Robert S. Hinz

Iontophoretic delivery of amino acids and amino acid derivatives across the skin in vitro.

The effects of penetrant properties (lipophilicity and charge) and of vehicle pH on the iontophoretically enhanced delivery of amino acids and their N-acetylated derivatives have been examined in vitro. The penetrants were nine amino acids (five were zwitterionic, two positively charged, and two negatively charged) and four N-acetylated amino acids, which carry a net negative charge at pH 7.4. Iontophoresis at constant current (0.36 mA/cm2), using Ag/AgCl electrodes, was conducted across freshly excised hairless mouse skin. Iontophoretic flux of the zwitterions was significantly greater than passive transport. Delivery from the anode was greater than from the cathode for all zwitterions. The level of enhancement was inversely proportional to permeant octanol/pH 7.4 buffer distribution coefficient. Cathodal iontophoresis of the negatively charged amino acids and of the N-acetylated derivatives produced degrees of enhancement which were significantly greater than those measured for the “neutral” zwitterions. Furthermore, the enhanced flux reached a steady-state level within a few hours for the negatively charged species, whereas the transport of the zwitterions continued to increase with time. Anodal iontophoresis of histidine and lysine, the two positively charged amino acids studied, induced substantial enhancement which was sensitive to the pH of the delivery vehicle. For example, the flux of histidine from an applied solution at pH 4 (where the amino acid carries a net positive charge) was significantly greater than that from a vehicle at pH 7.4 (where histidine is essentially neutral). The behavior of lysine was more complex and suggested a certain degree of neutralization of the skins net negative charge.

Metals and the Skin

Certain metals, and many metal-based compounds, are inherently toxic, and their presence in occupational and environmental settings raises appropriate questions concerning human exposure. Contact of these materials with the skin represents an important route of exposure, which is not well characterized. The purpose of this review, therefore, is to assemble the available, useful information pertinent to risk assessment following dermal contact. Specifically, we summarize here: (1) data relevant to the qualitative and (where possible) quantitative evaluation of metal compound permeation through the skin; (2) the role of each metal in metabolism, particularly with respect to the skin, and the potentially toxic effects that may result from dermal contact; and (3) the immunological characteristics (including allergenicity) of the metals and their derivatives. In total, information on 31 metals has been reviewed. It is clear that many diverse factors determine the ability of metal-based species to permeate biological membranes, not all of which have been fully defined. Therefore, considerably more experimentation, targeted at the development of high-quality transport data, will be required before the specification of practically useful structure-activity relationships are possible.

A New System for In Vitro Studies of Iontophoresis

This report describes a new iontophoretic diffusion cell that allows both electrodes to be applied to the same side of the same piece of skin. The cell permits a better approximation of the in vivo situation than do conventional side-by-side cells. The unique construction of the cell allows nonliquid material to be applied to the skin surface and makes it possible to investigate horizontal transport paths. Preliminary results utilizing the cell are described. Iontophoretic enhancement of morphine and clonidine delivery across full-thickness hairless mouse skin has been achieved. The importance of pH control in these experiments is apparent. Further experiments with morphine indicate that, for this drug at least, iontophoretically driven lateral transport within the skin is unimportant. Because the cell design allows significant parallels to the use of iontophoresis in vivo, we suggest that it will prove to be a useful tool in the determination of fundamental structure/transport relationships under the influence of an externally applied current.

lontophoretic Delivery of a Series of Tripeptides Across the Skin in Vitro

The iontophoresis of eight tripeptides, of the general structure alanine–X–alanine, has been measured across hairless mouse skin in vitro. The peptides were blocked (a) at the carboxyl terminus using the mixed anhydride reaction with t-butylamine and (b) at the amino terminus by acetylation with 14C-acetic anhydride. The nature of the central residue (X) was varied by selecting one of five neutral amino acids, two negatively chargeable moieties (aspartic and glutamic acids), and a positively chargeable species (histidine). Constant current iontophoresis at 0.36 mA/cm2, using Ag/AgCl electrodes, was performed for 24 hr in diffusion cells, which allowed both anode and cathode to be situated on the same (epidermal) side of a single piece of skin. Due to a combination of osmotic and electroosmotic forces, the anodal iontophoretic flux of neutral peptides was significantly greater than passive transport. Steady-state fluxes were not achieved, however, suggesting that time-dependent changes in the properties of the skin barrier may be occurring. Limited, further experiments confirmed that, on a 24-hr time scale, these changes were not fully reversible. The cathodal delivery of anionic permeants was well controlled at a steady and highly enhanced rate by the current flow. This behavior closely paralleled earlier work using simple negatively charged amino acids and N-acetylated amino acid derivatives. It appears that the normalized iontophoretic flux of these anionic species is independent of lipophilicity but may be inversely related to molecular weight. The positively charged peptide, Ac–Ala–His–Ala–NH(But), showed greater anodal iontophoretic enhancement when delivered from a donor solution at pH 4.0 than from a solution at pH 7.4. This was consistent with (a) the corresponding behavior of histidine alone and (b) the existence of a pKa for these compounds at ∼6. Steady-state delivery was not achieved, although the levels of enhancement, especially at pH 4, were the largest observed. A preliminary investigation of tripeptide stability to either (i) electrolysis in the donor compartment or (ii) cutaneous metabolism revealed very little degradation under the conditions of the experiment. Overall, this research supports the principle of enhanced peptide delivery across the skin by iontophoresis and indicates a number of areas (e.g., mechanism and extent of current-induced changes in skin barrier function, molecular size dependence, pathways of current flow) on which further work should be focused.

Controlled drug release from a novel liposomal delivery system. I: Investigation of transdermal potential

Abstract The in vitro release behavior of a novel liposome-based drug delivery device has been characterized. The system consists of a molded agarose matrix in which the model drug (progesterone) was dispersed either free or associated with one of four lipid formulations: egg-phosphatidylcholine (EPC) liposomes, EPC/cholesterol (2:1) liposomes, Intralipid® emulsion, and dipalmitoylphosphatidylcholine (DPPC) liposomes. Drug release rates from the devices into aqueous buffer were measured at 37° C. The free progesterone release rate decreased rapidly over 24 h with over 90% delivered. The liposomal patches, on the other hand, imposed apparent zero-order kinetics: for example, both the EPC and DPPC systems delivered their progesterone payloads at about 1%/h over 24 h. Further, the EPC and DPPC patches significantly slowed transdermal drug delivery across excised hairless mouse skin. The EPC device retarded throughput to one-half the control value, the DPPC system reduced the transport kinetics by an order of magnitude. The results support two hypotheses: (a) the liposomal-based reservoir system can modulate drug input via the skin, (b) the zero-order release of progesterone from liposomes is determined by slow interfacial transport out of the bilayer into the surrounding aqueous medium.

Cutaneous Metabolism of Nitroglycerin in Vitro. II. Effects of Skin Condition and Penetration Enhancement

The effects of skin storage, skin preparation, skin pretreatment with a penetration enhancer, and skin barrier removal by adhesive tape-stripping on the concurrent cutaneous transport and metabolism of nitroglycerin (GTN) have been studied in vitro using hairless mouse skin. Storing the skin for 10 days at 4°C did not alter barrier function to total nitrate flux [GTN + 1,2-glyceryl dinitrate (1,2-GDN) + 1,3-glyceryl dinitrate (1,3-GDN)]. However, metabolic function was significantly impaired and suggested at least fivefold loss of enzyme activity. Heating skin to 100°C for 5 min appreciably damaged hairless mouse skin barrier function. The ability to hydrolyze GTN was still present, however, and remained constant over the 10-hr experimental period, in contrast to the “control,” which showed progressively decreasing enzymatic function with time. Pretreatment of hairless mouse skin in vivo (prior to animal sacrifice, tissue excision, and in vitro transport/metabolism studies) with 1-dodecylazacyclo-heptan-2-one (Azone), a putative penetration enhancer, significantly lowered the skin barrier to nitrate flux (relative to the appropriate control). Again, barrier perturbation resulted in essentially constant metabolic activity over the observation period. The ratio of metabolites formed (1,2-GDN/1,3-GDN) was increased from less than unity to slightly above 1 by the Azone treatment. Adhesive tape-stripping gradually destroyed skin barrier function by removal of the stratum corneum. The effects of 15 tape-strips were identical to those of Azone pretreatment: a greatly enhanced flux, a constant percentage formation of metabolites over 10 hr (once again), and an increase in the 1,2-GDN/1,3 GDN ratio. Overall, the experiments caution that, for transdermal drug delivery candidates susceptible to skin metabolism, the status of barrier function (enhancer pretreated, skin damage or disease, etc.) may significantly affect systemic availability.

Transdermal iontophoresis of amino acids and peptides in vitro

Abstract The effects of penetrant properties (lipophilicity and charge) and of vehicle pH on the iontophoretically enhanced delivery of amino acids, their N-acetylated derivatives, and eight tripeptides, of the general structure alanine-X-alanine, have been examined in vitro. The penetrants were (a) 9 amino acids (five were zwitterionic, two positively charged and two negatively charged), (b) four N-acetylated amino acids, which carry a net negative charge at pH 7.4, and (c) peptides which were blocked both at the carboxyl terminus using the mixed anhydride reaction with t-butylamine, and at the amino terminus by acetylation with 14C-acetic anhydride; the central residue (X) was varied widely by selecting one of five neutral amino acids, two negatively chargeable moieties (aspartic and glutamic acids), and a positively chargeable species (histidine). Iontophoresis at constant current (0.36 mA/cm2), using Ag/AgCl electrodes, was conducted across freshly excised hairless mouse skin. The diffusion cells used were designed so that both anode and cathode were situated on the same (epidermal) side of a single piece of skin. Overall, it was found that the results of this research support the principle of enhanced peptide delivery across the skin by iontophoresis.

The Relationship between pKa and Skin Irritation for a Series of Basic Penetrants in Man

For a series of bases, which penetrate through human skin in vitro at similar rates (0.056-0.49 microM/cm2/hr), penetrant pKa is shown to correlate with erythema, edema, and color meter readings. As estimates of irritation, erythema, edema, and redness measurements are highly linearly correlated. For the selected series, irritation becomes significant for bases with a pKa greater than 8. The irritation potential of acids with pKa less than or equal to 4 has been previously reported; pKa appears highly predictive of acute skin irritation for acids and bases in man.

Cutaneous Metabolism of Nitroglycerin in Vitro. I. Homogenized Versus Intact Skin

The metabolism of nitroglycerin (GTN) to 1,2- and 1,3-glyceryl dinitrate (GDN) by hairless mouse skin in vitro has been measured. In the first set of experiments, GTN was incubated with the 9000g supernatant of fresh, homogenized tissue in the presence and absence of glutathione (GSH), a cofactor for glutathione-S-transferase. After 2 hr of incubation with GSH, 30% of the initially present GTN had been converted to 1,2- and 1,3-GDN; without GSH, less than 5% of the GTN was metabolized. The ratio of 1,2-GDN to 1,3-GDN produced by the homogenate was 1.8– 2.1. In the second series of studies, GTN was administered topically to freshly excised, intact hairless mouse skin in conventional in vitro diffusion cells. The concurrent transport and metabolism of GTN was then monitored by sequential analysis of the receptor phase perfusing the dermal side of the tissue. Three topical formulations were used: a low concentration (1 mg/ml) aqueous solution, a 2% ointment, and a transder-mal delivery system. Delivery of total nitrates (GTN + 1,2-GDN + 1,3-GDN) into the receptor phase was similar for ointment and patch formulations and much greater than that from the solution. The percentage metabolites formed, however, was greatest for the solution (61% and 2 hr, compared to 49% for the patch and 35% for the ointment). As has been noted before, therefore, the relative level of skin metabolism is likely to be greatest when the transepidermal flux is small. Distinct from the homogenate experiments, the 1,2/1,3-GDN ratios in the penetration studies were in the range 0.7– 0.9. It would appear that homogenization of the skin permits GTN to be exposed to a different distribution of enzymes than that encountered during passive skin permeation.

The Effects of Zwitterionic Surfactants on Skin Barrier Function

The action of five zwitterionic surfactants on the barrier function of hairless mouse skin has been studied in vitro. The surfactants considered were dodecylbetaine and hexadecylbetaine (C12BET and C16BET, respectively), hexadecylsulfobetaine (C16SUB), N,N-dimethyl-N-dodecylamine oxide (C12AO), and dodecyltrimethylammonium bromide (C12TAB). Excised skin was pretreated with each surfactant, at various concentrations, for 16 hr, following which the permeation of a model compound, nicotinamide, was measured. The action of the surfactants was assessed by comparing nicotinamide flux through surfactant-pretreated skin with that across control membranes which were exposed to buffer alone for 16 hr. All surfactants decreased skin barrier function to some extent. The degree of nicotinamide penetration enhancement induced was correlated with the ratio of the surfactant pretreatment concentration to the surfactant critical micelle concentration, suggesting that solubilization of stratum corneum lipids may be an important mechanism in explaining the effects observed. More detailed studies with 14C-radiolabeled C12BET and C16BET showed that the dodecyl analog was itself well absorbed, whereas the C16 compound partitioned into the skin favorably but then transferred only very slowly into the receptor phase. These observations were consistent with toxicity studies (albeit at much higher concentrations in a different animal model, the rat) which indicated that the dermal LD50 of C12BET was significantly less than that of C16BET (the value for which was so large that it could not be reliably determined). Overall, this study provides, we believe, useful information pertinent to the potential dermal toxicity of the surfactants considered following occupational or environmental exposure.