Dermatology Online Journal

Topical delivery of active principles: The field of dermatological research
Massimiliano Nino MD PhD, Gabriella Calabrò, Pietro Santoianni MD PhD
Dermatology Online Journal 16 (1): 4

Department of Systematic Pathology - Section of Dermatology, Naples, Italy. massimilianonino@yahoo.it

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Abstract

To be effective an active drug or principle must cross the stratum corneum barrier; this process can be influenced to obtain better functional and therapeutical effects. In spite of the wide variety of the methods studied in order to improve the transdermal transfer to obtain systemic effects, the applicability is limited in this field. Attention to the epidermal barrier and penetration of active principles has been reported mostly in studies concerning dermocosmetics. Studies regarding methods of penetration are gaining experimental and clinical interest. Cutaneous bioavailability of most commercially available dermatological formulations is low. Increase of intradermal delivery can relate to chemical, biochemical, or physical manipulations. Chemical enhancers have been adopted to: (a) increase the diffusibility of the substance across the barrier; (b) increase product solubility in the vehicle; (c) improve the partition coefficient. Moreover methods of interference with the biosynthesis of some lipids allow the modification of the structure of the barrier to increase the penetration. The main physical techniques that increase cutaneous penetration of substances are: iontophoresis (that increases the penetration of ionized substances), electroporation (that electrically induces penetration through the barrier), and sonophoresis, based on 20 to 25 KHz ultrasound that induces alterations of the horny barrier, allowing penetration of active principles. Recent development of these methods are here reported and underline the importance and role of vehicles and other factors that determine effects of partition and diffusion, crucial to absorption.

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Introduction

The skin represents an important barrier of the penetration of exogenous substances into the body and, on the other hand, a potential avenue for the transport of functional active principles into the skin and/or the body. Several studies have shown the modalities through which these molecules cross the horny layer, which represents the most important limiting factor of the process of diffusion and penetration, and have discussed how to increase the penetration of pharmacologically active substances [1, 2, 3].

The stratum corneum has a very peculiar structure: the corneocytes (the bricks: about 85% of the mass of horny mass) and intercellular lipids (15%) are arranged in approximately 15-20 layers. It consists of about 70 percent proteins, 15 percent lipids, and only 15 percent water.

In the corneocytes contain keratin, filagrin, and demolition products. The corneocyte lacks lipids, but is rich in proteins. The lipids are inside extracellular spaces, in a bilayer organization surrounding corneocytes [4]. The very low permeability of the horny layer to hydrosoluble substances is because of this extracellular lipid matrix. Cutaneous penetration of hydrophilic substances is limited because of the convoluted and tortuous intercellular space and hydrophobicity of three lipidic constituents: ceramides, cholesterol, and free fatty acids that are present in the molar ratio: 1: 1: 1 (weight ratio: ceramides 50%, cholesterol 35-40%, free fatty acids 10-15%) [5]. This ratio is critical: the diminution of the concentration of one of these types of lipids alters the molar ratio functional to the normality of the barrier and modifies its integrity [6].

The variations of this lamellar structure and/or its lipid composition are the structural and biochemical basis of permeability variations along with the thickness of the horny layer [7, 8].

The extracellular matrix forms also the so-called reservoir of the horny layer (some substances are partially retained in the corneous layer and are slowly released).

Various processes carried out serially or in parallel, are involved in cutaneous penetration of substances and these may cross the stratum corneum via an intercellular or a transcellular route. Moreover, entrance through pilosebaceous units and eccrine glands is possible.

Many efforts to obtain therapeutic effects in tissues far from the skin have been made. We may have: topical administration, with a pharmacological effect limited to skin, with some unavoidable systemic absorption; loco-regional delivery, when the therapeutic effect is obtained in tissues more or less deeply beneath the skin (muscles, articulations, vessels, etc.) with limited systemic absorption; and transdermic delivery that aims to obtain, through application of preparations on the skin, pharmacologically active levels for the treatment of systemic diseases through skin vascular network.

Stratum corneum barrier and intradermal delivery

The penetration through the stratum corneum involves partition phenomena of applied molecules between lipophilic and hydrophilic compartments. For many substances the penetration takes place through an intercellular way, more than transcellular, diffusing around the keratinocytes.

Intercellular movement. The lipid lamellae of the intercellular spaces (each one including 2 or 3 bilayers and made mainly of ceramides, cholesterol, and free fatty acids) are the intercellular structure of the horny layer, with the main role in barrier function. Most solute substances, non-polar or polar, penetrate across intercellular lipid avenues. The permeability of very polar solutes is constant and similar to the transport of ions (es. potassium ions). Lipophilic solute permeability increases according to specific lipophilic properties.

Transcellular movement. Stratum corneum intracellular components are essentially devoid of lipids and lack a functional lipid matrix around keratin and keratohyalin. This results in an almost impenetrability of corneocytes [9]. Degradation of the corneodesmosomes causes formation of a continuous lacunar dominio ("aqueous pore") allowing intercellular penetration; the lacunae formed are scattered and not continuous, and form as a result of occlusion, ionophoresis, and ultrasound waves. These may become larger and connect forming a net ("pore-way"). Various methods can induce this type of permeability increase [10].

Transport through follicular and gland structures. Movement through hair follicles, pilosebaceous units, and eccrine glands is limited. The orifices of the pilosebaceous units represent about 10 percent in areas where their density is high (face and scalp) and only 0.1 percent in areas where their density is low. This is a possible selective way for some drugs. Follicular penetration may be influenced by sebaceous secretion, which favors the absorption of substances soluble in lipids. The penetration through the pilosebaceous units is dependent upon the property of the substance and type of preparation.

a) Pharmacokinetic parameters. Vehicle/ corneous layer partition.

For the purpose of the study of the mechanisms of transport and the functions of the skin barrier, it can be considered as a membrane or a cluster of membranes (mathematical principles can be applied) [11]. On the whole, transport through the horny layer is mainly a molecular passive diffusion. The physico-chemical and structural properties of the substance determine the capacity of diffusion and penetration through the membrane: important determinants are solubility and diffusibility.

The diffusibility and the ability of a solute to penetrate through the barrier is influenced by several factors including the tortuosity of the intercellular route. This passive process of absorption follows Fick's law of diffusion: the velocity of absorption - flow - is proportional to the difference of concentration of the substance in relation to that within the barrier. It can finally be noted that the permeability coefficient relates flow and concentration, resulting from partition coefficient, diffusion coefficient, and length of diffusion route [12].

b) Role of the vehicle and excipients and interaction with the active principles

A vehicle is defined by the type of preparation (cream, ointment, gel) and the excipients (water, paraffin, propilen glycol); the terms "vehicle" and "excipient" refer to different entities.

Vehicle and excipients deeply influence the velocity and magnitude of absorption and consequently the bioavailability and efficacy. The excipients of the vehicle modulate the effects of partition and diffusion in the stratum corneum.

A lipid preparation that promotes occlusion may enhance the penetration of the drug, but ointments and lipid preparations and are not always more powerful than creams. Creams, gels and solutions may be formulated so as to obtain an effect equivalent to that of ointments. Topical corticosteroids, of different classes of potency, e.g., may show the same activity when formulated in different vehicles [13]. A gel preparation of kellin, obtaining better penetration, has demonstrated important results in the treatment of vitiligo [14]. Also transfollicular penetration is influenced by vehicle and excipients; in this case better results are given by lipophilic and alcoholic vehicles. Relevant factors include dimension and charge of the molecules of the solute [15, 16].

c) Conditions that modify the barrier function

During hydration the greater part of the water is associated with intracellular keratin; the natural factor of hydration or natural moisturizing factor (NMF) absorbs a noticeable amount of water (10% of the weight of the corneocyte). Corneocytes swell and the barrier properties of the stratum corneum are deeply altered. In the intercellular space the small amount of water linked to polar groups by hydration does not alter the organization of lipids and does not reduce of permeability [17]. The effect of the hydration however has a discontinuous effect; the increment may be by ten times for some substances and very limited for others [18]. Occlusion partially hinders the loss of humidity of the skin, increasing the content of water of the horny layer. However the NMF level in the horny layer is almost zero. It seems therefore that there is a homeostatic mechanism that prevents hyperhydration of the skin [9]. Occlusion may increase the absorption by several times, especially for hydrophilic compounds. However, in some conditions it may promote the formation of a reservoir effect. The acidity of the cutaneous surface, controlling homeostasis and enzymatic activities, influences permeability [19]; the metabolic activity of the skin (enzymatic oxidoreductive processes) may modify the substances applied, influencing permeability and effects.

Absorption is also influenced by other skin properties that vary at different cutaneous anatomical sites. For instance, the absorption diminishes greatly as one moves from the palpebral skin to the plantar surfaces [20].

Age influences skin absorption. Various biological activities are lower in the skin of the aged individual. Great variation is also noted for the premature infant and neonate, who have greater cutaneous permeability [21]. There are no experimental data confirming the validity of friction on transcutaneous absorption [6]. Alterations of the barrier induce modifications of TEWL [9]. In addition, the horny layer may be defined as a biosensor; alterations of external humidity regulate proteolysis of filaggrin, synthesis of lipids, DNA, and proteins within keratinocytes, which can lead also to inflammatory phenomena [22].

The cutaneous bioavailability of most commercial dermatological formulations is low (within 1-5% of applied dose) [23].

The active substances of topical formulations are generally absorbed in small quantities; only a reduced fraction passes from the vehicle into the stratum corneum. The greater part remains on the surface of the skin, subject to loss in several ways such as by sweating, chemical degradation, and removal. The absorption of the drug is on the order of 1-5 percent of the applied dose. Future standards would therefore aim to make formulations not merely high in concentration, but pharmaceutically optimized to have an elevated (50-100%) bioavailability. On the other hand, one must consider the marked variations of the different cutaneous areas and skin conditions that make uncertain the therapeutic equivalence when compared with other ways of administration in clinical conditions [24].

Biopharmaceutical appraisal of topical formulations

These methods are widely used for the development of topical dermatological formulations [25].

The in vivo methods include kinetic and dynamic models. The former are based on: a) selective removal of the horny layer; b) dissection techniques of the skin; c) methods of appraisal of the indirect percutaneous absorption (in blood secretions, etc.).

The dynamic models include: 1) determination of the variations of the skin color by various instruments (Minolta, X-rite, etc.), which determine also the degree of erythema or the variation of the color after UVB stimulation; 2) determination of the cutaneous blood flow (monitored by optical Doppler Laser procedures); 3) evaluation of the UVA-induced neutrophil infiltrate; 4) animal models [26].

Methods of modulation of cutaneous permeability

When a substance is applied on the skin with a simple vehicle the therapeutic result can be unsatisfactory because of the insufficient concentration obtained in the application area. In the last few years strategies have been developed in order to increase the efficacy of the vehicle. They may be of chemical, biochemical or physical order.

a) Chemical enhancers

In order to increase the penetration the vehicle may be integrated with enhancers that by interacting with intercellular lipids improve the diffusion coefficient of the substance in the stratum corneum. Chemical enhancers may: a) increase the diffusibility of the substance inside the barrier, b) increase the solubility in the vehicle or both, or c) improve the partition coefficient.

These substances may frequently have a not specific action. Enhancers of this type, that are not widely used, are Azone, Dermac SR-38, and oleic acid [27]. In some cases, however, these have an irritating effect and must be carefully evaluated in the various preparations [28].

Excipients like ethanol, propylene-glycol, and dimethylsulfoxide (DMSO) may increase the diffusion by altering the organization of lipids of the horny layer [29]. The interference with the biosynthesis of some lipids may alter the structure of the barrier and increase the penetration. Methods have also been studied that interfere with secretion and organization of lipids (e.g., brefeldine, monetine and cloroquine). In addition, enhancers that alter the supramolecolar organization of the bistratified lamellae (synthetic analogs of fats, inducing abnormalities of the organization of the membranes; complex precursors that can not be metabolized, etc.) have been studied. These methods produce an alteration of the critical molar ratio among ceramides, cholesterol, and fatty acids; if there is decrease or excess of one of these 3 key lipids, the the lamellar organization cannot be maintained. There may be separation of the phases with more permeable interestitial spaces and formation of a new way of penetration [30].

The efficacy of the enhancers may be increased by inhibition of the metabolic reaction of repair once the alteration of the barrier has been obtained. This would involve inhibiting metabolic sequences that can rebuild and maintain the barrier function. Inhibitors of enzymes with relevant functions (e.g., lovastatin) or specific inhibitors of enzymes synthesizing ceramides or fatty acids induces alteration of the molar ratio of the three critical lipids and leads to discontinuity in the lamellar layer system [31]. Other enhancements may be obtained by modifying the polarity [32].

The number of drugs for which transdermic methods for systemic use has been possible is very small and restricted to lipophilic and low molecular weight substances (nicotinic acid, nitroglycerin, clonidine, steroid hormones, scopolamine) [33].

b) Carrier vesicular systems

Liposome formulations can be very effective. However, they probably increase penetration only through the transappendigeal avenue [34]. Niosomes and transferosomes, formed by modified liposomes (phosphatidilcoline, sodium cholate, ethanol), are systems based on the ability of vesicles to cross the unaltered horny layer because of the osmotic gradient between external and internal layers of the barrier. These are "flexible" vesicles able to transport their contents through the intercellular tortuous route of the corneous layer.

Physical systems for vehicle enhancement

Several methods have been studied to increase the penetration for a systemic or a loco-regional effect. These systems aim to reach the target area at some depth, at an adequate drug concentration, in a selective way, without dispersion in the circulation (transdermic systems) [35].

a) Sonophoresis

Sonophoresis (phonophoresis, or ultrasonophoresis) may be applied to enhance penetration of drugs and other active principles in dermatology [36]. The low frequency ultrasound induces alterations in the structure of the stratum corneum and enhances permeability [37]. The useful frequencies have not been clearly identified; those used in diagnostic procedures (1-4 MHz) are insufficiently effective, whereas very high frequencies (10-20 MHz) increase penetration [38]. However, interestingly in dermatology there have been positive results with very low frequency sonophoresis, between 20 and 25 KHz [39]. Various parameters are responsible for the effect at 20 KHz [40, 41, 42]; the alteration of the barrier function is mainly the phenomenon of cavitation [43], although the increase of temperature induced by ultrasound accounts for about 25 percent of the effect in some experiments [44, 45, 46]. Transport by convection and mechanical impact also influence the results [47]. The mechanisms of penetration are linked to the formation of transitory modifications ("pores" approximately 20 micron) [48], sufficient to allow the penetration of high molecular weight drugs [49, 50].

The combination of ultrasound with use of some surfactants (e.g., sodiolauryl-sulfate) produces an increase in the transport and requires less energy [51]. Sonophoretic absorption varies according to specific drugs [52]. The biological effects on human skin and the possible applications have also been studied in the skin cancer field [53].

b) Ionophoresis and iontophoresis

Ionophoresis increases the penetration of ionizable substances using the force of a weak electric field (the applied direct or galvanic current ranges from 0 to 250 microamperes = 0.25 mA). Ions transport the applied current from an electrode to a second indifferent electrode [54]. Ionophoresis is widely used to control hyperhidrosis [55]. The time needed to allow the penetration of an ionized substance through the barrier is approximately 25-30 min. Transport takes place in the stratum corneum interstitially through the aqueous pores and also extracellularly at the lacunar level (less frequently at the appendices) [56].

The two main mechanisms responsible according to Santi [57] are: "ionophoresis", in which ions are rejected from the electrode of the same charge and "electrosmosis", convective movements of a solvent that takes place through a charged "pore" against the preferential passage of "counterions" under the influence of the electric field. Iontophoresis increases the transport through the barrier by three mechanisms: (a) interaction between ions and the electrical field supporting the ionic transfer; (b) permeability increase in the skin-membrane provoked by the flow of electric current; (c) electrosmosis, by which phenomenon there is a massive flow of ionized solvent, which transports ionized species as well as neutral ones [58, 59].

The possibility of iontophoretic transport of macromolecules has also been demonstrated [59].

Combining iontophoresis and passive diffusion has been observed to increase of the penetration of kellin at pH 7 through the horny layer, even if the drug is not ionized [60].

The transport of small molecules in general is lower than for large molecules [61] and the molecular structure is a key element for iontophoretic transport. Moreover, the chemical sequence within the molecular structure (as shown for oligonucleotides) is important.

Iontophoresis may also be an alternative to parenteral administration for systemic therapy for peptides and for macromolecular substances in a ionized state at physiological pH. On the other hand, these substances are poorly absorbed by other ways and rapidly degraded by proteolytic enzymes [62, 63].

It has been reported that iontophoresis may allow penetration of a drug to 1 centimeter depth or more. Once the current flow has transported the ions of the drug through the first 2 millimeters, there is a "reservoir" effect above the microcirculation area; the drug may diffuse from the "reservoir" in the dermis and be transported from the capillary network into the blood flow.

Iontophoresis has been studied mostly for loco-regional therapy (e.g., for muscle-skeletal disorders) or a few local applications (lidocaine, epinephrine, methylprednisolone, desametazone, antivirals, antibiotics, etc.).

Iontophoresis has also been studied for postherpetic neuralgia [64].

Iontophoresis may be improved by electronic instrumentation and by some particular devices (hydrogel pads) [65]. Some substances may function as cooperators, such as iontophoresis with hydrogel. Formulations with several polymers, hydrogels, have shown a capacity for transport greater than solutions [66].

Recently iontophoresis has been proposed in to be used in combination with low frequency ultrasound [67, 68].

c) Electroporation

With this modality a reversible electrical field, with short and high voltage pulses, "electropores" assaults the lipid double layer barrier and causes the formation of non-lamellar lipid phases and of subcellular pores; this promotes greater speed of substance transport [69]. Electroporation uses low intensity current from an electrode of the same charge of net polarity as the drug; the molecules are transported through the horny layer using extracellular avenues (probably also transappendigeal).

The use of electroporation to increase skin permeability for the penetration of a drug is in the early stages of development [70, 71]. Electroporation creates new routes and a more uniform distribution of the electric charge with low cutaneous irritation.

Further study is needed to further develop physical methods to promote transport, which may involve modifying the present methods of iontophoresis and/or sonophoresis.

d) Crioelectrophoresis

Cryoelectrophoresis is a technique of carriage of frozen drugs and active substances, allowing transdermic penetration of hydrosoluble, better ionizable substances, but also of substances with different physico-chemical properties. This modality allows the attainment of a deep level (6-8 cm) in appreciable concentrations, with very reduced systemic impact (0.04%). The alternating electric current is applied with frequency and polarity according to the specific drug; generally a variation of intensity is utilized [72]. The transport of the drug/substance probably takes place through: (a) diffusion (strongly enhanced by a poration effect due to the oscillating current used); (b) electrosmosis (electrosmosis enhances the diffusion and also electrophoretic action) [73]; (c) electrophoresis, that increases the diffusion of ionized drugs. The active substance is dissolved in ice and the electrodes are positioned on both sides of the areas to be treated. According to Aloisi [72] ionic flow toward deep tissues (e.g., articular caps), in local high concentration, may be obtained especially for antiinflammatory molecules. The maximum intensity and the total intensity of the current may be increased without damage and/or unpleasant sensations. The negative effects of the cold, of the electrical current, and of the drug become mutually neutralized. Moreover the vasocostrictive effect caused by the low temperature prevents the unwanted removal of the drug by the local blood flow.

e) Hydroelectrophoresis

Hydroelectrophoresis allows the active agent to penetrate about 0.5-10 cm in tissues, avoiding dispersion and creating a directional flow [73], allowing a loco-regional therapy. It permits management of pain with applications lasting 15-30 minutes and, possibility, to focus therapy to the regions involved.

f) Pressure waves by intense laser radiation (Transdermal drug delivery)

The photomechanical compression obtained by laser (Q-switched ruby laser) has been tested for modulating the permeation of the stratum corneum [74]. The material (polystyrene) including the solution with the active principle (e.g., δ-amino-levulinic acid) absorbs the laser radiation, while the solution increases the propagation in the stratum corneum. The penetration route is apparently extracellular, as it is for sonophoresis and ionophoresis. The effect is only temporary and the barrier function is restored [6].

Pressure waves, which are generated by intense laser radiation, can increase the permeability of the stratum corneum as well as the cell membrane. These pressure waves are compression waves and thus exclude biological effects induced by cavitation. Their amplitude is in the range of atmospheres (bar), whereass the duration is in the range of nanoseconds to a few microseconds. The pressure waves interact with cells and tissue in ways that are probably different from those of ultrasound. Furthermore, the interactions of the pressure waves with tissue are specific and depend on their characteristics, such as peak pressure, rise time, and duration. A single pressure wave is sufficient to make the stratum corneum permeable and allow the transport of macromolecules into the epidermis and dermis. In addition, drugs delivered into the epidermis can enter the vasculature and produce a systemic effect. For example, insulin delivered by pressure waves results in reducing the blood glucose level over many hours. The application of pressure waves does not cause any pain or discomfort and the barrier function of the stratum corneum always recovers.

Conclusions

Dermatologists should be aware of the expanding research into skin permeability enhancing techniques. These techniques are becoming more important methods to accomplish better drug delivery.

References

1. Dermatological and Transdermal Formulations. Marcel Dekker, Inc. New York 2002

2. Bronaugh RL, Maibach HI. Percutaneous Absorption: Drugs - Cosmetics - Mechanisms - Methodology (Drugs and the Pharmaceutical Sciences). Informa Healthcare; 4th Ed., 2005.

3. Smith EW, Maibach HI. Penetration percutaneous enhancers. Taylor and Francis, 2nd Ed, 2006.

4. Lampe MA, Burlingame AL, Whitney J, Williams ML, Brown BE, Roitman E. Human stratum corneum lipids: characterization and regional differences. J Lipid Res 1983; 24: 120-30.

5. Elias PM, Menon GK. Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv Lipid Res 1991; 24: 1-26. [PubMed]

6. Elias PM, Tsai JC, Menon GK. Skin barrier, percutaneous drug delivery and pharmacokinetics. Dermatology, 2003. Chap 125, p 1235-52.Mosby, editor.

7. Elias PM, Feingold KR, Menon JK. The stratum corneum, two compartments model and its functional implication. In Basel, Karger Shroot B, Shaefer H, editors. Skin Pharmacokinetics 1987. vol.1. p 1-9.

8. Surber C, Davis AF. Bioavailability and Bioequivalence of Dermatological Formulations p401-498. in: Dermatological and Transdermal Formulations.vol 119. Marcel Dekker, Inc. New York 2002.

9. Roberts MS, Cross SE, Pellett MA. Skin transport p 89-195 in: Dermatological and Transdermal Formulations.vol 119. Marcel Dekker, Inc. New York 2002.

10. Menon GK, Elias PM. Morphologic basis for a pore-pathway in mammalian stratum corneum. Skin Pharmacol 1997;10:235-46. [PubMed]

11. Watkinson AC, Brain KR. Basic mathematical principles in skin permeation p 61-88: in Dermatological and Transdermal Formulations.vol 119. Marcel Dekker, Inc. New York.

12. Franz TJ. Kinetics of cutaneous drug penetration. Int J Dermatol 1983; 499-505. [PubMed]

13. Franz TJ. Pharmacokinetics and skin in: Skin barrier, percutaneous drug delivery and pharmacokinetics. p 1969-78 in Dermatology,vol II. Mosby, 2003.

14. Orecchia G, Sangalli ME, Gazzaniga A, et al. Topical photochemotherapy of vitiligo with a new khellin formulation: preliminary clinical results. J Dermatol Treat 1998; 9: 65-9.

15. Roberts M, Cross SE, Pellett MA. Skin transport in Dermatological and Transdermal Formulations.vol 119. Marcel Dekker, Inc. New York 2002.

16. Middleton JD. The mechanism of water binding in stratum corneum. Br J Dermatol 1968; 80:437-50

17. Horii I, Nakajama Y, Obate Ml. Stratum corneum hydration and aminoacids contant in xerotic skin. Br J Dermatol Res 1989; 121: 588-64.

18. Imokawa G, Kuno H, Kawai M. Stratum corneum lipids serve as bound-water modulator. J Invest Dermatol 1991; 96: 845-51. [PubMed]

19. Mauro T, Hollerann WM, Grayson S, Gao WN, Man MQ, Kriehuber E, et al. Barrier recovery is impeded at neutal pH, independent of ionic effects: implications for extracellular lipid processing. Arch Dermatol Res 1998; 290: 215-22. [PubMed]

20. Rougier A, Lotte C, Corcuff TP. Relationship between skin permeability and cornecyte size according to anatomic site, age and sex in man. J Soc Cosmet Chem 1988; 39: 15-21. [PubMed]

21. Berardesca E, Maibach HI. Racial differences in skin pathophysiology. J Am Acad Dermatol 1996; 34: 667-72. [PubMed]

22. Menon GK, Elias PM, Feingold KR. Integrity of the permeability barrier is crucial for manteinance of the epidermal calcium gradient. Br J Dermatol 1994; 130: 139-47.

23. Surber C, Davis AF. Bioavailability and Bioequivalence of Dermatological Formulations p401-498. in: Dermatological and Transdermal Formulations.vol 119. Marcel Dekker,Inc. New York 2002. [PubMed]

24. Hauck WW. Bioequivalence studies of topical preparations: statistical considerations. Int J Dermatol 1992; 31 (suppl. 1): 29-33.

25. Skelly JP, Shah VP, Maibach HI. FDA and AAPS report of workshop on principles and practices of in vitro percutaneous penetration studies: relevance to bioavailability and bioequivalence. Pharm Res 1987; 4: 265-71.

26. Shah VP, Elkins J, Hanus J, Noorizadeh C, Skelly JP. In vitro release fo hydrocortisone from topical preparations and automated procedure. Pharm Res 1991; 8: 55-9.

27. Davis AF, Gyurik RJ, Hadgraft J. Formulation strategies for modulating skin permeation in Dermatological and Transdermal Formulations.vol 119. Marcel Dekker, Inc. New York 2002. [PubMed]

28. Patil S, Singh P, Szolar-Platzer C, Maibach HI. Epidermal enzymes as penetration enhancers in transdermal drug delivery? J Pharm Sci 1996; 85: 249-52.

29. Mitragotri S. Synergistic effects of enhancers for transdermal drug delivery. Pharm Res 2000; 17: 1354-9. [PubMed]

30. Tsai JC, Guy RH, Thornfeldt CR, Gao WN, Feingold KR, Elias PM. Metabolic approaches to enhance transdermal drug delivery. 1. Effect of lipid synthesis inhibitors. J Pharm Sci 1996; 85: 643-8. [PubMed]

31. Johnson ME, Mitragotri S, Patel A, Blankschtein D, Langer R. Synergistic effects of chemical enhancers and therapeutic ultrasounds on transdermal drug delivery. J Pharm Sci 1996; 85: 670-9. [PubMed]

32. Choi EH, Lee SH, Ahn SK, Hwang SM. The pretreatment effect of chemical skin penetration enhancers in transdermal drug delivery. Skin Pharmacol Appl Skin Physiol 1999; 12: 326-35.

33. Singh J, Maibach HI. Transdermal delivery and cutaneous reactions. in: Dermatological and Transdermal Formulations.vol 119. Marcel Dekker, Inc. New York 2002.

34. Korting HC, Stolz W, Schmid MH, Maierhofer G. Interaction of liposomes with human epidermis reconstructed in vitro. Br J Dermatol 1995; 132: 571-9. [PubMed]

35. Kassan DG Lynch AM, Stiller MJ. Physical enhancement of dermatologic drug delivery: iontophoresis and phonophoresis. J Am Acad Dermatol 1996; 34: 657-66. [PubMed]

36. Ka-yun Ng, Yang Liu. Therapeutic ultrasound: its application in drug delivery. Medicinal Research reviews 2002; 22: 204-8.

37. Merino G, Kalia YN, Guy RH. Ultrasound-enhanced transdermal transport. J Pharm Sci 2003, 92: 1125-37. [PubMed]

38. Bommannan D, Menon GK, Okuyama H, et al. Sonophoresis. II. Examination of the mechanism(s) of ultrasound-enhanced transdermal drug delivery. Pharm Res1992;9:1043-48. [PubMed]

39. Santoianni P, Nino M, Calabro' G. Intradermal drug delivery by low-frequency sonophoresis (25 KHz). Dermatology Online Journal 2004; 10: 24-32. [PubMed]

40. Terahara T, Mitragotri S, Kost J, Langer R. Dependence of low-frequency sonophoresis on ultrasound parameters: distance of the horn and intensity. Int J Pharm 2002; 235: 35. [PubMed]

41. Tezel A, Sens A, Tuchscherer J, Mitragotri S. Frequency dependence of sonophoresis. Pharm Res 2001; 18: 1694-700. [PubMed]

42. Fang J, Fang C, Sung KC, Chen H. Effect of low frequency ultrasound on the in vitro percutaneous absorption of clobetasol 17-propionate. Int J Pharm 1999; 191:33-42. [PubMed]

43. Mitragotri S, Farrell J, Tang H. Determination of threshold energy dose for ultrasound-induced transdermal drug transport. J Control Release 2000; 63: 41-48. [PubMed]

44. Machet L, Cochelin N, Patat F. In vitro phonophoresis of mannitol, oestradiol and hydrocortisone across human and hairless mouse skin. Int J Pharm 1998; 165: 169-74. [PubMed]

45. Merino G, Kalia YN, Delgado-Charro MB, Potts RO, Guy RH. Frequency and thermal effects on the enhancement of transdermal transport by sonophoresis. J Control Release 2003; 88: 85-94. [PubMed]

46. Machet L, Boucaud A. Phonophoresis: efficiency, mechanisms and skin tolerance. Int J Pharm 2002; 243: 1-15. [PubMed]

47. Tang H, Wang CC, Blankschtein D, Langer R. An investigation of the role of cavitation in low-frequency ultrasound-mediated transdermal drug transport. Pharm Res 2002; 19: 1160-9. [PubMed]

48. Tezel A, Sens A, Mitragotri S. Description of transdermal transport of hydrophilic solutes during low-frequency sonophoresis based on a modified porous pathway model. J Pharm Sci 2003; 92: 381-93. [PubMed]

49. Wu J, Chappelow J, Yang J. Defects generated in human stratum corneum specimens by ultrasound. Ultrasound In Med. & Biol.1998, Vol. 24, No. 5, p. 705-12.

50. Le L, Kost J, Mitragotri S. Combined effect of low-frequency ultrasound and iontophoresis: applications for transdermal heparin delivery. Pharm Res 2000; 17: 1151-4. [PubMed]

51. Mitragotri S, Ray D, Farrell J, Tang H, Yu B, Kost J, et al. Synergistic effect of low-frequency ultrasound and sodium lauryl sulfate on transdermal transport. J Pharm Sci 2000; 89: 892-900. [PubMed]

52. Mitragotri S, Blankschtein D, Langer R. An explanation for the variation of the sonophoretic transdermal transport enhancement from drug to drug. J Pharm Sci 1997; 86: 1190-2. [PubMed]

53. Tachibana K, Tachibana S. Application of Ultrasound Energy as a New Drug Delivery System. Japanese J Appl Phys 1999. Vol. 38 Part 1, No. 5B p.3014-3019.

54. Singh S, Singh J. Transdermal drug delivery by passive diffusion and iontophoresis: a review. Med Res Rev 1993; 13: 569-621. [PubMed]

55. Holzle E, Alberti N. Long-term efficacy and side effects of tap water iontophoresis of palmoplantar hyperhidrosis. The usefulness of home therapy. Dermatologica1987;175:126-33. [PubMed]

56. Menon GK, Elias PM. Morphologic basis for a pore-pathway in mammalian stratum corneum. Skin Pharmacol 1997; 10: 235-46. [PubMed]

57. Santi P, Volpato NM Colombo P. Iontophoresis enhances the transport of acyclovir through nude mouse skin by electrorepulsion d elecroosmosis. Pharm Res 1995; 12: 1623-7. [PubMed]

58. Pikal MJ. The role of elecroosmotic flow in transdermal iontophoresis. Adv Drug Del Rev 2001; 46: 281-305. [PubMed]

59. Grewal BS, Naik A, Irwin WJ, Gooris G, de Grauw CJ, Gerritsen HG, et al. Transdermal macromolecular delivery: real-time visualization of iontophoretic and chemically enhanced transport using two-photon excitation microscopy. Pharm Res 2000; 17: 788-95. [PubMed]

60. Marconi B, Mancini F, Colombo P, Allegra F, Giordano F, Gazzaniga A, et al. Distribution of khellin in excised human skin following iontophoresis and passive dermal transport. J Control Rel 1999; 60: 261-8. [PubMed]

61. Brand RM, Wahl A, Iversen PL. Effects of size and sequence on the iontophoretic delivery of oligonucleotides. J Pharm Sci 1998; 87: 49. [PubMed]

62. Volpato NM, Nicoli S, Laureri C, Colombo P, Santi P. In vitro acyclovir distribution in human skin layers after transdermal iontophoresis. J Control Release 1998; 50: 291-6. [PubMed]

63. Gangarosa LP Sr, Ozawa A, Ohkido M, Shimomura Y, Hill JM. Iontophoresis for enhancing penetration of dermatologic and antiviral drugs.J Dermatol 1995; 22: 865-75. [PubMed]

64. Gangarosa LP, Hill JM. Modern iontophoresis for local drug delivery. Int J Pharm 1995; 123:159-66. [PubMed]

65. Banga AK, Chien YW. Hydrogel-based iontotherapeutic delivery devices for transdermal delivery of peptide/protein drugs. Pharm Res 1993; 10: 697-702. [PubMed]

66. Fang JY, Kuo CT, Fang CL. Transdermal iontophoresis of sodium nonivamide acetate evaluated by in vivo microdialysis and histologic study. Drug Development Research 1999; vol. 46, issue 2, 87.

67. Wolf SL. Electrotherapy, clinics in physical therapy. Churchill Livingstone, 1981.

68. Le L, Kost J, Mitragotri S. Combined effect of low-frequency ultrasound and iontophoresis: applications for transdermal heparin delivery. Pharm Res 2000; 17: 1151-4. [PubMed]

69. Potts RO. Transdermal peptide delivery using electroporation. In: Proceedings of the Thrid TDS Technology Symposium: polymers and peptides in transdermal delivery. 47-67, 1993.

70. Sharma A, Kara M, Smith FR, Krishnan TR.Transdermal drug delivery using electroporation. I. Factors influencing in vitro delivery of terazosin hydrochloride in hairless rats. J Pharm Sci 2000;89:528-35. [PubMed]

71. Sharma A, M. Kara M, Smith FR, Krishnan TR. Transdermal drug delivery using electroporation. II. Factors influencing skin reversibility in electroporative delivery of terazosin hydrochloride in hairless rats. J Pharm Sci 2000; 89: 536-44. [PubMed]

72. Aloisi A, M. Matera M, Potenza M. Cryoelectrophoresis: Painless administration of drugs through a suitable association of thermal and electrical techniques. G Ital Dermatol Venereol 2005; 151: 345-8.

73. Misefari M, D' Africa A, Sartori M, Morabito F. Transdermal transport by hydroelectrophoresis: a novel method for delivering molecules. J Biol Regul Homeost Agents 2001; 15: 381-4. [PubMed]

74. Makoto O, Shunichi S, Masahiko K, et al. Transdermal delivery of photosensitizer by the laser-induced stress wave in combination with skin heating. Japanese J Appl Phys 2002. Vol. 41 Part2, pp. L814.

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