input kinetics with superior patient compliance and convenience. However, protective function of skin topmost membrane limits its application as a drug delivery route. Consequently, several techniques such as iontophoresis and fractional laser ablation have been developed to expand the horizons of drug candidates for transdermal administration in order to make the “undeliverable” deliverable.

The aim of this thesis was to investigate how different physical techniques could be used to enhance transdermal and cutaneous drug delivery.

First part of the thesis specifically investigates application of iontophoresis to control transdermal delivery kinetics of small molecules, in particular with applications in the treatment of neurodegenerative diseases. Transdermal iontophoresis is an interesting alternative for the controlled delivery of therapeutic agents to treat neurodegenerative diseases. One of its key properties is the ability to use the current profile to control drug delivery kinetics and to obtain complex drug input profiles that simulate the desired physiological profiles. Chapter 1 investigates temporal variation in the transport of pramipexole (PRA), rasagiline (RAS) and huperzine A (HUP) under continuous and multi-phasic current profiles and study the effects on cumulative permeation, transdermal flux and drug retention. Initial experiments with continuous current enabled establishment of correlation between the total delivery of PRA, RAS and HUP (i.e. sum of the cumulative permeation and skin deposition) and the amount of charge transferred. Subsequent experiments with multi-phasic current profiles validated the relationship established between amounts of charge transferred and total delivery to predict the total delivery of each drug. Experimental values were within ± 15% of the predicted values.

The effect of current density and the duration of current application on the skin biodistribution of PRA as a function of depth was also investigated and found to have significant impact on drug biodistribution. These results provided insight into the rate of formation of drug reservoirs in the skin.

Chapter 2 evaluates the transdermal delivery of the dopamine agonist, PRA, from an iontophoretic patch system, in vitro and in vivo. Preliminary in vitro experiments were conducted to optimize the design and development of iontophoretic patch. It was found that with a one-compartment patch model, the drug compartment in contact with electrode


8 Cl- led to decrease in the patch delivery efficiency (i.e. fraction of the amount applied that is delivered). A two-compartment model where the drug compartment was separated from the electrode compartment improved the DE of the patch with no precipitation of silver ions in the formulation compartment. In vivo studies were aimed to investigate the changes in plasma profiles upon modulation of the applied current densities in a single experiment. The plasma concentration of drug was found to vary under the influence of different current profiles.

Duration and density of applied current also influenced pramipexole delivery to the brain and cerebrospinal fluid. The plasma concentration profiles obtained using continuous and multi- phasic current profiles underlined the control provided by iontophoresis and its unique ability to rapidly change drug input rates.

After thoroughly investigating the iontophoretic delivery of PRA, RAS and HUP in vitro and also in vivo for PRA, the transdermal iontophoretic delivery of a new chemical entity, ARN14140, for Alzheimer’s disease (AD) was investigated in Chapter 3. ARN14140 is a conjugate of galantamine and memantine and modulates cholinergic and glutamatergic pathways simultaneously to manage AD. It has poor oral bioavailability and suboptimal pharmacokinetics because of which previous studies employed intracerebroventricular injection to administer ARN14140 directly to the brain. Preliminary experiments were performed using porcine skin in vitro and validated with human skin. ARN14140 was stable in the presence of skin and upon current application. Cumulative ARN14140 permeation across the skin increased with current density and concentration. Delivery efficiency reached an exceptional 76.9 %. Statistically equivalent delivery was obtained across human and porcine skin. In vivo studies in male Wistar rats revealed that 426.7 ± 42 nmol/cm2 and 1118.3 ± 73 nmol/cm2 of ARN14140 was delivered at 0.15 and 0.5 mA/cm2 after iontophoresis for 6h, respectively; more importantly ARN14140 was also detected in the brain. In conclusion, transdermal iontophoresis enabled non-invasive delivery of ARN14140 and it was demonstrated that drug was able to reach the brain.

Iontophoresis can also be useful to deliver peptides where stability needs and susceptibility to chemical and enzymatic degradation qualify them for only parenteral administration. Second part focuses on to investigate the feasibility of electric current to control the delivery of a decapeptide ‘degarelix’ across porcine skin and also from a subcutaneous depot. The ability of electric current to improve the delivery of gonadotropin releasing hormone antagonist,


9 across porcine skin. The results indicated that increasing the concentration did not increase degarelix delivery. This was attributed to its hydrophobic surface – as a lipophilic cation, degarelix is able to bind to negative charge sites in the skin and inhibit electroosmosis – determined from co-iontophoresis of acetaminophen and using heat separated dermis skin.

Electron microscopy revealed the time and concentration dependent aggregation behavior of degarelix that further impaired its electrotransport. In an approach to trigger the release of degarelix from a subcutaneous depot a new model was developed. Degarelix was found to form aggregates in the presence of negatively charged plasma proteins, thereby neutralizing its positive charge and thus shutting off its response to external electric current stimuli. Overall, this research highlights the complicated interaction between peptide structure, skin membranes and plasma proteins that must be judiciously considered in order to improve delivery.

Third part of the thesis investigates a novel approach of using fractional laser ablation as a means to create intraepidermal depots and so provide localized sustained delivery and avoid systemic off-target effects. In the Chapter 5 a fractionally ablative erbium:YAG laser was used to enable “needle-less” cutaneous deposition of polymeric microparticles containing triamcinolone acetonide (TA). The efficacy of some dermatological therapies might be improved by the use of “high dose” intraepidermal drug reservoir systems that enable sustained and targeted local drug delivery, e.g., in the treatment of keloids and hypertrophic scars. The microparticles were prepared using a freeze−fracture technique employing cryomilling that resulted in drug loading efficiencies of ∼100%. They were characterized by several different techniques, including scanning electron microscopy, powder X-ray diffraction and differential scanning calorimetry. TA was quantified by validated HPLC−UV and UHPLC−MS/ MS analytical methods. In vitro release studies demonstrated the effect of polymer properties on TA release kinetics. Confocal laser scanning microscopy enabled visualization of cryomilled microparticles containing fluorescein and Nile Red in the cutaneous micropores and the subsequent release of fluorescein into the micropores and its diffusion throughout the epidermis and upper dermis. The biodistribution of TA, i.e. the amount of drug as a function of depth in skin, following microparticle application was much more uniform than with a TA suspension and delivery was selective for deposition with less transdermal permeation. These findings suggest that this approach may provide an effective, targeted and minimally invasive alternative to painful intralesional injections for the treatment of keloid scars.


10 prepared using a freeze−fracture technique that resulted in nearly 100 % loading efficiencies.

Microparticles were characterized to confirm the incorporation and integrity of the antibody using nonspecific protein staining, western blotting and size exclusion chromatography and visualized using scanning electron microscopy. Cetuximab was quantified by using in-house developed ELISA method. In vitro dissolution studies demonstrated the effect of polymer viscosity on antibody release kinetics. Biodistribution experiments were carried out using sliced porcine skin. Shape of microparticles were different depending on the type of polymer used.

Non-specific protein staining and western blot demonstrated stability of antibody integrity and identity in the microparticle formulations. The biodistribution of cetuximab, i.e. the amount of antibody as a function of skin depth, following microparticles application resulted in less transdermal permeation with greater amounts present in the skin than the control solution formulation. These findings suggest that this approach may provide a targeted, effective and minimally invasive alternative to parenteral administration for the treatment of psoriasis.




13 Transdermal administration is an excellent non-invasive alternative for the delivery of systemically acting drugs. In addition to protecting the drug from the aggressive gastrointestinal environment, it provides controlled drug input kinetics with excellent patient compliance and convenience. Skin is body’s largest organ and one of its key functions is to keep endogenous material in and exogenous matter out of the body. The epidermis provides this protective function due to the brick and mortar like structure (multilamellar lipid covering proteinaceous corneocytes) of its outermost layer, the stratum corneum (SC) [1,2]. SC controls molecular transport so efficiently that only a select few highly potent molecules have the right balance of physicochemical properties to partition sufficiently into the lipidic SC and the hydrophilic deeper skin layers. As a result, the current transdermal market comprises products of only 18 molecules (buprenorphine, clonidine, estradiol, ethinyl estradiol, fentanyl, granisetron, levonorgestrol, methylphenidate, nicotine, nitroglycerin, norelgestromin, norethisterone, oxybutynin, rivastigmine, rotigotine, scopolamine, selegiline, and testosterone) [3].

The last 20-30 years have indeed seen the development of several techniques that have the potential to expand affectedly the horizons for transdermal administration – making the

“undeliverable” deliverable. These methods employ different mechanisms, including (i) a second driving force, e.g. iontophoresis [4], that creates an active transport process or, (ii) disruption of the SC and creation of new transport channels by mechanical (e.g. microneedles) [5] or energy-based (e.g. fractional laser ablation) [6] techniques – thereby, facilitating passive diffusion through these transient shunt pathways or (iii) application of energy to disrupt lipid organization and so render passive diffusion through an “intact” SC more facile (e.g.

electroporation [7] and sonophoresis [8]).

Iontophoresis among other techniques has been the most studied technique as it provides controlled delivery of actives and is the only active ‘physical’ transdermal technology to receive regulatory approval for commercial launch in the market. For topical delivery, fractional lasers to ablate the skin surface and improve the passive delivery of actives has found commercial and clinical acceptance by the dermatologists. This thesis has investigated (i) iontophoresis technique to control transdermal delivery and (ii) fractional laser ablation technique to provide sustained topical delivery.


14 Iontophoresis is a highly efficient non-invasive technique that drives charged and polar molecules into and across the skin under the influence of a small electric potential. The compounds of interest are placed in contact with either the anode or cathode depending on their charge, and are transported across the skin barrier by two primary mechanisms:

electromigration (EM) and electroosmosis (EO). EM is the orderly movement of ions under the controlled electric field and EO is the delivery of solute due to the convective solvent flow that is created in the anode-to-cathode direction under physiological conditions. Therefore, EO solvent flow opposes anion electromigration from the cathode.

A schematic representation of a transdermal iontophoretic system is depicted in Figure 1. A formulation containing drug (D+ with counter-ion) is present in the anodal compartment. The cathodal compartment is positioned at a distal site on the skin and completes the circuit. Anionic drugs (D-) can be delivered from the cathodal compartment. Ag/AgCl electrodes are more commonly used in iontophoretic systems because of several advantages over inert electrodes such as required low operating potentials and avoidance of changes in solution pH due to water hydrolysis [9]. Upon activation of the device, electron flow is converted to an ion flow at the respective electrode/formulation interface. At the anode, Ag+ ions react with Cl ions arriving from skin, if not present already in the formulation, to form insoluble AgCl at the electrode surface. Presence of insufficient amount of Cl leads to the deposition of Ag+ on the skin surface; therefore, Cl is added externally to the drug formulation. Electrochemistry at the cathode (AgCl) comprises reduction of the Ag salt and liberation of Cl- into the formulation; in this case, electroneutrality requires loss of an anion or addition of a cation in the cathodal compartment. In this case Cl ion can compete with the D- for electromigration .

The total iontophoretic flux (JT) is the sum of the fluxes due to EM (JEM) and EO (JEO) assuming negligible passive permeation.

𝐽𝑇 = 𝐽𝐸𝑀 + 𝐽𝐸𝑂 (1) 𝐽𝑇 (𝑚𝑜𝑙 𝑐𝑚⁄ 2𝑠) = 𝑢𝑑𝑐𝑑𝐼

𝑧𝑑 𝐹 . Σ1𝑖𝑢𝑖𝑐𝑖+ 𝑣 . 𝑐𝑑 = ( 𝑢𝑑𝐼

𝑧𝑑 𝐹 . Σ1𝑖𝑢𝑖𝑐𝑖+ 𝑣 ) × 𝑐𝑑 (2) As shown in equation (2), JEM depends on the product of mobility (ud), concentration of the drug ion (cd) in the membrane, and current intensity per unit area (I) and is inversely proportional to the sum of the corresponding values for the other ions (competitive) present in


15 the fraction of drug ions to total ion concentration [10]. JEO is known by the product of the linear velocity of solvent flow (𝑣) and drug concentration (cd).

Figure 1. A schematic representation of transdermal iontophoretic delivery. The anodal compartment contains a cationic drug (D+) and cathodal compartment contains an anionic drug (D-). Upon application of an electric potential electromigration takes place where D+ and D- transported into the skin from their respective compartments. Chloride ions (Cl) from the skin migrating towards the anode and sodium ions (Na+) migrating towards cathode. Electroosmotic (EO) flow from anode to cathode direction supporting electromigration from anodal compartment and counters the delivery from cathodal compartment.

Part 1: Transdermal iontophoretic delivery of small molecules

Drug physicochemical properties decide the candidacy of a drug for iontophoretic delivery. In addition to being stable in the skin (basic criteria for a drug for transdermal delivery), the drug should also be stable when exposed to the electric current without showing any sign of skin irritation. The SC is particularly effective in restricting the passive transport of polar or charged molecules [11,12] whereas iontophoresis is particularly effective in the delivery of drug substances with these very properties [4]. Iontophoresis shares all the benefits of passive delivery approach but being an active technique, iontophoresis enables a decrease in lag time and thus a faster onset of pharmacological effect. Furthermore, by providing high delivery efficiency, less residual amount of drug is left in the formulation by the end of the treatment.


16 enables tight control over drug input rates that provides highly individualized treatment regimens. This property can be of great importance for certain conditions, e.g. in neurodegenerative diseases, where dose ramping is an integral part of therapy thus improving patient compliance. Moreover, selection of an appropriate current intensity could be used to target different depths in the skin: epidermis, dermis or systemic delivery [13] .

Neurodegenerative disease such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) pose a significant burden on the healthcare system. The currently available treatments have several drawbacks that warrant the development of delivery systems that are more patient compliant and provide effectively controlled delivery of therapeutic agents. Investigations on to study the passive transdermal transport of drug molecules for neurodegenerative disorders such as pramipexole (PRA) [14], rasagiline (RAS) [15], and huperzine A (HUP) [10] led to the delivery in subtherapeutic amounts. PRA, RAS and HUP are low molecular weight compounds, readily soluble in aqueous solution and available in ionic forms (Table 1). PRA is widely prescribed both as monotherapy and as adjunct therapy with L-DOPA [16,17] in PD. RAS is a monoamine oxidase B (MAO-B) inhibitor that belong to a class of drugs that regulates the metabolism of dopamine [18-20]. Huperzine A (HUP) is an acetylcholinesterase inhibitor (AChEI) which is reversible in nature and is found naturally in Huperzia serrata, native to India and Southeast Asia [21,22]. It is used in AD and is reported to possess less antibutyrylcholinesterase activity and thus more selective to inhibit acetylcholinesterase than approved AChEIs; as a consequence, it may provoke fewer side effects in patients [23].

Table 1. Comparison of physicochemical properties of PRA, RAS and SEL.


Mw (Da) 302.26a 267.34b 242.32

Aqueous solubilitypH 6.0, 25 °C (g/L) 991 17 492

pKa 5.0, 9.6 7.2 6.97

log P25°C 2.35 1.67 0.83

log DpH 7.0, 25°C 0.02 1.4 -0.96

a Dihydrochloride monohydrate salt.

b Mesylate salt.


17 for iontophoresis and can be delivered in therapeutic amounts [10,14,15]. Current- and concentration-proportional increase in the transdermal delivery of these molecules was obtained with comparable delivery through porcine and human skin. This feature of iontophoresis is highly essential to individualize the treatment especially in AD and PD therapy where dose ramping is an integral part of the treatment regimens. The ability to modulate current density provides simple means to control electrotransport of drug using the same formulation to personalize dosing. However, the effect of variation in current density on the permeation is not immediate since the drug goes through the skin compartment before reaching the receiver compartment. Depending on the rate of drug electrotransport, which is influenced by the current density and duration, drug’s skin deposits are formed and the drug partitions into the systemic circulation. Formation of depots in the skin could be useful in permitting continuous drug delivery without the application of electric current for a certain duration during the treatment. Therefore, for the better understanding of transdermal iontophoretic delivery, it is important to investigate the changes in cumulative permeation, transdermal flux and drug retention in the porcine skin upon modulation of the applied current densities in a single experiment in vitro.

Given the rationale described above, an insight into the rate of formation of drug reservoirs in the skin was studied using PRA, RAS and HUP. Chapter 1 focuses on to investigate the temporal variation in the transport of PRA, RAS and HUP under continuous and multi-phasic current profiles. The effect of current density and the duration of current application on the skin biodistribution of PRA as a function of depth was investigated. Finally, the relationship between the amounts of charge transferred and the PRA total delivery was established.

Iontophoretic device formulation characteristics: compositions and challenges

A typical transdermal iontophoretic system comprises of four components: a power source generally a battery, electronic controller, electrodes (anode and cathode) and finally two reservoirs, one for each electrode, in the form of a hydrogel or sponge which is imbibed with electrolyte or drug solution before activation. Drug formulation composition should be such that it does not hinder the flow of ions into the skin and contain no other ions that may compete with the drug [15].

The electronic controller automatically regulates the applied potential for intra- and inter- individual variations in skin resistance to maintain constant current output, which delivers


18 the controller can also provide feedback on system operation to the patient and/or caregiver in the form of a visual or an audible signal. In addition, the system functional data can also be stored in the system to count the frequency of dose activation and patient compliance to the treatment.

As discussed earlier, Ag/AgCl electrodes are the most commonly used electrodes. Oxidation at anode requires Clto react with Ag+ to form AgCl at the anode formulation interface. Therefore, hydrochloride salt forms of drugs are ideal candidates since this avoids the need to add external Cl that brings with it another cation, normally Na+, which can compete with the drug to carry current and so reduce the delivery efficiency of the system [24,25]. The effect of competing ions can be reduced by increasing the proportion of drug content and also by selecting right formulation pH which influences the charge state as well as solubility of the drug [10,26-28].

Inert electrodes such as Pt-metal electrodes electrolyze water that can affect the pH of a formulation which can be irritating to the skin. Further, other ions generated in the process may compete with the drug to carry charge because of their smaller size and high mobility. Zinc (another source to prepare electrode) has good solubility in water and thus if used as an anode, during iontophoresis Zn+ will appear in the formulation to compete with the drug ions. An approach to separate the electrodes from the formulation using an ion-exchange resin can be useful to avoid addition of high proportion of drug to minimize the competition effect [29].

Earlier approved commercial devices

Several kinds of non-drug containing commercial iontophoretic devices are available from Travanti Pharma Inc. (St. Paul, MN), Iomed Inc. (Salt Lake City, UT) and Empi Corp. (St. Paul, MN). These systems are disposable and require transfer of drug solution by the prescriber in the clinic before application (Figure 2a-c). To avoid the step of adding drug solution technological advancements in the development of miniaturized electronic devices have facilitated the development of pre-filled iontophoretic therapeutic systems. Vyteris, Inc. (Fair Lawn, NJ) received FDA approval for ‘LidoSite’, first pre-filled iontophoretic system, in 2004 but was later discontinued due to undisclosed reasons even though its projected annual US market size was $200-$500 million. LidoSite contained lidocaine HCl and epinephrine to provide effective dermal anaesthesia for intravenous cannulation in 10 min [30]. As shown in Figure 2d, the LidoSite device consisted of a portable controller and a single use disposable patch component consisting of a circular drug reservoir (anode) and an oval return reservoir containing electrolytes (cathode). The controller consisted of a non-replaceable battery that


19 proceeded in three stages: ramp up, constant current, and ramp down. The embedded circuitry was capable of monitoring the current delivery and could signal its system status through visual and audio indicators.

In 2013, Teva Pharmaceuticals Inc. launched Zecuity, a sumatriptan iontophoretic transdermal system, to treat acute migraine headaches in adults. Unlike LidoSite and Ionsys (see below), Zecuity is disposable, single use iontophoretic transdermal system for outpatient self- administration to the upper arm or thigh. It consists of an iontophoretic electronic component and a drug reservoir card (Figure 2e). Two coin cell lithium batteries power the electronic circuitry that controls the amount of current applied to deliver 6.5 mg of sumatriptan over 4 h.

The reservoir card contains two non-woven pads to accommodate a sumatriptan succinate formulation and a sodium salt formulation in different pads. The sumatriptan succinate formulation and pad contains sumatriptan (86 mg), purified water, methylparaben, basic butylated methacrylate copolymer, lauric acid, adipic acid, and a non-woven viscose pad. The salt formulation is composed of purified water, hydroxypropylcellulose, sodium chloride, and methylparaben. The iontophoretic device consists of medical grade adhesive fabric and foam and a plastic dome that contains an activation button, batteries, and electronics. If required, a second Zecuity system can be applied to another site after 2 h of first treatment. Post marketing reports of burns and scars associated with the Zecuity patch led to the temporary suspension of sales, marketing, and distribution to investigate the cause. Similar to Zecuity, an iontophoretic system of Zolmitriptan HCl, a potent antimigraine drug, could be more successful since the operational current intensity to delivery therapeutic amounts would be much lower and the incidents of skin burns could be avoided.

Ionsys containing fentanyl was developed by Alza Corporation (Mountain View, CA), and approved by the FDA in 2006 for the short-term management of acute postoperative pain for use by patients under medical supervision in the hospital. Due to the corrosion found in one batch, the product was withdrawn from the market. However, since Ionsys had high market acceptance, it was relaunched in 2015 by The Medicines Company (Parsippany, NJ) after resolving the stability issue [31,32]. Ionsys consists of a plastic top housing that encloses the 3- volt lithium battery and electronics and a red plastic bottom housing for two hydrogel reservoirs and a polyisobutylene skin adhesive (Figure 2f). One of the hydrogels contains 10.8 mg of fentanyl hydrochloride along with the inactive ingredients for anode and the second hydrogel for cathode contains only inactive ingredients which are cetylpyridinium chloride, citric acid,


20 water. Upon each activation, Ionsys delivers a 40 mcg dose of fentanyl over a 10-minute. This system also provides audio and visual signals to provide the status of delivery and number of doses delivered. It is designed to deliver maximum 3.2 mg, equivalent to eighty 40-mcg doses) over 24h of period where maximum six 40-mcg doses per hour should be administered.

Sumatriptan was originally approved in 1992 and prescribed for the acute treatment of migraine.

NuPathe, Inc. had filed a 505(b)(2) NDA for Zecuity transdermal iontophoretic system as a new dosage form of sumatriptan. Section 505(b)(2) of the Federal Food, Drug and Cosmetic Act allows for faster development of new dosage forms of already approved drugs for the same indication. Therefore, this approach of obtaining regulatory approval may offer a faster route to commercialization and play a significant role in drug life-cycle management. Based on this approach, LidoSite and Ionsys were also approved via 505(b)(2) NDA pathway.

Figure 2. Commercial iontophoretic systems: (a) IontoPatch 80, Travanti Pharma Inc. (St. Paul, MN); (b) Companion 80, Iomed Inc. (Salt Lake City, UT); (c) Empi Action Patch, Empi Corp.

(St. Paul, MN); (d) LidoSite, Vyteris Inc. (Fair Lawn, NJ); (e) Zecuity, Teva Pharmaceuticals Inc. (North Wales, PA) (f) Ionsys, The Medicines Company (Parsippany, NJ).

Having understood the composition and characteristics of several iontophoretic systems, the







Portable controller

Disposable patch

Activation button

Medication pad Salt pad

Activation button

Display Top plastic housing

Preprogrammed electronic circuitry

Bottom housing with hydrogel


21 investigate PRA delivery kinetics to determine whether patch could deliver the active in therapeutic amounts. The first part of the study was conducted using porcine skin in vitro in order to optimize the transdermal iontophoretic patch system. The second part comprised two studies in rats in vivo. The first of these involved the application of continuous current profiles in order to study the pramipexole entry into the bloodstream at two current densities: 0.15 and 0.5 mA/cm2. The second, which involved application of three complex multi-phasic current profiles, investigated the potential for modifying the current intensity in a single experiment in order to regulate the pramipexole delivery kinetics to personalize the treatment (as might be required depending on the severity of the patients’ condition).

After thoroughly investigating the iontophoretic delivery of PRA, RAS and HUP in vitro and in vivo, the transdermal iontophoretic delivery of a new chemical entity, ARN14140, for AD was investigated. The current commonly employed treatment for AD is the combination of galantamine (acetylcholinesterase (AChE) inhibitor) and memantine (N-methyl-D-aspartate (NMDA) receptor antagonist) [33,34]. Galantamine, in addition to inhibiting AChE, also facilitates the effect of memantine by enhancing the synaptic NMDA receptor activity [35-37].

ARN14140 is a novel class of multi-target compound formed by linking of galantamine and memantine into a single entity to act on both targets [38] . Preliminary ARN pharmacokinetic and brain penetration data performed by Reggiani et. al., revealed suboptimal delivery via oral route that was consistent with predictions that suggested preferential accumulation in the liver [39]. Continuous inhibition of AChE and NMDA receptors is required to treat symptoms of AD. Considering the benefits offered by transdermal iontophoresis in providing controlled drug transport and improve drug’s pharmacokinetics, Chapter 3 investigates the feasibility of the iontophoretic transdermal delivery of ARN1410 and identify the mechanism governing electrotransport under in vitro conditions using porcine skin. In vivo studies performed in rats were performed to evaluate the feasibility of using iontophoresis to deliver ARN14140 in therapeutic amounts.

Part 2: Understanding the iontophoretic delivery of a decapeptide

Iontophoresis can also be useful to deliver peptides and moderately-sized proteins where stability needs and susceptibility to chemical and enzymatic degradation qualify them for only parenteral administration [40-45]. However, the transdermal delivery of large molecules is a complex challenge that should be carefully evaluated [46]. In addition to the solubility and charge required for the iontophoretic transport of peptides and proteins, the three dimensional


22 should also be taken into consideration. There remains a possibility of strong interaction of hydrophobic and positively charged regions of protein surface with the negatively charged skin membrane, which blocks the transport pathway and ultimately reduces protein electrotransport [47,48].

Degarelix, a gonadotropin-releasing hormone antagonist, is administered by subcutaneous injection for the treatment of advanced prostate cancer. Degarelix is a synthetic linear decapeptide amide with a molecular weight of 1632.26 Da and isoelectric point of 10.4. At a physiological pH 7.4, it acquires about +1 net charge, which is similar to other peptides such as triptorelin [43], nafarelin [47] and leuprolide [49]. Degarelix has different physicochemical properties with more hydrophobic regions then triptorelin, nafarelin and leuprolide and it forms a gel depot after subcutaneous (s.c.) administration that enables sustained drug release for over a month.

In an attempt to avoid s.c. administration, Chapter 4 focuses on to investigate the feasibility of iontophoresis to deliver degarelix across porcine skin and determine the effect of peptide on electroosmotic solvent flow. The aggregation behavior of degarelix and its impact on the skin biodistribution was also studied. Since degarelix is known to form a depot after s.c. injection, the secondary objective of this study was to evaluate the ability of electric current to trigger the release of degarelix from the in situ formed depot in the human s.c. tissue.

Part 3: Creation of intraepidermal depot for topical delivery using fractional laser ablation

Fractional laser ablation breaches the skin barrier by simply removing the skin tissue – energy of a specific wavelength is focused to create the micropore array. This energy can be controlled to enable selective removal of SC or the entire epidermis. At higher laser energies pores reaching deeper into dermis can also be created. Since the barrier is compromised intensively, formulation needs to be manufactured under sterile conditions. From a practical point of view laser skin ablation can provide faster, more reliable and reproducible results in comparison to solid microneedles. It has also been used to treat some skin conditions and diseases alone; plus can be used with tissues like nails, where the absorption of drugs is very limited [50]. Currently, the laser is principally used in aesthetics and odontology with different purposes, but recent studies on laser-assisted drug delivery into the skin appears very promising [6,51-54].


23 reaction and can differ depending on the laser used. The laser energy interacts with the tissue causing its vaporization; depending on the emission wavelength different structures can be affected. Mid-infrared lasers i.e. Er:YAG emits light at 2936 nm, produce a photomechanical reaction with the skin. The maximum absorption of the water molecules corresponds to the emission wavelength of these lasers. When the laser beam hits the skin, it produces an excitation of the water molecules, leading to an explosive evaporation that forms the craters or micropores.

Given the “water selectivity” of these lasers, the thermal damage to the nearby tissue is reduced.

Far infrared lasers i.e. CO2 emits at 10600 nm, produce a thermomechanical reaction that can affect the dermal proteins and vessels [55,56]. Similar to emission wavelength, the pulse duration is an important factor too. Applying a pulse duration shorter than the water relaxation time, the heat transfer to the surrounding tissue is reduced [6]. The energy applied in function of the area (J/cm2), known as fluence, is a very important parameter that allows controlling the depth of the micropores. The more energy applied in the same spot, the deeper the pore will be.

It is important to highlight that the lasers currently used for drug delivery are fractional ablation lasers that can ablate from 5 to 20% of the skin surface that means there are regions of intact skin between ablated area (total >80 % of the total treated area) which expedites skin healing and closure of pores [57]. The specificity of these lasers allows minimizing the skin damage, reducing the risks of scarring, irritation and erythema. Several commercially available laser instruments are presented in Figure 3.

Figure 3. Laser instruments: (a) Epiture Easytouch by Norwood Abbey, Ltd. [58]; (b) P.L.E.A.S.E. Professional (painless laser epidermal system) device by Pantec Biosolutions [59]; (c) P.L.E.A.S.E. Private device by Pantec Biosolutions [59]; and (d) PinPointe FootLaser by PinPointe USA, Inc [60].

Work to-date has demonstrated that the technique can increase the rate and the extent of the delivery of small molecules [6,61-63], peptides and proteins [51,53] into and across the skin

a. b. c. d.


24 ablation by definition completely removes the SC at the very least; hence the formulation approach must be re-considered. Once the skin barrier is gone, drug can be continuously delivered from any kind of formulation. This enables the delivery to be controlled by varying the formulation characteristics. In principle, formulation application could be repeated at the same microporation site as long as the pores remained open − a conservative estimate would be for 48 to 72 h – beyond that time interval, a new micropore array must be created. However, from a clinical perspective, it would be of considerable interest to be able to introduce an intraepidermal drug depot where the natural healing process after microporation would close the skin around it. The depot would have different drug release kinetics to a formulation applied topically to the skin surface and would enable sustained drug release over a more prolonged time frame. This intraepidermal drug depot is clearly analogous to the well-known subcutaneous and intramuscular depots that are frequently used in therapy to enable prolonged systemic release of peptide therapeutics, e.g. gonadotropin-releasing hormone agonists and antagonists [65].

The efficacy of some dermatological therapies might be improved by the use of “high dose”

intraepidermal drug reservoir systems that enable sustained and targeted local drug delivery.

The Chapter 5 and Chapter 6 investigates an interesting approach of creating intraepidermal depot of triamcinolone acetonide for the management of keloids and hypertrophic scars and depot of cetuximab, a monoclonal antibody, for treating psoriasis. The specific objectives of this study were to develop and to optimize a new technique to prepare stable drug loaded microparticles with high drug loading. The sustained delivery of drugs from the developed and optimized microparticles were then evaluated using the confocal laser scanning microscopy (CLSM) technique (qualitatively) and finally, quantitative biodistribution (the amounts of drug as a function of position) in the skin following release from the skin deposited microparticles was also evaluated.


25 [1] R.O. Potts, M.L. Francoeur, The influence of stratum corneum morphology on water permeability, J. Invest. Dermatol., 96 (1991) 495-499.

[2] A. Naik, Y.N. Kalia, R.H. Guy, Transdermal drug delivery: overcoming the skin's barrier function, Pharm. Sci. Technolo. Today, 3 (2000) 318-326.

[3] M.N. Pastore, Y.N. Kalia, M. Horstmann, M.S. Roberts, Transdermal patches: history, development and pharmacology, Br. J. Pharmacol., 172 (2015) 2179-2209.

[4] Y.N. Kalia, A. Naik, J. Garrison, R.H. Guy, Iontophoretic drug delivery, Adv. Drug Deliv.

Rev., 56 (2004) 619-658.

[5] E. Larraneta, M.T. McCrudden, A.J. Courtenay, R.F. Donnelly, Microneedles: A New Frontier in Nanomedicine Delivery, Pharm. Res., 33 (2016) 1055-1073.

[6] Y.G. Bachhav, S. Summer, A. Heinrich, T. Bragagna, C. Bohler, Y.N. Kalia, Effect of controlled laser microporation on drug transport kinetics into and across the skin, J. Control.

Release, 146 (2010) 31-36.

[7] E.P. Spugnini, A. Melillo, L. Quagliuolo, M. Boccellino, B. Vincenzi, P. Pasquali, A. Baldi, Definition of novel electrochemotherapy parameters and validation of their in vitro and in vivo effectiveness, J. Cell Physiol., 229 (2014) 1177-1181.

[8] G. Merino, Y.N. Kalia, M.B. Delgado-Charro, R.O. Potts, R.H. Guy, Frequency and thermal effects on the enhancement of transdermal transport by sonophoresis, J. Control. Release, 88 (2003) 85-94.

[9] C. Cullander, G. Rao, R.H. Guy, Why silver/silver chloride? Criteria for iontophoresis electrodes, in: K.R. Brian, V.J. James, K.A. Walters (Eds.) 3rd International conference on prediction of percutaneous penetration, STS Publishing, Cardiff (Wales), La Grande Motte, France, 1993, pp. 381-390.

[10] D.R. Kalaria, P. Patel, V. Merino, V.B. Patravale, Y.N. Kalia, Controlled iontophoretic transport of huperzine A across skin in vitro and in vivo: effect of delivery conditions and comparison of pharmacokinetic models, Mol. Pharm., 10 (2013) 4322-4329.

[11] R.J. Scheuplein, I.H. Blank, Permeability of the skin, Physiol. Rev., 51 (1971) 702-747.

[12] S. Monash, Location of the superficial epithelial barrier to skin penetration, J. Invest.

Dermatol., 29 (1957) 367-376.

[13] Y. Chen, I. Alberti, Y.N. Kalia, Topical iontophoretic delivery of ionizable, biolabile aciclovir prodrugs: A rational approach to improve cutaneous bioavailability, Eur. J. Pharm.

Biopharm., 99 (2016) 103-113.


26 delivery of pramipexole: electrotransport kinetics in vitro and in vivo, Eur. J. Pharm.

Biopharm., 88 (2014) 56-63.

[15] D.R. Kalaria, P. Patel, V. Patravale, Y.N. Kalia, Comparison of the cutaneous iontophoretic delivery of rasagiline and selegiline across porcine and human skin in vitro, Int.

J. Pharm., 438 (2012) 202-208.

[16] M.F. Piercey, Pharmacology of pramipexole, a dopamine D3-preferring agonist useful in treating Parkinson's disease, Clin. Neuropharmacol., 21 (1998) 141-151.

[17] M. Horstink, E. Tolosa, U. Bonuccelli, G. Deuschl, A. Friedman, P. Kanovsky, J.P. Larsen, A. Lees, W. Oertel, W. Poewe, O. Rascol, C. Sampaio, Review of the therapeutic management of Parkinson's disease. Report of a joint task force of the European Federation of Neurological Societies and the Movement Disorder Society–European Section. Part I: early (uncomplicated) Parkinson's disease, Eur. J. Neurol., 13 (2006) 1170-1185.

[18] L.W. Elmer, J.M. Bertoni, The increasing role of monoamine oxidase type B inhibitors in Parkinson's disease therapy, Expert Opin. Pharmacother., 9 (2008) 2759-2772.

[19] P. Jenner, Preclinical evidence for neuroprotection with monoamine oxidase-B inhibitors in Parkinson’s disease, Neurology, 63 (2004) S13-S22.

[20] P.A. LeWitt, MAO-B inhibitor know-how: back to the pharm, Neurology, 73 (2009) 2048.

[21] R. Wang, H. Yan, X.C. Tang, Progress in studies of huperzine A, a natural cholinesterase inhibitor from Chinese herbal medicine, Acta Pharmacol. Sin., 27 (2006) 1-26.

[22] X.C. Tang, P. De Sarno, K. Sugaya, E. Giacobini, Effect of huperzine A, a new cholinesterase inhibitor, on the central cholinergic system of the rat, J. Neurosci. Res., 24 (1989) 276-285.

[23] B.S. Wang, H. Wang, Z.H. Wei, Y.Y. Song, L. Zhang, H.Z. Chen, Efficacy and safety of natural acetylcholinesterase inhibitor huperzine A in the treatment of Alzheimer's disease: an updated meta-analysis, J. Neural. Transm. (Vienna), 116 (2009) 457-465.

[24] N. Abla, A. Naik, R.H. Guy, Y.N. Kalia, Contributions of electromigration and electroosmosis to peptide iontophoresis across intact and impaired skin, J. Control. Release, 108 (2005) 319-330.

[25] J. Cazares-Delgadillo, C. Balaguer-Fernandez, A. Calatayud-Pascual, A. Ganem-Rondero, D. Quintanar-Guerrero, A.C. Lopez-Castellano, V. Merino, Y.N. Kalia, Transdermal iontophoresis of dexamethasone sodium phosphate in vitro and in vivo: effect of experimental parameters and skin type on drug stability and transport kinetics, Eur. J. Pharm. Biopharm., 75 (2010) 173-178.



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