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We examined uptake of the model therapeutic agent, minoxidil, into appendages, stratum corneum (SC), and through human skin, under the influence of different vehicles. Quantitative estimation of therapeutic drug deposition into all three areas has not previously been reported. Finite doses of minoxidil (2%, w/v) in formulations containing varying amounts of ethanol, propylene glycol (PG), and water (60:20:20, 80:20:0, and 0:80:20 by volume, respectively) were used. Minoxidil in SC (by tape stripping), appendages (by cyanoacrylate casting), and receptor fluid was determined by liquid scintillation counting. At early times (30 min, 2 h), ethanol-containing formulations (60:20:20 and 80:20:0) caused significantly greater minoxidil retention in SC and appendages, compared to the formulation lacking ethanol (0:80:20). A significant increase in minoxidil receptor penetration occurred with the PG-rich 0:80:20 formulation after 12 h. We showed that deposition of minoxidil into appendages, SC, and skin penetration into receptor fluid were similar in magnitude. Transport by the appendageal route is likely to be a key determinant of hair growth promotion by minoxidil. Refereed/Peer-reviewed

Expression plasmids encoding chloramphenicol acetyltransferase (CAT) or human interferon-alpha2 cDNA were formulated in water-in-oil nanoemulsions and applied to murine skin. The histological location of transfected cells was assessed by in situ DNA PCR and showed that the deposition of plasmid DNA was primarily in follicular keratinocytes. Transgene expression in the skin was monitored for 24-72 h, following topical application of either single or multiple daily doses by quantitative RT-PCR and ELISA. It was found that transgene expression was optimal at 24 h following topical application of a single dose of water-in-oil nanoemulsion containing plasmid DNA. Dose-response studies using a total dose of 3, 10 or 30 microg of plasmid DNA suggested that topical transfection using nanoemulsions is subject to both threshold and saturation effects. None of the cationic liposome formulations tested as controls mediated transgenic protein expression at levels higher than background values of the ELISAs used to assay transgenic protein. Single and multiple dose experiments using human interferon-alpha2 as a transgene indicated that the efficiency of nanoemulsion mediated transfection was most effective in the context of normal versus atrophic hair follicles. In addition, the total amount of human interferon-alpha2 present in skin appeared to accumulate as a consequence of multiple dosing. Histologic evaluation of treated skin showed no overt signs of toxicity or irritation associated with the short-term application of the nanoemulsions. The results suggest that water-in-oil nanoemulsions can be used to facilitate transfection of follicular keratinocytes in vivo.

Our previous reports using hairless mouse skin model showed that topical delivery of cyclosporin-A (CsA) could be achieved and controlled with nonionic liposomal and nonionic lipid-based formulations composed of glyceryl dilaurate (GDL), cholesterol (CH), and polyoxyethylene-10-stearyl ether (POE-10) at varying weight ratios (1,2). However, the distribution profiles were obtained using radiolabeled CsA and thus could represent intact drug as well as its metabolites. The present studies aim to investigate if the previously reported profiles actually represented intact CsA. The studies were carried out using a “high-pressure” liquid chromatography (HPLC) method and in vitro skin deposition studies with a metabolic inhibitor (sodium azide) incorporated in the receptor fluid. The results indicated that skin metabolism of CsA and hence its effect on percutaneous absorption of CsA is minimal and that the CsA distribution profiles obtained using radiolabeled CsA represented distribution of intact drug an...

In an earlier report (1) we described the controlled follicular delivery of hydrophobic macromolecules from nonionic lipid-based formulations composed of glyceryl dilaurate (GDL), cholesterol (CH), and polyoxyethylene-10-stearyl ether (POE-10). However, the influence of lipid composition on topical delivery of marginally hydrophobic and hydrophilic drugs from these nonionic lipid-based systems has not been investigated. In this report we describe the effect of variation of GDL to POE-10 ratio in the nonionic lipid-based formulations on the extent and route of delivery of hydrocortisone and mannitol, a marginally hydrophobic and hydrophilic model drug, respectively, into and through hairless mouse skin mounted on Franz diffusion cells. The results indicate that the extent of hydrocortisone uptake increased with increasing GDL to POE-10 weight ratio whereas mannitol uptake was quite the opposite and decreased with increasing GDL to POE-10 weight ratio. The diametrically opposite trends for the two d...

The hair follicle, hair shaft and sebaceous gland collectively form what is recognized as the pilosebaceous unit. This complex, 3-D structure within the skin, possesses a unique biochemistry, metabolism and immunology. Recent studies have focused on the hair follicle as a potential pathway for both localized and systemic drug delivery. Targeted drug delivery may enhance current therapeutic approaches to treating diseases of follicular origin. In a growing number of topical studies, liposomes have been shown to target drug delivery to the pilosebaceous unit. Presented here is a summary of some of our recent work with a particular focus on studies which have demonstrated targeted follicular delivery via liposomes.

In a previous report (1), we showed that the rate and extent of uptake of cyclosporin-A (CsA) following topical application of nonionic liposomal formulations composed of glyceryl dilaurate (GDL), cholesterol (CH), and polyoxyethylene-10-stearyl ether (POE-10) into and through hairless mouse skin mounted on Franz diffusion cells could be controlled by varying the ratios of GDL to POE-10 (CH being held constant at 15 wt%). However, the pathways of transport as well as the dominant factors that control drug delivery from these formulations are not well understood. In this report, we describe results from studies similar in design to that reported earlier but using the melted form of the lipid components as a vehicle for transport of CsA into and through hairless mouse skin. The results suggest that the transport of CsA from liposomal formulations into and through the skin occurs as a result of dehydration of the liposomes followed by melting of the lipid components on the skin. Microautoradiographic...

Expression plasmid DNA for the human interleukin-1 receptor antagonist (IL-1ra) protein was formulated with nonionic:cationic (NC) liposomes or phosphatidylcholine:cationic (PC) liposomes and applied to the auricular skin of hamsters in single- and multiple-dose protocols. Confocal microscopy identified delivery of plasmid DNA proximal to perifollicular cells, and successful transfection of perifollicular cells was identified by immunohistochemistry and ELISA. Skin treated for 3 days with the NC liposomes had statistically significant levels of transgenic IL-1ra present for 5 days post-treatment. Expression of transgenic IL-1ra was specific to areas of skin treated with NC liposomes but not PC liposomes. The results indicate that the NC liposomes can deliver expression plasmid DNA to perifollicular cells and mediate transient transfection in vivo.

Previous studies in our laboratories showed that a novel nonionic liposome formulation composed of glyceryl dilaurate (GDL), cholesterol (CH), and polyoxyethylene-10-stearyl ether (POE-10) at a weight ratio composition of 57:15:28 delivered more cyclosporin-A (CsA) into and through the skin than phospholipid-based liposomal formulations and more conventional formulations that were tested. Since only a single GDL:CH:POE-10 composition was tested, we initiated studies to determine if it would be possible to control the rate and extent of drug uptake by varying the ratios of the liposome-forming components of the formulation. This report describes how the GDL to POE-10 ratio (CH being held constant at 15 wt%) influences the rate and extent of uptake of CsA following topical application of nonionic liposomal formulations to hairless mouse skin mounted on Franz diffusion cells. The results indicate that the rate and extent of CsA uptake is highest between GDL/POE-10 ratios of about 1 to 1.5 and decreas...

Asymmetric liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP2) predominantly on the inner leaflet of the bilayer were prepared by means of a non-penetrating enzyme, phosphoinositide-specific phospholipase C isolated from adult human platelets. Symmetric liposomes prepared by a modification of the reverse-phase evaporation method were incubated with partially purified enzyme in the presence of Ca2+ at 37°C. The resultant liposomes were collected by high-speed centrifugation. Hydrolysis of PIP2 on the outer leaflet of the membrane was completed after approximately 4 h of incubation. Since PIP2 is predominantly located in the inner leaflet of biological membranes, these asymmetric liposomes should be suitable for investigation of structural and functional roles of PIP2 in biomembranes.

The hamster ear has been shown to be an appropriate model for human pilosebaceous units. In this study, we report the deposition of retinoic acid (RA) into pilosebaceous units and other strata of the hamster ear 8 h after topical in vivo application of two hydroalcoholic gel formulations containing RA with and without polyolprepolymer-2 (PP-2). PP-2, a TopiCare Delivery Compound (Penederm, Inc.), consists of a mixture of high-molecular-weight oligomers, ranging in molecular mass from 1700 to 10,000 daltons (average of 4000), that can modify drug delivery by retaining drug on and in the upper layers of the skin. The formulations tested were (A) RA (0.025 wt%) in Penederm Gel with PP-2 and (B) RA (0.025 wt %) in Penederm Gel without PP-2. It was observed that the amount of RA found in the sebaceous glands was roughly fivefold greater from formulation B than from formulation A. Although total RA delivery was less from formulation A, the results suggest that formulation A containing PP-2 may target de...

The physical stability of the four liposomal systems was determined on storage at 4 and 25°C over a 6-month period. A correlation of the mean volume diameter, zeta potential and pH lead to the conclusion that stability follows the order of egg lecithin (PC)/cholesterol (CH)/stearylamine (SA) < PC/CH/phosphatidylserine (PS) < bovine brain ceramides (CM)/CH/palmitic acid (PA)/CS < PC/CH/cholesteryl sulphate (CS) at 4°C, as well as at 25°C, after a 6-month storage period. Large unilamellar vesicles (REV) proved to be superior to multilamellar liposomes (MLV) and dehydration/rehydration liposomes (DRV) systems as far as physical stability was concerned. Instability was exaggerated in the systems stored at 25°C as compared to storage at 4°C.

The significance of the pilosebaceous unit (hair follicle and sebaceous glands) as a route for passive drug delivery into the skin remains a controversial subject. The pilosebaceous unit is a complex, dynamic structure, which is the site of unique biochemical, metabolical and immunological events. Recent topical delivery studies have employed increasingly systematic, quantitative methodologies in attempts to define the follicular pathway. Targeting specific sites of the hair follicle may represent a feasible therapeutic approach, as several dermatological abnormalities are known to originate at the hair follicle. In a growing number of topical delivery studies, liposomes have been shown to target drug delivery to the pilosebaceous unit. This review will describe general aspects of follicular delivery, with a particular focus on studies which have demonstrated targeted follicular delivery via liposomes.

Purpose. Topical delivery has been suggested to reduce systemic side effects while targeting cytokines for the treatment of certain skin conditions. Liposomes have been proposed as an enhancing agent for such a delivery. We have tested the potential of liposomes to augment the uptake of biologically active recombinant human interferon-gamma (rhIFN-γ) into human skin lacking adnexa in an in vivo model.

The results of this study clearly demonstrates the utility of novel non-ionic liposomal systems in facilitating transfer of GHRP-6 into and across deeper strata of skin following topical application. These findings indicate that it may be possible to deliver therapeutic doses of a wide variety of peptides to local skin tissue via topical application. The results also suggest the possibility of controlled enhancement of skin penetration or metered polypeptide deposition through appropriate choice of liposomal lipid components. The pronounced enhancement of GHRP-6 and mannitol transport from emulsions containing the nonionic lipids suggests a promising delivery system for hydrophilic drugs in general.

We have previously established that the relative concentrations of propylene glycol and ethanol as a binary solvent system have a significant effect on the skin penetration of 2% solutions of minoxidil at 50 microL/cm2. The present work extends these studies and investigates the penetration of minoxidil from the different vehicle combinations as functions of application volume and occlusion. Decreasing the application volume has a variable effect which depends on vehicle composition. Penetration of minoxidil from 100% ethanol solutions decreased linearly with application volume. Generally, irrespective of the volume applied, the penetration of minoxidil increased with increasing ethanol fraction with a maximum penetration at 90% ethanol. Penetration from all the formulations was enhanced upon occluding the skin, with greatest increase evident in solutions with higher volatile fraction. Penetration of minoxidil in vivo showed trends similar to those seen in vitro.

The major influence on the rate of drug transfer out of its vehicle and into the skin is the thermodynamic activity of the drug within its formulation. This study addresses certain thermodynamic dependencies of topical delivery in a model system. Prototypical water-in-oil (W/O) and oil-in-water (O/W) emulsions and their component phases are used as the test vehicles, polydimethylsiloxane is the membrane, and three homologous n-alkyl p-aminobenzenzoate esters are the test permeants. In an emulsion, the interaction of the compound between the water and oil phase can determine the extent of lowering of the thermodynamic activity in the external phase in contact with the membrane. The emulsifiers (surfactants) impact strongly on partitioning and permeation as a result of the extra solubilizing capacity contributed by the surfactant micelles. The lower flux in the aqueous phase of the O/W emulsion is the result of micellar solubilization, and this solubilization increased with increasing ester chain length. Solubilization is also an influence in nonaqueous phases, but permeant hydrophobicity is without specific influence; therefore, transport become less dependent upon the structure of the compound.

The hair follicle, hair shaft, and sebaceous gland collectively form what is recognized as the pilosebaceous unit. This complex, three-dimensional structure within the skin possesses a unique biochemistry, metabolism and immunology. Recent studies have focused on the hair follicle as a potential pathway for both localized and systemic drug delivery. Greater understanding of the structure and function of the hair follicle may facilitate rational design of drug formulations to target follicular delivery. Targeted drug delivery may enhance current therapeutic approaches to treating diseases of follicular origin. Presented here is a review of follicular drug delivery and a discussion of the feasibility of the pilosebaceous unit as a target site.

The deposition of a-interferon into living skin strata of hairless mouse following topical in vivo application of a glyceryl dilaurate: POE-10: cholesterol liposomal formulation was much higher than from a glyceryl distearate: POE-10: cholesterol liposomal formulation or from an aqueous solution. The kinetics and extent of drug deposition from the liposomal formulations appear to correspond to the extent of release of bilayer lipid components following topical application and subsequent action of the lipid components as skin permeation enhancers.

Purpose. The purpose of this study was to test the hypothesis that nonionic liposomes facilitate the topical delivery of peptide drugs into pilosebaceous units. Methods. The hamster ear was used as a model for human pilosebaceous units. The deposition of a hydrophilic protein, alpha-interferon (α-IFN), into pilosebaceous units and other strata of the hamster ear 12 hours after topical in vivo application of three nonionic liposomal formulations, one composed of glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether (Non-1), the second composed of glyceryl distearate/cholesterol/ polyoxyethylene-10-stearyl ether (Non-2) and the third composed of polyoxyethylene-10-stearyl ether/cholesterol (Non-3), a phospho-lipid-based liposomal formulation (PC) and an aqueous control solution (AQ) was determined. We also determined the deposition of a hydrophobic peptide, cyclosporin-A (CsA), into pilosebaceous units and other strata of the hamster ear after topical in vivo application of these liposomal formulations and a hydroalcoholic control solution (HA). Results. The deposition of α-IFN into the pilosebaceous units was in the order: Non-1 ≫ PC > Non-2 > Non-3 = AQ. The deposition of CsA into the pilosebaceous units was in the order: Non-1 ≫ HA > PC > Non-2 = Non-3. Conclusions. Despite differences in the hydrophobicities and size of the drug molecules, deposition into the various ear strata was significantly enhanced by the Non-1 liposomal system.

There are numerous kinetic and thermodynamic processes which occur during the transport of a topically applied drug from within its vehicle to the site of action in the skin. The present study compares the release and permeation of a model compound, ethyl p-aminobenzoate, from water, mineral oil, a mineral oil in water (O/W) emulsion, and a water in mineral oil (W/O) emulsion. These formulations are compared for differences in transport characteristics using in vitro diffusion studies and a synthetic polydimethylsiloxane membrane. Two factors appear prominent in influencing the release kinetics of the drug from its formulation. The first is the observation that the drug is released 2 times slower from mineral oil than from water at approximately equivalent thermodynamic activities, and the second is the appreciable lowering of the thermodynamic activity as a result of substantial micellar solubilization of the drug in the aqueous phase of the O/W emulsion. The thermodynamic activity effect predominants, resulting in a lower release rate from the O/W emulsion as compared to the W/O emulsion.

The delivery of active agents to the skin by liposome carriers is an interdisciplinary topic of great interest today. Data accumulated over the last decade strongly point to important advantages of these drug delivery systems. A symposium devoted to classic and new approaches in the use of liposomal systems was organized and chaired by M. Mezei and E. Touitou as a part of the Jerusalem Conference on Pharmaceutical Sciences and Clinical Pharmacology, held on May 24–30, 1992, in Jerusalem, Israel. The presentations focused on liposomes as tools in the mechanistic study of absorption promoters (T. Nagai), drug liposomal delivery in the skin strata and structures (N. Weiner), interaction of liposomes and niosomes with the human skin (H. E. Junginger), and design and characterization of caffeine liposomal systems for use in hyperproliferative diseases (E. Touitou). Mezei reviewed biodisposition and clinical studies on liposomal dosage forms containing various drugs.

The effect of particle size of liposomes on the deposition of drugs into the strata of skin was evaluated using hairless mice, hamster and pig skin. In vitro diffusion studies were performed in an attempt to find an optimum formulation for topical drug delivery as well as to try to explain the mechanism of topical drug delivery by liposomes. The results indicate that an optimum particle size for optimal drug delivery exists. The study proved that the follicular route play an important role in determining the kinetics of drug transfer from liposomes into the skin.

Hairless mice were treated in vivo with empty phospholipid and ‘skin lipid’ liposomes, respectively. The deposition of a hydrophobic drug (hydrocortisone) and a hydrophilic drug (inulin) was investigated in both treated and untreated skin. It was found that drugs should be encapsulated in liposomes or at least be administered together with the lipid, to achieve targeted drug delivery. Pretreatment of the skin with liposomes did not show the advantage of liposomally encapsulated drugs.

This review will highlight work done in our laboratories evaluating topical liposomal delivery in a wide variety of animal models and human skin using both in vivo and in vitro techniques. The mechanism by which liposomes facilitate deposition of drugs into the skin and some potential applications of topically applied liposomes will be discussed. Particular emphasis will be placed on the development of analytical techniques that allow the quantification of drug levels in the various skin strata and in pilosebaceous structures. Appropriate models of skin representing the two cases, as well as those wherein both routes are available for skin deposition, were examined using a wide variety of liposomal preparations containing radiolabeled and fluorescent molecules as marker compounds.

The effect of formulation on cimetidine delivery to the pilosebaceous unit and other skin phases was studied. In vitro and in vivo deposition determinations as well as a pharmacodynamic antiandrogenic sebaceous gland bioassays were made. A complex variety of factors influence how the formulation affects both the degree of drug deposition and its pharmacological activity in the pilosebaceous unit. When cimetidine was applied in formulations at pH values where it was predominately unionized, the thermodynamic driving force proved the dominant factor in influencing the extent of drug deposition into the pilosebaceous unit. Although more cimetidine was deposited into the pilosebaceous unit in vivo from the phospho lipid-based liposomal formulation when cimetidine was ionized, this formulation was also the only one devoid of significant antiandro genic action. Of great importance, it is clear from the studies that deposition from complex formulations, such as liposomes, where bilayer/drug interactions can persist in the skin, may give a false impression of the activity of a drug within a tissue. Moreover, data for cimetidine in 50% alcohol solution show that one can maintain local effects while reducing systemic activity by simply manipulating drug concentration in the application.

The effect of viscoelastic properties on mucociliary transport rate was investigated using the frog palate ciliated model. Mucociliary transportability of several hydrophilic polymeric gels with widely different viscoelastic characteristics were tested on the frog palate mucociliary model. An apparent negative relationship is observed between the relative transport rate (TR) and storage (G1) or loss (G2) modulus. However, a minimum in relative transport rate is observed at an apparent loss tangent (tan delta) value of between 0.7 and 0.9. A theoretical model for mucociliary transport is presented. The model predicted a minimum in transport rate at tan delta equal to 1.74 after adjustment for primary variation due to storage modulus (G1), which is in agreement with the observed frog palate transport rate. The model isolates the loss tangent (tan delta) and the magnitude of the complex modulus (magnitude of G*) as the important viscoelastic parameters for mucociliary transport. Optimum rheological characteristics with respect to slow transport rate can be achieved by using hydrophilic polymer gels with a large complex modulus and simultaneously with a loss tangent equal to 1.74.

The cutaneous bioavailability of topical 2% minoxidil solution was verified in live hairless mice. Minoxidil and propy-lene glycol deposition on the skin surface, epidermis and der-mis from the single-dose in vivo study were compared with the results from previous in vitro studies. A distinct difference is apparent in the epidermis where the in vitro values are 11–22 times higher than the in vivo values for minoxidil and 8–16 times higher for propylene glycol. The differences were not as great in the dermis. Percutaneous absorption of the drug appeared to be a very small fraction of the applied dose. Similarly shaped stratum corneum and plasma concentration profiles and the relatively constant dermal profiles of minoxidil and propylene glycol open the possibility of transappendageal routes being involved in percutaneous absorption. The greater amount of drug and vehicle found in the dermis from in vitro studies can be explained by the absence of dermal clearance. The overestimation in the amount of drug found in the epidermis in vitro may also be attributable to poor dermal clearance. On the whole, the study raises questions about the use of in vitro tissue dispositions for bioavailability assessment and bioequivalence demonstration.

Summary Minoxidil dissolved in propylene glycol/ethanol/water (20: 60: 20; v/v) was more efficiently delivered into hairless mouse skin from a 0.02% solution than from a 2% formulation during in vitro mass balance experiments. Evaporation of the vehicle after application of the 2% minoxidil solution led to compositional changes so drastic that saturation is attained quickly. The shifting compositions of the vehicle were experimentally profiled and the drug’s solubility was measured in media reflecting these changes over time. The original 2% formulation has excess capacity to dissolve minoxidil but, following its application, the solution rapidly saturates through evaporation, reaching saturation within 30 minutes. While the 2% formulation definitely functions at a proportionally higher thermodynamic activity than the 0.02% formulation at early times, most of this thermodynamic advantage is lost abruptly upon precipitation of the drug. The driving force following drug precipitation only reflects the dissolved minoxidil, and thus the relative delivery rate of the 2% formulation eventually drops to only several multiples of the delivery rate of the 0.02% solution which never quite attains saturation.

The compatibility of cyclosporine with fat emulsion was studied. Admixtures consisted of cyclosporine injection at final drug concentrations of 0.5 and 2.0 mg/mL in 10% and 20% fat emulsion. Fat emulsion stability was determined by visual inspection, pH testing, and particle-size measurements at 0, 24, and 48 hours. Cyclosporine stability was measured with high-performance liquid chromatography at the same intervals. The admixtures were stored in glass containers at 23-25 degrees C under fluorescent light. Mean particle diameters remained relatively constant in all the admixtures throughout the study period. No particles larger than 6 microns were observed. Cyclosporine retained more than 96% of its initial concentration in all the admixtures. There were no changes in color or consistency, and pH did not vary by more than 0.11 pH unit. Cyclosporine 0.5 or 2 mg/mL was compatible with 10% or 20% fat emulsion at room temperature in glass containers for up to 48 hours.

The influence of formulation variables on the rheology of polyanionic formulations and the relationships between viscoelastic properties and mucociliary transport rate were investigated. Polymeric samples were oscillated from 0.001 to 5 Hz using either a "cone and plate” or a "coaxial cylinder” measuring system. The mucociliary transport rates of polymeric samples were determined and compared movement of charcoal powder on the frog palate. For the linear polymeric solutions, sodium carboxymethylcellulose and sodium alginate, the elastic modulus (G′) increased with increasing amplitudes during frequency scan. However, the G′ or viscous modulus (G″) of partially cross-linked polyacrylic acid (cPAA) samples did not change significantly under oscillation. Both G′ and G″ of cPAA samples were significantly influenced by the amount of salt present in the formulation. The rheology of 2% (w/w) cPAA in 90:10 (w/w) propylene glycol:alcohol changed from a viscous fluid to a coarse suspension after neutralization. The pH increased gradually when the nonaqueous formulation reacted with water and the maximum dynamic moduli were obtained after incorporating 20% (w/w) water in the formulation. A negative correlation was found between the G′ of linear polyanionic samples and the relative transport rate. However, the lowest mucociliary transport rate was observed when the loss tangent (G″/G′) was around 0.4–0.5.

The delivery of active agents to the skin by liposome carriers is an interdisciplinary topic of great interest today. Data accumulated over the last decade strongly point to important advantages of these drug delivery systems. A symposium devoted to classic and new approaches in the use of liposomal systems was organized and chaired by M. Mezei and E. Touitou as a part of the Jerusalem Conference on Pharmaceutical Sciences and Clinical Pharmacology, held on May 24–30, 1992, in Jerusalem, Israel. The presentations focused on liposomes as tools in the mechanistic study of absorption promoters (T. Nagai), drug liposomal delivery in the skin strata and structures (N. Weiner), interaction of liposomes and niosomes with the human skin (H. E. Junginger), and design and characterization of caffeine liposomal systems for use in hyperproliferative diseases (E. Touitou). Mezei reviewed biodisposition and clinical studies on liposomal dosage forms containing various drugs.

Evidence suggesting liposomal delivery into the pilosebaceous unit of the male Syrian hamster ear membrane was found using two fluorescent techniques, quantitative fluorescence microscopy (QFM), and a scraping method where the various tissue strata of treated skin are analyzed using fluorescence spectrophotometry. Whole ears were mounted on Franz diffusion cells and treated for 24 h with 40 microliters of the following test formulations, each containing approximately 100 micrograms/ml carboxyfluorescein (CF): i) multilamellar phosphatidylcholine: cholesterol: phosphatidylserine liposomes; ii) HEPES buffer (pH, 7.4); iii) 5% propylene glycol; iv) 10% ethanol; v) 0.05% sodium lauryl sulfate; and vi) a suspension of the same lipids used to form the liposomes that were not processed so as to produce a bilayer configuration. Topical application of the liposomally based formulation resulted in a significantly higher accumulation of CF in the pilosebaceous units than the application of any of the other non-liposomal formulations. There was excellent correlation between the two analytical methods used to determine CF deposition into the sebaceous glands.

The extent of uptake of gamma interferon (gamma-IFN) in various strata of hairless mouse, human and hamster skin upon application of a liposomal formulation and an aqueous solution were determined by in vitro diffusion cell experiments. For each of the animal species studied, 70-80% of the liposomally entrapped IFN was deposited onto or penetrated into the skin as determined 24 h after in vitro application. However, a significant fraction of this total amount (approximately 0.25-0.30) is either adsorbed to or associated with the stratum corneum. The drug content found in the deeper skin strata, where the receptor sites reside, suggests that drug deposition is strongly influenced by the skin species tested. The percent of applied drug found in this strata 24 h after application followed the order: hamster (6.1) much greater than human (0.9) greater than hairless mouse (0.3), although the amounts of drug in the total skin of each species tested were approximately the same. This indicates that the deposition of drug into the living epidermis and/or dermis cannot be predicted by determination of the amount of drug in the total skin. The amounts in the deeper skin strata were also in the order of increasing number of follicles/hair in the skin species, suggesting that the transfollicular route is an important pathway for liposomal topical therapeutics.

The partitioning of a homologous series of n -alkyl p -aminobenzoales into the lipid bilayer is dependent not only on their physicochemical properties but also on liposome type. Partitioning studies with this homologous series demonstrate the effect of alkyl chain length on partitioning in DPPC multilamellar liposomes (MLV) and DPPC MLV extruded through 1or 0.4 μm polycarbonate filters. A general trend was observed wherein increasing alkyl chain length increased partitioning of drug into the bilayer. The distribution coefficients of the esters were found to be strongly influenced by the physical structure of the membrane. Factors which decrease the surface density of the bilayer, such as temperature, will also increase solute partitioning into the bilayer. The amounts of the alkyl p -aminobenzoates included in the bilayers of DPPC multilamellar liposomes at 23° C were quite low because the high surface density of the bilayer at 23° C precludes the incorporation of solute. The extruded liposomes studied were much smaller and are believed to have greater bilayer fluidity and disorder than their MLV counterparts, thus allowing greater incorporation of the alkyl p -aminobenzoates in the extruded DPPC bilayer. Hence, higher distribution coefficients and lower free energies of partitioning are achieved with extruded liposomes. The effect of extrusion on solute partitioning was found to be similar to the effects caused by increasing temperature.

The effectiveness of three adhesive tapes, 3M's No. 810, No. 845 and No. 850, in stripping stratum corneum from hairless mouse skin, was compared by measuring transdermal water loss (TEWL) and distribution profiles of a marker, minoxidil, from a solution formulation. While No. 850 tape failed to increase TEWL perceptively even after 24 strippings, both No. 810 and 845 tapes effectively removed the whole stratum corneum, at least as reflected in TEWL measurements. TEWL specifically increased incrementally from somewhere between 4 and 8 g/m 2 per h to 120 g/m 2 per h. From both TEWL measurements and distribution profiles of minoxidil in skin 2 h post-application of its formulation, it appears that the 3M No. 845 tape more efficiently removes stratum corneum than 3M No. 810 tape. The difference between No. 810 and No. 845 tapes in stripping formulation-treated skin held in a diffusion cell was application-time dependent, since the No. 845 tape harvested no more minoxidil than the No. 810 tape at 12 and 24 h following application of the formulation.

The partitioning of the n -alkyl- p -aminobenzoates into the lipid bilayer is dependent not only on their physicochemical properties, but also on temperature and bilayer composition. Partitioning studies with this homologous series demonstrate the effect of alkyl chain length on partitioning in pure DPPC liposomes at 23 and 50 °C and in DPPC: Chol liposomes at 23 °C. A general trend is observed wherein increasing alkyl chain length increases partitioning of the drug into the bilayer. It is also shown that inclusion of cholesterol in the bilayer or increasing the temperature increases the partitioning of the n -alkyl p -aminobenzoates into the bilayer. The distribution coefficients are strongly influenced by the physical structure/state of the membrane. Therefore, factors which increase the fluidity of the bilayer will also increase solute partitioning into the bilayer. Compounds that are highly lipid soluble, such as butyl p -aminobenzoate, will preferentially partition into the bilayer, and are relatively insensitive to bilayer composition or to structural changes within the bilayer.

125 μl of a propylene glycol/ethanol/water vehicle were applied for various lengths of time to 1.77 cm 2 area of excised hairless mouse skin sections held in in vitro diffusion cells. After removal from the cell, each skin section was stripped repeatedly with a non-hygroscopic polypropylene tape. The amount of tissue removed in each strip was determined after allowing the volatile solvents to evaporate. Weights were corrected for residual propylene glycol and water, the amounts of which were determined radioisotopically. More tissue was harvested in the first and second strips from skin conditioned with the vehicle for more than 12 h. The effect of vehicle treatment on stripping properties precludes one from determining drug and vehicle concentration gradients in the stratum corneum at different treatment times by direct comparison of corresponding strips. While it does not appear that a penetrant's deposition as a function of time can be followed easily and directly by stripping and then quantifying the drug (or solvent) in the respective layers, the stripping technique may still be useful in separating stratum corneum (or epidermis) from dermis.

The objective of the present study is to apply response surface methodology to the design and analysis of composite experiments containing independent covariate(s). The approach is illustrated here by the study of viscosity characteristics of a polymeric mucoadhesive formulation in multicomponent solvent vehicles. The nonaqueous formulation will produce a gel network with significant rheological change when in contact with body fluids. The process of water inclusion will induce not only solvent compositional change of the mixture but also concomitant dilution of the polymer concentration. To study the viscosity change over the solvent compositions and polymeric concentrations of interest, an experimental design is utilized consisting of a 10-point simplex-centroid lattice augmented with three interior points at each polymeric concentration. The contour patterns are compared with the experimental data using the variance and lack of fit, starting with the Scheffe linear model and building up to the full cubic model including the covariate terms. The fitted model provides information needed to predict optimum formulations, i.e., initial viscosity of less than 100 cP, but yielding rheological profiles commensurate with high degrees of substantivity when diluted with water. For illustrative purposes, the Carbopol resins neutralized with a 1:1 molar equivalent ratio of triethanolamine in three primary solvents, propylene glycol, glycerol formal, and water, were chosen for this study.

This report describes a novel nonaqueous polymeric formulation that exhibits low-viscosity fluid behavior for ease of spraying with conventional nebulizer, which when sprayed into the nasal cavity, transforms to a high-viscosity gel for efficient retention and drug absorption. The transformation occurs because of the rheological changes induced by a change in the solvent composition of the polymeric formulation in the moist nasal cavity. Such a rheological change would then facilitate enhanced residence time of the drug at the site of administration in order to avoid drainage losses. This study reports the results of the effects of a variety of factors such as solvent composition and polymer concentration on the rheological properties of a polyacrylic acid polymer. An attempt to correlate viscosity enhancement effects with enhanced and sustained-release behavior of propranolol, a drug that undergoes extensive first-pass effects, from such formulations via nasal administration in beagle dogs is also described.

In the past decade, liposomal formulations have been extensively employed to enhance the efficiency of drug delivery via several routes of administration. In a number of instances, liposomal drug formulations have been shown to be markedly superior to conventional dosage forms, especially for intravenous and topical modes of administration of drugs. The major advantages of topical liposomal drug formulations accrue from their demonstrated ability: (i) to reduce serious side effects and incompatibilities that may arise from undesirably high systemic absorption of drug; (ii) to enhance significantly the accumulation of drug at the site of administration as a result of the high substantivity of liposomes with biological membranes; and (iii) to readily incorporate a wide variety of hydrophilic and hydrophobic drugs. Liposomes are also non-toxic, biodegradable and are readily prepared on a large scale. This paper presents a review of topically applied liposomal formulations with emphasis on the evaluation of liposomal systems in a wide variety of animal models and human skin using both in-vivo and in-vitro techniques. The mechanism by which liposomes facilitate deposition of drugs into the skin and potential applications of topically applied liposomes are discussed.

Stainless-steel templates of various thicknesses (75, 200, 800, and 1600 µm) were used to apply propylene glycol/water gels containing methyl or propyl p-aminobenzoates to silicone rubber membranes, and drug delivery was studied with the use of the Bronaugh diffusion cell under conditions in which the drug was initially in thermodynamic equilibrium with respect to the application and membrane. Theoretical diffusion profiles were generated with the use of a model which assumes that diffusional gradients exist within the application. To use the model equation, previously derived for the initial condition in which the drug is in thermodynamic equilibrium with respect to the application and membrane, drug diffusivity in both the application and the membrane and the drug's membrane/ vehicle partition coefficient were independently determined. In general, agreement between experimental and theoretical results was within 25%.

Topical products containing erythromycin, a macrolide antibiotic with poor aqueous solubility, are usually formulated as high alcohol content solutions or gels. In this study, we evaluated the deposition of erythromycin base into various strata of hairless mouse skin following topical in vivo application from various low- and nonalcoholic formulations. The formulations tested included nonionic liposomal formulation composed of glyceryl dilaurate, cholesterol, and polyoxyethylene-10-stearyl either at a weight ratio of 57:15:28, two nonionic oil-in-water (o/w) liposomal emulsions containing isopropyl myristate or light mineral oil as the oil phase, a conventional o/w emulsion, a 40% hydroalcoholic solution, and two commercially available topical products. Eight hours after topical administration of these formulations, the efficiency of uptake of erythromycin into the living skin strata was in the order: liposomal isopropyl myristate emulsionliposomal mineral oil emulsionnonionic liposomes approximately Emgel approximately Theramycin-Zconventional emulsionhydroalcoholic solution. Alcohol-free liposomal systems are shown to be as efficient as high alcohol content products in facilitating permeation of erythromycin through the stratum corneum into living skin tissue.

Traditionally, the prime pathway for the topical delivery of active agents across the skin was thought to be through intercellular routes and transcellular routes of the stratum corneum. However, alternative means such as via appenageal transport, i.e., follicular transport, is gaining more acceptances in the scientific community. Targeting specific sites of the hair follicle may represent a feasible therapeutic approach to skin diseases such as hair loss. It is therefore an object of this research to develop novel liposomal formulations for enabling the topical delivery of difficult-to-absorb agents for localized action, specifically to the hair follicles and sebaceous glands. We examined small and large molecules. The small molecule chosen was minoxidil, a known hair growth stimulator. The large molecular weight molecule was plasmid DNA encoded with interleukin-1 receptor antagonist protein (IL-1ra).

A variety of water-in-oil nanoemulsions were prepared using sorbitan monooleate (Span80), polyoxyethylene 20 sorbitan monooleate (Tween80), olive oil and water. The nanoemulsions were tested for their ability to facilitate transport of a model hydrophilic solute, inulin, across hairless and hairy mouse skin and hairy rat skin following topical in vitro application. The transport of inulin incorporated in water-in-oil nanoemulsions was found to be significantly higher (5- to 15-fold) than that obtained with micellar dispersions or aqueous controls. The rate and extent of inulin transport across hairy mouse skin was found to be highly dependent on the hydrophile-lipophile balance (HLB) of the surfactant mixture in the nanoemulsion. Nanoemuslions prepared using mixtures with lower HLB exhibited significantly higher rate and extent of transport. It was also found that nanoemulsion-mediated transport was independent of molecular size of the hydrophilic solute and the nature of the aqueous phase. More importantly, transport of inulin from nanoemulsions was independent of animal skin characteristics such as stratum corneum thickness and follicle-type. The combined results suggest that water-in-oil nanoemulsions that are compatible with the lipophilic sebum environment of the hair follicle facilitate efficient transport of incorporated hydrophilic solutes and imply that such transport is predominantly transfollicular in nature.

In this study a general description of the use of solid support membranes as the device for DNA delivery mediated by PAMAM dendrimers is presented. In contrast to the other DNA carriers, dendrimer/DNA complexes retain the ability to transfect after drying, which enabled coating or incorporation of complexes into poly(DL-lactide-co-glycolide) or collagen-based bioerodable membranes. These studies provide support for the use of this technology for in vitro and in vivo transfection of skin cells. Expression of luciferase or green fluorescent protein from pCF1-Luc and pEGFP1 plasmids indicated that dendrimer/DNA complexes can mediate transfection after dissociation from the solid support and/or when retained on the surface of the membranes. Modification of the membranes by incorporation of an anionic lipid, phosphatidyl glycerol (PG) at 1-5% concentrations, resulted in more efficient in situ transfection, particularly with dendrimer/DNA complexes formed at the low charge ratios (1-5). We also report data supporting the feasibility of membrane-based dendrimer/DNA complexes, particularly formed at lower than neutralizing conditions, for topical in vivo delivery of DNA to hairless mouse skin.