IL109036A - Compositions for disrupting the epithelial barrier function and use of agents for preparing such compositions - Google Patents

Abstract

A method for inducing phase separation of the epithelial multilayered lipid bilayers within the intercellular spaces of the stratum corneum in a host in need of the topical administration of a physiologically active substance delivered percutaneously or transdermally which comprises applying to the epithelium of the host, an effective amount of one or more intercellular phase-separating agents, as well as a topical composition useful therefor. [WO9421271A1]

Description

109036/2 ya> OVPYJWI iwsiib t.>j>a»i& Ώ>ι>νΜ Compositions for disrupting the epithelial barrier function and use of agents for preparing such compositions Cellegy Pharmaceuticals, Inc. and The Regents of the University of California C.92873 -1- 109036/ 2 BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates generally to a novel method for enhancing penetration of physiologically active substances through epithelium which comprises the stratum corneum/epidermis and keratinizing mucous membranes. More specifically, it relates to a method and composition far disrupting the epithelial barrier function in a host which employs at least one agent selected from the group consisting of inhibitors of ceramide synthesis,, an inhibitor of glucosyl-ceramide synthesis, an inhibitor of acylceramide synthesis, an inhibitor of fatty acid synthesis, an inhibitor of cholesterol synthesis, inhibitors of phospholipid, glycosphingolipid, acylceramide and sphingomyelin degradation, a degradation enzyme of free fatty acid, ceramide, acylceramide, or glucosylceramides and both inhibitors and stimulators of metabolic enzymes of free fatty acids, ceramide, and cholesterol. 2. Description of Related Art The major function of the epithelium is that of a barrier to prevent the excessive loss of bodily fluids. If this barrier is disrupted or perturbed, it stimulates a variety of metabolic changes in the epithelium leading to repair of the barrier defect. While the barrier protects against external damage induced by such agents as ultraviolet radiation, desiccation, chemicals, and frictional or blunt trauma, it impedes the penetration of topically applied medicaments, nutrients, or other xenobiottcs.

The epithelial barrier is a system of multiiayered membrane lipid bilayers that exist throughout the intercellular spaces of the stratum corneum in epidermis and keratinizing mucous membranes. The bilayers in stratum corneum of epidermis consist of approximately equimolar ratios of three major lipid species: ceramides, free fatty acids, and cholesterol, as well as small, but critical, amounts of acylceramides. Keratinizing mucous membrane multiiayered bilayers consist of approximately equimolar ratios of glucosylceramides, free fatty acids and cholesterol. These lipid species are synthesized in the subjacent nucleated cell layers of the epithelium. Following any type of barrier perturbation, an increase in lipid biosynthesis occurs, which leads to the recovery of barrier structure and function. The more extensive the perturbation of the barrier, the more active is the subsequent lipid biosynthetic response.

In addition to the long-standing approaches of hydration and occlusion, currently available percutaneous and transmucosal penetration enhancement technology relies on physical-chemical methods, such as solvents or detergents, and physical approaches, such as iontophoresis, electroporation, or sonophoresis. Typical solvents or detergents alter the physical properties of the multiiayered lipid bilayers. Such agents include dimethylsulfoxide (DMSO), oleyi alcohol (OA), propylene glycol (PG), methyl pyrrolidone and AZONE® (dodecylazyl cycloheptan 2-one). For example, U.S. Patent No. 4,177,267 discloses topical steroid compositions containing dimethylsulfoxide as an epithelial penetration enhancer. It is generally believed that many of these epithelial penetration enhancers fluidize the polar head group (e.g., DMSO) and/or nonpolar tail group (e.g., OA) domains within the multiiayered lipid bilayers. Yet, some compounds with significant fluidizing effect have been shown to be incapable of substantially increasing epithelial permeability. While these methods enhance penetration of certain compounds by three- to fivefold, these methods are only relatively effective for smaller lipophilic and amphiphathic molecules. Hydrophilic compounds such as proteins or peptides do not penetrate in pharmaceutically useful quantities through the epithelia even when most of these permeation technologies are utilized.

Accordingly, there is a need for epithelial penetration enhancers capable of allowing and/or increasing the penetration of large and/or water-soluble molecules in therapeutically effective quantities. This invention addresses this need by providing methods and topical compositions for disrupting the epithelial barrier thereby facilitating the penetration of therapeutic known or potential molecules.

SUMMARY OF ΤΉΕ INVENTION ft has been discovered that a formulation comprising at least one agent selected from the group consisting of inhibitors of ceramide synthesis, an inhibitor of giucosyiceramide synthesis, an inhibitor of acylceramide synthesis, an inhibitor of fatty acid synthesis, an inhibitor of cholesterol synthesis, inhibitors of phospholipid, glycosphingoiipid, acylceramide and sphingomyelin degradation, a degradation enzyme of free fatty acid, ceramide, acylceramide, or gjucosylceramides and both inhibitors and stimulators of metabolic enzymes of free fatty acids, ceramide, and cholesterol is very effective for disrupting epithelial barrier function in a host,, and thereby enhancing penetration of a physiologically active substance administered topically.

In one aspect thereof, this invention provides use of a barrier disrupting amount of at least one agent selected from the group consisting of an inhibitor of ceramide synthesis, an inhibitor of giucosyiceramide synthesis, an . inhibitor of acylceramide synthesis, an inhibitor of fatty acid synthesis, an inhibitor of cholesterol synthesis, inhibitors of phospholipid, glycosphingoiipid, acylceramide and sphingomyelin degradation, a degradation enzyme of free fatty acid, ceramide, sphingomyelin, acylceramide, orglucosylceramides and both inhibitors and stimulators of metabolic enzymes of free fatty acids, ceramide, and cholesterol for the preparation of a topical composition for disrupting the epithelial barrier function in a host in need of the topical -administration of a physiologically active substance, substantially as described in the specification.

In another aspect, this invention provides a topical composition for disrupting epithelial barrier function in a host in need of topical administration of a. physiologically active substance which comprises an epithelial barrier-disrupting amount of at least one agent selected from the group consisting of an inhibitor of ceramide synthesis, an inhibitor of acylceramide synthesis, an inhibitor of giucosyiceramide synthesis, an inhibitor of free fatty acid synthesis, and an inhibitor of cholesterol synthesis, an inhibitor of phospholipid or glycosphingolipid degradation, a stimulator of steps of ceramide and cholesterol metabolism distal to these compounds, and a degradation enzyme of ceramides, acylceramide, or glucosylceramides, together with a physiologically acceptable carrier.

The above features and advantages of this invention will be more fully understood by reference to the following detailed description and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 schematically shows a biosynthetic and degradation pathway of ceramides, acylceramides and glucosylceramides.

FIGURE 2 schematically shows a biosynthetic pathway of free fatty acids.

FIGURE 3 schematically shows a biosynthetic and degradation pathway of cholesterol.

FIGURE 4A shows the propylene glycol concentrations at an epidermal surface of control animals when they were treated with a vehicle. O, VBL; O, VBR; □, VCL; ■, VCR.

FIGURE 4B shows the propylene glycol concentrations at an epidermal surface of tested animals when they were treated with fiuvastatin (fluindostatin). O, FBL; O, FBR; □, FCL; ■, FCR.

FIGURE 5A shows the cyclophenol concentrations at an epidermal surface of control animals when they were treated with a vehicle. O, VBL; O, VBR; □, VCL; ■, VCR.

FIGURE 5B shows the cyclophenol concentrations at an epidermal surface of tested animals when they were treated with fluindostatin. O, FBL; O, FBR; □, FCL; ■, FCR.

FIGURE 6A shows TEWL when animals were treated with oleic acid followed by fluindostatin.

FIGURE 6B shows TEWL when animals were treated with oleic acid followed by 5-{tetradecyloxy)-2-furoic acid (TOFA).

DETAILED DESCRIPTION OF THE INVENTION This invention is based on the discovery that when the biosynthesis of one or more of the epithelial lipids, ceramides, acylceramide, glucosylceramides, cholesterol and free fatty acids is inhibited, or their distal utilization is increased, the lipid species is depleted perturbing the normal mole ratio, resulting in incompetent epithelial barrier function. Inhibition of enzymes involved in a biosynthetic pathway or inhibition of the degradation enzymes for the precursors of each of these key lipid constituents have also been specifically targeted according to the present invention. Further, it has been discovered that the inhibition of enzymes aiming at two or more lipid constituents may be additive or synergistic in the opening of the epithelial barrier for percutaneous or transmucosal delivery of physiologically active substances.

The composition of this invention principally employs an epithelial barrier-disrupting amount of at least one agent selected from the group consisting of inhibitors of ceramide synthesis, an inhibitor of glucosylceramide synthesis, an inhibitor of acylceramide synthesis, an inhibitor of fatty acid synthesis, an inhibitor of cholesterol synthesis, inhibitors of phospholipid, glycosphingolipid, acylceramide and sphingomyelin degradation, a degradation enzyme of free fatty acid, ceramide, acylceramide, or glucosylceramides and both inhibitors and stimulators of metabolic enzymes of free fatty acids, ceramide, and cholesterol.

As used herein, the term "an epithelial barrier-disrupting amount" means that the amount of enzyme inhibitor(s), stimulator(s) or degradation enzymes are of sufficient quantity to disrupt the barrier when these compounds are topically applied to the skin or mucous membrane of a host. The amount can vary according to the effectiveness of each enzyme inhibitor or stimulator, or degradation enzyme, as a percutaneous or transmucosal penetration enhancer, the host age, and response of the host. More importantly, the therapeutic amount should be determined based on the penetration efficiency of a given physiologically active substance when that substance is administered in conjunction with a particular combination of the enzyme inhibitors or stimulators, or degradation enzymes. The required quantity to be employed in this invention can be determined readily by those skilled in the art.

As used herein, the term "host" includes humans and non-human mammals. Non-human mammals of particular interest are domesticated species such as dogs, cats, monkeys, cows, horses, llamas, sheep, pigs, and goats.

The term "penetration enhancement' or "permeation enhancement' as used herein relates to an increase in the permeability of skin to a physiologically active substance, i.e., so as to increase the rate at which the substance permeates through the epithelium and enters the bloodstream.

As applied in this invention, the term "physiologically active substance" is intended to encompass any substance that will produce a physiological response when topically administered to a host. In general, the terms include therapeutic or prophylactic agents in all major therapeutic/prophylactic areas of medicine as well as nutrients, cofactors, enzymes (endogenous or foreign), antioxidants or other defensive principals, and xenobiotics. Suitable substances include, but are not restricted to, antifungals such as amphotericin B, griseofulvin, miconazole, ketoconazole, tioconazol, itraconazole, and fluconazole; antibacterials such as penicillins, cephalosporins, tetracyclines, aminoglucosides, erythromicin, gentamicins, polymyxin B; anti-cancer agents such as 5-fluorouracii, bleomycin, methotrexate, hydroxyurea; antiinflammatories such as hydrocortisone, glucocorticoids, colchicine, ibuprofen, indomethacin, and piroxicam; antioxidants, such as tocopherols, retinoids, carotenoids, ubiquinones, metal chelators, and phytic acid; antihypertensive agents such as prazosin, nifedipine, and diltiazem; analgesics such as acetaminophen and aspirin; anti-viral agents such as acyclovir, ribavarin, and trifluorothyridine; antiandrogens such as spironolactone; androgens such as testosterone; estrogens such as estradiol; progestins such as modified progestogens; opiates; muscle relaxants such as papaverine; vasodilators such as nitroglycerin, vasoactive intestinal peptide and calcitonin related gene peptide; antihistamines such as cyproheptadine; antitussives such as dextromethorphan; neuroleptics such as Clozaril; antiarrhythmics; antiepileptics; and other polypeptides and neuropeptides such as somatostatin, various cytokines, insulin, gastrin, substance P, and capsaicin; and enzymes, such as superoxide dismutase and neuroenkephalinase. Other useful drugs, in approved commercially available formulations, and their recommended dosages are listed in the annual publication of the Physicians' Desk Reference, published by Medical Economics Company, a division of Litton Industries, Inc. More than one physiologically active substance may be included, if desired, in the topical composition of this invention.

The active substance may be water-soluble or water-insoluble and may include higher molecular weight proteins, peptides, carbohydrates, glycoproteins, lipids, and glycolipids. Such proteinaceous active substances which can be included are immunomodulators and other biological response modifiers. Examples of immune response modifiers include such compounds as lymphokines. Lymphokines include tumor necrosis factor, the interieukins, lymphotoxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, and interferon. Interferons with which the monoclonal antibodies of the invention can be labeled include alpha-interferon, beta-interferon and gamma-interferon and their subtypes.

The active substance will be present in the composition in an amount sufficient to provide the desired physiological effect with no apparent toxicity to the host.

Of course, the appropriate dosage levels of all the physiologically active substances, without the use of the epithelial barrier-disrupting agents of the present invention, are known to one skilled in the art. These conventional dosage levels correspond to the upper range of dosage levels for compositions, including a physiologically active substance and an epithelial barrier- disrupting agent. However, because the delivery of the active substance is enhanced by the epithelial barrier-disrupting agent of this invention, dosage levels significantly lowering the conventional dosage levels may be used with success. In general, the active substance will be present in the composition -*in an amount of from about 0.0001% to about 60%, more preferably about 0.01 % to about 20% by weight of the total composition depending upon the particular substance employed.

Ceramides, including acylceramides, account for 40-50% by weight of the stratum corneum lipids while glucosylceramides account for a similar percent in mucosal membranes. Representative in vivo biosynthetic pathways for these lipid species are shown in Fig. 1. Among many enzymes involved in these biosynthetic pathways, serine palmitoyl transferase is the rate limiting enzyme.

The inhibitors of ceramide, acyiceramide, and giucosyiceramide synthesis and metabolism include inhibitors of serine palmitoyl transferase such as jS- chloroalanine, fluoropalmitate, and j3-fluoroalanine, and inhibitors of ceramide synthetase such as fumonisins. Inhibitors of sphingomyelinase include agents such as tricyclodecan-9-yl-xanthogenate, ethylisopropylamiloride, N-palmitoyl- DL-dihydrosphingosine, methylene-dioxybenzapine, tricyclodecan-9yl-xantho- genate; aminoglycosides including gentamicin and neomycin; ethyliso- proplylamiloride; tricyclic antidepressants, including desipramine and imipra- mine; and phenothiazines including chlorpromazine and perchlorperazine. Inhibitors of giucosyiceramide synthesis further include inhibitors of UDP- glucose-ceramide glucosyl transferase (glucosyl transferase), such as 1 -phenyl- 2-deanoylamino-3-morphalino-1-propanol (PD P), its analogs, σ- and β-xylosides; and alpha xylosides including p-nitro-phenyl- r-xyloside and beta xylosides including 4-methyl umbeHiferyl-/3-0-xyloside, O-and p-nitrophenyl-3-0-xyiopyaranoside. Inhibitors of acylceramide synthesis further include inhibitors of σ-hydroxylation, N-acyl chain length elongation, and ω-acyl transferase, alpha xylosides including p-nitro-phenyl-cr-xyloside and beta xylosides including 4-methyl umbelliferyl-/3-o-xyloside, 0- and p-nitrophenyl-/3-0-xylopyaranoside. Inhibitors of acylceramide synthesis further include inhibitors of σ-hydroxylation, N-acyl chain length elongation, and ω-acyi transferase. Inhibitors of acid lipase include the boronic acids, including phenylboronic acid, tetrahydrolipstatin and esterasin.

In keratinizing mucous membrane, glycosphingolipids, including glucosylceramide, are not metabolized into ceramide, as occurs in the stratum corneum. Therefore, inhibitors of glycosphingolipids including inhibitors of β-glucosidase such as N-hexylglucosyl-sphingosine, bromoconduritoi B-epoxide, conduritol, cyclophellitol, conduritol B-epoxide, and deoxynojirimycin will effectively perturb the barrier in the stratum corneum, but not in the keratinized mucous membranes.

D-cycloserine, 3-Chioroalanine, L-cycloserine, jS-fluoroalanine, fluoropalmrtate, and fumonisins are preferred inhibitors of epithelial sphingolipid synthesis, with /3-chloroalanine and fumonisins being most preferred. Stimulators of glucosyl transferase, a-hydroxylation, N-acyl chain length elongation, and ω-acyl transferase and phosphotidylcholine-ceramide phosphorylcholine transferase will effect epithelial ceramide and glucosylceramide concentrations. An effective concentration range for these inhibitors and stimulators in the topical composition of this invention is generally from about 0.01% to about 5% by weight of the total.

I Free fatty acids account for 20-25% of the epithelial lipids by weight The free fatty acids are synthesized and metabolized in vivo as shown in Fig. 2. The two rate limiting enzymes in the biosynthesis of the free fatty acids are acetyl CoA carboxylase and fatty acid synthetase. Through a series of steps, free fatty acids are metabolized into phospholipids.

The inhibitors of free fatty acid synthesis and metabolism include inhibitors of acetyl CoA carboxylase such as 5-tetradecyloxy-2-furancarboxylic acid (TOFA); inhibitors of fatty acid synthetase; inhibitors of phospholipase A such as gomisin A, 2-(p-amylcinnamyl)amino-4-chlorobenzoic acid, bromophenacyl bromide, onoaiide, 7,7-dlmet y\-5,8-e]cosad enoic acid, nicergoline, cepharanthine, quercetin, dibutyryl-cyclic AMP, R-24571 , N-oleoylethanolamine, N-(7-nitro-2, 1 ,3-benzoxadiazol-4-yl) phosphostidyiserine, verapamil, diltiazepam, nifedipine, quinacrine, cyclosporine A, dibucaine, prenylamine, retinoids, such as all-trans and 13-cis-retinoic acid, propanalol, W-7, trifluoperazine, R-24571 (calmidazolium), 1 -hexadocyl-3-trifluoroethyl glycero-sn-2-phosphomenthol (MJ33); calcium channel blockers including verapamil, diltiazem, nifedipine, and nimodipine; antimalarials including quinacrine, mepacrine, chloroquine and hydroxychloroquine; beta blockers including propanalol and labetaloi; calmodulin antagonists; EGTA; thimersol; glucocorticosteroids including dexamethasone and prednisolone; and nonsteroidal antiinflammatory agents including indomethacin and naproxen. TOFA is the preferred inhibitor of free fatty acid synthesis. An effective concentration range for the fatty acid inhibitor in the topical composition of this invention is generally from about 0.01 % to about 5% by weight of the total.

Free sterols, primarily cholesterol, account for 20-25% of the epithelial lipids by weight. The free sterols are synthesized and metabolized in vivo as shown in Fig. 3. The rate limiting enzymes in the biosynthesis of cholesterol is 3-hydroxy-3-methylglutaryl (HMG) CoA reductase.

The inhibitors of cholesterol synthesis include competitive inhibitors of (H G) CoA reductase such as simvastatin, lovastatin, fluindostatin (fluvastatin), pravastatin, mevastatin, as well as other HMG CoA reductase inhibitors, such as cholesterol sulfate and phosphate, and oxygenated sterols, such as 25-OH-and 26-OH-cholesterol; inhibitors of squalene synthetase; inhibitors of squalene epoxidase; inhibitors of Δ7 or Δ24 reductases such as 22,25-diazacholesterol, 20,25-diazacholestenol, AY9944, and triparanol. The preferred inhibitors are fluindostatin, simvastatin, lovastatin, cholesterol sulfate, and 25-OH-cholesterol. An effective concentration range for the cholesterol inhibitor in the topical composition of this invention is generally from about 0.01% to about 5% by weight of the total.

The degradation enzyme of ceramide is ceramidase. The degradation enzymes of acylceramides are acid lipase followed by ceramidase. The degradation enzymes of glucosylceramide are j3-glucosidase followed by ceramidase. The degradation enzyme of sphingomyelin is sphingomyelinase. An inhibitor of ceramidase is N-oleoyl-ethanolamine. The degradation enzyme for sphingomyelin is sphingomyelinase. An effective concentration range for these degradation enzymes is generally from about 0.01% to about 5% by weight of the total.

The term "stimulators of steps of ceramide, free fatty acid, and cholesterol metabolism distal to these molecules" means molecules capable of shunting cholesterol, free fatty acid or cholesterol to more distal metabolites, such as glucosylceramide, acylceramide, sphingomyelin, phospholipids; and steroid hormones, respectively. This has the effect of depleting either cholesterol or ceramides. An effective concentration range for such stimulators is generally from about 0.01% to about 5% by weight of the total.

Several of the metabolic pathways mentioned herein have as yet no known Inhibitors for the enzymes, such as alpha-hydroxylation, N-acyl chain length elongation, omega-acyl transferases, phosphatidylcholine-ceramidephosphoryl-choline transferases, fatty acid synthetase, squalene synthetase, cholesterol phosphate synthetase, cholesterol sulfate synthetase. However, should such inhibitors be discovered, the use of such inhibitors is contemplated within the scope of this invention.

These enzyme inhibitors, degradation enzymes, or enzyme stimulators can be co-applied to the skin or mucous membrane of a host in a formulation with any combination of these compounds with or without conventional penetration enhancers or other drug delivery technology, including transdermal patches, iontophoretic and electrophoretic devices, and sonicators. Alternatively, they can be applied concurrently as separate formulations. Still further, one agent can be applied before, simultaneously with, or after application of the other agent(s) provided that the time interval between the two (or more) is not too lengthy (e.g. typically, not more than about 24 hours). The physiologically active substance can be co-administered to the host with a topical composition which contains these inhibitors or stimulators. Alternatively, the active substance may be administered after application of the topical composition of the invention. It is, however, preferred to use the enzyme inhibitors or stimulators, or degradation enzymes, as a single composition or formulation.

Preferably and conveniently, the combined or single inhibitor is applied to the skin in combination with a physiologically acceptable carrier. The carrier may comprise any conventional topical formulation base such as those described in Remington's "Pharmaceutical Sciences," 17th Edition (Mack Publishing Co., Pa), the disclosure of which is incorporated by reference. A lotion, solution, cream, ointment, paste, gel, suppository, aerosol, or nebulized formulation are representative of the topical compositions of this invention.

Additional ingredients may be added to the topical composition, as long as they are physiologically acceptable and not deleterious to the epithelial cells and function. Such additives should not adversely affect the epithelial penetration efficiency of the above-noted enzyme inhibitors or stimulators, or degradation enzymes, nor cause the stability of the composition to deteriorate. Examples of ingredients which can be added to the compositions of the invention include stabilizers, preservatives, buffering agents, surfactants, emulsifiers, fragrances, humectants, and the like.

In one embodiment of this invention, a known percutaneous penetration-enhancing compound may be included in the composition to be additive or synergistic with the above enzyme inhibitors or stimulators, or degradation enzymes. Some of such penetration-enhancing compounds are described in U.S. Patent Nos. 4,424,210 and 4,316,893, the disclosures of which are incorporated by reference. The preferred compounds include propylene glycol, methyl pyrrolidone, oleyl alcohol, D SO and A20NE®. The particularly preferred penetration-enhancing compound is 1-dodecylazacycloheptan-2-one (AZONE®) (Stoughton, Arch. Dermatol.. 118, 1982). The use level of the additional penetration-enhancing compounds is not significantly different from that of the enzyme inhibitors or stimulators, or degradation enzymes, and is in the range of from about 0.01% to about 10.0% and preferably about 0.01 % to about 5.0% by weight of the topical composition.

Topical lovastatin, an H G CoA reductase inhibitor, is shown to impair the recovery of barrier function, assessed as transepidermal water loss (K.R. Feingold, et ai., J. Clin. Invest 88, 1338-1345, 1991). Also, 3-chloro-L-alanine, an irreversible inhibitor of serine-palmitoyl-transferase, is shown to slow barrier recovery, assessed as transepidermal water loss (W.M. Holleran, et al., J. Clin. Invest. 88:1338, 1991 ). However, these references neither teach nor suggest that either compound is capable of disrupting epidermal barrier function sufficient for percutaneous drug delivery. It is recognized by one skilled in the art that the inhibition of barrier recovery to excess water loss (inside to outside) and the disruption of epidermal barrier function sufficient for delivery of molecules much larger than water from the outside to the inside are not correlated.

In another embodiment of this invention, phase separation of the stratum corneum membrane bilayers is induced by application of inactive synthetic analogs of the critical lipids to significantly increase transdermal delivery of a drug or other compound of interest.

The effectiveness of the topical compositions of this invention to enhance penetration of a physiologically active substance at an epithelial site can be determined by their ability to disrupt the normal diffusion profile of marker compounds such as cyclophenol or propylene glycol through the skin.

While the present invention has been described with respect to preferred embodiments thereof, it will be understood that various changes and modifications will be apparent to those skilled in the art and that it is intended that the invention encompass such changes and modifications as falling within the scope of the appended claims. The following non-limiting examples are provided to further illustrate the present invention.

EXA PLE 1 The following ingredients were combined and blended uniformly together to produce a gel formulation: INGREDIENTS PERCENT BY WEIGHT Fluindostatin 2.0 j3-chioroalanine 1.5 Carboxyvinyl polymer 940 1.0 Ethanol 30.0 Propylene glycol 30.0 Triethylamine 1.5 Distilled water Remaining part A solution was prepared by mixing all the ingredients except triethylamine. Neutralization of the aqueous solution with triethylamine furnished a viscous gel.

Pharmaceutically active substances such as hydroxycortisone can be added to this gel for anti-inflammatory therapy.

EXA PLE 2 The following ingredients were combined and blended uniformly together to produce an ointment formulation: INGREDIENTS PERCENT BY WEIGHT Fluindostatin 1.5 β-chloroalanine 1.0 Plastibase 50W Remaining part (mineral oil 95%, polyethylene 5%) Blends of the active ingredients in ointment base were mixed together for 30 minutes at 40 rpm followed by 60 minutes at 25 rpm under vacuum to prevent aeration.

Pharmaceutically active substances such as erythromicin can be added to this ointment for antibacterial therapy.

EXAMPLE 3 The following ingredients were combined and blended uniformly together to produce a cream formulation: INGREDIENTS PERCENT BY WEIGHT TOFA 1.5 Fluindostatin 1.0 S-chloroalanine 1.0 Cetyl/stearyl alcohol 25.0 Glycerin 5.0 Oleic acid oleyl ester 3.0 Distilled water Remaining part Cetyi/stearyl alcohol (25 g), 10 g of an aqueous suspension of the active ingredient and 3 g of oleic acid oleyl ester were heated to 80· C and emulsified by stirring at that temperature with a mixture of 5 g of glycerin and 57 ml of water.

Pharmaceutically active substance such as ketoconazole can be added to this cream for antifungal therapy.

EXAMPLE 4 The following ingredients were combined and blended uniformly together to produce a cream formulation: INGREDIENTS PERCENT BY WEIGHT TOFA 1.0 Cetyl/stearyl alcohol 40.0 Polysorbate 80 10.0 Distilled water Pharmaceutically active substance such as nifedipine can be added to this cream for antihypertensive therapy.

EXAMPLE 5 EPIDERMAL BARRIER DISRUPTION To determine the epidermal barrier disruption by application of fluindostatin, an animal testing was conducted in the manner as follows: Hairless mice (two animals, B and C) were topically pretreated with fluindostatin for 7 days. In a control group, animals (two) were only treated with a vehicle. The vehicle used was a mixture of propylene glycol (PG) and ethanol (0.5 ml of 5% w/v per deuterated PC in ethanoi). After treatment, the skin surface was cleaned and stratum corneum allowed to recover to its normal state of hydration for 2 hours. An IR spectrum of the hydrophilic marker compound, propylene glycol, was recorded, and a single tape-stripping was conducted at the treated skin site. Another spectrum was recorded followed by the removal of a second tape strip. The same sequence was repeated 10 times. Figs. 4A, 4B show the test results. In Fig. 4A, control data indicate a gradual decrease of the PG concentration, while the data in Fig. 4B show lower absolute levels of PG in the stratum corneum, and a constant concentration level of PG, indicative of loss of the diffusional barrier to drug penetration. These results demonstrate that there is an absolute reduction of the permeability function and loss of the diffusion gradient with fluindostatin treatment, resulting in enhanced percutaneous transport of the marker compound, propylene glycol.

In essentially the same manner, cyclophenol (CP) was used as a lipophilic marker. Tape-stripping was repeated 7 times after application of fluindostatin. Test results are shown in Figs. 5A, 5B. In Fig. 5A, control data indicate a gradual decrease of the CP concentration, while the data in Fig. 5B show a lower, constant concentration level of CP.

Both sets of data demonstrate that fluindostatin caused significant disruption of the stratum corneum barrier, and enhanced drug delivery as illustrated both by loss of the diffusion gradient, and by the lower absolute concentrations of the marker compounds in the stratum comeum.

EXAMPLE 6 TEWL (TRANS-EPIDERMAL WATER LOSS) A female hairless mouse, aged 8 weeks, was treated with oleic acid. After oleic acid treatment, fluindostatin and/or 5-tetradecyloxy-2-furancarboxylic acid (TOFA) was applied to the animal.

TEWL (trans-epidermal water loss) was measured before and after treatment at convenient time intervals when the animal was alert. An Evaporime ter (Servo ed) was used.

First, TEWL of the left (LHS) and right (RHS) dorsal sides was measured. Then, the animal was anesthetized by 0.25 ml chloral hydrate (CH) IP injection. At t=0, an aliquot of 30 μ\ oleic acid solution (5% in propylene glycol/ethanol 7:3 (v/v)) was applied directly to both sides of the animal (~ 1.5 x 3 cm) using a Hamilton syringe. Two hours after oleic acid treatment, the TEWL rates of both sides were measured, and the RHS was treated with 30 μΙ of fluindostatin (1.5% fluindostatin in propylene glycol/ethanol 7:3 (v/v)) or TOFA (0.25% TOFA in propylene glycol/ethanol 7:3 (v/v)). TEWL was measured thereafter every 2 or 3 hours.

Test results are shown in Figs. 6A and 6B. These data demonstrate that both fluindostatin and TOFA delay the recovery of the barrier, after prior barrier disruption by the oleic acid treatment.

EXAMPLE 7 TEWL fTRANS-EPtPERMAL WATER LOSS) To prove that disruption of the permeability barrier as shown by increased TEWL results in increased delivery of known cutaneously active pharmacologic agents, the plasma and total body concentrations of lidocaine after its topical application were measured using the following protocol.

Mice were anesthetized with chloral hydrate as described in Example 6 and treated with acetone to disrupt the lipid barrier until the desired TEWL level was attained as monitored by a Meeco Evaporimeter. The mice were kept under anesthesia until the harvest of tissue samples and then sacrificed. At time zero, a penetration enhancer was applied to the whole treated flank of each mouse. After four hours TEWL was measured again and a drug formulation containing lidocaine and LHRH in a vehicle of 60% ethanol, 20% propylene glycol and 20% water. Residual formulation was removed from the skin surface with cotton balls three times and the cotton balls were put into vial #1. Five tape-strippings were conducted at the treated skin sites, and each tape was put into an individual vial numbered 2-6. At the same time, blood was drawn and stored under refrigeration. Urine was collected from the mice during the two hour drug application period and placed into vial #8.

After an additional two hours, the treated skin was cut off, the subcutaneous fat was removed, and the whole skin was placed into vial #7 to which 1 ml of tissue solubilizer was added, and the mixture was allowed to digest overnight at 55 eC. The corpse was digested in 100 ml of saponification mix at 55 °C overnight.

For analysis, 10 ml of Scintisafe (30%) was added to vials 1-6, and 8. The blood samples were centrifuged, and an aliquot of 100-200 μΐ was placed into vial #9 and 10 ml of Scintisafe (30%) was added. Similar aliquots of the corpse digest were placed into vials #10 and #11. To vials #9, 10, and 11 , 100 Ι of 30% hydrogen peroxide was added to decolorize the contents for about two hours. Then 16 ^l of Scintisafe (30%) and 150 μ\ of acetic acid were added and the mixture was let stand overnight. All vials were subjected to scintillation counting as described above.

EXAMPLE 8 By testing pairs of inhibitors or stimulators, each from a different metabolic pathway, faster screening could be achieved. Individual compounds within successful pairs were then tested to confirm barrier disruption and increased drug delivery activity of the sole compound. Two compounds from each chemical class were tested. The inhibitors and metabolic pathways are listed in Table 1 below: EXA PLE 9 Studies were conducted using the same protocols as in Example 8 to inhibit rate-limiting synthetic enzymes for the three lipids that are critical for stratum corneum barrier function - free fatty acid, ceramide, and cholesterol. Inhibition disrupts the critical mole ratio of 1 :1 :1 , thereby perturbing barrier function. Acetyl CoA Carboxylase (ACC) is the rate-limiting synthetic enzyme for free fatty acids, and its only known inhibitor is 5-tetradecyloxy-2-furancarboxylic acid (TOFA). Serine palmitoyl transferase (SPT) is the rate-limiting synthetic enzyme for ceramide, and its inhibitors betachloroanine and L-cycloserine were evaluated. HMG CoA Reductase is the rate-limiting synthetic enzyme for cholesterol and is inhibited by three compounds that were tested - fluindostatin, lovastatin, and cholesterol sulfate.

As shown in Table 2 below, when the six combinations of any two of these inhibitors were applied together, a statistically significant increased delivery of Iidocaine and of barrier permeability occurred, as indicated by increased TEWL, except in the case of the combination of TOFA plus lovastatin, which produced a statistically significant trend only in the latter parameter. In this assay, fluindostatin alone did not increase TEWL or transdermal delivery of Iidocaine. TOFA plus betachloroanlanine produced the greatest delivery of Iidocaine by increasing its delivery by nine-fold. TOFA alone did effect a statistically significant increase in transdermal delivery of Iidocaine, but the effect on TEWL achieved only a statistical trend.

TABLE 2 Compounds N Cone TEWL P Plasma Cone.

(%) (T=4 hours) (%dose /ml plasma) ΊΌΙ'Λ 1 fluiwlostalm 4 0.5/1.5 475 ± 34 0.78 ± 0.11 0. ΤΟΓ'Λ -1- cholesterol sulfate 4 0.5/1.0 588 ± 54 1.52 ± 0.35 0. 'ΓΟΙ'Λ 1 i-chloroalanine 4 0.5/1.0 520 ± 1 2.47 ± 0.22 0.

TOI'A I lovastatin 4 0.5/2.5 440 ± 79 1.35 ± 0.34 .0. fl chloroalaiiinu h cholesterol sulfate 4 1.0/1.0 525 ± 47 0.68 ± 0.10 0. l luindostatin 0-chloroalani»e 4 1.5/1.0 513 ± 41 0.48 ± 0.06 0; EXAMPLE 10 In this study, phase separation of the stratum corneum membrane bilayers was induced by application of inactive synthetic analogs of the critical lipids, as shown by significantly increased delivery of lidocaine. The delivery protocol and the barrier permeability protocol used were those of Example 7 above. The inactive analogs applied were epicholesterol, which were substituted for cholesterol, and transvaccenic acid, which was substituted for free fatty acid. The results are shown in Table 3 below.

TABLE 3 Compounds N COHC EWL P Hasina Cone (%) (T=4 un) (%dosc /ml plasma) ΊΌΙ'Λ 4 1.0 520 ± 31 1.20 ± 0.16 0.0 Huindoslalin 4 1.5 403 ± 105 0.57 ± 0.19 0.6 ! As shown by the results in Table 3, lidocaine delivery was increased nearly seven-fold with the composition containing epicholesterol and transvaccenic acid. Surprisingly, the barrier permeability was significantly decreased, indicating this composition stimulated repair of the stratum corneum barrier. t=XAMPLE 11 In this study, the same protocols as used in Example 9 above were employed to test the effect upon transdermal delivery of lidocaine by a combination of a rate-limiting enzyme inhibitor used in Example 9 with a nonrate-limiting synthetic enzyme of another critical lipid. As shown in Table 4 below, the results of this study were varied.

TABLE 4 Compounds N Coitc TEWL 1» Plasma Cone. P (%) (1=4 h(M.rs) (%< ise An! plasma) coiuiuritol /i-cpoxidc -1- fluindoslalin 4 0.5/1.5 583 ± 128 0.84 ± 0.17 0.01 t owliuilol /i cfx>xidc 1- ΤΟΡΛ 4 2.0/0.5 448 ± 80 1.27 ± 0.44 0.06 lu 1 non i. sin 1 lliiiridostatin 4 0.5/1.5 513 1.03 ± 0.25 >.I iiiiH>f lisi -1· TOPA 4 0.5/0.5 453 0|85 ± 0.15 >.1 N p:diniloyl-DL-liydroxys|>liingosiiie 3 0.5/0.5 148 ± 75 0.83 ± 0.25 0.00 1 Ί ΠΛ N |>;ilfnitoyl-I)L- ydroxysj)liiii]osiiic 4 0.5/1.5 650 ± 68 0.29 ± 0.04 0.00 1 flutnd slaliri EXAMPLE 12 Conduritol B epoxide plus TOFA, an inhibitor of β glucocerebrosldase, plus fluindostatin caused a statistically significant increase in lidocaine delivery and in barrier permeability. The combination of Conduritor B epoxide plus TOFA resulted in a statistical trend of increased delivery of lidocaine and barrier permeability. The statistical trend of barrier permeability also occurred with application of TOFA or fluindostatin with fumonisin B1 , an inhibitor of ceramide synthetase. However, neither of these two combinations resulted in a statistically significant increase in delivery of lidocaine. When TOFA or fluindostatin was combined with N-palmitoyl-DL-hydroxysphingosine, which inhibits sphingomyelinase, significantly increased delivery of lidocaine occurred. Barrier permeability markedly decreased with the former combination and minimally increased with the latter.

Thus, it can be concluded that inhibiting one or more rate limiting synthetic enzyme for the three critical lipids will increase stratum corneum permeability and deliver drugs transdermally. This activity is also achieved when a rate-limiting synthetic enzyme is combined with a synthetic enzyme for a different critical lipid. When two nonrate-limiting enzymes are combined, the results are more variable, as shown in Table 5.

TABLE 5 Compounds N Cone TEWL I» Hasina Cone.

(%) (T=4 lioure) (%dasc /ml plasma) i.ondut ilol /i epoxide + 4 0.5/0.05 398 ± 23 0.31 ± 0.04 0. hrnniophcnacylbroniidc comlurilol /J-cpoxide + 3 2.0/0.25 753 ± 149 1.60 ± 0.42 0. bionioplieiiacylbroiuide dcsipramine -1- 4 0.5/0.1 595 ± 89 .64 ± 0.09 0. broiitoplicnacylbromide lunionisia II , -1- 4 0.5/0.1 682 ± 83 0.57 ± 0.06 bromopltmiacylbroinide fiiiiuMUsin B, -I- MJ33 3 0.5/1.5 573 ± 101 0.4 ± 0.03 < N palmiW>yi-DL-liydroxyspltingosiiic 4 0.5/0.1 1000 ± 0 0.47 ± 0.O7 < 1 biumophcnacylbromide EXAMPLE 3 The importance of inhibitor concentration is shown by the failure of conduritol B epoxide alone at low doses to increase permeability or lidocaine delivery. Yet when conduritol B epoxide was frequently applied to increase its concentration, even to intact skin, statistically significant increases occurred in both lidocaine delivery and permeability, as shown by the data in Table 6 below.

TABLE 6 CniiipoiiiMls N Cone TE L P Plasma Cone.

(%) (T=4 houre) (%dosc /ml plasma) i:oi lmilol-/?-epoxi(le (intact skin) - 3 4.0 184 ± 39 0.82 ± 0.22 0 I II ), l il 7-8 doses uuliiriiol /3 -epoxide 4 4.0 253 ± 33 0.30 ± 0.04 0 .'unionism Π, 3 0.5 440 ± 149 0r24 ± 0.03 Fumonisin B1 individually had no effect upon stratum corneum barrier permeability and drug delivery.

EXAMPLE 14 The critical mole ratio of the three lipids in the stratum corneum barrier is also disrupted if normal degradation does not occur due to metabolic enzyme inhibition. As shown in Table 7 below, this was proven when lidocaine delivery was increased by a statistically significant amount by blocking two ceramide metabolic pathways. N-oleoylethanolamine, a ceramidase inhibitor, combined with either PDMP or 4-methylumbelliferyl-^-O-xyloside inhibitors of glucosyl transferase, were both successful, but only the former combination produced, in addition, a statistical trend toward increased barrier permeability. Thus, accumulation of ceramide, disrupting the critical ratio, perturbs the stratum corneum barrier.

TABLE 7 It will be apparent to one of ordinary skill in the art that many changes and modifications can be made in the invention, now being fully described, without departing from the spirit or scope of the invention. - 40 - 109036/2

Claims (1)

1. CLAIMS Use of a barrier disrupting amount of at least one agent selected from the group consisting of an inhibitor of ceramide synthesis, an inhibitor of glucosylceramide synthesis, an inhibitor of acylceramide synthesis, an inhibitor of fatty acid synthesis, an inhibitor of cholesterol synthesis, inhibitors of phospholipid, glycosphingolipid, acylceramide and sphingomyelin degradation, a degradation enzyme of free fatty acid, ceramide, sphingomyelin, acylceramide, orglucosylceramides and both inhibitors and stimulators of metabolic enzymes of free fatty acids, ceramide, and cholesterol for the preparation of a topical composition for disrupting the epithelial barrier function in a host in need of the topical administration of a physiologically active substance, substantially as described in the specification. The use according to claim 1 , wherein the inhibitor of ceramide, acylceramide, or glucosylceramide synthesis is selected from the group consisting of inhibitors of serine ' palmitoyl transferase,, inhibitors of ceramide synthetase, inhibitors of sphingomyelinase, inhibitors of jS- glucosidase, inhibitors of acid lipase, inhibitors of cr-hydroxylation, N-acyl chain length elongation, ω-acyl transferases, inhibitors of glucosyl transferase, and inhibitors of phosphatidylcholine-ceramide phosphorylcholine transferase. The use according to claim 1 , wherein the stimulator of ceramide metabolism distal to ceramide is selected from the group consisting of ω-acyl transferase, glucosy!transferase, and phosphatidylcholine- ceramide phosphorylcholine transferase. -41 " 109036 / 2 The use according to claim 2, wherein the inhibitor of serine palmitoyl transferase is selected from the group consisting of D-cycloserine, /3-chlarolanine, f!uoropaimitate, L-cycloserine, and β-fluoroalanine. The use according to claim 2, wherein the inhibitor of ceramide synthetase is a fumonisin. The use according to claim 2, wherein the inhibitor of sphingomyelinase is selected from the group consisting of tricyclodecan-9yl-xanthogenate, gentamicin, neomycin, ethylisopropylamiloride, desipramine, imipramine, chlorpromazine, perchlorperazine, N-palmitoyl- DL-dihydroxysphingosine and methylene-dioxybenzapine. The use according to claim 2, wherein the inhibitor is selected from the group consisting of N-hexylglucosyl-sphingosine, j3-glucosidase is bromoconduritol-B-epoxide, condurttol, cyclopheilitot, conduritol-B- epoxide, and deoxynojirimycin. The use according to claim 2, wherein the inhibitor of acid lipase is selected from the group consisting of boronic acid, phenylboronic acid, tetrahydrolipstatin and esterasin. The use according to claim 2, wherein the inhibitor of glucosyl transferase is selected from the group consisting of PD P, its analogs, p-nitro-phenyl-a-xyloside, 4-methyl umbelliferyl-j3-0-xylaside, and O-and p-nitrophenyl-S-O-xylopyaranoside and the inhibitor of ceramidase is N- oleoyl-ethanolamine. "^" 109036 The use according to claim 1 , wherein the inhibitor of free fatty acid synthesis is selected from the group consisting of inhibitors of acetyl CoA carboxylase, inhibitors of fatty acid synthetase, and inhibitors of phospholipase. The use according to claim 10, wherein the inhibitor of acetyl CoA carboxylase is 5-tetradecyloxy-2-furancarboxylic acid (TOFA). The use- according to claim 10, wherein the inhibitor of phospholipase is selected from the group consisting of gomisin A, 2-(p- amylcinnamyl) amino-4-chlorobenzoic acid, bromophenacylbromide, moniliid, monoaloque morioalide, 7,7-dimethyl-5,8-eicosadienoic acid, nicargoline, cepharanthine, quercetin, dibutyryl-cyclic AMP, diaminoethoxyhexesterol, R-24571 , N-oleoylethanolamine, N-(7-nitro- 2,1 ,3-benzoxadiazol-4-yl)phosphostidylserine, verapamil, diltiazam, nifedipine, quinacrine, cyclosporine A, dibucaine, prenylamine, retinoids, such as all-trans and 13-cis-retinoic acid, propana(al,_ W-7, trifluoperazine, R-24571 (calmidazolium), 1 -hexadecyl-3-trifluoroethyl glycero-sn-2-phosphomenthol (MJ33), calcium - channel blockers, verapamil, diltiazem, nifedipine, nimodipine, antimalarials, quinacrine, mepacrine, chloroquine hydroxychloroquine, beta blockers, propanaiol, labetalol, calmodulin antagonists, EGTA, thimersol, glucocorticosteroids, dexamethasone, prednisolone, nonsteroidal antiinflammatory agents, indomethacin and naproxen; and wherein the stimulator of fatty acid . metabolism is an enzyme selected from the group comprising the fatty acid to phospholipid metabolic pathway. 109036 / 2 The use according to claim 1 , wherein the inhibitor of cholesterol synthesis is selected from the group consisting of inhibitors of HMG CoA reductase, squalene epoxidase, squalene synthetase, cholesterol sulfate, phosphate synthetase, and Δ7 or Δ24 reductase. The use according to claim 1 , wherein the stimulator of cholesterol metabolism distal to cholesterol is a synthetic enzyme of a steroid hormone. The use according to claim 13, wherein the inhibitor of HMG CoA reductase is selected from the group consisting of simvastatin, lovastatin, fluindostatin, pravastatin, mevastatin, cholesterol sulfate, cholesterol phosphate, and 25-OH or 26-OH cholesterol. The use according to claim 13, wherein the inhibitor of Δ7, Δ24 reductase is selected from the group consisting of 22,25- diazacholesterol, 20,25-diazacholesterol, AY9944 and triparanol. -44" 109036 / 2 The use according to claim 1 , wherein the inhibitor of phospholipid degradation is selected from the group consisting of gomisin A, 2-(p-amylcinnamyt) amino-4-chiorQbenzoic acid, bromophenacylbromide, moniliid, monoaloque monoalide, 7,7-dimethyl-5,8-eicosadienoic acid, nicergoline, cepharanthine, quercetin, dibutyryl-cyclic AMP, diaminoethoxyhexesterol, R-24571 , N-oleoylethanolamine, N-(7-nitro-2,1 ,3-benzoxadiazol-4-yl)phosphastidylserine, verapamil, diltazepam, " nifedipine, quinacrine, cyclosporine A, dibucaine, prenylamine, retinoids, such as all-trans and 13-cis-retinoic acid, propanalol, W-7, R-24571 (calmidazolium) , 1 -hexadecyl-3-trifIuoroethyl giycero-sn-2-phospho- menthol (MJ33); calcium channel blockers, verapamil, diltiazem, nifedipine, nimodipine; antimalarials, quinacrine, mepacrine, chloroquine hydroxychloroquine, beta blockers, propanalol labetalol, calmodulin antagonists, EGTA, thimersol, glucocorticosteroids, dexamethasone and prednisolone; nonsteroidal antiinflammatory agents, indomethacin, naproxen and trifluoperazine. The i use according to claim 1-, wherein the inhibitor of glycosphingolipid degradation is selected from the group consisting of bromoconduritol-B-epoxide; conduritol-B-epoxide, and cyclophellitol. The use according to claim 1 , wherein the degradation enzyme of ceramide is ceramidase. The , use according to claim 1 , wherein the degradation enzymes of acy!ceramide are acid lipase and ceramidase. The use according to claim 1 , wherein the degradation enzymes of glucosylceramide are 3-glucostdase and ceramidase, .and the enzyme of sphingomyelin is sphingomyelinase. -45- 109036 / 2. The use according to claim 1 , wherein the inhibitor of ceramide synthesis, acylceramide synthesis, glycosylceramide synthesis, sphingomyelin synthesis, free fatty acid synthesis, cholesterol synthesis, acylceramide synthesis, and glucosylceramide synthesis, if present, is present at a concentration of from about 0.01 % to about 5.0% by weight of the total. The use according to claim 1 , wherein the composition is a lotion, cream, ointment, solution, paste, suppository, aerosol, nebulized formulation, or gel. The use according to claim 1 , wherein the composition further contains a known epithelial penetration enhancer. The use according to claim 24, wherein the penetration enhancer is selected from the group consisting of 1 -dodecylazacycloheptan-2-one, D SO, propylene glycol, oieyl alcohol, and methyl pyrrolidone. . The use according to claim 1 , wherein the composition further contains an effective amount of a physiologically active substance. A topical composition for disrupting the epithelial barrier function in a host in need of topical administration of a physiologically active substance, which comprises an epithelial barrier disrupting amount of at least one agent selected from the group consisting of an inhibitor of ceramide synthesis, an inhibitor of fatty acid synthesis, an inhibitor of cholesterol synthesis, inhibitors of phospholipid, glycosphingolipid, acylceramide and sphingomyelin degradation, a degradation enzyme of free fatty acid, ceramide, sphingomyelin, acylceramide, or glucosyl- ceramides and both inhibitors and stimulators of metabolic enzymes of free fatty acids, ceramide, and cholesterol, metabolism distal to ceramide and cholesterol, respectively, together with a pharmaceutically acceptable carrier. 28. The composition according to claim 27, wherein the active substance is present at a concentration of about 0.001% to about 60% by weight of the total. 29. The composition according to claim 27, wherein the inhibitor of ceramide, acylceramide, or glucosylceramide synthesis is selected from the group consisting of inhibitors of serine palmitoyl transferase, inhibitors of ceramide synthetase, inhibitors of sphingomyelinase, inhibitors of j3-glucosidase, inhibitors of phospholipase, inhibitors of acid lipase, inhibitors of ω-acyl transferases, inhibitors of glucosyl transferase, and inhibitors of phosphatidylcholine-ceramide phosphorylcholine transferase. 30. The composition according to claim 27, wherein the stimulator of ceramide metabolism distal to ceramide is selected from the group consisting of ω-acyl transferase, glucosyltransferase, and phosphatidylcholine-ceramide phosphorylcholine transferase. 3. . The composition according to claim 29, wherein the inhibitor of serine paJmitoyl transferase is selected from the group consisting of D-cycioserine, 3-chlorolanine, fluoropalmitate, L-cycloserine, and β- fluoroalanine. 32. The composition according to claim 29, wherein the inhibitor of ceramide synthetase is a fumonisin. 33. The composition according to claim 29, wherein the inhibitor of sphingomyelinase is selected from the group consisting of tricyclodecan- 9yl-xanthogenate, gentamicin, neomycin, ethylisopropylamiloride, desipramine, imipramine, chlorpromazine, perchlorperazine.N-palmitoyl- DL-dihydroxysphingosine and methylene-dioxybenzapine. 34. The composition according to claim 29, wherein the inhibitor is selected from the group consisting of N-hexyiglucosyl-sphingosine, 3-glucosidase is bromoconduritol-B-epoxide, conduritol, cyclophellitol, conduritol-B- epoxide, and deoxynojirimycin. 35. The composition according to claim 29, wherein the inhibitor of acid lipase is selected from the group consisting of boronic acid, phenyl- boronic acid, tetrahydrolipstatin and esterasin. 36. The composition according to claim 29, wherein the inhibitor of glucosyi transferase is selected from the group consisting of PD P, its analogs, a- or /3-xylosides, p-nitro-phenyl-cr-xyloside, 4-methyl umbelliferyl-j3-0- xyloside, and O-and p-nitrophenyl-jS-O-xylopyaranoside, and the inhibitor of ceramidase is N-oleoy!-ethanolamine. 37. The composition according to claim 27, wherein the inhibitor of free fatty acid synthesis is selected from the group consisting of inhibitors of acetyl CoA carboxylase, inhibitors of fatty acid synthetase, and inhibitors of phospholipase. 38. The composition according to claim 37, wherein the inhibitor of acetyl CoA carboxylase is 5-tetradecyloxy-2-furancarboxylic acid (TOFA). 39. The composition according to claim 37, wherein the inhibitor of phospholipase is selected from the group consisting of gomisin A, 2-(p- amylcinnamyl) amino-4-chlorobenzoic acid, bromophenacylbromide, moniliid, monoaloque monoalide, 7,7-dimethyl-5,8-eicosadienoic acid, nicergoiine, cepharanthine, quercetin, dibutyryl-cyclic AMP, diaminoethoxyhexesterol, R-24571, N-oleoylethanolamine, N-(7-nitro- 2,1,3-benzoxadiazol-4-yl)phosphostidylserine, verapamil, diltiazam, nifedipine, quinacrine, cyclosporine A, dibucaine, prenylamine, retinoids, such as all-trans and 13-cis-retinoic acid, propanalol, W-7, trifluoperazine, R-24571 (calmidazolium), 1-hexadecyl-3-trifIuoroethyl giycero-sn-2-phosphomenthol (MJ33), calcium channel blockers, verapamil, diltiazem, nifedipine, nimodipine, antimalarials, quinacrine, mepacrine, chloroquine hydroxychloroquine, beta blockers, propanalol, labetalol, calmodulin antagonists, EGTA, thimersol, glucocorticosteroids, dexamethasone and prdnisolone, nonsteroidal antiinflammatory agents, indomethacin and naproxen. 40. The composition according to claim 27, wherein the inhibitor of cholesterol synthesis is selected from the group consisting of inhibitors of HMG CoA reductase, squalene epoxidase, squalene synthetase, cholesterol sulfate, phosphate synthetase, and Δ7 or Δ24 reductase. 41. The composition according to claim 27, wherein the stimulator of cholesterol metabolism distal to cholesterol is a synthetic enzyme of steroid hormone. 42. The composition according to claim 41 , wherein the inhibitor of HMG CoA reductase is selected from the group consisting of simvastatin, lovastatin, fluindostatin, pravastatin, mevastatin, cholesterol sulfate, cholesterol phosphate, and 25-OH or 26-OH cholesterol. 43. .The composition according to claim 41 , wherein the inhibitor of Δ7, Δ24 reductase is selected from the group consisting of 22,25- diazacholesterol, 20,25-diazacholesterol, AY9944 and triparanol. The composition according to claim 27, wherein the inhibitor of phospholipid degradation is selected from the group consisting of gomisin A, 2-{p-amylcinnamyl) amino-4-chlorobenzoic acid, bromophenacylbromide, moniliid, monoaloque monoalide, 7,7-dimethyl-5,8-eicosadienoic acid, nicergoline, cepharanthine, quercetin, dibutyryl-cyclic AMP, diaminoethoxyhexesterol, R-24571 , N-oleoylethanolamine, N-(7-nitro-2,1 ,3-benzoxadia2ol-4-yl)phosphostidylserine, verapamil, diltazepam, nifedipine, quinacrine, cyclosporine A, dibucaine, prenylamine, retinoids, such as all-trans and 13-cis-retinoic acid, propanalol, W-7, trifluoperazine, R-24571 (caimidazolium), 1 -hexadecyl-3-trifluoroethyl glycero-sn-2-phosphomenthol (MJ33), calcium channel blockers, verapamil, di\tiazem, nifedipine, nimodipine; antimalarials, quinacrine, mepacrine, chloroquine hydroxychloroquine, beta blockers, propanalol, labetalol, calmodulin antagonists, EGTA, thimersol, glucocorticosteroids, dexamethasone and prednisolone, nonsteroidal antiinflammatory agents, indomethacin and naproxen; and wherein the stimulator of fatty acid metabolism is an enzyme selected from the group comprising the fatty acid to phospholipid metabolic pathway. The composition according to claim 27, wherein the degradation enzyme of ceramide is ceramidase. The composition according to claim 27, wherein the degradation enzymes of acylceramide are acid lipase and ceramidase. The composition according to claim 27, wherein the degradation enzymes of glucosylceramide are 3-glucosidase and ceramidase, and the enzyme of sphingomyelin is sphingomyelinase. The composition according to claim 27, wherein the inhibitor of ceramide synthesis, free fatty acid synthesis, cholesterol synthesis, acylceramide synthesis, sphingomyelin synthesis and glucosylceramide synthesis, if present, is present at a concentration of from about 0.01% to about 5.0% by weight of the total. The composition according to claim 27, wherein the composition is a lotion, cream, ointment, solution, paste, suppository, aerosol, nebulized formulation, or gel. 50. The composition according to claim 27, further comprising an epithelial penetration enhancing compound. 51. The composition according to claim 50, wherein the epithelial penetration enhancing compound is selected from the group consisting of 1 - dodecylazacycloheptan-2-one, DMSO, propylene glycol, oleyl alcohol, and methyl pyrrolidone. 52. The composition according to claim 27, further comprising a therapeutically effective amount of the physiologically active substance. A topical composition comprising: (a) 0% to about 5.0% by weight of an inhibitor of ceramide synthesis; (b) 0% to about 5.0% by weight of an inhibitor of free fatty acid synthesis; (c) 0% to about 5.0% by weight of an inhibitor of cholesterol synthesis; (d) 0% to about 5.0% by weight of an inhibitor of degradation selected from the group consisting of phospholipid, glyco- sphingolipid, sphingomyelin, and acylceramide ; (e) 0% to about 5.0% by weight of a degradation enzyme for ceramide or free fatty acid; (f) 0% to about 5.0% by weight of stimulators of steps of metabolism of ceramide, free fatty acid and cholesterol metabolism distal to ceramide, free fatty acid, and cholesterol, respectively; and (g) a sufficient amount of a physiologically acceptable carrier to total 100%. The composition according to claim 53, further comprising about 0.001% to about 20% by weight of a physiologically active substance. The composition according to claim 54, wherein the physiologically active substance is selected from the group consisting of an antimicrobial, anti-inflammatory, anti-oxidant, antineoplastic, hormonal, and nutritional agent. 56. The composition according to claim 51 , wherein the inhibitor of ceramide, acylceramide, or glucosylceramide synthesis is selected from the group consisting of inhibitors of serine palmitoyl transferase, inhibitors of ceramide synthetase, inhibitors of sphingomyelinase, inhibitors of jS-glucosidase, inhibitors of acid lipase, inhibitors of omega- acyl transferases, inhibitors of glucosyl transferase, and inhibitors of phosphatidylcholine-ceramide phosphorylcholine transferase. 57. The composition according to claim 53, wherein the stimulator of ceramide metabolism distal to ceramide is selected from the group consisting of ω-acyl transferase, glucosyltransferase, and phosphatidylcholine-ceramide phosphorylcholine transferase. 58. The composition according to claim 53, wherein the inhibitor of free fatty acid synthesis is selected from the group consisting of inhibitors of acetyl CoA carboxylase, inhibitors of fatty acid synthetase, and inhibitors of phosphoiipase. 59. The composition according to claim 53, wherein the inhibitor of cholesterol synthesis is selected from the group consisting of inhibitors of H G CoA reductase, squalene epoxidase, squalene synthetase, as well as oxygenated sterols, cholesterol sulfate or phosphate, and Δ7 or Δ24 reductase. 60. The composition according to claim 53, wherein the stimulator of cholesterol metabolism distal to cholesterol is a synthetic enzyme of a steroid hormone. 61. The composition according to claim 53, further comprising an epithelial penetration enhancing compound. The composition according to claim 61 , wherein the epithelial penetration enhancing compound is selected from the group consisting of 1-dodecylazacycloheptan-2-one, DMSO, propylene glycol, oleyl alcohol, and methyl pyrrolidone. For Ihe A ilican DR. REIN! IN AND PARTNERS By: