Treatments for androgenetic alopecia and alopecia areata: current options and future prospects.

Androgenetic alopecia and alopecia areata are common disorders of the hair follicle which may heavily influence self esteem and self image. Androgenetic alopecia is caused by the heightened sensitivity of scalp follicles to dihydro- testosterone whereas alopecia areata is induced by an autoimmune reaction. Current drug treatment approaches include the use of regrowth stimulators such as topical minoxidil and oral finasteride for androgenetic alopecia, as well as topical minoxidil, dithranol (anthralin), corticosteroids, contact sensitisers, and psoralen plus ultraviolet A irradiation (PUVA) therapy for alopecia areata. Combination regimens are also proposed. However, extreme cases of either type of alopecia do not generally respond well to these existing treatments. For this reason, new therapeutic strategies are directed towards both improving the targeting of existing agents, as well as the development of novel hypertrichotic modalities.

Carriers for skin delivery of trihexyphenidyl HCl: ethosomes vs. liposomes.

The purpose of this work was to characterize a novel ethosomal carrier containing trihexyphenidyl HCl (THP) and to investigate the delivery of THP from ethosomes versus classic liposomes. THP-ethosomal systems were shown by electron microscopy to contain small, phospholipid vesicles. As the THP concentration was increased from 0 to 3%, the size of the vesicles decreased from 154 to 90 nm. This is most likely due to the surface activity of THP (critical micelle concentration of 5.9 mg/ml), as measured in this work. In addition, the ethosome zeta potential value increased as a function of THP concentration, from -4.5 to +10.4 when the THP concentration was increased from 0 to 3%. In contrast, THP liposomes were much larger and their charge was not affected by THP. When compared with standard liposomes, ethosomes had a higher entrapment capacity and a greater ability to deliver entrapped fluorescent probe to the deeper layers of skin. The flux of THP through nude mouse skin from THP ethosomes (0.21 mg/cm2 h) was 87, 51 and 4.5 times higher than from liposomes, phosphate buffer and hydroethanolic solution, respectively (p < 0.01). The quantity of THP remaining in the skin at the end of the 18-h experiment was statistically significantly greater from the ethosomal system than from liposomes or a control hydroethanolic solution. Our results indicate that the ethosomal THP system may be a promising candidate for transdermal delivery of THP.

Intracellular delivery mediated by an ethosomal carrier.

The goal of this work was to investigate the efficiency of transcellular delivery into Swiss albino mice 3T3 fibroblasts of molecules with various physico-chemical characteristics from ethosomes, phospholipid vesicular carriers containing ethanol. The probes chosen were: 4-(4-diethylamino) styryl-N-methylpyridinium iodide (D-289), rhodamine red dihexadecanoylglycerophosphoethanolamine (RR) and fluorescent phosphatidylcholine (PC*). The penetration of these fluorescent probes into fibroblasts and nude mice skin was examined by CLSM and FACS. CLSM micrographs showed that ethosomes facilitated the penetration of all probes into the cells, as evident from the high-intensity fluorescence. In comparison, when incorporated in hydroethanolic solution or classic liposomes, almost no fluorescence was detected. The intracellular presence of each of the three probes tested, was evident after 3 min of incubation. Furthermore, with ethosomal D-289, fluorescence was also seen in the fibroblast nucleus. Enhanced delivery of molecules from the ethosomal carrier was also observed in permeation experiments with the hydrophilic calcein and lypophilic RR to whole nude mouse skin. Calcein penetrated the skin to a depth of 160, 80 and 60 microm from ethosomes, hydroethanolic solution and liposomes, respectively. Maximum fluorescence intensities measured for RR delivered from ethosomes, hydroethanolic solution and liposomes were 150, 40 and 20 AU, respectively. Fibroblast viability tests showed that the ethosomal carrier is not toxic to the cultured cells.

Mechanism of bacitracin permeation enhancement through the skin and cellular membranes from an ethosomal carrier.

The main objective of the present work was to investigate the dermal and intracellular delivery of bacitracin, a model polypeptide antibiotic, from ethosomes. Bacitracin and fluorescently labeled bacitracin (FITC-Bac) ethosomes were characterized for shape, lamellarity, fluidity, size distribution and entrapment capacity by scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), dynamic light scattering (DLS) and ultracentrifugation, respectively. Confocal laser scanning microscopy (CLSM) experiments revealed that ethosomes facilitated the copenetration of antibiotic and phospholipid into cultured 3T3 Swiss albino mice fibroblasts. These results, confirmed by data obtained in fluorescent-activated cell sorting (FACS) experiments, suggest that ethosomes penetrate cellular membrane releasing the entrapped molecule within cells. Additional work was focused on skin permeation behavior of FITC-Bac from ethosomal systems in in vitro and in vivo experiments through human cadaver and rat skin, respectively. These studies demonstrated that the antibiotic peptide was delivered into deep skin layers through intercorneocyte lipid domain of stratum corneum (SC). Occlusion had no effect on the permeation profile of the drug from ethosomes in in vitro experiments. Efficient delivery of antibiotics to deep skin strata from ethosomal applications could be highly beneficial, reducing possible side effects and other drawbacks associated with systemic treatment. Furthermore, ethosomal delivery systems could be considered for the treatment of a number of dermal infections, requiring intracellular delivery of antibiotics, whereby the drug must bypass two barriers: the SC and the cell membrane.

Methods for quantitative determination of drug localized in the skin.

The quantification of drugs within the skin is essential for topical and transdermal delivery research. Over the last two decades, horizontal sectioning, consisting of both tape stripping and parallel slicing through the deeper tissues has constituted the traditional investigative technique. In recent years, this methodology has been augmented by such procedures as heat separation, qualitative autoradiography, isolation of the pilosebaceous units and the use of induced follicle-free skin. The development of skin quantitative autoradiography represents an entirely novel approach which permits quantification and visualization of the penetrant throughout a vertical cross-section of skin. Noninvasive strategies involve the application of optical measuring systems such as attenuated total reflectance Fourier transform infrared, fluorescence, remittance or photothermal spectroscopies.

Oleic acid, a skin penetration enhancer, affects Langerhans cells and corneocytes.

Permeation enhancers (PE) are frequently used in the field of dermal research and for the development of transdermal delivery products. However, their influence on skin epidermal Langerhans cells (LC) has not yet been investigated. In this work we studied the effect of four PE, oleic acid (OA), propylene glycol (PG), ethanol, and diethylene glycol monoethyl ether (DGME), and an iontophoretic treatment on the morphometric parameters of epidermal Langerhans cells (LC). Retinoic acid (RA) was used as a positive control. Test solutions were applied to the footpad of Sabra mice. The area, perimeter, density and shape factor (SF) were the morphometric parameters evaluated following ATPase staining of LC. Application of RA led to a large decrease in cell density (-50.2%, P<0.01) and dendritic shape (19.8%, P<0.01). Treatment with 10% OA in ethanolic solution caused a severe decrease in LC density (-69.0%, P<0.01), accompanied by a decrease in dendricity as measured by the changes in SF. Ethanol had no statistically significant effect on the LC morphologic parameters tested. All other PE had a mild, if any, effect on LC morphology. SEM micrographs of the skin of IOPS hairless rats demonstrated that 24 h in vivo treatment with 10% OA in ethanolic solution resulted in the generation of pores on the surface of epidermal corneocytes.

Ethosomes - novel vesicular carriers for enhanced delivery: characterization and skin penetration properties.

This work describes a novel carrier for enhanced skin delivery, the ethosomal system, which is composed of phospholipid, ethanol and water. Ethosomal systems were much more efficient at delivering a fluorescent probe to the skin in terms of quantity and depth, than either liposomes or hydroalcoholic solution. The ethosomal system dramatically enhanced the skin permeation of minoxidil in vitro compared with either ethanolic or hydroethanolic solution or phospholipid ethanolic micellar solution of minoxidil. In addition, the transdermal delivery of testosterone from an ethosomal patch was greater both in vitro and in vivo than from commercially available patches. Skin permeation of ethosomal components, ethanol and phospholipid, was demonstrated in diffusion-cell experiments. Ethosomal systems composed of soy phosphatidylcholine 2%, ethanol 30% and water were shown by electron microscopy to contain multilamellar vesicles. 31P-NMR studies confirmed the bilayer configuration of the lipids. Calorimetry and fluorescence measurements suggested that the vesicular bilayers are flexible, having a relatively low T(m) and fluorescence anisotropy compared with liposomes obtained in the absence of ethanol. Dynamic light scattering measurements indicated that ethanol imparted a negative charge to the vesicles. The average vesicle size, as measured by dynamic light scattering, was modulated by altering the ethosome composition. Experiments using fluorescent probes and ultracentrifugation showed that the ethosomes had a high entrapment capacity for molecules of various lyophilicities.

Cannabidiol-transdermal delivery and anti-inflammatory effect in a murine model.

Cannabidiol (CBD) is a new drug candidate for treatment of rheumatic diseases. However, its oral administration is associated with a number of drawbacks. The objective of this study was to design a transdermal delivery system for CBD by using ethosomal carriers. CBD ethosomes were characterized by transmission electron microscopy, confocal laser scanning microscopy and differential scanning calorimetry. Results indicated that CBD and phosphatidylcholine form an eutectic mixture. In vivo application of ethosomal CBD to CDI nude mice produced a significant accumulation of the drug in the skin and in the underlying muscle. Upon transdermal application of the ethosomal system to the abdomen of ICR mice for 72 h, steady-state levels were reached at about 24 h and lasted at least until the end of the experiment, at 72 h. Furthermore, transdermal application of ethosomal CBD prevented the inflammation and edema induced by sub-plantar injection of carrageenan in the same animal model. In conclusion, ethosomes enable CBD's skin permeation and its accumulation in a depot at levels that demonstrate the potential of transdermal CBD to be used as an anti-inflammatory treatment.

Activation and inhibition of mast cells degranulation affect their morphometric parameters.

Activation of mast cells, the key cells of allergic inflammation, causes typical morphological changes associated with an increase in volume, that is a function of area and perimeter. The purpose of this study was to evaluate the effect of mast cell activation to degranulate, carried out by the secretagogue Compound 48/80, and of inhibition of this activation carried out by Nedocromil sodium, a mast cell stabilizing drug, on mast cell area, perimeter and shape factor by a computerized image analyzer. Mast cells were isolated and purified by peritoneal lavage of rats (purity >98%) and co-cultured with mouse 3T3 fibroblasts to which they adhere. Cultures were incubated for 10 min at 37 degrees C with culture medium alone (Enriched Medium) or Enriched Medium containing either Nedocromil (10(-4) M) or Compound 48/80 (0.3 microg/ml) or Compound 48/80 and Nedocromil (0.3 microg/ml and 10(-4) M respectively). Supernatants were then assessed for histamine release, as a marker of mast cell activation and the cell monolayers were fixed and stained with an alcoholic-acidic toluidine blue solution and examined with a computerized image analyzer connected with a light microscope. Mast cells incubated in Enriched Medium or Nedocromil possessed similar morphometric parameters. Mast cells activated with Compound 48/80 (70% histamine release) had a significant increase in area and perimeter and a decrease in shape factor in comparison to mast cells in Enriched Medium alone. Simultaneous incubation of mast cells with Compound 48/80 and Nedocromil significantly inhibited their histamine release (36% histamine release) and the increase in area and perimeter, but did not affect significantly their shape factor, in comparison with mast cells incubated with Compound 48/80 alone. These data clearly show that there is a relationship between mast cell activation, consequent histamine release and changes in cell area, perimeter and shape factor and that Nedocromil not only inhibits mast cell histamine release but also the activation induced morphometric changes in mast cells.

Estimation of dissolution rate of salicylamide in complexing media using a theoretical diffusion model.

Dissolution rates of salicylamide in water and caffeine solutions under perfect sink conditions were predicted by theoretical diffusion equations applicable to dissolution in complexing media. Experimental dissolution rates were measured using a compartmentalized rotating-basket apparatus under two sets of conditions. Agreement was found between experimental and predicted rates. Use of the theoretical equation for estimating dissolution rates involves simple calculations of diffusion coefficients and diffusion layer thickness under the operative dissolution conditions. The increase in dissolution rate caused by addition of the complexant can be calculated for diffusion-controlled dissolution directly if the stability constant and the drug solubility in water are known or measured.

Prevention of molecular self-association by sodium salicylate: effect on methylene blue.

Sodium salicylate improves the rectal absorption of drugs which exhibit molecular self-association; it is suggested that salicylate may improve drug bioavailability by altering the drug self-association pattern. Methylene blue was chosen as a model molecule for investigating the interference of salicylate with drugs undergoing self-association. The effect of sodium salicylate on the concentration-dependent association of methylene blue as expressed by metachromasy was observed and compared with the effects of other additives: urea, sodium chloride, sodium acetate, sodium sulfate, and sodium benzoate. By increasing the methylene blue concentration from 10(-5) M to 2 X 10(-3) M, the lambda max peak shifts from the longer wavelength region (approximately 660 nm) of the monomer toward the shorter (approximately 600 nm) indicating the presence of dimers and other oligomers. Addition of increased concentrations of sodium salicylate had a deaggregative effect on a 10(-3) M methylene blue solution, shifting the peaks toward the monomer region. On the other hand, the addition of 0.5 M of any of the following salts: sulfate, acetate, or chloride, to a 10(-3) M, aqueous solution of methylene blue had the opposite effect, eliminating the lambda max peak at 660 nm and generating a spectrum with one peak at approximately 600 nm, which indicated a high degree of self-association. The sodium salicylate effect is concentration dependent, with a high excess (approximately 450 times on a molar scale) being necessary to reduce the self-association. At lower concentrations of salicylate, precipitation occurs in the system.(ABSTRACT TRUNCATED AT 250 WORDS)

Testosterone skin permeation enhancement by menthol through formation of eutectic with drug and interaction with skin lipids.

The aim of this investigation was to elucidate the mechanism of skin permeation enhancement of the lipophilic drug, testosterone, by menthol. Menthol was found to form eutectic mixtures with testosterone, cholesteryl oleate, and ceramides. DSC measurements showed that menthol drastically lowers the Tm of testosterone, from 153.7 to 39.9 degrees C. The decrease in Tm resulted in an increase in the solubility of testosterone in an aqueous ethanol vehicle by 2.8-fold, which caused a corresponding 2.8-fold increase in the flux of testosterone. A further increase in skin flux, to eight times the base line, could be attributed to the effect of menthol on the skin barrier properties. This assumption is supported by DSC results showing that menthol decreases the Tm of cholesteryl oleate and ceramides and modifies the thermogram profile of isolated stratum corneum. The results of this investigation indicate that menthol affects skin permeation by a dual mechanism: by forming a eutectic with the penetrating compound, thereby increasing its solubility, and by altering the barrier properties of the stratum corneum. Moreover, this study indicates that both types of interactions must be taken into consideration when using chemical enhancers and that decreasing the melting temperature of the permeant through formation of a eutectic could be one approach for increasing solubility and permeation rates.

Promoting effect of n-decylmethyl sulfoxide on the penetrability of the optic nerve for lidocaine HCl in cats.

We examined the effect of n-decylmethyl sulfoxide (n-decylMSO) on the resistance of the optic nerve sheath to the penetration of lidocaine hydrochloride. Three series of experiments were carried out in living cats, administering lidocaine with and without n-decylMSO. The visual evoked potential (VEP) and optic nerve response (ONR) were measured. The results obtained indicated that n-decylMSO increased the penetration of lidocaine. The presence of lidocaine (1%) and n-decylMSO (2.5%) decreased the ONR amplitude from 120 to 15 microV after a time lag of 105 min, and VEP decreased from 9.0 to 5.5 microV. This effect was preserved during the experiment. Histological examination showed only minimal changes in the optic nerve and the eye globe and its contents. Some infiltration of mononuclear cells was found around the optic nerve sheath.

Prevention of molecular self-association by sodium salicylate: effect on insulin and 6-carboxyfluorescein.

The effect of sodium salicylate on the concentration-dependent self-association of insulin and 6-carboxyfluorescein (CF), as expressed by metachromasy, fluorescence, and changes in aqueous solubility, was learned. By decreasing the CF concentration from 12 to 0.48 microgram.ml-1, lambda max peaks shift from the shorter wavelengths (451, 474 nm), indicating the presence of oligomers, toward the monomer wavelength region (484 nm). Sodium salicylate shifts the peaks of a 12 micrograms.mL-1 CF solution towards the monomer region, eliminating the peak at the lower wavelengths and generating a spectrum with one peak at 490 nm, the effect being concentration dependent. The fluorescence of insulin and CF solutions increases with their concentration. Quenching of these solutions was observed, up to complete elimination of fluorescence, when various concentrations of salicylate were added. The water solubility of both molecules, CF and insulin, was considerably increased with the addition of increasing concentrations of salicylate to the solutions: at 37 degrees C, 2.5 M sodium salicylate solution increases the CF solubility 532 times from 12.2 to 6.5 mg.mL-1, and 1.5 M salicylate increases the solubility of insulin 7875 times, thus an aqueous solution containing 630 mg.mL-1 of insulin may be prepared. The results obtained here, together with our previously reported data, indicate that the interference between sodium salicylate and drug self-association behavior, by increasing drug solubility, may substantially contribute to the improved drug bioavailability mediated by salicylate.

Dyphylline liposomes for delivery to the skin.

Delivery of dyphylline to the skin using liposomes was investigated. Xanthines are inhibitors of cAMP phosphodiesterase and have been considered for treatment of psoriasis. Dyphylline was chosen because of its solubility in water, which should allow for incorporation of higher concentrations within the liposomes. Liposomes containing dyphylline were prepared by a method using sonication. Transmission electron micrography (TEM) visualization showed small particles ranging from 40 to 100 nm, and particle size distribution determined by light scattering showed the vesicles to have an average diameter of 360 nm. The transdermal delivery of free dyphylline and dyphylline incorporated in unilamellar liposomes was measured from polyethylene glycol (PEG), Carbopol gel, a PEG enhancer base, and water. For comparison, similar experiments were carried out with theophylline as well. When the drugs were incorporated in Carbopol gel, a large difference was seen between their fluxes, with free dyphylline having the highest permeation, followed by liposomal dyphylline, and then theophylline. With the PEG enhancer base, a very high permeation of theophylline was observed relative to dyphylline and liposomal dyphylline. From the PEG base, liposomal dyphylline exhibited the lowest skin permeation flux relative to other bases. Using the PEG base for dyphylline incorporated in liposomes, a high skin partitioning of the drug, along with low transdermal permeation, was measured. These results may indicate that the drug is localized in the skin.

Liposomes as carriers for topical and transdermal delivery.

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 dissolution mechanism in a system undergoing complexation: salicylamide in caffeine solution.

The dissolution rate of compressed salicylamide discs has been measured in water and in caffeine solutions of increasing concentration at 15, 25, 37 and 45 degrees in an apparatus rotating at 48 rev min-1 or more. Dissolution rate profiles showed breaks indicative of a shift in the mechanism of dissolution from interfacial towards transport control. The shifts occurred at higher caffeine concentrations on increasing the agitation rate or temperature. The dependencies of dissolution rates on agitation rates typified the intermediate type of dissolution and Arrhenius plots indicated that interfacial and transport processes participated in salicylamide dissolution.

Effective intestinal absorption of insulin in diabetic rats using a new formulation approach.

Insulin injected intra-jejunally together with the non-ionic surfactant cetomacrogol was effective in streptozocin-induced diabetes in the rat, as measured by the hypoglycaemic effect. The reduction in blood sugar was maximal at about 2 h after administration but continued at a high level for the 4 h of the experiment. No hypoglycaemic effect was observed in controls injected with insulin or saline alone. Intestinal absorption of insulin has thus been effected by the addition of cetomacrogol, which appears to enhance membrane-permeability to insulin rather than to function as a protective agent preventing insulin degradation, as in liposome-encapsulation. In support of this, a significant hypoglycaemic action was still obtained when the insulin injection was given half-hour after that of the cetomacrogol, both intra-jejunally. Furthermore, oral administration of the surfactant followed by intra-jejunal injection of the insulin also gave a hypoglycaemic effect. The use of this agent to enhance insulin absorption offers the possibility of a new approach to oral insulin therapy.

Stability of salicylamide-caffeine complex at different temperatures and its thermodynamic parameters.

The stability constants for formation of complexes of salicylamide with caffeine have been measured between 15 and 45degrees, by means of the solubility method. There was a linear solubility increase at all temperatures but phase diagrams indicated that at 15 and 25degrees an additional phase was formed which was found to be an insoluble 1 : 1 complex. The enthalpies and entropies of interactions were evaluated. They indicate that the interaction is exothermic and enthalpy controlled.

A clinical evaluation of a novel liposomal carrier for acyclovir in the topical treatment of recurrent herpes labialis.

In a 2-armed, double-blind, randomized clinical study, the efficacy in the treatment of recurrent herpes labialis of 5% acyclovir in a novel liposomal carrier (ethosome) was evaluated in comparison with that of a commercial 5% acyclovir cream (Zovirax cream) and that of a drug-free vehicle. Data were based on 61 herpetic episodes in 40 subjects. In a crossover arm in which the 2 active preparations were compared, the time to crusting of lesions was significantly shorter (P < .025) with the ethosomal acyclovir (1.8 days) than with the cream (3.5 days). Time to loss of crust was also significantly shorter (4.2 vs 5.9 days; P < .05). In a parallel arm in which all 3 preparations were compared, the time to crusting with the ethosomal acyclovir (1.6 days) was significantly shorter than the time with the acyclovir cream (4.3 days; P < .02) and the time with the drug-free vehicle (4.8 days; P < .005); in this arm, the shorter time to loss of crust for the ethosome (3.5 days), in comparison with the times for the cream (6.4 days) and the drug-free vehicle (6.1 days), did not reach statistical significance. Approximately 30% of all episodes treated with the ethosome were clinically abortive; this compared with 10% of those treated with the cream or the drug-free vehicle. No adverse effects were reported, other than minor burning sensations at the application site that lasted a few seconds after application and were evenly distributed between the investigated preparations. This pilot study suggests the improved clinical efficacy of the new liposomal preparation in comparison with Zovirax cream in the treatment of recurrent herpes labialis.