Drug Delivery Applications with Ethosomes

Copyright © 2010 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Biomedical Nanotechnology Vol. 6, 558–568, 2010 Drug Delivery Applications with Ethosomes D. Ainbinder1 , D. Paolino2 , M. Fresta3 , and E. Touitou1
∗ 1 The Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, PO Box 12065, Jerusalem, 91120, Israel 2 Department of Experimental and Clinical Medicine, Faculty of Medicine, University “Magna Græcia” of Catanzaro, Campus Universitario “Salvatore Venuta”-Building of BioSciences, Viale Europa, I-88100 Germaneto (CZ), Italy 3 Department of Pharmacobiological Sciences, Faculty of Pharmacy, University “Magna Græcia” of Catanzaro, Campus Universitario “Salvatore Venuta”-Building of BioSciences, Viale Europa, I-88100 Germaneto (CZ), Italy REVIEW Ethosomes are specially tailored vesicular carriers able to efficiently deliver various molecules with different physicochemical properties into deep skin layers and across the skin. This paper reviews the unique characteristics of the ethosomal carriers, focusing on work carried out with drug containing ethosomal systems in animal models and in clinical studies. The paper concludes with a discussion on the safety of the ethosomal system applications. Keywords: Transdermal, Dermal, Skin, Ethosome, Lipid Carrier, Nanovesicle. CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Applications of Ethosomes for Deep Dermal and Transdermal Delivery: In Vivo Studies . . . . . . . . . . . . . . . . . . . . . . . 2.1. Treatment of Microbial and Viral Skin Infections . . . . . 2.2. Anti-Inflammatory Ethosomal Systems . . . . . . . . . . . . 2.3. Ethosomal Systems for Menopausal Syndromes . . . . . . 2.4. Management of Erectile Dysfunction . . . . . . . . . . . . . 2.5. Analgesic and Antipyretic Ethosomal Systems . . . . . . . 3. Safety of Ethosomal Systems . . . . . . . . . . . . . . . . . . . . . 4. Conflict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . 558 . . . . . . . . . 561 561 563 564 565 565 566 567 567 1. INTRODUCTION In spite of the ongoing research in the field of dermal and transdermal drug delivery, efficient administration of drugs by topical application remains a challenge. In order to overcome the barrier properties of the skin and make this route of administration useful for more drugs, many approaches have been proposed, including the use of microinvasive devices, electrically-assisted methods (iontophoresis, electrophoresis and sonophoresis), chemical permeation promoters and vesicular systems.1 Ethosomes are specially tailored vesicular carriers which were invented by Touitou in response to the need for efficient delivery of drugs by topical application on the skin.2 This system is composed mainly of phospholipids, ethanol (up to 50%) and water. Ethosomes, unique vesicular carriers, are distinguished from other lipid nanocarriers by a number of important characteristics, such as ∗ Author to whom correspondence should be addressed. 558 J. Biomed. Nanotechnol. 2010, Vol. 6, No. 5 the vesicle’s bilayer fluidity, its mechanism of permeation enhancement (attributed to the entire system), simple mode of preparation and a lack of side effects. These special characteristics of the ethosomal systems will be further discussed. Comprehensive work on the characterization of the ethosomal systems was carried out by Touitou and her group. The existence of vesicles in the system was demonstrated by 31 P-nuclear magnetic resonance studies and electron microscopy.3–5 Further examination of the vesicular structure by transmission electron microscopy showed that ethosomes are unilamellar or multilamellar vesicles, dependent on system composition (Fig. 1).3–6 Differential scanning calorimetry was applied to measure the transition temperature of lipids in the ethosomal systems. 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.3–5 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. Further characterization of ethosomes by measuring the vesicular size distribution of systems with various compositions has indicated that the size of the vesicles ranges between 30 nm to several microns and that it can be adjusted by changes in the system’s composition. A systematic investigation of the effect of system composition on vesicular size showed that increases in the percent of lipid in the system result in larger vesicles, while increases in the percent of ethanol at the same lipid concentration 1550-7033/2010/6/558/011 doi:10.1166/jbn.2010.1152 Drug Delivery Applications with Ethosomes Ainbinder et al. decrease the size of the ethosomes.3 This correlation implies that 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. Furthermore, special attention should be paid to the dilutions of the tested system. D. Ainbinder is a post-doctoral fellow in the Dermal, Transdermal and Transmucosal Delivery group at the Institute for Drug Research at the Hebrew University of Jerusalem, Israel. She has extensive experience in the field of transdermal delivery and in dermal systems for skin cancer. As a group member of professor Touitou, she has been involved in many projects in cooperation with the industry. She is a recipient of the outstanding teacher award and award for excellence in M.Sc. studies. M. Fresta was born in Catania on 13.05.1967. Massimo Fresta is Full Professor of Pharmaceutical Technology and Advanced Drug Delivery at the Department of Pharmacobiological Sciences, Faculty of Pharmacy, University “Magna Græcia” of Catanzaro. He is Coordinator of the Doctorate Course in Pharmaceutical Sciences at the University of Catanzaro, Italy. From the University of Catania, he received the degree in Pharmacy (110/110 cum laude) and “Federfarma” award in 1990 and the specialization degree in Chemistry and Technology of Foods (50/50 cum laude) in 1992. From 1992 to 1994 he was visiting scientist at the Institut für Polymere, ETH-Zentrum, Zurich (Switzerland), where he got the master in Supramolecular Chemistry. In 1996 he received his Doctorate in Technology of Biologically Active Substances from the University of Palermo. In 1997 he joined the Faculty of Pharmacy at the University of Catania as assistant professor. From 2000 up to now, he joined the Faculty of Pharmacy at the University “Magna Græcia” of Catanzaro. His teaching responsibilities are in undergraduate and Ph.D. programmes, as well as European Union exchange activities. He is responsible of various Long-life learning programs on behalf of the Italian Ministery of Health. He has published more than 100 peer reviewed articles in international scientific journals and was co-author of various chapters in scientific books. He is the author of more than 170 abstracts of congress/conference/workshop/meeting presentations. He has filed 3 patents on pharmaceutical products. Professor Massimo Fresta is the recipient of various awards and honors. He is a member of various national and international scientific societies. He is member of the editorial board of various scientific journals. He acts as an active reviewer for the most important and prestigeous scientific journals in the field of drug delivery and pharmaceutical nanotechnology. The current research interests of Professor Massimo Fresta are in the design, preparation, characterization and evaluation of innovative colloidal drug delivery systems for the selective delivery to cancer tissues and CNS. Topical administration of colloidal carriers is another research field under investigation for ophthalmic, dermal, transdermal, mucosal and transmucosal applications. J. Biomed. Nanotechnol. 6, 558–568, 2010 559 REVIEW D. Paolino was born in Modica. From the University of Catania, she received the degree in Pharmaceutical Chemistry and Technology (110/110) in 1999. In 2003 she received his Doctorate in Pharmaceutical Technology from the University of Palermo. She is specialist in Hospital Pharmacy (50/50 cum laude). She is a member of various national and international scientific societies. She is member of the editorial board of various scientific journals. She acts as an active reviewer for the most important and prestigeous scientific journals in the field of drug delivery and pharmaceutical nanotechnology. The current research interests of Donatella Paolino, Ph.D. are in the design, preparation, characterization and evaluation of innovative colloidal drug delivery systems for the selective delivery of drugs. In particular her research fields are: ophthalmic, dermal, and brain delivery. She has published 47 peer reviewed articles in international scientific journals, 2 chapters in scientific books and 1 international patent. She is the author of more than 100 abstracts of presentations. She is a member of various national and international scientific societies. At today she is permanent researcher at the Department of Experimental and Clinical Medicine, Faculty of Medicine, University Magna Graecia of Catanzaro REVIEW Drug Delivery Applications with Ethosomes Ainbinder et al. E. Touitou is a Professor of Pharmaceutical Sciences and Head of the Dermal, Transdermal and Transmucosal Drug Delivery Group at the Institute for Drug Research of the School of Pharmacy, The Hebrew University of Jerusalem, Israel. She is an internationally recognized authority in the drug delivery field. Her primary research interest is in the field of enhanced drug absorption and design of novel carriers for efficient drug delivery. She is the inventor of leading technologies and has authored numerous patents, including the patent on Ethosomes® , a recognized nanocarrier for enhanced dermal and transdermal delivery. Based on her new technologies two start-up companies; Novel Therapeutic Technologies and Shimnei Eden, were established. Professor Touitou has been a visiting Professor at a number of pharmaceutical companies and universities in Europe and US including Hoffmann La Roche, American Cyanamid, the University of Rome and the Chulalongkorn University of Bangkok. She has broad experience in collaborating with the pharmaceutical industry in the design of new formulations. She is a member of the editorial board of the Drug Delivery and Translational Research Journal and serves as a reviewer for major pharmaceutical international scientific journals as well as for research grants for foundations and grant agencies in Israel, Italy, Netherlands, UK, Austria, US, Hong Kong, Europe Union. Professor Touitou has published over 200 scientific works, including original research papers, reviews, book chapters and the books Enhancement in Drug Delivery and Novel Cosmetic Delivery Systems. She has served as President of the Israeli Chapter of Controlled Release Society (CRS) (2003–2007), Member of CRS Board of Directors (2007–2010) and a CRS Fellow. She is the recipient of a number of awards, including the Kaye Award for Innovation (2006) and the Jorge Heller Outstanding Paper Award (2000), and has been elected numerous times as the Best Teacher of the year at The Hebrew University of Jerusalem. Another important characteristic of this carrier is its ability to efficiently entrap molecules with a wide range of physicochemical characteristics, including lipophilic, hydrophilic and high molecular weight entities.3
7
8 Examination by CLSM of the intra-vesicular distribution of lipophilic (fluorescently labelled phospholipid) and hydrophilic (FITC-bacitracin) fluorescent probes showed that both molecules fill up 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.7 This high encapsulation of both hydrophilic and lipophilic probes in ethosomes could be explained by the multilamellar structure of the vesicle as well as by the presence of a hydroethanolic environment between the bilayers in the vesicle. Fig. 1. Visualization of ethosomal vesicles. TEM (magnification 315 000) of ethosomal vesicles composed of 2% PL, 30% ethanol and water. Reprinted with permission from [3], E. Touitou et al., Ethosomes—novel vesicular carriers for enhanced delivery: Characterization and skin penetration properties. J. Controlled Release 65, 403 (2000). © 2000, Elsevier. 560 However, it is important to bear in mind that in contrast to liposomes and other nanovesicles, the permeation enhancing property of ethosomes is attributed to the entire system, composed of vesicles with fluid bilayers and ethanol. Therefore, for these systems both encapsulated and free drug present in the ethosomal system will permeate the skin. Thus, drug encapsulation and vesicular size do not play key roles in determination of the penetration enhancement magnitude. Interestingly, in a work published by Verma and coworkers, investigating the effect of deformable liposomes on skin penetration of entrapped and non-entrapped hydrophilic substances into human skin, a similar pattern was found when delivery of calcein from liposomal system containing the probe both inside and outside the vesicles resulted in the highest penetration enhancement effect, as compared to liposomal systems containing the probe only inside or outside the vesicles.9 Previous work has shown that ethosomal systems can efficiently enhance skin penetration of a wide range of molecules.3
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11 A CLSM study examining the penetration of the lipophilic probe Rhodamine Red into nude mice skin (Fig. 2), clearly indicated that both the depth of skin penetration and the fluorescence intensity in different skin layers increased, when the probe was delivered by the ethosomal system.3
12 Later, in another CLSM study, Dubey et al. confirmed this enhanced skin penetration effect of Rhodamine Red by ethosomes.13 The proposed mechanism for penetration enhancement by ethosomes is based on the dual effect of ethanol on both the lipid bilayers in the stratum corneum and in the vesicle: ethanol enables fluidization of the lipids in the ethosomal structure on one side, along with changes in the J. Biomed. Nanotechnol. 6, 558–568, 2010 Drug Delivery Applications with Ethosomes Ainbinder et al. (A) (B) (c) Fig. 2. Penetration of rhodamine red from ethosomes into nude mouse skin. Ethosomes (A), hydroethanolic solution (B) or liposomes (C) each containing 0.5% rhodamine red were applied nonocclusively to the back skin of 8-week old male nude mice. At the end of the experiment, the skin was excised and analyzed by CSLM, xz scans. Reprinted with permission from [3], E. Touitou et al., Ethosomes—novel vesicular carriers for enhanced delivery: Characterization and skin penetration properties. J. Controlled Release 65, 403 (2000). © 2000, Elsevier. 2. APPLICATIONS OF ETHOSOMES FOR DEEP DERMAL AND TRANSDERMAL DELIVERY: IN VIVO STUDIES Delivery of various molecules with different physicochemical characteristics into the deep skin layers or across the skin by the ethosomal carrier has been tested widely. This paper reviews work carried out in animal models or in clinical studies. J. Biomed. Nanotechnol. 6, 558–568, 2010 2.1. Treatment of Microbial and Viral Skin Infections Ethosomal systems containing antibiotic drugs have been investigated in the treatment of various skin infections. Bacitracin and erythromycin ethosomal systems were formulated and tested for their efficiency in animal models of deep skin infections.5
18 The pharmacodynamic effect of the tested topical treatments was measured in immunocompetent ICR male mice, injected intradermally with S. aureus, by isolation of S. aureus colonies from the skin wounds 7 and 10 days after the beginning of the experiment. Results showed that no S. aureus bacteria was found in the inoculation sites in mice treated with ethosomal erythromycin system compared to 090 × 107 and 057 × 107 cfu/g tissue on days 7 and 10, respectively, in untreated mice. Furthermore, histological examination of the wounded skin tissue on days 7 and 10 of the treatment revealed no dermatonecroses and preservation of normal skin structures. In contrast, inspection of the wounded areas from untreated mice and those treated with erythromycin hydroethanolic solution showed progression of the infection, resulting in a significant dermatonecroses of the skin and adjacent tissues and initial crust formation over the necrotic area (Fig. 3). These results indicate that erythromycin ethosomal system is able to efficiently eradicate the bacteria at the site of inoculation in the deep skin strata. Thus, in the treatment of deep skin infections topical application of antibiotic ethosomal system could be a good alternative to systemic administration of the drug by injection.5
18 Another antibiotic containing ethosomal system was tested in a pilot clinical study. In this study, carried out on forty patients, the efficacy of a new clindamycin ethosomal gel (CLSA) for the treatment of mild to moderate acne vulgaris was investigated.19 CLSA contains a mixture of clindamycin phosphate and salicylic acid in ethosomes. Twice a day treatment with CLSA gel during eight weeks resulted in considerable improvement of the acneic condition, significantly decreasing the number of comedones, pustules and total number of lesions compared with 561 REVIEW arrangement of the lipids in the skin barrier on the other side. This allows the soft vesicles to penetrate the altered structure of the stratum corneum and to release the active in the deeper layers of the skin.3
8 Thus, the enhanced dermal delivery of molecules by ethosomal systems can be attributed to a possible synergistic effect on the stratum corneum lipids of ethanol and the nanovesicles with fluid bilayers. It is also important to note, that in contrast to deformable vesicular systems such as transfersomes for which skin permeation enhancement is observed only under non-occlusive conditions,14–16 for ethosomes, occlusion does not affect the skin permeation profile. This was shown by the examination of the human cadaver skin permeation profiles of FITC-Bac following occlusive versus nonocclusive application resulting in similar quantities permeated the skin during the 24 h experiment.7 Examination of the stability of ethosomes containing various drug entities done by measuring the vesicular size distribution, along with structure visualization, showed that ethosomes are stable for long periods of time.3
4 Additionally, Fresta and his group evaluated the stability of ethosomes containing linoleic acid using a Turbiscan optical analyzer for determination of the long-term stability of colloidal systems. No modification of the backscattering profiles of colloidal suspensions and no coalescence, sedimentation, flocculation or clarification occurred when ethosomes with different amounts of linoleic acid were tested.17 Drug Delivery Applications with Ethosomes 0 3 Ainbinder et al. Induction of the infection with S. aureus 29213 Treatment started Untreated control Ethosomal erythromycin Erythromycin hydroethanolic soln. REVIEW 7 10 Time, days Fig. 3. Histological images taken from skin of mice intradermally inoculated with 0.1 mL × 108 cfu/mL (107 cfu/mouse) S. aureus ATCC 29213 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). Reprinted with permission from [18], B. Godin et al., A new approach for treatment of deep skin infections by an ethosomal antibiotic preparation: An in vivo study. J. Antimicrob. Chemother. 55, 989 (2005). © 2005, Oxford University Press. placebo. Seventy one percent of the participants indicated improvement of the condition, with no reports on worsening. Furthermore, fourteen of the seventeen participants with a history of previous topical treatment preferred the clindamycin ethosomal gel compared with prior commercial topical medications, based on improved tolerability and fewer side-effects.19 An ethosomal system containing a synthetic acyclic nucleoside analog, acyclovir (ACV), was designed and tested for the treatment of another skin infection, Herpes labialis.20 In this randomized double-blind clinical study, the efficiency of an ethosomal formulation, a commercial acyclovir cream (Zovirax® , GlaxoSmithKline S.p.A.) and a solution of the free drug was compared in forty participants, experiencing 61 assessable episodes. 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. Time (in days) to crust formation, time (in days) to loss of crust, the proportion of abortive lesions of all assessable lesions, time (in days) 562 to first reduction of reported pain intensity, time (in days) to absolute resolution of pain and the proportion of lesions in which reported pain intensity was reduced from day 1 to day 2 and from day 1 to day 3 were assessed in this study. Application of ethosomal acyclovir system resulted in a significant improvement of all the evaluated clinical parameters. Comparison with Zovirax cream showed, that in the parallel arm on the third day from the beginning of herpetic episode, 80% of lesions crusted after treatment with ethosomal drug system versus only 10% in the Zovirax 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 (Fig. 4). 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.20 The findings of this clinical study, indicating an improved clinical efficacy of ethosomal acyclovir compared to ZC, resulted in the release of a new topical J. Biomed. Nanotechnol. 6, 558–568, 2010 Drug Delivery Applications with Ethosomes Ainbinder et al. 100 Crusted lesions (%) 90 80 Parameter V subgroup (n=9) ZC subgroup (n=10) EA subgroup (n=12) Days to crust formation (SD) 4.8 (2.1) 4.3 (1.9) 1.6 (1.4)* Days to loss of crust (SD) 6.1 (3.1) [n=9] 6.4 (3.2) [n=7] 3.5 (3.1) [n=11] No. of abortive lessions(%) 1 (11) 1 (10) 4 (33) 70 60 50 40 30 EA (Ethosomal ACV) 20 ZC (Zovirax cream) 10 V (Vehicle) 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 *P<0.05 Days from beginning of episode Fig. 4. Days to crust formation: parallel arm. Day 0: 33% of lesions aborted in EA subgroup, 10% in ZC subgroup, 11% in V subgroup. Day 3: 80% of lesions crusted in EA subgroup, 10% in ZC subgroup, 11% in V subgroup. Day 4: 100% of lesions crusted in EA subgroup, 60% in ZC subgroup, 44% in V subgroup. Time to crusting of all lesions: 4 days in EA subgroup, 7 days in ZC subgroup, 7.5 days in V subgroup. Reprinted with permission from [20], E. Horwitz et al., 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. 87, 700 (1999). © 1999, Elsevier. Ammonium glycyrrhizinate (AG) ethosome was tested by Paolino and colleagues for the treatment of inflammatory-based skin diseases on human volunteers with methyl-nicotinate chemically induced erythema.10 The anti-inflammatory effect of ethosomal AG system following either pre-treatment or treatment of skin erythema was compared to aqueous or hydroethanolic drug solutions and evaluated by a reflectance visible spectrophotometer used for the quantification of the erythema index ( EI). Results showed that AG ethosomes induced a significant reduction in the intensity and the duration of erythema with respect to the other formulations (Fig. 5). 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. Pre-treatment of the skin with ethosomal AG system for 1, 3 and 5 h resulted in decreased erythema index, indicating that the system was able to antagonize the appearance of the erythema. The most profound effect was observed following pre-treatment with the system for 5 h.10 Besides deep skin delivery, ethosomal systems have been investigated for transdermal delivery of antiinflammatory drugs. Cannabidiol (CBD), a highly lipophilic molecule, is a new drug candidate for treatment of rheumatic diseases. CBD ethosomal system was designed and tested for in vivo skin permeation by measurement of drug accumulation in the skin and other body organs.21 The results showed a significant accumulation of the drug in the skin and underlying tissue. After 24 h of treatment, CBD was detected in the hip skin (3743 ± 13.58 g/cm2 , abdominal skin (11007 ± 24.15 g/cm2  J. Biomed. Nanotechnol. 6, 558–568, 2010 40 AG ethosomes-H AG Hydroethanolic solution AG Aqucous solution Saline 35 30 25 20 15 10 5 0 1 2 3 4 Time (h) Fig. 5. In vivo anti-inflammatory activity in human volunteers of various formulations containing ammonium glycyrrhizinate evaluated as the ability to reduce a chemically induced skin erythema. Results are expressed as a mean value (6 different volunteers) of the erythema index variation ±S.D. as a function of the time. Reprinted with permission from [10], D. Paolino et al., Ethosomes for skin delivery of ammonium glycyrrhizinate: In vitro percutaneous permeation through human skin and in vivo anti-inflammatory activity on human volunteers. J. Controlled Release. 106, 99 (2005). © 2005, Elsevier. 563 REVIEW 2.2. Anti-Inflammatory Ethosomal Systems and abdominal muscle (11.537 g CBD/g muscle), as well as in the hip muscle, liver and pancreas (Fig. 6). Drug plasma concentrations measured during 72-h system application in ICR mice indicated that steady state levels of the drug were achieved after 24 h and lasted until the end of the experiment 72 h. 43.33% of the initial drug’s dose penetrated the skin into systemic circulation. Furthermore, the anti-inflammatory effect of ethosomal CBD system applied topically 19 h before the carrageenan injection completely prevented the development of the edema, as evaluated in carrageenan-induced aseptic paw edema in male ICR mice by hourly measurements of paw thickness for up to 4 h.21 ∆ΕΙ acyclovir cream based on the ethosome technology, the Supra-Vir (Trima, Israel). Drug Delivery Applications with Ethosomes REVIEW Fig. 6. Anti-inflammatory effect of CBD transdermal patch, applied 19 h prior to the injection, is compared to no pretreatment:  (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 < 001; ∗ p < 005. Reprinted with permission from [21], M. Lodzki et al., Cannabidiol-transdermal delivery and anti-inflammatory effect in a murine model. J. Controlled Release 93, 377 (2003). © 2003, Elsevier. These results show that CBD can be efficiently administrated from transdermal ethosomal system, thus making it possible to overcome the drawbacks 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 (CBD is very lipophilic with Ko/w ∼ 8) and to increase patient compliance. 2.3. Ethosomal Systems for Menopausal Syndromes Ethosomal compositions have been tested for their efficiency in the treatment of androgen deficiency associated with menopause in men and menopausal syndromes in women.3
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11 A testosterone ethosomal patch system, Testosome, was designed for the treatment of androgen Ainbinder et al. deficiency in men.3 An in vivo study, comparing testosterone serum levels in rabbits, following single or multiple (once a day for five days) application from either Testosome or Testoderm® patch (Alza) was carried out. Results of single patch application showed no significant differences between the tested groups. However, following daily application of the patches to rabbit pinna skin for 5 consecutive days, the AUC and Cmax values obtained for Testosome were 2.2 and 2.4 times higher, as compared to Testoderm® .3 Further, transdermal testosterone ethosomal gels were formulated and tested.6 Pharmacokinetic studies in rats resulted in significantly higher Cmax (1970 ± 251 vs. 601±88 ng ∗ dL−1  and AUC (9313 ± 385 vs. 5678 ± 719 ng∗ dL−1 ∗ h) values for the ethosomal gel in comparison to AndroGel® (Unimed). For calculation of the required application area for achieving efficient hormone levels in men, the drug flux through human skin was further measured. These studies gave a 6.4 times higher testosterone skin permeation for the ethosomal gel relative to AndroGel® . A Css value of 911 ng/dL for application area of 40 cm2 was predicted by using the experimental data.22 These results show that ethosomes can be used for an improved efficient systemic delivery of testosterone, with a smaller application area sufficient to achieve therapeutic hormone blood levels (300–1000 ng/dL).6 An interesting recent finding is that buspirone HCl (BH), an anxiolytic drug, could affect hot flashes, the most common menopausal syndrome in women. The pharmacodynamic effects of the transdermal ethosomal BH system in the treatment of menopausal syndromes were investigated in both hot flashes and anxiety animal models by Fig. 7. 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 < 005 compared to untreated OVX group. Reprinted with permission from [11], M. Shumilov and E. Touitou, Buspirone transdermal administration for menopausal syndromes, in vitro and in animal model studies. Int. J. Pharm. 387, 26 (2010). © 2010, Elsevier. 564 J. Biomed. Nanotechnol. 6, 558–568, 2010 Drug Delivery Applications with Ethosomes Ainbinder et al. 2.4. Management of Erectile Dysfunction In an “in-office” pilot clinical study, carried out on 16 men with 17 episodes of erectile dysfunction, patients were treated with ethosomal prostaglandin E1 (PGE1  systems applied on the glans penis.23 The patients were asked to evaluate their ability to have sexual intercourse by scoring the erectile response, in addition to erection assessment by a physician (Fig. 8). The effect was further tested by Duplex examination of the cavernous arteries 15 minutes following the application, in order to assess Peak-Systolic Velocity (PSV) and Pulsative index (PI) of both left and right cavernous arteries (Fig. 9). The duration of the erection was recorded. Results of this study Response 3.5 Response (AU) 3 Placebo (n=2) Ethosomal PGE1 A (n=8) Ethosomal PGE1 B (n=7) 2.5 2 1.5 1 Fig. 8. Patients’ scores of erectile response following topical application of Ethosomal formulations to the penile glans area. The assessment was made using the following scoring of erectile response: 1. no erection, no tumescence; 2. partial tumescence not likely to be sufficient for penetration; 3. tumescence sufficient for penetration; 4. full rigidity; 5. excessive rigidity. showed that following a single topical application of PGE1 ethosomal system, enhanced penile rigidity and improved peak systolic velocity were observed in 12 patients out of 15 men tested. No erectile response or changes in penile blood flow were found following the application of the empty ethosomal vehicle. The duration of erection varied between 10 to 60 minutes. It is noteworthy, that 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 prostaglandin E1 (PGE1  ethosomal systems could be a promising approach for the local treatment of erectile dysfunction.23 2.5. Analgesic and Antipyretic Ethosomal Systems A recent study investigated the in vivo analgesic and antipyretic therapeutic effects of transdermal ethosomal ibuprofen in two animal models, the Brewer’s yeast induced fever rat and tail flick nociception mice.24 Application of ibuprofen gel on the animal skin resulted in a gradual decrease in the body temperature of fevered rats, Table I. Effect of BH administration on inhibitory avoidance in elevated T-maze rat model. Withdrawal latency inhibitory avoidance System Untreated control BH transdermal ethosomal system 4 h after application 12 h after application BH oral solution 12 h after application Drug dose (mg/kg) Baseline (S) Avoidance 1 (S) Avoidance 2 (S) n — 30 ± 33 289 ± 185 573 ± 51 8 15 15 26 ± 20 50 ± 27 165 ± 115 68 ± 44 316 ± 130∗ 205 ± 118∗∗ 4 5 3 33 ± 21 263 ± 132 576 ± 42 ∗ 4 ∗∗ BH: buspirone hydrochloride Mean ± S.D. p < 001, significant difference from untreated control (ANOVA with Tukey-Kramer multiple comparisons test). p < 0001, significant difference from untreated control (ANOVA with Tukey-Kramer multiple comparisons test). Source: Reprinted with permission from [11], M. Shumilov and E. Touitou, Buspirone transdermal administration for menopausal syndromes, in vitro and in animal model studies. Int. J. Pharm. 387, 26 (2010). © 2010, Elsevier. J. Biomed. Nanotechnol. 6, 558–568, 2010 565 REVIEW Shumilov et al.11 For the hot flashes animal model, the estrogen deficiency-associated thermoregulatory dysfunction rat model was produced by bilateral ovariectomization (OVX rats). These animals exhibited elevated tail skin temperature (TST) during the active phase of the day, as compared to intact (sham-operated) rats. 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. Subcutaneous injection of the drug resulted in a faster decrease in TST (2 h); however, it remained normal for only 3 h followed by an increase of the temperature (Fig. 7). Interestingly, the transdermal BH ethosomes resulted in a prolonged presence of the drug in the rat plasma as compared to oral administration.11 Since BH is an anxiolytic drug, the effect of transdermal BH ethosomes was also studied using an elevated T-maze anxiety model. The results showed a significant decrease of avoidance latency 4 and 12 hours after BH transdermal system application in contrast to the orally treated animals (no effect, p < 0001, Table I). The results of the study suggest that by transdermal delivery of the drug from the ethosomal system, a non-fluctuated and continuous delivery of BH into the bloodstream might be achieved, offering sustained efficacy with reduced side effects.11 Drug Delivery Applications with Ethosomes The analgesic effect of ethosomal ibuprofen gel was compared to oral treatment by tail flick test in mice. A statistically significant higher effect was obtained for the ethosomal ibuprofen system 120 and 360 min after administration. The duration of effect was at least 6 h.24 Data obtained in this work suggests that the designed ethosomal ibuprofen gel can be further investigated in humans for its antipyretic and analgesic effect. Besides enabling a convenient and efficient treatment, with continuous drug input to the systemic circulation and avoidance of possible gastrointestinal ulceration and bleeding, transdermal delivery could also be beneficial for pediatric patients who often refuse to take the full dose of the medication orally or suffer from vomiting. PSV-Rt 60 Placebo (n=2) Ethosomal PGE1 A (n=8) Ethosomal PGE1 B (n=7) 50 PSV-Rt, cm/s Ainbinder et al. 40 30 20 10 0 Pre-treatment 60 Placebo (n=2) Ethosomal PGE1 A (n=8) Ethosomal PGE1 B (n=7) PSV-Lt, cm/s 50 40 3. SAFETY OF ETHOSOMAL SYSTEMS 30 20 10 0 Pre-treatment Post treatment Fig. 9. Duplex examination of peak systolic velocity (PSV) in the right (Rt, left graph) and left (Lt, right graph) cavernous arteries following topical application to the penile glans area of two different Ethosomal formulations in patients suffering from erectile dysfunction. achieving normal values within three hours. The body temperature remained low (370 ± 02  C) until the end of the tested time period, at least 12 h. In contrast to this, oral administration resulted in the rat’s body temperature returning to baseline after 1 h, but remaining low for only 7 h, followed by a rise to 380 ± 04  C. A correlation between the plasma drug concentrations following ibuprofen transdermal ethosomal gel application and the pharmacodynamic effect of decrease in body temperature is shown in Figure 10. Ethosomes are composed of ingredients generally regarded as safe (GRAS). The safety of ethosomal systems applied topically to the skin has been tested in numerous works, both in vitro and in vivo. In vitro studies on cell cultures showed that ethosomal systems are safe to skin cells.5
12 Histological observations of the skin at the site of the treatment following both single and chronic application of ethosomal systems containing various molecules (e.g., BH, ibuprofen, testosterone, CBD, etc.) showed no changes in the structure and the thickness of the horny layer, and no infiltration of inflammatory cells to the skin. Both acute and 14-day repeated ethosomal patch applications in 38.4 Ibuprofen plasma concentration Body temperature 80 38.2 38 37.8 60 37.6 40 37.4 37.2 20 Body temperature, ºC 100 Ibuprofen plasma concentration, µg/ml REVIEW Post treatment PSV-Lt 37 0 0 5 10 15 20 Time after administration, h 36.8 25 Fig. 10. Correlation between the pharmacokinetic profile and pharmacodynamic effect after transdermal administration of ibuprofen from ethosomal gel in male Wistar rats. Mean ± SD (n = 6 for drug plasma concentration; n = 4 for body temperature). 566 Fig. 11. In vivo human skin tolerability of various topical formulations after 6, 24 or 48 h of treatment. Results are expressed as a mean value of EI (the variation of the erythema index) (n = 6 ± S.D. Reprinted with permission from [10], D. Paolino et al., Ethosomes for skin delivery of ammonium glycyrrhizinate: In vitro percutaneous permeation through human skin and in vivo anti-inflammatory activity on human volunteers. J. Controlled Release. 106, 99 (2005). © 2005, Elsevier. J. Biomed. Nanotechnol. 6, 558–568, 2010 Drug Delivery Applications with Ethosomes Ainbinder et al. SAFETY STUDIES CARRIED OUT WITH ETHOSOMES In vitro studies on cultured cells Ethosomal carriers were not toxic to 3T3 fibroblasts and the cultured cells kept their viability as assessed in Live/dead viability/cytotoxicity test Studies in animals No acute skin irritation or erythema in rabbits was observed following single 48 h or cumulative 14-days repeated ethosomal patch application Data from clinical trials No adverse skin reactions were associated with the treatment in three clinical trials with ethosomal acyclovir, clindamycin and PGE1 Studies in humans No signs of erythema following 12, 24 and 48 h applications REVIEW Post-marketing information No reported adverse reaction for marketed ethosomal formulations Fig. 12. Safety studies with ethosomes. rabbits showed no signs of skin irritation.3 The results of biochemical analysis of rat’s blood after 5 days of treatment with transdermal ethosomal ibuprofen gel revealed no statistically significant differences between the treated group and the control group with regard to liver, kidney and muscle function parameters.24 Skin tolerability of ethosomal systems on healthy human subjects was assessed by reflectance spectrophotometry in a study carried out by the group of Paolino and Fresta.10 Their results showed that the systems did not induce skin erythema 12, 24 and 48 h after application. In contrast to this, application of hydroethanolic solution with an equal water/ethanol ratio to that of ethosomes resulted in significant skin erythema (Fig. 11).10 Furthermore, application of various ethosomal systems containing clindamycin and salicylic acid, acyclovir or PGE1 to the skin of human volunteers in three clinical studies has not shown adverse skin reactions. Moreover, products formulated with ethosomal carriers have been in use for a number of years, without any reports on skin irritation or safety issues (Fig. 12). 4. CONFLICT OF INTEREST The authors have no conflict of interest. References and Notes 1. E. Touitou, Drug delivery across the skin. Expert Opin. Biol. Ther. 2, 723 (2002). 2. E. Touitou, Compositions for applying active substances to or through the skin, US Patent 5,716,638 (1998). J. Biomed. Nanotechnol. 6, 558–568, 2010 3. E. Touitou, N. Dayan, L. Bergelson, B. Godin, and M. Eliaz, Ethosomes—novel vesicular carriers for enhanced delivery: Characterization and skin penetration properties. J. Controlled Release 65, 403 (2000). 4. N. Dayan and E. Touitou, Carriers for skin delivery of trihexyphenidyl HCl: Ethosomes versus liposomes. Biomaterials 21, 1879 (2000). 5. B. Godin and E. Touitou, Erythromycin ethosomal systems: Physicochemical characterization and enhanced antibacterial activity. Curr. Drug Deliv. 2, 269 (2005). 6. D. Ainbinder and E. Touitou, Testosterone ethosomes for enhanced transdermal delivery. Drug Deliv. 12, 297 (2005). 7. B. Godin and E. Touitou, Mechanism of bacitracin permeation enhancement through the skin and cellular membranes from an ethosomal carrier. J. Controlled Release 94, 365 (2004). 8. B. Godin and E. Touitou, Ethosomes: New prospects in transdermal delivery. Crit. Rev. Ther. Drug Carrier Syst. 20, 63 (2003). 9. D. D. Verma, S. Verma, G. Blume, and A. Fahr, Liposomes increase skin penetration of entrapped and non-entrapped hydrophilic substances into human skin: A skin penetration and confocal laser scanning microscopy study. Eur. J. Pharm. Biopharm. 55, 271 (2003). 10. D. Paolino, G. Lucania, D. Mardente, F. Alhaique, and M. Fresta, Ethosomes for skin delivery of ammonium glycyrrhizinate: In vitro percutaneous permeation through human skin and in vivo antiinflammatory activity on human volunteers. J. Controlled Release 106, 99 (2005). 11. M. Shumilov and E. Touitou, Buspirone transdermal administration for menopausal syndromes, in vitro and in animal model studies. Int. J. Pharm. 387, 26 (2010). 12. E. Touitou, B. Godin, N. Dayan, C. Weiss, A. Piliponsky, and F. Levi-Schaffer, Intracellular delivery mediated by an ethosomal carrier. Biomaterials 22, 3053 (2001). 13. V. Dubey, D. Mishra, T. Dutta, M. Nahar, D. K. Saraf, and N. K. Jain, Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes. J. 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