Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C327/00—Thiocarboxylic acids
  • C07C327/20—Esters of monothiocarboxylic acids
  • C07C327/32—Esters of monothiocarboxylic acids having sulfur atoms of esterified thiocarboxyl groups bound to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
  • C07C327/34—Esters of monothiocarboxylic acids having sulfur atoms of esterified thiocarboxyl groups bound to carbon atoms of hydrocarbon radicals substituted by carboxyl groups with amino groups bound to the same hydrocarbon radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
  • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
  • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated

Definitions

• This invention is a method for the treatment of hyperkeratosis and diseases mediated by proteases that includes the topical or systemic administration of an effective amount of N- acetylcysteine or a derivative or salt thereof.
• Hyperkeratosis is an increase in the thickness of the stratum corneum (the outermost layer of the epidermis) which can be the result of either the increased production or decreased loss of keratinized cells.
• the general term "hyperkeratosis” encompasses both ort-hokeratosis (an increase in fully cornified cells) and parakeratosis (an increase in partially cornified cells) .
• keratin filament proteins are deposited in their reduced thiol forms and are subsequently oxidized to disulfides during the cornification process (Sun TT, Green, H., Cell , 1976: 9(4) 511-21; Sun TT, Green, H., J. Biol Chem, 1978, 253(6) 2053-60; Steinert, P.M., et al, Proc . Natl . Acad . Sci USA , 1981: 78(7) 4097-101).
• Examples of diseases in which hyperkeratosis are displayed range from simple warts, corns and calluses, which are examples of focal or localized lesions associated with hyperkeratosis, to more severe disorders including ichthyosis, keratoderma, lichen planus and psoriasis, which can involve large areas or, in some cases, all of the skin surface.
• Ichthyoses comprise a family of disorders characterized by the abnormally increased accumulation of scale on the skin.
• Ichthyosis vulgaris is the most common form of the dise.ase, occurring in one out of three hundred people (Baden, H.P. in Dermatology in General Medicine, Fitzpatrick, T.B. , et al eds . , McGraw- Hill: New York, 1987. pp 506). It is characterized by very rough skin with fine scales that are white or translucent with turned up edges. Although ichthyosis vulgaris is a lifelong condition, it generally first presents before age five. The severity of the disease has been reported to improve with age. Histologically, this disease is characterized by mild to moderate hyperkeratosis and occasional parakeratosis. The granular cell layer usually is either absent or very thin.
• ichthyosis vulgaris is thought to be caused by hyperproliferation (overproduction) of keratinocytes, the precise pathogenesis is unclear (Littzi, S.J. in The Manual of Clinical Dermatology, Olbricht, S.M., Bigby, M.E., Arndt, K.A. eds, Little Brown, Boston, 1992, p 73-76).
• X-linked ichthyosis effects one in six thousand males (Baden, H.P. in Dermatology in General Medicine , Fitzpatrick, T.B., et al eds., McGraw-Hill: New York, 1987. pp 506).
• the scaling is usually more severe than in ichthyosis vulgaris and the majority of cases present before age one.
• the ears, neck and scalp are commonly affected by X-linked ichthyosis.
• the scales in X-linked ichthyosis are usually large and dark due to the oxidation of keratin.
• Light microscopy of the ichthyotic skin reveals a thickened stratum corneum, a normal to slightly thickened granular layer, acanthosis (loss of intercellular adhesion) and a slight perivascular lymphocyte infiltrate (inflammation localized around blood vessels).
• Lamellar ichthyosis presents at birth as a tight non-pliable film of the stratum corneum enveloping the newborn infant, and develops into large thick scales over most of the body. It occurs in one of every hundred thousand people (Baden, H.P. in Dermatology in General Medicine , Fitzpatrick, T.B., et al eds., McGraw-Hill: New York, 1987. pp. 506). Lamellar ichthyosis is characterized histologically as hyperkeratosis with focal parakeratosis, a normal to slightly thickened granular layer, acanthosis and follicular plugging.
• Epidermolytic hyperkeratosis Bullous congenital ichthyosiform erythroderma occurs shortly after birth as erythematous, moist skin that blisters easily.
• the incidence of this form of ichthyosis is similar to that of lamellar ichthyosis, one in one hundred thousand infants (Baden, H.P. in Dermatology in General Medicine , Fitzpatrick, T.B., et al eds., McGraw-Hill: New York, 1987. pp 506).
• the condition is aggravated during birth and, as the stratum corneum heals, dry scaly skin is formed.
• This disease may involve the entire surface of the skin, and is characterized by epidermolytic hyperkeratotic scales which usually are dark and which may be associated with an unpleasant odor. While the blistering, or epidermolysis, occurs mainly in children, the condition may reoccur in adulthood.
• epidermolytic hyperkeratosis displays variable hyperkeratosis, papillomatosis (formation of a microscopically undulating projection), and acanthosis (loss of intercellular adhesion) with distinctive vacuolization of the granular and upper spinous layers of the epidermis.
• Treatments for the various ichthyotic disorders primarily include hydration and removal of the scale.
• Application of petrolatum has been reported to improve the condition, as does ⁇ - hydroxy acid or urea-containing creams. While such creams have limited benefit for ichthyosis vulgaris, they do not improve other forms of ichthyosis.
• keratolytic agents can diminish corneocyte adhesion and thereby decrease scaling in these conditions.
• Oral synthetic retinoids may dramatically effect many of these conditions, but these drugs are associated with a number of adverse side effects including teratogenicity, night blindness, and bone spurs.
• Psoriasis is a disease of the skin that has unique clinical and pathogenetic features and which is characterized by its chronic nature (Baden, H.P. in Dermatology in General Medicine , Fitzpatrick,
• the disease may occur in up to two percent of the U.S. population. It is characterized by excess but controlled (non-neoplastic) keratinocyte proliferation associated with cutaneous inflammation. Once psoriasis occurs, it will persist throughout life, with periods of disease activity which occur at unpredictable intervals. The marked tendency of the disease to wax and wane, as well as its tendency to occur in greater frequency in certain families makes it unique. Psoriasis can be diagnosed by the characteristic skin lesions (Baden, H.P.
• psoriasis vulgaris psoriasis vulgaris, plaque type psoriasis
• the redness and scale of this type often persist for months and years.
• the plaques tend to maintain a constant size and usually occur on the elbows, knees, scalp, and lumbar areas.
• Other types of psoriasis which can be recognized by their constellation of clinical and histological features, include eruptive (guttate) psoriasis, generalized pustular psoriasis, psoriatic erythroderma, localized and annular pustular psoriasis.
• Microscopic changes in the skin include a thickened (3-5 times normal) epidermis mass, thin elongated papillae in the dermis, and the presence of lymphocytes, histocytes and neutrophils around the blood vessels.
• the inflammatory features are more prominent than in plaque type psoriasis (Baden, H.P. in Dermatology in General Medicine , Fitzpatrick, T.B., et al eds., McGraw-Hill: New York, 1987. pp. 529).
• psoriasis may have an autoimmune/inflammatory component, in which antigen-antibody complexes cause infiltration of lymphocytes and other inflammatory cells (leukocytes) into the stratum corneum.
• leukocytes lymphocytes and other inflammatory cells
• neutrophils release inflammatory mediators such as serine proteases and oxygen radicals which contribute to tissue damage.
• proteases There are a number of diseases that affect the skin and mucosal membranes which have been found to be mediated by proteases.
• protease mediated disorders include lichen planus, canker sores (aphthous ulcers), and a number of bullous diseases, including but not limited to pemphigus, bullous pemphigoid and cicatricial pemphigoid.
• Lichen Planus is a relatively common disease that results in cutaneous lesions and often oral lesions. Its prevalence averages between 0.5 and 1.0 percent in patients in large dental clinics (Arndt, K. , in Fitzpatrick, Eisen, Wolff, Freedberg and Austen, Dermatology in General Medicine , 1987, Vol.l, McGraw-Hill, Inc., New York, pp. 967-973). The disease occurs primarily after the age of 20, with most cases presenting in 40 to 60 year old patients (Arndt, K. in Fitzpatrick, Eisen, Wolff, Freedberg and Austen, Dermatology in General Medicine , 1987, Vol. 1, McGraw-Hill, Inc., New York, pp. 967-973).
• lichen planus Although squamous cell carcinoma can arise in lesions of chronic oral lichen planus, lichen planus is often self-limiting and requires treatment only if it is symptomatic (Bleicher, P.A. in Manual of Clinical Problems in Dermatology, Olbricht, Bigby and Arndt, eds., 1992, Little, Brown & Co., Boston, pp. 85-89). In certain instances, however, lichen planus results in significant morbidity, and in the case of severe or chronic lesions involving mucosal surfaces, potentially debilitating pain. Ulcerating lesions can be very persistent, sometimes lasting months or years.
• Systemic corticosteroid therapy may be of some benefit for the treatment of lichen planus (Arndt, K. in Fitzpatrick, Eisen, Wolff, Freedberg and Austen, Dermatology in General Medicine , 1987, Vol. 1, McGraw-Hill, Inc., New York, pp. 967-73).
• the most reliable method of treating ulcerative lichen planus symptoms is with intra-lesional steroid injections, which is often repeated at frequent intervals.
• Potent topical steroids such as beta- methasone dipropionate and clobestol propionate are also be helpful, but the medication must be applied very frequently (every hour or so) .
• Bullous Diseases A number of the protease mediated diseases are classified as bullous disorders. Bullae, more commonly known as blisters, are circumscribed, fluid containing elevated lesions which are usually more than 5 mm in diameter. Diseases exhibiting bullae can affect the skin or the mucous membranes. These diseases are very debilitating and potentially fatal. Patients can succumb to fluid or electrolyte imbalance or infection if serious bullous disease is not adequately treated. Bullous diseases include, but are not limited to, pemphigus, bullous pemphigoid, and cicatricial pemphigoid. These three typical examples of bullous conditions are briefly described below. Pemphigus
• Pemphigus is an auto-immune disease of the skin which is manifested by the loss of intercellular adhesion between the keratinocytes (cells) of the epidermis, resulting in bulla (blister) formation (Sharpe, R.J. in Manual of Clinical Problems in Dermatology, Olbricht, Bigby and Arndt eds., Little Brown & Co., Boston, 1992, pp. 56-60). Pemphigus can be further categorized by the specific site of the blisters in the various layers of the epidermis.
• blister formation occurs just below the stratum corneum (i.e., higher in the epidermis).
• Pemphigus vulgaris can affect all age groups. Lesions usually occur in the mouth, as well as on the chest, scalp, periumbilical, and intertriginous areas of the skin. Oral lesions frequently occur and may be the sole manifestation of the disease. This form of the disease can involve the oropharynx and other mucosal surfaces, sometimes extending into the esophagus and cardia of the stomach. Pemphigus vulgaris is characterized by intra- epidermal blister formations due to acantholysis (i.e., loss of intercellular adhesions) in the superbasilar epidermis and intact basal cells that histologically resemble a row of tombstones at the base of the blister.
• acantholysis i.e., loss of intercellular adhesions
• Pemphigus vegetans is clinically manifested by vegetating legions and sometimes by pustules. The latter may represent super-infection at the edges of the broken bullae. Intertriginous regions are more commonly affected. The histology of this form co monly shows abscess formation within the epidermis. Eosinophils are present in moderate numbers. Hyperkeratosis, pseudoepitheliomatous hyperplasia, and papillomatosis also occurs. The blisters formed in pemphigus foliaceus are superficial and easily ruptured. Primary symptoms often include crusting, scales, erosion, and excoriations.
• Pemphigus erythematosus is similar to pemphigus foliaceus histologically, and represents a localized form of pemphigus. Lesions of this type are characterized by a lupus-like butterfly rash as well as bullous and seborrheic dermatitis ⁇ like lesions. This type of pemphigus can be associated with other auto-immune diseases including rheumatoid arthritis, thymoma, myasthenia gravis and systemic lupus erythematosus.
• Bullous pemphigoid is the most common bullous disease of the skin. It is more prevalent in elderly patients than in younger patients.
• Clinical signs frequently include large tense blisters, on erythematous or non-erythematous skin or on urticarial plaques. Bullae often occur on the joints of the extremities, lower abdomen, groin, and inner thighs. The blisters do not rupture easily; after they rupture, however, the lesions usually heal without scarring. A mortality rate of 10 to 20 percent is reported for the disease, largely due to side-effects from the use of systemic steroid therapy.
• treatments for the various forms of bullous pemphigoid include systemic glucocorticosteroids. Often treatment will include an immuno-suppressive agent in addition to the steroids. Intra-lesional steroids may be beneficial in preventing scarring and may be used to treat mucous membrane disease. Topical treatments including steroid creams and Burows' solution baths are used to prevent secondary infection and scarring. Cicatricial Pemphigoid
• Cicatricial pemphigoid also called benign mucous membrane pemphigoid or ocular pemphigoid
• ocular pemphigoid is an uncommon chronic subepidermal bullous dermatosis which involves primarily the mucous membranes (Baden, L.A., Manual of Clinical Problems in Dermatology, Little, Brown & Co., Boston, 1992, pp. 54). Its chronic lesions often cause scarring. It frequently leads to blindness in the case of the ocular lesions. Oral and ocular membranes are frequently involved, but other mucous membranes including the nasal mucosa, pharynx, larynx, esophagus, genitalia, anus, and the skin may also be affected. In many patients the disease begins with desquamative gingivitis (loss of the surface of the gums), with fragility, pain, and easy bleeding of the gingivae.
• Cyclophosphamide, methotrexate, dapsone and azathioprine have been beneficial to some patients, while others have shown little improvement with these agents.
• Topical and intra-lesional steroids seem to be less effective in cicatricial pemphigoid than in oral lichen planus.
• a common feature of lichen planus, pemphigus, bullous pemphigoid, cicatricial pemphigoid and lichen planus is the role of proteases in their pathogenesis.
• proteases were identified in the blister fluid of pemphigus and pemphigoid patients (Grando, Glukhenky, Drannik, Kostromin and Chernyavsky, Int . J. Tissue React . 1989, Vol. 11, pp. 195-201).
• protease inhibitors ⁇ >-guanidino ester gabexate mesylate, camostat mesylate and nafomastat mesylate inhibit the induction of acantholysis in an organ culture system, but have little or no effect on lesion formation in a neonatal mouse model of pemhigus (Naito, Morioka, Nakajima, Ogbawa, J. Invest . Dermatol . , 1989, Vol. 93 pp. 173-77).
• the natural plasma proteinase inhibitor, alpha-1-proteinase inhibitor completely inhibited acantholysis formation in the mouse model.
• NAC N-acetyl cysteine
• a thioester of N-acetylcysteine or its pharmaceutically acceptable salt optionally in a pharmaceutically- acceptable diluent or carrier.
• Lipophilic NAC thioesters and thioethers, or their derivatives or salts are preferred for the treatment of cutaneous disorders because they are able to permeate the lipid rich stratum corneum at a much higher rate than native NAC.
• NAC thioester traverses the stratum corneum, simple hydrolysis of the thioester, by thioesterases or other mechanisms, enables the fatty acid and NAC to separate into two components. Likewise, NAC thioethers are degraded to NAC and the ether residue.
• the NAC thiol group thought to be primarily responsible for a variety of reactivities associated with NAC, is then in the proper anatomical location to treat cutaneous inflammatory or immunologically mediated conditions, including those which are discussed herein.
• fatty acid which is released upon hydrolysis of the thioester, or alcohol or other residue released upon biodegradation of the thioether is by selection an innocuous, biocompatible compound that is free to diffuse from the site of administration.
• the thioester or thioether residue that is cleaved in vivo exhibits an independent beneficial biological activity.
• a method and composition is provided for the treatment of a cutaneous condition characterized by hyperkeratosis, including ichthyosis, keratoderma, lichen planus, and psoriasis, in a human or other mammal.
• the method includes the topical administration of an effective amount of N-acetyl cysteine ("NAC”) or its derivative or salt, and preferably, a thioester of N-acetylcysteine or its pharmaceutically acceptable salt, optionally in a pharmaceutically-acceptable diluent or carrier suitable for topical delivery.
• NAC N-acetyl cysteine
• a thioester of N-acetylcysteine or its pharmaceutically acceptable salt optionally in a pharmaceutically-acceptable diluent or carrier suitable for topical delivery.
• the active compounds are administered topically in an appropriate carrier in an amount and for a period of time sufficient to effectively treat the patient at the site of application. Because the application is topical, i.e., local, relief of hyperkeratosis is achieved with minimal systemic effects.
• NAC derivatives include, but are not limited to the thioesters of oleic acid (S-oloeyl- N-acetyl-L-cysteine) , lauric acid (S-lauryl-N- acetyl-L-cysteine) , myristic acid (S-myristoyl-N- acetyl-L-cysteine) , capric acid (S-caprolyl-N- acetyl-L-cysteine) , retinoic acid (S-3,7-dimethyl- 9-(2,6,6-trimethyl-l-cyclohexen-l-yl)-2,4,6, 8- nonatetraenoyl-N-acetyl-L-cysteine
• a method for the topical or systemic treatment of disorders mediated by proteases that cause skin or mucosal lesions and in particular, pemphigus, cicatricial pemphigoid, bullous pemphigoid, lichen planus, and canker sores (aphthous ulcers)
• the host is treated with an effective amount of N- acetylcysteine ("NAC") or a derivative thereof, or its pharmaceutically acceptable salt, optionally in a pharmaceutically acceptable diluent or carrier for systemic or topical delivery.
• NAC N- acetylcysteine
• the active compound or its derivative is administered for a sufficient time period to alleviate the undesired symptoms and or the clinical signs associated with the disorder.
• Oral lesions associated with these disorders can be treated, for example, with an mouthwash rinse that contains an effective amount of N- acetylcysteine or its derivative or salt.
• the mouthwash is used as often as necessary to obtain amelioration of symptoms, and typically from one to several times a day until the desired benefit is achieved.
• the rinse is swished and expectorated or swallowed by the patient.
• an additive such as lemon or peppermint oil.
• Skin or mucosal lesions on non-oral membranes can be treated with an effective amount of N-acetylcysteine or its derivative or salt in a carrier for topical delivery.
• the active compound is administered in an effective dosage range to cause suppression of the symptoms.
• a wet gauze compress soaked with a solution of N-acetylcysteine or its derivative or salt is maintained on the lesion for a period of time, from one to several times a day.
• an effective amount of N-acetylcysteine or its derivative or salt is applied to the lesion in a cream, gel, ointment, diluent, foam or paste, from one to several times a day.
• a method for the treatment of disorders mediated by proteases includes administering an effective amount of N-acetylcysteine, or its derivative or pharmaceutically acceptable salt thereof, in a carrier for systemically delivery.
• NAC or its derivative or salt can be useful in interrupting the cascade of events which result in pathological tissue injury and thus should assist in accelerating the healing of painful lesions associated with aphthous ulcers and preventing the formation of new lesions.
• alkyl refers to a saturated straight, branched, or cyclic (or a combination thereof) hydrocarbon of C, to C 22 , and specifically includes methyl, ethyl, propyl, isopropyl, cyclopropylmethyl, cyclobutylmethyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, heptyl, octyl, nonyl, and decyl.
• aryl refers to phenyl, or substituted phenyl, wherein the substituent is halo, alkyl, alkoxy, alkylthio, haloalkyl, hydroxyalkyl, alkoxyalkyl, methylenedioxy, cyano, C(0) (alkyl), carboxylie acid, C0 2 alkyl, amide, amino, alkylamino or dialkylamino, and wherein the aryl group can have up to 3 substituents.
• aralkyl refers to an aryl group with an alkyl substituent.
• alkaryl refers to an alkyl group that. has an aryl substituent, including benzyl, substituted benzyl, phenethyl or substituted phenethyl, wherein the substituents are as defined above for aryl groups.
• fatty acid refers to a long chain (C 6 to C 22 ) aliphatic carboxylic acid
• enantiomerically enriched composition or compound refers to a composition or compound that includes at least 95%, and optimally, 98 or 99% by weight of a single enantiomer of the compound.
• inorganic cation refers to a charged moiety in which an atom other than carbon carries a positive charge, and includes, but is not limited to, H + , potassium, sodium, ammonium, mono, di, or trialkylammonium, quaternary amine, specifically including but not limited to the quaternary ammonium salt of the formula NR 4 + Z " , wherein R is independently alkyl or benzyl and Z is a counteranion, including chloride, bromide, iodide, -O-alkyl, tolunesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, succinate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphylacetate) ; or a multivalent ion such as calcium, barium
• Cysteine is an amino acid with one chiral carbon atom. It exists as an L-enantiomer, a D- enantiomer, or a racemic mixture of the L and D enantiomers.
• the L-enantiomer is the naturally occurring configuration.
• NAC acetamido-mercaptopropionic acid
• NAC is the N-acetylated derivative of cysteine. It also exists as an L-enantiomer, a D- enantiomer, or a racemic mixture of the L and D enantiomers. Any of these three forms of NAC, or its derivative or salt, can be used in the treatment of pathological conditions associated with hyperkeratosis or inflammatory conditions described herein.
• an enantiomerically enriched composition of a thioester of NAC or its salt, and most preferably, the naturally occurring L-enantiomer is used in the treatment process.
• NAC is known to be an effective mucolytic agent (i.e., an agent which reduces the viscosity of mucus) (Lightowler and Lightowler, Arch . Int . Pharmacodyn . Ther . 1971, Vol. 189, pp. 53-8).
• the mucolytic activity is related to the reactive sulfhydryl group in the molecule.
• the sulfhydryl group probably opens sulfide linkages in mucus, thereby lowering mucosal viscosity.
• NAC is also used for the treatment of acetaminophen overdoses (Smilkstein, Knapp, Kulig and Rumack, N. Engl . J. Med . 1988, Vol. 319, pp. 1557-62).
• N-acetylcysteine when administered within the first few hours of overdose, protects the liver by acting as an alternate substrate for conjugation with and detoxification of the reactive metabolite.
• NAC has been reported to be an effective collagenase inhibitor (Lemp and Roddy, Ann. Qphthalmol. 1974, Vol. 6, pp. 893-5) and an antioxidant in vivo (Knight, K.R. , MacPhadyen, K. , Lepore, D.A., Kuwata, N., Eadie, P. ., O'Brien, B. Clinical Sci . , 1991, Vol. 81, pp. 31-36; Ellis, E.F., Dodson, L.Y., police, R.J. , J. Neurosurg . , 1991, Vol. 75, pp. 774-779).
• NAC reduces the activity of the proteolytic porcine enzymes, leukocyte elastase and pancreatic elastase by greater than 55% in vitro (Morrison, Burnett and Stockley, Biol . Chem . Hoppe Seyler 1986, Vol. 367, pp. 177-82). Given the complexity of disorders such as pemphigus, cicatricial pemphigoid, bullous pemphigoid, lichen planus, and canker sores, however, one could not predict from this report whether NAC would be an effective treatment in vivo for these diseases.
• N-acetylcysteine can act as an inhibitor of tumor necrosis factor production in vivo (Peristeris, P. et al, Cell . Immunol . 1992, Vol. 140, pp. 390-99).
• pharmaceutically acceptable derivative or salt of N-acetylcysteine refers to either:
• R 1 is hydrogen, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, an amino acid salt formed by the reaction of the amino group of a naturally occurring amino acid with the carboxylic acid group of the N-acetylcysteine or derivative thereof; an amine salt formed by the reaction of an amine-containing antibiotic with the carboxylic - acid group of the N-acetylcysteine, or an inorganic cation; and wherein the term amino acid includes but is not limited to alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl,
• R 2 is hydrogen, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, C(0 or S)alkyl, C(0 or S)aryl, C(0 or S)alkaryl, C(0 or S)aralkyl, C(0 or S)alkyloxyalkyl, C(0 or S)acyloxyalkyl, or phosphate.
• R 2 can also be the residue of a saturated or unsaturated fatty acid, including but not limited to lauric, oleic, caproic, linoleic, linolenic, caprylic, capric, perlargonic, neononanoic, neodecanoic, palmitelaidoic, myristic, palmitic, stearic, arachidic, behenic, lignoceric, heptanoic, nonanoic, undecanoic, tridecanoic, pentadecanoic, heptadecanoic, nonadecanoic, heneicosanoic, tricosanoic, arachidonic, docosahexanoic, elaidic, erucic, nervonic, palmitoleic or petriselinic acid.
• a saturated or unsaturated fatty acid including but not limited to lauric, oleic, caproic, linole
• R 2 can be the residue of lactic acid, retinoic acid, or ascorbic acid (to form the thioester) or other ⁇ -hydroxy acid (including but not limited to glycolic acid) , or the residue of a dicarboxylic acid (wherein N-acetylcysteine is bound through either or both carboxylic acid groups), including but not limited to cromolyn, nedocrimil, or other mast cell stabilizers, azelaic acid, or methotrexate.
• R 2 is the residue of sebacic acid, phthalic acid, terephthalic acid, isophthalic acid, adipic acid, 1, 10-dodecanoic acid, bis(p-carboxyphenoxyalkane) , fumaric acid, 1,4-diphenylenediacrylic acid, branched monomers such as 1,3,5- benzenetricarboxylic acid, azeleic acid, pimelic acid, suberic acid (octanedioic acid), itaconic acid, biphenyl-4,4'-dicarboxylic acid, and benzophenone-4,4'-dicarboxylic acid, p- carboxyphenoxyalkanoic acid, hydroquinone-0,0- diacetic acid, 1,4-bis-carboxymethyl benzene, 2,2- bis-(4-hydroxyphenyl)propane-0,O-diacetic acid, 1,4-phenylene-dipropionic acid, and cycl
• Non-limiting examples of amine-containing antibiotics that can be used to form a salt with N- acetylcysteine include, but are not limited to, erythromycin, propionylerythromycin, neomycin, gentomycin, mechlocyclin, tobramycin, and kanamycin.
• a dimer or oligomer of NAC or its derivative is provided.
• the dimer can be formed by joining two -SR 2 moieties to form a disulfide bridge (with the elimination of the R 2 groups), or by joining two carboxylate moieties to form an anhydride.
• the dimer can be formed by combining an -SR 2 moiety of NAC or its derivative with a COjR 1 moiety of another NAC molecule or derivative to form a thioester dimer.
• two or more NAC molecules or derivatives are combined through thioester linkages.
• compositions of NAC Humans, equine, canine, bovine, feline and other animals, and in particular, mammals, suffering from hyperkeratosis or diseases mediated by proteases can be treated by administering to the patient or animal an effective amount of NAC or a pharmaceutically acceptable derivative or salt thereof in a pharmaceutically acceptable carrier or diluent for systemic, topical, or transdermal delivery.
• the active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid, cream, gel or solid form.
• pharmaceutically acceptable salts or complexes refers to salts or complexes that retain the desired biological activity of the above-identified compounds and exhibit minimal undesired toxicological effects.
• Pharmaceutically acceptable carboxylic acid and mercaptyl salts are known to those skilled in the art, including inorganic salts with cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed with a nitrogenous base such as ammonia, N,N-dibenzylethylene-diamine, D-glucosamine, or ethylenediamine.
• the derivatives of N- acetylcysteine disclosed herein are "prodrugs" of N-acetylcysteine, that are either active in the prodrug form or are cleaved in vivo to the parent, active compound.
• Modifications of NAC can affect the bioavailability and rate of metabolism of the active species, thus providing control over the delivery of the active species.
• modifications of the active molecule such as alteration of charge, can effect water and lipid solubility and thus alter the potential for percutaneous absorption.
• NAC lipophilicity of NAC by formation of the thioester with a long chain fatty acid will enhance its ability to cross certain biological membranes (i.e., skin, mucous membranes, and ocular membranes).
• modifications can affect the bioactivity of the compound, in some cases increasing the activity over the parent compound. This can easily be assessed by preparing the derivative and testing its activity according to the methods described herein, or other method known to those skilled in the art for the target indication.
• N-acetylcysteine or its derivative or salt is applied in the form of a topical composition.
• composition can be formulated in a variety of ways known to those skilled in the art, for example, in a solid form such as a powder; a liquid form such as a solution or a suspension in an aqueous or oily medium; or a semi-liquid formulation such as a cream, jelly, paste, ointment, or salve.
• the compound is applied in the form of a solution, gel, ointment, cream, lotion or foam, in a 1-100%, for example a 10-20% by weight, aqueous solution.
• Acetylcysteine is currently available in 10 and 20% aqueous solutions (Mucomyst, Mucosil) .
• the active ingredient or its derivative or salt or composition thereof is incorporated into a lesion cover such as a plaster, bandage, dressing, gauze pad or the like.
• a lesion cover such as a plaster, bandage, dressing, gauze pad or the like.
• the active ingredient or its derivative or salt or composition thereof can be administered by transdermal patch.
• Solutions or suspensions used for parenteral, intra-dermal, subcutaneous, or topical application can include, for example, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; anti-bacterial agents such as benzyl alcohol or methyl parabens; anti-oxidants such as ascorbic acid, BHA or BHT, or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
• anti-bacterial agents such as benzyl alcohol or methyl parabens
• anti-oxidants such
• the vehicle, or carrier can be modified to enhance cutaneous absorption, enhance the reservoir effect, or minimize potential irritancy or neuropharmacological effects of the composition.
• penetration enhancers include N- methylpyrrolidine and SEPAs (1,3-dioxolanes) . SEPAs are available from MacroChem Corporation and are described in U.S. Patent No. 4,861,764 and European Pat. No. 0 268 460. These enhancers are designed to assist in the penetration of drugs into the skin. For other examples, see, in general, Arndt, K.A. , Mendenhall, P.V., "The Pharmacology of Topical Therapy", Dermatology in General Medicine. 1987; Fitzpatrick, Eisen, Wolff, Freeberg, Austen, eds., 3d ed. , McGraw Hill, Inc., New York, pp. 2532-2540.
• the active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a host a therapeutically effective amount of the drug for the target indication without causing serious toxic effects in the patient treated.
• a typical topical dosage will range from 0.5 or 1 to 30 weight percent in a suitable carrier.
• a preferred systemic dose of the active compound for all of the above-mentioned conditions is in the range from about 10 to 8000 mg/kg, preferably 100 to 1500 mg/kg per day, more generally 300 to about 1200 mg per kilogram body weight of the recipient per day.
• the effective dosage range of the pharmaceutically acceptable derivatives and salts thereof can be calculated based on the weight of the parent compound to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the derivative, or by other means known to those skilled in the art.
• the compound is conveniently administered in any suitable unit dosage form, including but not limited to pills, tablets, troches, and caplets containing 50 to 3000 mg, preferably 250 or 500 to 3000 mg of active ingredient per unit dosage form.
• a oral dosage of 50 to 1500 mg is usually convenient.
• the active ingredient can be administered by the intra-venous injection of a solution or formulation of the active ingredient, optionally in physiological saline or phosphate buffered saline (PBS), or an aqueous medium or administered as a bolus of the active ingredient.
• concentration of active compound in the drug composition will depend on absorption, distribution, de-activation, and excretion rates of the drug as well as other factors known to those of skill in the art. Dosage values will also vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
• concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
• the active ingredient can be administered at once, or can be divided into a number of smaller doses to be administered at varying time intervals.
• Natural or artificial flavorings or sweeteners can be used to enhance the taste and odor of topical preparations applied for local effect to mucosal surfaces.
• Inert dyes or colors can also be added, particularly for compositions designed for application to oral and mucosal surfaces.
• Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets.
• the active compound can be used in the form of tablets, troches, or capsules.
• Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
• the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose, saccharin or Nutrasweet
• dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.
• the active compound or pharmaceutically acceptable salt or derivative thereof can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
• a syrup may contain, in addition to the active compounds, sucrose, Nutrasweet (phenylalanine) or saccharrin as a sweetening agent and certain preservatives, dyes and colorings and flavors.
• the active compound or pharmaceutically acceptable derivatives or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as anti ⁇ biotics, anti-fungals, anti-inflammatories, disinfectants, or anti-viral compounds.
• the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• a controlled release formulation including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as poly(ethylene vinyl acetate), polyanhydrides, poly(glycolic acid), collagen, polyorthoesters, and poly(lactic acid), and others known to those skilled in the art. Methods for preparation of such formulations will be apparent to those skilled in the art. IV. Methods for the Evaluation of Effectiveness of NAC in the Treatment of Disorders Characterized by Hyperkeratosis.
• N-acetylcysteine or its derivative or salt in the treatment of hyperkeratotic disorders can be evaluated in humans or in another appropriate model.
• the ability of the active compound to treat a hyperkeratotic disorder can be evaluated in humans as follows. Single, thick psoriatic plaques are identified on each patient's arms or legs. The plaques are initially evaluated prior to therapy to provide a "baseline evaluation" . The lesions are then washed and dried thoroughly. A topical solution of desired test concentration (for example, 20%) of the active compound is applied to a single thick psoriatic plaque on each patient's arm or leg. A matched vehicle placebo is applied to a similar plaque on the corresponding limb on the opposite side of the body. The solution of the active compound or vehicle (control) is applied by dripping one or the other onto the test lesion until it is thoroughly wet, and then occluded for four hours.
• desired test concentration for example, 20%
• Drug (or vehicle) application is repeated four times daily and left under occlusion for four hours after each application. Lesions are evaluated weekly throughout the course of the study. The lesions are scored by an observer who is not aware of which lesions are receiving drug or vehicle treatment. Erythema, induration and scaling are evaluated for each test lesion on a scale of 0- 3. Methods for the Evaluation of Effectiveness of NAC in the Treatment of Pemphigus in Model Systems.
• N-acetylcysteine or its derivative or salt in the treatment of any of the forms of pemphigus described above can be evaluated by one or more of the following methods: (a) in an established organ culture model where the degree of acantholysis can be measured, after introduction of exogenous pemphigus antibody; (b) in a neonatal mouse model where disease can be induced, and evidence of clearing can be monitored; and or (c) in humans with pemphigus.
• the pemphigus antibodies to be used in the analysis are purified and prepared in the following manner (Anhalt, Till, Diaz, Labib, Patel and Eaglstein, J. Immunol . 1986, Vol. 137, pp. 2835- 40). Serum is obtained from human patients with the clinical, histologic and immunologic features of pemphigus.
• the IgG fractions of the sera are purified by 40% ammonium sulfate precipitation, followed by ion exchange chromatography. IgG fractions prepared in this manner are free of significant protease contamination when assayed.
• acantholysis in vitro can be carried out as follows (Lever, J. Am . Acad . Dermatol . 1979, Vol. 1, pp. 2-31). Normal human skin is maintained in organ cultures to which sera of patients with pemphigus is added. Direct IF staining of the explants with fluorescein-labeled goat anti-human IgG shows that, after incubation, binding of the pemphigus IgG has occurred in the intercellular cement substance of the epidermis. Suprabasal acantholysis is observed which progresses to extensive acantholysis.
• NAC or its derivative or salt to lessen or eliminate acantholysis in vitro caused by exposure to pemphigus-IgG the following experiment can be evaluated as follows.
• Normal human skin is cultured according to the method described by Naito, et al., (Naito, Morioka, Nakajima, Ogawa, J. Invest . Dermatol . 1989, Vol. 93, 173-77). Skin is sliced into 2 x 2 mm pieces thick. The skin is then floated on top of a total volume of 1.0 mL culture medium with the assistance of paraffin edged lens paper. The cultures are kept in humid atmosphere containing C0 2 in air for 24, 48 and 72 hours.
• the culture medium should contain approximately 7 mg/mL of pemphigus IgG with or without the NAC or its derivatives or salts. After each culture period, the skin explants are examined by routine histologic (hemotoxylin and eosin staining) methods. The final concentration of NAC, its derivative, or its salt should range from 0.1 to 20 mg/mL. The skin can be preincubated (1-24 hours) with NAC, its derivative or salt prior to addition of pemphigus IgG.
• Acantholysis is evaluated on a scale of (-), (+), (++), or (+++), where (-) is no acantholysis, (+) is positive on 10-30%, (++) is positive on 30-70%, and (+++) is positive on 70-100% of the epidermis in the histologic section.
• NAC or its derivative or salt to reduce the symptoms of pemphigus in vivo can be evaluated in a neonatal mice model (Anhalt, Labib, Voorhees, Beals and Diaz, N. Engl . J. Med . 1982, Vol. 306, pp. 1189-96).
• Purified IgG fractions are injected i.p. into neonatal mice using a 30 gauge needle in a single administration of 10 mg IgG per gram body weight according to an established model (Takahashi, Patel, Labib, Diaz, Anhalt, J " . Invest . Dermatol . 1985, Vol. 84, pp. 41-46).
• Skin and serum samples are obtained from animals receiving injections of either normal human IgG (control) or human pemphigus IgG. Skin samples from the flank region, where lesions most often occur are processed for direct immuno-fluorescence. Human pemphigus antibodies are also monitored in the animals' serum, to confirm transfer of the pemphigus antibodies.
• One group of mice is treated with topical administration of the test compound and monitored for disease improvement by sampling the skin and assessing its appearance by histology and/or by clinical appearance.
• the neonatal mice receive injections of NAC, its salt, or its derivative prepared in PBS.
• the administered dosages of NAC, its derivative, or its salt range from 13 ⁇ g/g of mouse body weight to 2 mg/g mouse body weight.
• Each of the solutions to be injected are sterilized by filtration through an 0.45 ⁇ m millipore filter.
• mice 24 hours after pemphigus IgG is injected.
• biochemical analysis 24 hours after pemphigus IgG injection the mice are sacrificed and the whole skin of each animal removed. At least five different sites from each removed skin are then examined for histologic analysis.
• the extracts are centrifuged at 750 g for 10 min, and the supernatant dialyzed against 0.12 M glycine-NaOH, pH 8.5. Plasminogen activity is determined spectroscopically according to literature procedures (Naito, Morioka, Nakajima, Ogawa, H. J Invest Dermatol , 1989, vol. 93, 173-177). VI. Methods for the Evaluation N-acetylcysteine in Humans with oral lesions.
• Clinical evaluations are performed by the same physician for the duration of the experiment. Each patients disease is measured on a scale of 1 to 4, with 1 indicating minimal disease, and 4 indicating severe lesions. The degree of erosion, erythema, and reticulation of each lesion is separately scaled over time for a period ranging from 1 day to 6 months, as desired. In addition, the patients evaluate lesion discomfort on a scale of 1 to 4.
• diseases involving other mucosal membrane surfaces or the skin can be treated topically or systemically via oral or i.v. injection with N-acetylcysteine or its derivative or salt and the results compared with placebo.
• this invention is a method for the treatment of cutaneous, ocular, or mucosal hypersensitivity and/or hyperproliferative conditions induced by or associated with an immune response or inflammation, that includes the topical or systemic administration of an effective amount of a lipid soluble thioester or thioether of N- acetylcysteine or a derivative or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier.
• the thioester and thioether derivative of N- acetylcysteine, or the salt thereof, is administered as a general immunosuppressive and/or anti-inflammatory agent.
• the compounds may be useful as specific topical agents in treating contact dermatitis, atopic dermatitis, eczematous dermatitis, psoriasis, Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcers, ulceris, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, and drug eruptions.
• the active compounds may also be useful in reducing the infiltration of skin by malignant leukocytes in diseases such as mycosis fungoides. These compounds may also be effective to treat an aqueous-deficient dry eye state (such as immune mediated keratoconjunctivitis) in a patient suffering therefrom, by administering the compound topically to the eye.
• an aqueous-deficient dry eye state such as immune mediated keratoconjunctivitis
• N-Acetylcysteine derivatives or salts are considered to be immunosuppressants if they exhibit immunosuppressant activity in any accepted model, for example, if they suppress the ear swelling associated with an experimental contact hypersensitivity response in mice after specific antigen challenge using an assay such as that described in J. Invest . Derm . , Vol 99(5), November 1992.
• the ability of N-acetylcysteine or its derivative or salt to impart antiinflammatory activity can be evaluated in any of a wide variety of accepted models.
• the compounds can be evaluated for antiinflammatory activity by their ability to suppress the inflammation of mouse ears arising from over-exposure to ultraviolet (UVA or UVB) light, for example, as described in Arch . Biochem . Biophys . , 298:87-90 (1992); J. Invest . Dermatol . , 89:1410-1414 (1987); Arch . Dermatol . Ref . , 278:445-448 (1986).
• UVA or UVB
• mice are exposed to various levels of ultraviolet radiation via external light sources (FS40 T12 fluorescent sunlamps for UVB source, and FS40/350BL fluorescent sunlamps for UVA source) .
• the mice should have their dorsal region shaved with small animal clippers if not a hairless strain.
• Sufficient ultraviolet radiation should be used to simulate the exposure to sunlight causing a first degree burn and optimize the amount of radiation to maximize ear swelling.
• the UV light source should be located 24.25 inches from the mice.
• the UVB radiation can range between 150 and 250 mJ/cm 2 .
• the mice are subjected to radiation for 3 to 5 minutes during which period they are contained in a plastic box with no cover.
• the test compound is applied to the animals' UV-light treated skin.
• the test compound is provided systemically, for example, orally, intravenously or interperitoneally.
• the active compound, metabolite, prodrug, or metabolite of the prodrug of NAC preferably suppresses the ear swelling in mice associated with an experimental contact hypersensitivity response by a significant amount (for example at least 40%) at 24 hours after specific antigen challenge, using an assay such as that set out below.
• the compounds can also be evaluated in vivo in humans, by evaluating its ability to inhibit contact hypersensitivity responses to patch test allergens in patients hypersensitive to a given allergen, using procedures generally accepted by those of skill in the art. Oxazolone-Induced Contact Hypersensitivity 1.
• Sensitization and challenge for contact hypersensitivity can be measured as follows. The abdomens of mice are shaved with electric clippers, and then 50 ⁇ l of a 4% (w/w) solution of oxazolone in 4:1 (v:v) acetone:olive oil is applied to the shaved abdomen, and 5 ⁇ l of the same solution is applied to each hind footpad.
• mice Five to seven days later, the mice are challenged for contact hypersensitivity by applying 10 ⁇ l of a 0.5% (w:w) solution of the hapten, oxazolone, in 4:1 (v:v) acetone:olive oil to both the inner and outer surface of the right ear of each mouse (in the case of mice treated systemically with active compound) or to both ears (in the case of mice treated topically with the active compound), except in the case where sensitization phase suppression is studied. 2.
• Topical Treatment Both ears of each mouse are challenged for elicitation of contact hypersensitivity by the application of oxazolone (as appropriate) to the inner and outer surfaces of both ears.
• mice Two hours before, or twenty-four hours after application of hapten, the right ears of some of the mice are treated with the active compound in a suitable vehicle, applied epicutaneously to both surfaces.
• the right ears of control mice are similarly treated, but with vehicle alone.
• the thioester or thioether derivative of N- acetylcysteine, its derivatives or salts only the right ear is to be challenged. Evaluation of Ear Swelling Response - Immediately before and 24 or 48 hours after application of oxazolone, the ear thicknesses are determined with an engineer's micrometer.
• the increment (delta) in ear thickness (ear swelling) is calculated as the 24- or 48-hour value minus the baseline (pre-challenge) value and is expressed in units of micrometers. Mice are euthanized by C0 2 inhalation after the last experimental time point, and the ears are then processed for histologic examination.
• the number of leukocytes/mm 2 of dermis is calculated by counting all of the leukocyte cells in an area of at least 0.14 mm 2 of dermis.
• oxazolone can be applied to both ears of all mice at different times either pre- or post-drug treatment, and the change in ear thickness can be measured at a specified interval thereafter. For example, two hours before oxazolone challenge, 100 mg/ml of test drug in a suitable vehicle can be applied to both surfaces of the right ears of some mice, whereas vehicle alone is applied to both surfaces of the ears of the control (0% active compound) animals. The ears are to be measured 24 hours after oxazolone challenge. This experiment tests the ability of the active compound to interfere with the elicitation of the contact hypersensitivity reactions.
• phorbol-12-myristate- 13-acetate induces edema (tissue swelling), leukocyte infiltration, and eventually, epidermal hyperplasia in the ears of mice.
• This model of immunologically non-specific inflammation is used to measure the effectiveness of lipid soluble N- acetylcysteine thioesters in reduction of ear swelling and leukocyte infiltration.
• the study of the inhibition of chemically induced inflammatory response can be carried out as follows.
• the extent of subsequent inflammation is measured as described above, primarily by measuring the change in ear thickness by an engineer's micrometer, and by counting leukocytes (neutrophils) in a 50 ⁇ m section of full thickness ear tissue after H & E staining.
• leukocytes neutrophils
• the decrease in immunologically non-specific ear swelling and leukocyte infiltration induced by the chemical PMA is measured after either topical or systemic treatments with the active compound.

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