Ethosomes: Enhanced Delivery of Drugs to and Across the Skin

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1 Ethosomes: Enhanced Delivery of Drugs to and Across the Skin 4 Denize Ainbinder, Biana Godin, and Elka Touitou Contents 4.1 Introduction Definition, Structure, and the Main Properties of Ethosomes Mechanism of Skin Permeation Enhancement by Means of Ethosomes Ethosomal Systems as Efficient Carriers for Enhanced Drug Delivery into and Across the Skin Skin Penetration Performance by Ethosomal Carrier: In Vitro Studies Proof of Concept In Vivo and in Clinical Trials Safety Evaluation In Vitro, in Animals, and in Human Studies Summary 73 References 74 D. Ainbinder E. Touitou (*) Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, 12065, Jerusalem 91120, Israel denizekib@gmail.com; elka.touitou@mail.huji.ac.il B. Godin Department of Nanomedicine, The Methodist Hospital Research Institute, Houston 77030, TX, USA bianagodinv@gmail.com 4.1 Introduction In spite of the tremendous research carried out in the field of dermal and transdermal drug delivery, efficient administration of drugs by topical application to the skin is still challenging. In response to the need for efficient delivery of drugs into/across the skin, Touitou has designed a phospholipid vesicular carrier named ethosome (Touitou 1998 ; Touitou et al. 2000a ). This vesicular carrier is distinguished from other lipid nanocarriers by a number of important characteristics, such as the vesicle s bilayer fluidity, its mechanism of permeation enhancement (attributed to the entire system), simple mode of preparation, and lack of side effects. These special features of the ethosomal systems will be further discussed in this chapter. Since their introduction ethosomes were thoroughly investigated for dermal and transdermal delivery of a variety of actives. Following Touitou and colleagues, other groups have carried out research on the delivery enhancement properties of the ethosomal systems and their safety. It is noteworthy that in a number of publications describing work with ethosomes, some misleading nomenclature is used, such as elastic liposomes and ethanolic liposomes. This chapter presents the properties of ethosomes emphasizing some unique features and reviews the work carried out with this vesicular carrier from the beginning till now. Springer-Verlag Berlin Heidelberg 2016 N. Dragicevic, H.I. Maibach (eds.), Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement: Nanocarriers, DOI / _4 61

2 Definition, Structure, and the Main Properties of Ethosomes D. Ainbinder et al. Ethosomal systems are specially tailored vesicular carriers for enhanced delivery of active agents into the deep layers of the skin and through the skin (Touitou 1998 ; Touitou et al. 2000a ). Due to the possible interdigitation effect of ethanol on lipid bilayers, it was commonly believed that vesicles cannot coexist with ethanol (Chin and Goldstein 1997 ; Harris et al ; Pang et al ). Phosphorus nuclear magnetic resonance (PNMR) spectra of ethosomal systems showed a typical configuration of the phospholipid bilayer generally observed in phosphatidylcholine vesicles in water. Furthermore, the paramagnetic-ion NMR spectra indicated that the phospholipid in ethosomes is in a more fluid state and the membrane is more permeable to cations, in comparison to liposomes (Touitou et al. 2000a ). Further confirmation of the fluid nature of ethosomal bilayers was obtained by differential scanning calorimetry (DSC). A comparison of the lipid transition temperatures in ethosomes and in conventional liposomes revealed lower values for the lipids in ethosomes, suggesting that they possess a higher degree of fluidity (Touitou et al. 2000a ; Dayan and Touitou 2000 ; Godin and Touitou 2005 ). These differences of up to 30 C in the transition temperatures of ethosomal versus liposomal lipids confirm the fluidizing effect of ethanol on the lipid bilayers in ethosomal systems. Negatively stained transmission electron (TE) micrographs showed that the ethosomes could vary from unilamellar (Godin and Touitou 2005 ; Shumilov and Touitou 2010 ; Shumilov et al ) structures to multilamellar (Touitou et al. 2000a ; Godin and Touitou 2004 ; Ainbinder and Touitou 2005 ) vesicles with bilayers throughout the vesicle (Fig. 4.1 ). The size of the vesicles ranges between 30 nm to several microns and can be adjusted by changes in the system s composition. A systematic investigation of the effect of system composition on vesicular size showed that increase in the percent of lipid results in larger vesicles, while increase in the percent of ethanol at the same lipid concentration decreases the size of ethosomes (Touitou et al. 2000a ). This correlation implies that a b Fig. 4.1 Visualization of uni and miltilamellar ethosomes by TEM

3 4 Ethosomes: Enhanced Delivery of Drugs to and Across the Skin 63 alterations in the concentration of one of the system s components may result in overall modification of the system s characteristics. Therefore, unlike with any other lipid vesicle, in the characterization of ethosomal systems, it is important to use methods adequate for work with soft vesicles. Additionally, special attention should be paid to the dilution of the tested system. Furthermore, the size of the vesicles in the system is also affected by the vehicle and drug physicochemical characteristics and the concentration of the drug. For example, increase in the concentration of trihexyphenidyl hydrochloride (THP) in the ethosomal system resulted in a decrease in the vesicular size (Dayan and Touitou 2000 ). Similar effect was observed for bacitracin ethosomal system (Godin and Touitou 2004 ). Since the characteristics of ethosomal systems depend on the system composition and ratio between the system components, in their work with ethosomes, researchers should be aware in preserving system s properties. For example, in a number of studies, carried out by researchers without experience in work with ethosomes, the physical characterization protocols were wrong resulting in artifacts and misleading results and conclusions. An important property of this carrier is its ability to entrap compounds of various hydrophilicities. Visualization of the intra-vesicular distribution of lipophilic (fluorescently labeled phospholipid) and hydrophilic (fluorescein isothiocyanate (FITC)-bacitracin) fluorescent probes by means of confocal laser scanning microscopy (CLSM) showed that both molecules filled the entire volume of ethosomes. These findings were in contrast to those for liposomes, where the lipophilic probe was localized only in the vesicle membrane and the water-soluble FITC-bacitracin filled the core (Godin and Touitou 2004 ). These results could be explained by the liposomes structure, in which a small number of bilayers surround an aqueous core. The unique structure of ethosomes, due to the presence of ethanol together with vesicle lamellarity, resembles a fingerprint in the case of multilamellar vesicles allowing for efficient entrapment of lipophilic and amphiphilic molecules (Touitou and Godin 2005 ). Stability of vesicular systems could be evaluated by measuring changes in vesicle size distribution and visualization of the vesicles. The stability of ethosomal systems incorporating various drugs was assessed in a number of studies by comparing the average diameter and the structure of the vesicles during the 2-year period at room temperature (Touitou et al. 2000a ; Dayan and Touitou 2000 ). No changes in the mean size of empty and cationic THP loaded vesicles were observed during the storage interval. Moreover, visualization by negative-stain TEM confirmed that the vesicular structure of the ethosomes persisted after 2 years and no significant structural changes over that time occurred in the systems. In another study with ethosomes (comprising 30 % ethanol, 2 % Phospholipon 90) and the lipophilic antibiotic erythromycin, no significant variations in the vesicle were observed throughout 1-year storage at room temperature (123 ± 15 nm vs. 117 ± 18 nm). TE micrographs confirmed that erythromycin unilamellar ethosomes kept their configuration during the stability evaluation experiment (Godin and Touitou 2005 ). Evaluation of the stability of linoleic acid ethosomes by using a Turbiscan optical analyzer showed no modification of the backscattering profiles of colloidal suspensions and no coalescence, sedimentation, flocculation, or clarification (Celia et al ). Data on SupraVir cream (Trima, Israel), a marketed ethosomal formulation of acyclovir, indicate that the formulation and the drug have long shelf lives with no stability problems. In this formulation, the drug acyclovir was stable for at least 3 years at 25 C. Furthermore, skin permeation experiments showed that the initial penetration enhancing capacity of the carrier was retained for at least 3 years ( Trima Israel ). 4.3 Mechanism of Skin Permeation Enhancement by Means of Ethosomes The proposed mechanism of permeation enhancement by ethosomes is based on the dual effect of ethanol, on the lipid bilayers in the stratum

4 64 D. Ainbinder et al. corneum (SC) and in the vesicles (Touitou et al. 2000a, b ; Touitou and Godin 2005 ; Godin and Touitou 2003 ; Ainbinder et al ). The presence of ethanol in ethosome composition enables fluidization of the lipid bilayers in the vesicle together with changes in the arrangement of the lipids in the SC. The ethosome penetrates the altered SC barrier releasing the active agent in the deeper skin layers. A scheme of the proposed model of penetration enhancement by the ethosomal system through the SC lipids is given in Fig This synergistic effect of the ethanol on the SC lipids and vesicular lipid bilayers was suggested based on the results obtained in fluorescent anisotropy and DSC experiments as well as in skin permeation studies. In order to gain insights into the characteristics of ethosomes that might allow them to efficiently enhance drug delivery into the skin, free energy measurements of the vesicle bilayers were assessed by DSC and fluorescence anisotropy studies (Touitou et al. 2000a ). The transition temperature value of phospholipids in ethosomes was C lower than in liposomes. This can be explained by the fluidity of the phospholipid bilayers in ethosomes (Touitou et al. 2000a ; Godin and Touitou 2004 ). Further confirmation to this fluidity of lipids in ethosomal bilayers was found in fluorescent anisotropy measurements of 9-Antrivinyl labeled analog of phosphatidylcholine (AVPC), where a 20 % lower value was measured in comparison to liposomes (Touitou et al. 2000a ). Skin permeation studies measuring the vesicle components transport through the skin shed further light on understanding the mechanism of permeation enhancement by ethosomes (Touitou et al. 2000a, b ). It was reported that a significant amount (10.5 % of initial) of phosphatidylcholine permeated the skin during a 24-h experiment from an ethosomal system (Touitou et al. 2000a ). These results suggest that the vesicles may have entered the skin strata. Furthermore, skin permeation studies with ethosomes containing a phospholipid fluorescent probe rhodamine red dihexadecanoyl glycerophosphoethanolamine (RR) showed high fluorescence intensity up to a depth of 150 μm (Touitou et al. 2000a ). Since this lipophilic probe is used as an indicator of lipid fusion and does not usually cross lipid bilayers, these findings suggest penetration of ethosomes into the deep skin layers. Experiments with the fluorescently labeled polypeptide, bacitracin (FITC-Bac), showed penetration of the probe through the inter-corneocyte pathways following in vivo application on rat abdomen from ethosomes (Godin and Touitou 2004 ). The high fluorescence intensity in the intercellular space due to the penetration of FITC-Bac allowed for visualization of clear shadows of hexagonal and pentagonal corneocytes. Examination Ethosome Ethanol Ethosomal system Stratum corneum lipids Fig. 4.2 Scheme of the proposed mechanism of enhanced permeation of molecules from ethosomal system across the lipid domain of SC

5 4 Ethosomes: Enhanced Delivery of Drugs to and Across the Skin 65 of the permeation profiles of FITC-Bac from ethosomes through human cadaver skin revealed that occlusion has almost no effect (0.34 ± 0.03 and 0.29 ± 0.02 mg/cm 2 h for occlusive and nonocclusive conditions, respectively) on skin permeation (Godin and Touitou 2004 ). 4.4 Ethosomal Systems as Efficient Carriers for Enhanced Drug Delivery into and Across the Skin Skin Penetration Performance by Ethosomal Carrier: In Vitro Studies Touitou and her group reported that ethosomes are capable to enhance skin delivery of compounds with a wide spectrum of physicochemical properties as compared to many control systems (Touitou et al. 2000a ; Dayan and Touitou 2000 ; Shumilov and Touitou 2010 ; Godin and Touitou 2004 ; Lodzki et al ). Other research groups have further reported the superior skin permeation enhancement ability of ethosomes as compared to various controls, including liposomes and hydroethanolic solutions (Elsayed et al ; Rao et al ; Dubey et al. 2007a, b, 2010 ; Jain et al ). The ability of ethosomes (containing % of ethanol) to enhance the penetration deep into the skin of lipophilic molecules was first evaluated by using the phospholipid fluorescent probe RR (Touitou et al. 2000a ). CLS micrographs of nude mice skin after 8-h application of RR from ethosomes, hydroethanolic solution, and liposomes showed the highest intensity of fluorescence up to a depth of 150 μm with ethosomal systems. No deep penetration of RR from liposomal dispersion was visualized. The application of the hydroethanolic solution containing the same concentration of ethanol resulted in very low fluorescence intensity at the same skin depth as from ethosomes. The results obtained in these experiments, showing delivery of RR to deep skin layers with ethosomes, suggest that ethosomes entered the skin strata to a high depth, in contrast to liposomes, which remained on the skin surface. One of the first drugs investigated for skin permeation enhancement by ethosomes was THP, a cationic molecule with antimuscarinic activity used for the treatment of Parkinson and dystonia (Dayan and Touitou 2000 ). Due to the strong evidence that SC is much more permeable to neutral molecules than the salts of weak acids or bases (Swarbrick et al ; Green et al ), delivery into and through the skin of charged molecules is challenging. The results of skin permeation studies carried out in side-by-side diffusion cells showed that THP flux from ethosomes was 87, 51, and 4.5 times higher than from liposomes, phosphate buffer, and hydroethanolic solution, respectively ( p < 0.01). The quantity of THP in the skin was significantly higher (586 ± 77μg/cm 2, p < 0.01) for delivery from ethosomes, while lower amounts were obtained for liposomes (416 ± 27μg/cm 2 ), hydroethanolic solution (415 ± 21μg/cm 2 ), and phosphate buffer (127 ± 15μg/cm 2 ). Another cationic molecule tested for skin permeation enhancement by ethosomes was buspirone hydrochloride (BH) (Shumilov and Touitou 2010 ). Delivery of BH from ethosomes (containing 38 % ethanol and 2.5 % phospholipid) across full-thickness porcine ear skin resulted in significantly higher drug amounts in the receiver, relative to drug aqueous solution. The calculated fluxes were ± 4.4 and 4.74 ± 0.60 μg/cm 2 h for ethosomes and aqueous solution, respectively. In order to examine the efficiency of ethosomes in delivery of BH into the skin, a cationic hydrophilic fluorescent probe with a similar molecular weight, rhodamine 6G (R6G), was used as a model. R6G skin penetration versus skin depth profiles showed a significantly higher average fluorescence intensity at various skin depths following application from the ethosomal system ( p < 0.01). Moreover, increased penetration depth was observed (120 μm vs. 80 μm). Due to the lipophilic nature of the stratum corneum (SC), highly lipophilic molecules (log P > 5) generally show low transdermal absorption. These compounds accumulate within this layer and might encounter problems at the SC viable

6 66 D. Ainbinder et al. epidermis interface where they must partition into a predominantly aqueous environment. The delivery of a lipophilic drug, minoxidil, into and across the skin was measured after 24-h application of four different compositions all containing 0.5 % w/w of the drug: ethosomal system, phosphatidylcholine ethanolic solution, hydroethanolic solution, and absolute ethanolic solution (Touitou et al. 2000a, b ; Godin and Touitou 2003 ). Delivery of the drug from the ethosomal system resulted in 10, 45, and 35 times higher drug quantity that permeated the skin and 2, 7, and 5 times higher drug quantity accumulated in the skin, compared to phosphatidylcholine ethanolic solution, hydroethanolic solution, or absolute ethanolic solution of the drug, respectively. Lodzki et al examined the skin permeation of cannabidiol (CBD), a molecule with logp ~8, mediated by an ethosomal carrier (containing 40 % ethanol) (Lodzki et al ). The data from in vitro permeation experiments through nude mice skin indicated that following 24-h application of 100 mg ethosomal composition containing 3 % CBD, a significant amount of the drug permeated the skin (559 μg/cm 2 ) and CBD skin reservoir (845 μg/cm 2 ) was generated. This study demonstrated that ethosomes possess the ability not only to enhance the partitioning into the lipophilic layers of the skin but also to enhance the clearance of the drug into the hydrophilic environment leading to transdermal delivery. Rao and colleagues investigated the percutaneous permeation and skin accumulation of the lipophilic 5α-reductase inhibitor, finasteride, from ethosomal system (Rao et al ). The amount of finasteride permeated through cadaver human skin from ethosomal system was significantly higher ( p < 0.01), as compared to liposomes, aqueous solution, and hydroethanolic solution. Drug transdermal flux from the ethosomal carrier was 7.4, 3.2, and 2.6 times higher than in other formulations. Moreover, higher drug skin accumulation was measured both in the whole skin and in the dermis. It is well documented in the literature that polypeptides, due to their size and hydrophilicity, generally do not permeate through the intact skin. A work by Godin and Touitou described the ethosomal delivery of FITC-Bac, a polypeptide antibiotic (MW ~ 1.4 kda), through human cadaver skin. Application of FITC-Bac ethosomes resulted in efficient delivery of the active through the skin with a flux value of around 300 μg/cm 2 h (Godin and Touitou 2004 ). Jain and colleagues have investigated ethosomes for dermal and transdermal delivery of methotrexate (MTX), as an antipsoriatic agent (Dubey et al. 2007a ). The steady-state transdermal flux of MTX across human cadaver skin was 57.2 ± 4.3 μg/cm 2 h, as compared to the significantly lower flux of 14.6 ± 1.6, 22.4 ± 0.2, and 2.2 ± 0.7 μg/cm 2 h for liposomes, hydroethanolic solution, and drug solution, respectively. Moreover, the lag time following application of MTX ethosomal formulation was the shortest (0.9h), and high skin deposition of the drug was obtained (31 % vs. 8, 6, and 2 in other formulations, respectively). Similar superior skin permeation enhancement and deposition of ethosomes was observed with melatonin (Dubey et al. 2007b ). The transdermal flux through human cadaver skin for melatonin- loaded ethosomes was found to be 59.2 ± 1.2 μg/cm 2 h versus 22.4 ± 0.2 μg/cm 2 h for hydroethanolic solution and 10.9 ± 1.6 μg/cm 2 h for liposomes. Enhanced transdermal delivery of lamivudine from ethosomes was reported by Jain and colleagues (Jain et al ). In their study, 5-, 8-, 12-, 15-, and 25-fold higher drug skin flux from ethosomes was obtained as compared to liposomal formulation, phospholipid solution in ethanol, hydroalcoholic solution, and PBS solution. In a recent study, the evaluation of the effect of ethosomes on transdermal permeation of the anti- HIV agent, indinavir, carried out by Jain and his group demonstrated again the superior effect of this carrier (Dubey et al ). At least two times higher transdermal flux of the drug was obtained from ethosomal system as compared to drug hydroethanolic solution, liposomes, and aqueous solution. Drug-loaded ethosomes facilitated better skin drug deposition (28.3 %) as compared with ethanolic solution (16.1 %), liposomes (11.2 %), and drug solution (4.6 %).

7 4 Ethosomes: Enhanced Delivery of Drugs to and Across the Skin 67 The results summarized above show superior skin permeation enhancement by the ethosomal system as compared to ethanol, hydroalcoholic solutions with same ethanol concentration as in ethosomes, ethanolic phospholipid solutions, or classic liposomes. The enhanced skin permeation effect was shown for molecules with various properties, including lipophilic, charged, and large molecular weight (MW) actives Proof of Concept In Vivo and in Clinical Trials Since their invention, many pharmacokinetic and pharmacodynamic studies on animals and in humans were carried out with ethosomal systems. Some are further overviewed categorized by their therapeutic use (Touitou et al. 2000a, 2007 ; Godin and Touitou 2005 ; Shumilov and Touitou 2010 ; Shumilov et al ; Ainbinder and Touitou 2005 ; Lodzki et al ; Godin et al. 1999, 2005 ; Horwitz et al ; Chertin et al ; Paolino et al ; Dkeidek and Touitou 1999 ; Meidan and Touitou 2001 ) Skin Infections Treatment with Ethosomal Systems Erythromycin ethosomal systems (containing 30 % ethanol and 2 % Phospholipon 90) were investigated in the treatment of various skin infections (Godin and Touitou 2005 ; Godin et al ). Measurement of the pharmacodynamic effect in immunocompetent Hsd:ICR (CD-1 ) male mice (Harlan, Israel) injected intradermally with Staphylococcus aureus, by isolation of Staphylococcus aureus colonies from the skin wounds, showed that no bacteria were found in the inoculation sites in mice treated with ethosomal erythromycin system compared to and cfu/g tissue on days 7 and 10 after the beginning of the experiment in untreated mice. Histological examination of the wounded skin tissue revealed no dermatonecrosis and preservation of normal skin structures. Wounded areas from untreated mice and mice treated with erythromycin hydroethanolic solution showed progression of the infection, resulting in significant necrosis of the skin and adjacent tissues and initial crust formation over the necrotic area (Fig. 4.3 ). These findings demonstrate the ability of erythromycin ethosomal 0 Induction of the infection with S. aureus Fig. 4.3 Histological images taken from skin of mice intradermally inoculated with 0.1 mlx10 8 cfu/ml (10 7 cfu/ mouse) S. aureus ATCC on days 0, 3, 7 and 10 after challenge. Mice groups: untreated control ( left panels ), ethosomal erythromycin applied on the infected skin ( middle panels ) and hydroethanolic erythromycin solution applied on the infected skin ( right panels ). (From Godin et al. 2005, with permission from Oxford Journals.) 3 Treatment started Untreated control Ethosomal erythromycin Erythromycin hydroethanolic soln Time, days

8 68 D. Ainbinder et al. t (days) a Days to crust formation Parallel arm 6.4 Days to lost of crust b t (days) Days to crust formation Crossover arm Days to lost of crust ZC EA ZC EA c No. of abortive lesions, % Parallel arm 33 Abortive lesions 7 27 Crossover arm ZC EA Fig. 4.4 Parameters assessed in a two-armed, randomized, double-blind clinical study in recurrent herpes labialis patients with two formulations containing 5 % acyclovir: ethosomal acyclovir (EA) versus Zovirax cream (ZC) (From Horwitz et al. 1999, with permission from Elsevier) system to eradicate the bacteria at the site of inoculation in the deep skin strata, thus suggesting this system as a good alternative to systemic administration of the drug by injection in the treatment of deep skin infections. A pilot clinical study was carried out to test the efficacy of a new clindamycin phosphate and salicylic acid (CLSA) ethosomal gel for the treatment of mild to moderate acne vulgaris (Touitou et al ). In this study, carried out on 40 patients, the efficacy of CLSA ethosomal gel in the treatment of mild to moderate acne vulgaris was investigated. A considerable improvement of the acneic condition, significantly decreasing the number of comedones, pustules, and total number of lesions, was found following twice-a-day treatment with CLSA ethosomal gel during 8 weeks. Seventy-one percent of the participants indicated improvement of their condition, with no reports on worsening. Furthermore, 14 of the 17 participants with a history of previous topical treatment preferred the CLSA ethosomal gel over prior commercial topical medications, based on improved tolerability and fewer side effects. An ethosomal system containing the synthetic acyclic nucleoside analog, acyclovir (ACV), was designed and tested for the treatment of herpes labialis (Horwitz et al ). In a randomized double-blind clinical study, the efficiency of ACV ethosomal formulation was compared to a commercial acyclovir cream (Zovirax, GlaxoSmithKline) and a solution of the free drug (Fig. 4.4 ). The parallel arm consisted of 31 participants of whom 12 received ethosomal acyclovir (EA), 10 Zovirax cream (ZC), and 9 vehicle (V). In the crossover arm, 8 participants were treated with EA followed by ZC and 7 participants were treated with ZC followed by EA. Application of EA system resulted in a significant improvement of time (days) to crust formation, time (days) to loss of crust, the proportion of abortive lesions of all assessable lesions, time (days) to first reduction of reported pain intensity, time (days) to absolute resolution of pain, and the proportion of lesions in which reported pain intensity was reduced from day 1 to 2 and from day 1 to 3. Comparison with ZC showed that in the parallel arm on the third day from the begin-

9 4 Ethosomes: Enhanced Delivery of Drugs to and Across the Skin 69 ning of herpetic episode, 80 % of lesions crusted after treatment with EA versus only 10 % in the ZC group. The time to crust formation was 1.6 days in the EA group versus 4.3 and 4.8 in the ZC and V groups, respectively. Moreover, 33 % of the lesions in the EA group were abortive compared to only 10 % in the ZC group. In the crossover arm, the number of days to crust loss was significantly reduced in the EA group from 4.2 to 5.9 in ZC group on day 2. Sixty percent of the lesions in the EA group crusted versus only 15 % of lesions treated with ZC. The findings of this clinical study showed improved clinical efficacy of ethosomal acyclovir compared to ZC and lead to the release of a new topical acyclovir cream based on the ethosome technology, the SupraVir (Trima, Israel) Hypogonadal and Menopausal Syndromes and Erectile Dysfunction Transdermal Treatments with Ethosomal Systems Transdermal delivery of testosterone from an ethosomal patch named Testosome was compared to Testoderm patch (Alza, Palo Alto, CA, USA), in an in vivo study on rabbits following daily applications for five consecutive days (Touitou et al. 2000a, b ). At least twice higher area under the concentration-time curve (AUC) and the highest concentration measured at any time (Cmax) values were calculated for the ethosomal system following the collection of blood samples and their analysis by radioimmunoassay at the end of the experiment. In a later study in rats, plasma testosterone levels following topical application from ethosomal formulation were measured (Ainbinder and Touitou 2005 ). Significantly higher ( p < 0.05) Cmax and AUC values for ethosomal formulation were obtained as compared to a commercially available preparation, AndroGel (Unimed, USA). Skin permeation studies through human cadaver skin enabled to calculate the flux of the drug through the skin ( mgh 1 / cm 2 ). Theoretical calculation of testosterone plasma concentrations expected in humans showed that application area of 40 cm 2 would be sufficient to achieve plasma values within the normal hormone range in men. These results show that ethosomes allow for enhanced in vivo transdermal absorption of the steroid hormone testosterone enabling to achieve therapeutically efficient plasma levels. Another investigation was carried out for BH ethosomal system in the treatment of hot flashes, the most common menopausal syndrome in women (Shumilov and Touitou 2010 ). The authors studied the pharmacodynamic effect in both hot flashes and anxiety animal models. For hot flashes, a decrease in the elevated tail skin temperature (TST) was measured in the estrogen deficiency-associated thermoregulatory dysfunction rat model, produced by bilateral ovariectomization (OVX rats) (Berendsen et al ). Application of BH ethosomal system on the skin of OVX rats caused a decrease in the elevated tail temperature 3 h after administration, which continued for a total period of 6 h, until the end of the experiment (Fig. 4.5 ). A faster (2 h) but shorter (3 h) decrease in TST was obtained following subcutaneous injection of the drug. Pharmacokinetic studies have shown a prolonged presence of the drug in the rat plasma following application of the transdermal BH ethosomes as compared to oral administration. These findings suggest that transdermal delivery of BH from the ethosomal system may result in a continuous delivery of BH into the bloodstream, possibly offering sustained efficacy with reduced side effects. The effect of prostaglandin E1 (PGE1) ethosomal systems on erectile dysfunction was tested in an in-office pilot clinical study, carried out on 16 men with 17 episodes of erectile dysfunction (Chertin et al ). Patients, treated with ethosomal systems topically applied on the glans penis, were asked to evaluate their ability to have sexual intercourse by scoring the erectile response, in addition to erection assessment by a physician. Furthermore, duplex examination of the cavernous arteries 15 min following the application, in order to assess peak systolic velocity (PSV) and pulsative index (PI) of both left and right cavernous arteries, was measured. The results show that following a single topical application of PGE1 ethosomal system, enhanced penile rigidity and improved peak systolic

10 70 D. Ainbinder et al. Tail skin temperature, C * Time after treatment (h) Untreated control BH administrated SC Tail skin temperature, C Untreated control Time after treatment (h) * BH transdermal vesicular system Buspirone administrated SC Buspirone transdermal ethosomal system Dose 5 15 Onset Duration Mean DTST Max DTST ± >3 1.6 ± Fig. 4.5 Effect of buspirone (BH) following: (A) subcutaneous (n=4) and (B) transdermal administration (n=5) on elevated TST in OVX rats. TST are measured in the active phase 1, 2, 3, 4, and 5 h after treatment ( TST tail skin temperature; OVX ovariectomized). Mean ± S.D.; * p<0.05 compared to untreated OVX group (From Shumilov and Touitou 2010, with permission from Elsevier) velocity were observed in 12 out of 15 patients. The duration of erection varied between 10 and 60 min. Empty ethosomal system had no effect on the erectile response or the penile blood flow. No penile erythema or other adverse events have been reported by any of the participants in any of the study groups. The results of this pilot study show that topical application of PGE1 ethosomal systems could be a promising approach for the local treatment of erectile dysfunction Anti-inflammatory, Antipyretic, and Analgesic Treatments with Ethosomal Systems Cannabidiol (CBD) is a potent anti-inflammatory agent in the treatment of rheumatoid arthritis (Malfait et al ). Lodzki and colleagues have studied the anti-inflammatory potential of transdermal ethosomal CBD system (Lodzki et al ). A significant accumulation of the drug in the skin and in the underlying muscle was measured following a 24-h application of CBD ethosomes to the abdominal skin of nude mice. CBD was detected in the hip skin (37.43 ± μg/ cm 2 ), abdominal skin ( ± μg/cm 2 ), and abdominal muscle (11.54 μg CBD/g muscle), as well as in the hip muscle, liver, and pancreas. Drug plasma concentrations measured during 72-h system application in ICR mice indicated that steady-state levels of the drug (0.67 μg/ml) were achieved after 24 h and lasted until the end of the experiment (72 h). The anti-inflammatory effect of ethosomal CBD system was evaluated using carrageenan-induced aseptic paw inflammation in ICR mice (Sammons et al ). In these experiments, the effect was measured as delta calculated by deduction of the thickness of saline-injected paw from that of the carrageenaninjected inflamed paw in each mouse. Hourly measurements of mice paw thickness for up to 4 h demonstrated a significant difference in the pharmacodynamic profiles of CBD treated and untreated animals, indicating that the inflammation was prevented by transdermal delivery of ethosomal CBD (Fig. 4.6 ). These results show that efficient delivery of CBD can be achieved by transdermal administration from ethosomal system, enabling to overcome the drawbacks

11 4 Ethosomes: Enhanced Delivery of Drugs to and Across the Skin 71 Delta paw thickness (mm) CBD pretreatment ** ** No Treatment * * Time post injection (h) Fig. 4.6 Anti-inflammatory effect of CBD transdermal ethosomal patch, applied 19 h prior to the injection, is compared to no pretreatment: Δ (mean±s.e.m.) between the thickness of carrageenan injected and saline injected paws of the same mouse at different time points post injection. ** p <0.01; *p <0.05 (From Lodzki et al. 2003, with permission from Elsevier) associated with oral drug delivery, including low oral bioavailability, extensive first-pass hepatic metabolism, instability in the acidic gastric ph, and/or low water solubility. Paolino and colleagues have evaluated the anti-erythemal efficacy of a natural antiinflammatory agent, ammonium glycyrrhizinate (AG), delivered from ethosomes (containing 1 3 % Phospholipon 90 and % ethanol) versus same concentration ethanolic solution and aqueous solution, in humans (Paolino et al ). The research was carried out on 12 healthy volunteers pretreated with AG systems 1, 3, and 5 h prior to application of methyl nicotinate (MN), a vasodilator agent, and tested the induced erythema at six sites on the ventral surface of each forearm. A significant reduction in the intensity and duration of erythema, monitored for 8 h by using a reflectance visible spectrophotometer, was induced by ethosomal AG. The maximal erythema index ( E.I.) following 5-h pretreatment with AG ethosomal system was 29.6 % versus 62.7 and 60.7 %, for ethanolic and aqueous controls, respectively. No erythema was observed in sites treated with AG ethosomes 3 h after topical application, while sites treated with aqueous or hydroethanolic solutions of the drug showed presence of chemically induced erythema. It is noteworthy that an examination of the possible effect of empty ethosomes showed no anti-inflammatory activity. These results show that pretreatment of the skin with ethosomal AG system for 1, 3, and 5 h was able to antagonize the appearance of the erythema. A recent study by Touitou and her group investigated the analgesic and antipyretic therapeutic effects of transdermal ethosomal ibuprofen system in two animal models, the Brewer s yeast-induced fever rat and tail flick nociception mice (Shumilov et al ). Application of transdermal ethosomal ibuprofen gel resulted in a gradual decrease in the body temperature of fevered rats, achieving normal values within 3 h (37.0 ± 0.2 C) and remaining low for at least 12 h. The oral administration resulted in rat s body temperature returning to baseline after 1 h, but remaining low for only 7 h and followed by a rise to 38.0 ± 0.4 C. A significant ( p < 0.05) analgesic effect of ethosomal ibuprofen gel 120 and 360 min after administration compared to oral treatment was observed by tail flick test in mice. The data obtained in this work suggest that ethosomal ibuprofen gel might allow for an efficient antipyretic and analgesic treatment, with continuous drug input to the systemic circulation and avoidance of possible gastrointestinal ulceration and bleeding. Moreover, transdermal ibuprofen gel could be beneficial for pediatric patients who often refuse to take the full dose of the medication orally or suffer from vomiting.

12 Insulin Delivery with Ethosomal Systems Transdermal delivery of proteins from the ethosomal system was tested by measuring the effect of insulin on lowering blood glucose levels (BGL) (Touitou et al. 2000b ; Dkeidek and Touitou 1999 ; Dkeidek 1999 ). Insulin delivered transdermally from an ethosomal patch caused a significant reduction (up to 60 %) in BGL in both normal and diabetic rats. On the other hand, insulin skin application from a control non-ethosomal formulation was not able to decrease the blood levels of glucose. The results of this study show that the ethosomal system enables an efficient delivery of insulin through the skin resulting in a significant pharmacodynamic response. The prolonged plateau effect, lasting for at least 8 h, demonstrates the advantage of ethosomes for transdermal delivery of insulin Pilosebaceous and Hair Follicle Treatments with Ethosomal Systems Efficient delivery of actives to pilosebaceous and hair follicular units could highly improve therapies targeting skin appendage-related disorders such as seborrhea, hair loss, and acne. In order to test the efficiency of ethosomes in delivering drugs to skin appendages, minoxidil, a lipid- soluble drug administered topically on the scalp for alopecia treatment, was incorporated into ethosomes, and the system was evaluated for localization of the drug into the pilosebaceous units (Meidan and Touitou 2001 ). Localization of H 3 -minoxidil within the pilosebaceous units was observed (Godin and Touitou 2003 ; Touitou et al. 1998, 2000b ) and measured by quantitative skin autoradiography (Fabin and Touitou 1991 ; Godin et al ) following the application of compositions containing 0.5 % minoxidil and 50 μci tritiated drug to the dorsal region of hairless rats for up to 24 h. Comparison to liposomes demonstrated that the ethosomal system was superior in delivery of minoxidil to the pilosebaceous units (22 vs. 4.5 nmol/g tissue, p < 0.005) (Godin and Touitou 2003 ; Touitou et al. 1998, 2000b ). Thus, administration of minoxidil from ethosomal system could D. Ainbinder et al. result in drug targeting to the hair follicles and improvement of the low efficiency of the current minoxidil therapy for alopecia. The findings of the studies reported above suggest that ethosomes are able to efficiently enhance skin permeation of various molecules leading to improved dermal and transdermal treatments of deep skin and systemic ailments. 4.5 Safety Evaluation In Vitro, in Animals, and in Human Studies Many penetration enhancers besides interfering with the barrier function of the skin cause also damage to the skin cells and induce local irritation. In contrast to these materials, ethosomal carriers composed of inactive ingredients for pharmaceutical use approved by FDA enable efficient skin permeation enhancement on a safe mode. The safety of ethosomal systems was tested in numerous works, both in vitro and in vivo (Fig. 4.7 ). Some of them are outlined below. An in vitro live/dead viability/cytotoxicity viability test carried out in cultured fibroblasts for various vesicular systems and controls indicated that the ethosomal carrier (containing 2 % Phospholipon 90 and 30 % ethanol) is not toxic to 3T3 fibroblasts and that cultured cells kept their viability. Studies in animals have evaluated the safety of ethosomes to the skin following single or chronic application (Shumilov and Touitou 2010 ; Shumilov et al ; Ainbinder and Touitou 2005 ; Lodzki et al ; Dayan and Touitou 2000 ). Histological observations of the skin at the site of treatment showed no changes in the skin structure and in the thickness of the horny layer and no infiltration of inflammatory cells to the skin. Furthermore, biochemical analysis of rat s blood after 5-day treatment with transdermal ethosomal ibuprofen gel revealed no statistically significant differences between the treated and the control groups (Shumilov et al ). Paolino and Fresta carried out a study testing the skin tolerability of ethosomal systems in

13 4 Ethosomes: Enhanced Delivery of Drugs to and Across the Skin 73 Post-marketing data No adverse reactions with marketed formulations In-vitro No toxicity to 3T3 fibroblasts ETHOSOMES SAFETY Clinical trials No adverse skin reactions in clinical trials with Acyclovir, Clindamycin and PGE 1 Animal studies No skin irritation or erythema in rabbits following single 48h or repeated applications Fig. 4.7 Safety studies with ethosomes human volunteers utilizing a noninvasive technique of reflectance spectrophotometry (Paolino et al ). No signs of erythema following 12-, 24-, or 48-h application of ethosomal carrier containing 2 % Phospholipid and 45 % ethanol were reported. In contrast to this, application of hydroethanolic solution with an equal water/ ethanol ratio to that of ethosomes resulted in significant skin erythema. Moreover, no significant difference in erythema index (ΔEI) was measured between skin areas treated with ethosomes and saline. In three separate studies in humans, application of ethosomal systems containing clindamycin, acyclovir, or PGE 1 to the skin of human volunteers has shown no adverse skin reactions, and products based on ethosomal carriers have been marketed for a number of years without any reports on skin irritation or safety issues. 4.6 Summary Ethosomes are phospholipid vesicular systems for dermal and transdermal delivery of actives. The proposed permeation enhancement mechanism suggests that ethanol present in the system has a fluidizing effect both on the phospholipid bilayers of the vesicle and on the SC lipid bilayers of the intercellular pathway in the skin. The vesicle penetrates through the disorganized SC lipids into the deep skin strata where it releases its contents. In vitro, in vivo, and clinical studies summarized in this chapter show that this passive delivery system is able to efficiently enhance skin penetration of molecules with various physicochemical characteristics and structures. In terms of safety, no local irritation was detected following skin application of ethosomes. The enhanced penetration of drugs deep into and across the skin

14 74 by means of ethosomal carrier could be valuable in a variety of existing and new emerging therapies. References Ainbinder D, Touitou E (2005) Testosterone ethosomes for enhanced transdermal delivery. Drug Deliv 12: Ainbinder D, Paolino D, Fresta M, Touitou E (2010) Drug delivery applications with ethosomes. J Biomed Nanotechnol 6: Berendsen HH, Weekers AH, Kloosterboer HJ (2001) Effect of tibolone and raloxifene on the tail temperature of oestrogen-defficient rats. Eur J Pharmacol 419: Celia C, Trapasso E, Cosco D, Paolino D, Fresta M (2009) Turbiscan Lab Expert analysis of the stability of ethosomes and ultradeformable liposomes containing a bilayer fluidizing agent. Colloids Surf B Biointerfaces 72: Chertin B, Touitou E, Godin B, Pollack A, Shemesh D, Farkas A (2006) The efficiency of topical treatment with ethosomal PGE1 formulation in erectile dysfunction patients. Annual meeting of Israeli Urological Association, Eilat Chin JM, Goldstein DB (1997) Membrane disordering action of ethanol: variation with membrane cholesterol content and depth of the spin label probe. Mol Pharmacol 13:435 Dayan N, Touitou E (2000) Carriers for skin delivery of trihexyphenidyl HCl: ethosomes vs. liposomes. Biomaterials 21: Dkeidek I (1999) Transdermal transport of macromolecules. MSc thesis, The Hebrew University of Jerusalem, Jerusalem Dkeidek I, Touitou E (1999) Transdermal absorption of polypeptides. AAPS Pharm Sci 1:S202 Dubey V, Mishra D, Dutta T, Nahar M, Saraf DK, Jain NK (2007a) Dermal and transdermal delivery of an antipsoriatic agent via ethanolic liposomes. J Control Release 123: Dubey V, Mishra D, Jain NK (2007b) Melatonin loaded ethanolic liposomes: physicochemical characterization and enhanced transdermal delivery. Eur J Pharm Biopharm 67: Dubey V, Mishra D, Nahar M, Jain V, Jain NK (2010) Enhanced transdermal delivery of an anti-hiv agent via ethanolic liposomes. Nanomedicine 6: Elsayed MM et al (2006) Deformable liposomes and ethosomes: mechanism of enhanced skin delivery. Int J Pharm 322:60 66 Fabin B, Touitou E (1991) Localization of lipophilic molecules penetrating rat skin in vivo by quantitative autoradiography. Int J Pharm 74:59 65 Godin B, Touitou E (2003) Ethosomes: new prospects in transdermal delivery. Crit Rev Ther Drug Carrier Syst 20: D. Ainbinder et al. Godin B, Touitou E (2004) Mechanism of bacitracin permeation enhancement through the skin and cellular membranes from an ethosomal carrier. J Control Release 94: Godin B, Touitou E (2005) Erythromycin ethosomal systems: physicochemical characterization and enhanced antibacterial activity. Curr Drug Deliv 2: Godin B, Alcabez M, Touitou E (1999) Minoxidil and erythromycin targeted to pilosebaceous units by ethosomal delivery systems. Acta Technol Legis Medicament 10:107 Godin B, Touitou E, Rubinstein E, Athamna A, Athamna M (2005) A new approach for treatment of deep skin infections by an ethosomal antibiotic preparation: An in vivo study. J Antimicrob Chemother 55: Green PG, Hadgraft J, Ridout G (1989) Enhanced in vitro permeation of cationic drugs. Pharm Res 6: Harris RA et al (1987) Effect of ethanol on membrane order: fluorescence studies. Ann N Y Acad Sci 492:125 Horwitz E, Pisanty S, Czerninski R, Helser M, Eliav E, Touitou E (1999) A clinical evaluation of a novel liposomal carrier for acyclovir in the topical treatment of recurrent herpes labialis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 88: Jain S, Tiwary AK, Sapra B, Jain NK (2007) Formulation and evaluation of ethosomes for transdermal delivery of lamivudine. AAPS PharmSciTech 8:E1 E9 Lodzki M, Godin B, Rakou L, Mechoulam R, Gallily R, Touitou E (2003) Cannabidiol transdermal delivery and anti-inflammatory effect in a murine model. J Control Release 93: Malfait AM, Gallily R, Sumariwalla PF, Andreakos E, Mechoulam R, Feldmann M (2000) The nonpsychoactive cannabis constituent cannabidiol is an oral antiarthritic therapeutic in murine collagen-induced arthritis. Proc Natl Acad Sci 97: Meidan V, Touitou E (2001) Treatments for androgenetic alopecia and alopecia areata: current options and future prospects. Drugs 61:53 69 Pang KYY et al (1980) The perturbation of lipid bilayers by general anesthetics: a quantitative test of the disordered lipid hypothesis. Mol Pharmacol 18:84 Paolino D, Lucania G, Mardente D, Alhaique F, Fresta M (2005) Ethosomes for skin delivery of ammonium glycyrrhizinate: in vitro percutaneous permeation through human skin and in vivo anti-inflammatory activity on human volunteers. J Control Release 106: Rao Y, Zheng F, Zhang X, Gao J, Liang W (2008) In vitro percutaneous permeation and skin accumulation of finasteride using vesicular ethosomal carriers. AAPS PharmSciTech 9: Sammons M, Raval P, Bingham S, Davey P, Parsons AA (1997) A method for determining thermal hyperalgesia and inflammation in the mouse hind paw. Br J Pharmacol 122:334 Shumilov M, Touitou E (2010) Buspirone transdermal administration for menopausal syndromes, in vitro and in animal model studies. Int J Pharm 387:26 33 Shumilov M, Bercovich R, Duchi S, Ainbinder D, Touitou E (2010) Ibuprofen transdermal ethosomal gel: char-

15 4 Ethosomes: Enhanced Delivery of Drugs to and Across the Skin 75 acterization and efficiency in animal models. J Biomed Nanotechnol 6:1 8 Swarbrick J, Brom J, Gensmantel NP (1984) Drug permeation through human skin. II. Permeability of ionizable compounds. J Pharm Sci 73: Touitou E (1998) Composition for applying active substances to or through the skin. US Patent 5,716,638 Touitou E, Godin B (2005) Enhanced skin permeation using ethosomes. In: Smith EW, Maibach HI (eds) Percutaneous penetration enhancement, 2nd edn. New York, CRC Press, pp Touitou E, Meidan V, Horwitz E (1998) Methods for quantitative determination of drug localized in the skin. J Control Release 56:7 21 Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M (2000a) Ethosomes- novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. J Control Release 65: Touitou E, Godin B, Weiss C (2000b) Enhanced delivery of drugs into and across the skin by ethosomal carriers. Drug Dev Res 50: Touitou E, Godin B, Shumilov R, Bishouty N, Ainbinder D et al (2007) Efficacy and tolerability of clindamycin phosphate and salicylic acid gel in the treatment of mild to moderate acne vulgaris. J Eur Acad Dermatol Venerol 21:1 2 Trima Israel Pharmaceutical Products Maabarot Ltd., data on SupraVir cream file.