Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0014—Skin, i.e. galenical aspects of topical compositions

Definitions

• This invention relates generally to a novel method for enhancing penetration of physiologically active substances for cutaneous or transdermal delivery through epithelium which comprises the stratum corneum/epidermis and keratinizing mucous membranes. More specifically, it relates to a method and composition for 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 glucosylceramide synthesis, an inhibitor of acylceramide synthesis, an inhibitor of sphingomyelin synthesis, an inhibitor of fatty acid synthesis, an inhibitor of cholesterol synthesis, inhibitors of phospholipid, glycosphingolipid, including glucosylceramide, acylceramide and sphingomyelin degradation, a degradation enzyme of free fatty acid, ceramide, acylceramide, or glucosylceramides and sphingomyelin, and both inhibitors and stimulators of metabolic enzymes of free fatty acids, cer
• 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 xenobiotics.
• the epithelial barrier is a system of multilayered 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 multilay ⁇ ered 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.
• Typical solvents or detergents alter the physical properties of the multilayered lipid bilayers.
• solvents or detergents include dimethylsulfoxide (DMSO), oleyl alcohol (OA), propylene glycol (PG), methyl pyrrolidone and AZONE ® (dodecylazyl cycloheptan 2-one).
• DMSO dimethylsulfoxide
• OA oleyl alcohol
• PG propylene glycol
• AZONE ® dodecylazyl cycloheptan 2-one
• 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 multilayered 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 five ⁇ fold, 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.
• polar head group e.g., DMSO
• nonpolar tail group e.g., OA
• a formulation comprising 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, is very effective for disrupting epithelial barrier function in a host, and thereby enhancing penetration of a physiologically active substance administered topically.
• this invention provides a method for disrupting epithelial barrier function in a host in need of topical administration of a physiologically active substance which comprises applying to the skin of the host a 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 glucosylceramide synthesis, an inhibitor of free fatty acid synthesis, an inhibitor of cholesterol synthesis, an inhibitor of phospholipid or glycosphingolipid, acylceramide and sphingomyelin degradation, both inhibitor and a stimulator of steps of free fatty acid ceramide and cholesterol metabolism distal to these compounds, and a degradation enzyme of free fatty acids, ceramides, acylceramide, and glucosylceramides or sphingomyelin or glucosylceramides and both inhibitors and stimulators of metabolic enzymes of free fatty acids, ceramide, and cholesterol.
• an agent selected from the group consisting of an inhibitor of ceramide synthesis
• 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 glucosylceramide synthesis, an inhibitor of sphingomyelin synthesis, an inhibitor of free fatty acid synthesis, and an inhibitor of cholesterol synthesis, inhibitors of phospholipid, glycosphingolipid, including glycosylceramide, acylceramide and sphingomyelin degradation, a degradation enzyme of free fatty acid, ceramide, acylceramide, sphingomyelin or glucosylceramides and both inhibitors and stimulators of metabolic enzymes of free fatty acids, ceramide, and cholesterol
• 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.
• FIGURE 4B shows the propylene glycol concentrations at an epidermal surface of tested animals when they were treated with fluvastatin (fluindostatin).
• fluvastatin fluindostatin
• FIGURE 5A shows the cyclophenol concentrations at an epidermal surface of control animals when they were treated with a vehicle.
• FIGURE 5B shows the cyclophenol concentrations at an epidermal surface of tested animals when they were treated with fluindostatin.
• 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).
• This invention is based on the discovery that when the biosynthesis of one or more of the epithelial lipids, ceramides, acylceramide, glucosylceramides, sphingomyelin, 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.
• the inhibition of biosynthetic enzymes or stimulation of degradative 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.
• 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 sphingomyelin synthesis, an inhibitor of glucosylceramide synthesis, an inhibitor of acylceramide synthesis, an inhibitor of sphingomyelin synthesis, an inhibitor of fatty acid synthesis, an inhibitor of cholesterol synthesis, inhibitors of phospholipid, glycosphingolipid, including glucosylceramide, acylceramide and sphingomyelin degradation, a degradation enzyme of free fatty acid, ceramide, acylceramide, or glucosyl ⁇ ceramides and sphingomyelin and both inhibitors and stimulators of metabolic enzymes of free fatty acids, ceramide, and cholesterol.
• agent selected from the group consisting of inhibitors of ceramide synthesis, an inhibitor of sphingomyelin synthesis, an inhibitor of glucosylceramide synthesis, an inhibitor of
• 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.
• 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.
• 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.
• physiologically active substance is intended to encompass any substance that will produce a physiological response when topically administered to a host.
• 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 substanc ⁇ es 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-fluorouracil, bleomycin, methotrexate, hydroxyurea; anti- inflammatories 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, verapamil, nifedipine, and diltiazem; analgesics such as acetaminophen and aspirin; anti-viral agents such as acyclovir, rib
• the active substance may be water-soluble or water-insoluble and may include higher molecular weight proteins, peptides, carbohydrates, glycoproteins, lipids, and glycolipids.
• proteinaceous active substances which can be included are immunomodulators and other biological response modifiers.
• immune response modifiers include such compounds as cytokines, including tumor necrosis factors, interleukins, growth factors, colony stimulating factors, and interferons.
• 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.
• the appropriate dosage levels of all the physiologically active substances are known to one skilled in the art. These conventional dosage levels correspond to the upper range of dosage levels for composi- tions, including a physiologically active substance and an epithelial barrier- disrupting agent.
• dosage levels significantly lowering the conventional dosage levels may be used with success.
• 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, acylceramide, sphingomyelin, and glucosylceramide synthesis and metabolism include inhibitors of serine palmitoyl transferase such as / 9-chloroalanine, fluoropalmitate, and ⁇ -fluoroalanine, and inhibitors of ceramide synthetase such as fumonisins.
• Inhibitors of sphingomyelinase include agentssuch as tricyclodecan-9-yl-xanthogenate,ethylisopropylamiloride, N-palmitoyl-DL-dihydrosphingosine, methylene-dioxybenzapine, tricyclodecan- 9yl-xanthogenate; aminoglycosides including gentamicin and neomycin; ethyliso-proplylamiloride; tricyclic antidepressants, including desipramine and imipramine; and phenothiazines including chlorpromazineand perchlorperazine.
• agents such as tricyclodecan-9-yl-xanthogenate,ethylisopropylamiloride, N-palmitoyl-DL-dihydrosphingosine, methylene-dioxybenzapine, tricyclodecan- 9yl-xanthogenate; aminoglyco
• Inhibitors of glucosylceramide synthesis further include inhibitors of UDP- glucose-ceramide glucosyl transferase (glucosyl transferase), such as 1-phenyl- 2-deanoylamino-3-morphalino-1 -propanol (PDMP), its analogs, ⁇ - and ⁇ - xylosides; and alpha xylosides including p-nitro-phenyl- ⁇ -xyloside and beta xylosides including 4-methyl umbelliferyl-/9-0-xyloside. 0-and p-nitrophenyl-yS-O- xylopyaranoside.
• Inhibitors of acylceramide synthesis further include inhibitors of ⁇ -hydroxylation, N-acyl chain length elongation, and ⁇ -acyl transferase.
• Inhibitors of acid lipase include the boronic acids, including phenylboronic acid, tetrahydrolipstatin and esterasin.
• glycosphingolipids including glucosylceramide, are not metabolized into ceramide, as occurs in the stratum corneum. Therefore, inhibitors of glycosphingolipids including inhibitors of ⁇ - glucosidase such as N-hexylglucosyl-sphingosine, bromoconduritol 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.
• inhibitors of glycosphingolipids including inhibitors of ⁇ - glucosidase such as N-hexylglucosyl-sphingosine, bromoconduritol B-epoxide, conduritol, cyclophellitol, conduritol B-epoxide, and deoxynojirimycin will effectively perturb the barrier in the stratum corn
• D-cycloserine, ⁇ -chloroalanine, L-cycloserine, /9-fluoroalanine, fluoropalmitate, and fumonisins are preferred inhibitors of epithelial sphingolipid synthesis, with / 3-chloroalanine and fumonisins being most preferred.
• Stimulators of glucosyl transferase, ⁇ -hydroxylation, N-acyl chain length elongation, and ⁇ -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 0.0001 % to about 20% by weight of the total, with about 0.01% to about 5% preferred.
• 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, monoalide, 7,7-dimethyl-5,8-eicosadienoic acid, nicergoline, cepharanthine, quercetin, dibutyryl-cyclicAMP, R-24571 , N-oleoylethanolamine, N-(7-nitro-2,1 ,3-benzoxadiazol-4-yl) phosphostidyl serine, cyclosporine A, topical anesthetics, including dibucaine, prenylamine, retinoids, such as all-trans and 13-cis
• 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 0.0001 % to about 20% by weight of the total, with a preferred range of about 0.01 % to about 5%.
• 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 (HMG) 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 ⁇ 7 or ⁇ 24 reductases such as 22,25-diazacholesterol, 20,25-diazacholestenol, AY9944, and triparanol.
• HMG HMG CoA reductase
• simvastatin lovastatin
• fluindostatin fluindostatin
• pravastatin pravastatin
• mevastatin mevastatin
• 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 0.0001% to about 20% by weight of the total, with a preferred range of about 0.01% to about 5%.
• the degradation enzyme of ceramide is ceramidase.
• the degradation enzymes of acylceramides are acid lipase followed by ceramidase.
• the degradation enzymes of glucosylceramide are -glucocerebrosidase followed by ceramidase.
• the degradation enzyme of sphingomyelin is sphingomyelinase.
• An inhibitor of ceramidase is N-oleoyl-ethanolamine.
• An effective concentration range for these degradation enzymes is generally from about 0.0001% to about 20% by weight of the total, with a preferred range of about 0.01% to about 5%.
• 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 free fatty acid, cholesterol, or ceramides.
• An effective concentration range for such stimulators is generally from about 0.0001% to about 20% by weight of the total, with a preferred range of about 0.01 % to about 5%.
• 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 pharmacologically 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, with the pharmacologically active compound as a single composition or formulation.
• 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.
• ingredients which can be added to the compositions of the invention include stabilizers, preservatives, buffering agents, surfactants, emulsifiers, fragrances, humectants, and the like.
• 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, DMSO and AZONE®.
• 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 0.0001% to about 20.0% and preferably about 0.01% to about 5.0% by weight of the topical composition.
• Topical lovastatin an HMG CoA reductase inhibitor
• ⁇ -chloro-L-alanine an irreversible inhibitor of serine-palmitoyl-transferase
• W.M. Holleran ef al., J. Clin. Invest. 88:1338, 1991.
• 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.
• 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.
• a solution was prepared by mixing all the ingredients except triethylamine. Neutralization of the aqueous solution with triethylamine furnished a viscous gel.
• compositions such as hydroxycortisone can be added to this gel for anti-inflammatory therapy.
• 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.
• compositions such as erythromicin can be added to this ointment for antibacterial therapy.
• Cetyl/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.
• ketoconazole can be added to this cream for antifungal therapy.
• compositions such as nifedipine can be added to this cream for antihypertensive therapy.
• Hairless mice two animals, B and C were topically pretreated with fluindostatin for 7 days.
• 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 ethanol).
• PG propylene glycol
• ethanol 0.5 ml of 5% w/v per deuterated PC in ethanol.
• Figs. 4A, 4B show the test results.
• control data indicate a gradual decrease of the PG concentration
• 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.
• cyclophenol was used as a lipophilic marker. Tape-stripping was repeated 7 times after application of fluindostatin. Test results are shown in Figs. 5A, 5B.
• control data indicate a gradual decrease of the CP concentration, while the data in Fig. 5B show a lower, constant concentration level of CP.
• fluindostatin and/or 5-tetradecyloxy-2-furancarboxylic acid (TOFA) was applied to the animal.
• TEWL trans-epidermal water loss
• TEWL was measured also using the following protocol.
• mice Four hairless 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 electrolytic water analyzer (Meeco Inc., Warrington, PA). The mice were kept under anesthesia until the harvest of tissue samples and then sacrificed. At time zero, the tested drug delivery compound listed in Tables 2 - 7 below was applied to the whole treated flank of each mouse. After four hours TEWL was measured again and two drug formulations, one containing lidocaine and the other containing Luteinizing Hormone Releasing Hormone (LHRH) in a vehicle of 60% ethanol, 20% propylene glycol and 20% water.
• LHRH Luteinizing Hormone Releasing Hormone
• 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.
• 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.
• 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° C.
• the corpse was digested in 100 ml of saponification mix at 55° C overnight.
• the vehicle for these studies is an effective drug delivery agent in its own right comprising propylene glycol ethanol, and water.
• Fumonisin B FUM Ceramide Synthetase l-Hexadecyl-3-trifluoroethyl glycero-sn-2- MJ33 Phospho- Free Fatty Acid, phosphomenthol lipase A 2 Ceramide
• N-Oleoylethanolamine NOEA Ceramidase, Ceramide, Phospho-lipase Free Fatty Acid
• Acid lipase ALP Degredation Acylceramide Degredation l-PhenyI-2-decanoylamino-3-morpholino-l- PDMP Glucosyl- Ceramide propanol transferase Degradation
• ACC Acetyl CoA Carboxylase
• TOFA 5-tetradecyloxy-2-furancarboxylic acid
• SPT Serine palmitoyl transferase
• HMG CoA Reductase is the rate-limiting synthetic enzyme for cholesterol and is inhibited by three compounds that were tested - fluindostatin and lovastatin, which are chemically related, and cholesterol sulfate.
• the critical lipid rate-limiting enzyme inhibitors were tested as solitary agents as shown in Table 3.
• TOFA, fluindostatin, and cholesterol sulfate as the solitary inhibitors each produced a statistically very significant increase in lidocaine delivery while beta chloroalanine produced a significant increase.
• TOFA very significantly increased water permeability while fluindostatin and cholesterol sulfate significantly increased it.
• Beta chloroalanine did not increase stratum corneum water permeability in this assay.
• Example 9 the same protocols as used in Example 9 above were employed to test the effect upon transdermal delivery of lidocaine and LHRH by a combination of a rate-limiting enzyme inhibitor used in Example 9 with a nonrate-limiting synthetic enzyme of another of the three critical lipids. As shown in Table 4 below, all seven of these combinations successfully delivered lidocaine, with five producing a very significant increase. Fumonisin B1 combined with TOFA or fluindostatin, significantly increased lidocaine delivery.
• Conduritol B epoxide an inhibitor of ⁇ glucocerebrosidase, applied with fluindostatin very significantly increased stratum corneum water permeability but when combined with TOFA a significant increase in water permeability resulted.
• Fumonisin B1 an inhibitor of ceramide synthetase, when combined with cholesterol sulfate, very significantly increased water permeability.
• fumonisin B1 was applied with TOFA a significant increase of water permeability resulted.
• N-palmitoyl-DL-dihydroxysphingosine an inhibitor of sphingomyelinase
• fluindostatin an inhibitor of sphingomyelinase
• Inhibitors of phospholipase A such as bromophenacylbromide (BPB) and MJ33 are particularly active because they actually inhibit both free fatty acid and ceramide synthesis. All eight of the tested combinations comprise one of these two inhibitors.
• BPB bromophenacylbromide
• MJ33 MJ33
• All eight of the tested combinations comprise one of these two inhibitors.
• BPB When fumonisin B1 is applied with either BPB or MJ33, a very significant increase in lidocaine delivery, with a significant increase in water permeability occurs.
• BPB When BPB is applied with deoxynojirimycin, an inhibitor of B glucocerebrosidase, or NPHS, very significant increases of lidocaine delivery and water permeability resulted.
• BPB combined with NPHS most successfully delivered LHRH by increasing this peptide's delivery by 7.6 times greater than the amount delivered by the vehicle as shown in Table 8.
• BPB combined with deoxynojirimycin, and the combination of fumonisin B1 and MJ33 each increased delivery of LHRH by 2.0 and 1.7 times respectively as compared to vehicle.
• Fumonisin B1 and N-oleolyethanolamine an inhibitor of both phospholidase A and ceranidase, each very significantly increased lidocaine delivery while NPHS significantly increased it. Both NPHS and N-oleolyethanolamine very significantly increased water permeability. Fumonisin B1 successfully delivered LHRH as shown in Table 8 by increasing its delivery by 3.8 times greater than the vehicles.
• 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 resulting in critical lipid accumulation as shown in Table 7. This concept was proven when lidocaine was successfully delivered and water permeability increased when ceramide metabolic pathways were blocked, resulting in its accumulation.
• Acid lipase metabolizes acylceramide to ceramide. Its application also results in significantly increased lidocaine delivery and water permeability.
• Acid lipase as a solitary delivery compound increased LHRH plasma concentration by 2.2 times as compared with its vehicle, Morpholinoethane Sulfonic Acid (MES), as shown in Table 8.
• MES Morpholinoethane Sulfonic Acid
• N-palmitoyl-DL-hydroxysphingosine 3 0.5/0.5 148 ⁇ 75 >.l 0.83 + 0.25 ⁇ .0001 56.58 ⁇ 14.48 ⁇ .0001 + TOFA
• N-palmitoyl-DL-hydroxysphingosine 4 0.5/1.5 650 ⁇ 68 ⁇ .003 0.29 ⁇ 0.04 ⁇ .0001 18.67 ⁇ 1.38 ⁇ .0001 + fluindostatin fumonisin B, + cholesterol sulfate 3 0.5/1.0 950 ⁇ 43 ⁇ .0001 0.52 ⁇ 0.03 ⁇ .0001 27.57 ⁇ 6.93 ⁇ .001
• N-palmitoyl-DL-hydroxysphingosine 0.5/0.1 1000 ⁇ 0 ⁇ .0001 0.47 + 0.07 ⁇ .0001 33.24 + 2.07 ⁇ .0001 + bromophenacylbromide bromophenacylbromide + 0.2/4.0 753 + 153 ⁇ .001 0.65 + 0.11 ⁇ .001 34.60 + 3.27 ⁇ .001 deoxynojirimycin
• N-palmitoyl-DL-hydroxysphingosine 4 0.5 793 ⁇ 124 ⁇ .0001 0.21 ⁇ 0.03 0.012 6.88 ⁇ 0.69 > . l
• N-oleoylethanolamine 4 0.5 720 ⁇ 229 ⁇ .001 1.2 ⁇ 0.32 ⁇ .0001 42.58 ⁇ 2.4 ⁇ .0001 bromophenacylbromide (intact skin) 3 0.15 98 ⁇ 47 0.127 0.19 ⁇ 0.10 0.165 14.73 ⁇ 5.86 0.092 bromophenacylbromide 4 0.2 690 ⁇ 177 ⁇ .001 0.84 ⁇ 0.07 ⁇ .001 39.42 ⁇ 11.09 ⁇ .001

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• 1994
  • 1994-03-18 IL IL10903794A patent/IL109037A/he active IP Right Grant
  • 1994-03-21 AU AU65894/94A patent/AU6589494A/en not_active Abandoned
  • 1994-03-21 WO PCT/US1994/003030 patent/WO1994021230A1/en not_active Application Discontinuation

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