CA2164955A1 - Method for treating hyperkeratosis and diseases mediated by proteases - Google Patents

Abstract

A method for the topical or systemic treatment of hyperkeratosis or disorders mediated by proteases that includes the administration of an effective amount of N-acetylcysteine or a pharmaceutically acceptable salt or derivative thereof, and preferably, a lipophilic thioester or thioether of N-acetyl cysteine. In an alternative embodiment, the invention is a method for the treatment of inflammatory conditions or conditions that benefit from immunosuppression that includes the administration of an effective amount of a lipophilic thioester or thioether of N-acetyl cysteine.

Description

~09~/00~6 PCT~S94/06949 ~ 216~95~
Method for Treating Hyperkeratosis and Diseases Mediated by Proteases 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.

Background of the Invention I. Hyperkeratosis 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 orthokeratosis (an increase in fully cornified cells) and parakeratosis (an increase in partially cornified cells). In the process of keratinization, keratin filament proteins are deposited in their reduced thiol forms and are subsequently oxidized to disulfides during the cornification process (Sun Tr~ Green, H., Cell, 1976: 9(4) 511-21; Sun TT, G~een, 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. Below is a description of the more severe and uncommon of the hyperkeratotic disorders.

W095/00~6 . x PCT~S94/069 ~
. ;
216~95~ -2-Ichthyoses Ichthyotic diseases comprise a family of disorders characterized by the abnormally increased accumulation of scale on the skin. There are four distinct types of ichthyosis: vulgaris, recessive X-linked, lamellar, and epidermolytic hyperkeratosis. The four types can be distinguished by age of onset, sex of the patients, clinical and histological findings, and pattern of inheritance.
Ichthyosis vulgaris is the most common form of the disease, occurring in one out of three hundred people (Baden, H.P. in Dermatology in General Medicine, Fitzpatrick, T.B., et al eds., McGraw-15 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 toimprove 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. While 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, 30 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 ~edicine, 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 :
~ W095/00~6 21 6 ~ 9 ~ PCT~S94tO6949 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 ocal parakeratosis, a normal to slightly thickened granular layer, acanthosis and follicular plugging.
There is an increase in the number of keratinosomes and an increase in intercellular substance, but is associated with a decrease in the number of Langerhans cells. Like ichthyosis vulgaris, lamellar ichthyosis is thought to be an epi~erm hyperproliferative state.
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 WO95/00~6 ~6 ~9~ PCT~S94/069 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.
Histologically, 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 a-hydroxy acid or urea-cont~;n;ng creams. While such creams have limited benefit for ichthyosis vulgaris, they do not improve other forms of ichthyosis. It has been reported that keratolytic agents can ~i m; n; sh 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 = 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, T.B., et al eds., McGraw-Hill: New York, 1987. pp.
529). 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 ~o gs/oo~ 2 1 6~4 ~ ~ PCT~S94/06949 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. in Dermatology in General Medicine, Fitzpatrick, T.B., et al eds., McGraw-Hill: New York, 1987. pp. 529).
E'our signs indicative of psoriasis include: (1) sharply outlined borders of affected areas; (2) scales which are non-coherent and scaly; (3) glossy homogeneous reddening under the scale; (4) upon scratching the hyperkeratotic, red scaly lesions of psoriasis, small blood droplets appear on the reddened surface (this does not occur in inverse or pustular psoriasis, or other conditions with other features similar to those of psoriasis).
Chronic common psoriasis (psoriasis vulgaris, plaque type psoriasis) is the most common form of the disease. 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 psorlasls .
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. In the pustular and eruptive types of psoriasis, the inflammatory features are more prominent than in plaque type psoriasis (~aden, H.P. in Dermatology in General Medicine, W095/00~6 ~ ~ ~ PCT~594/~

Fitzpatrick, T.B., et al eds., McGraw-Hill: New York, 1987. pp. 529).
The pathogenesis of psoriasis is poorly understood, and there is no good experimental model useful for laboratory study of the disease (Baden, H.P. in Dermatology in General Medicine, Fitzpatrick, T.B., et al eds., McGraw-Hill: New York, 1987. pp. 529). Analysis of the actual lesions of psoriasis has indicated hyperproliferation of keratinocytes, evidence for a shortening of the epidermal cycle, and an increase in the population of proliferating keratinocytes.
More recent research has suggested roles for inflammatory cells and epidermal-dermal interactions in certain aspects of the disease;
particular attention is focused on the possibility that cytokines and other products of the lymphocytes present in the lesions might contribute to the epidermal hyperproliferation which is characteristic of the disease.
It has recently been hypothesized that psoriasis may have an autoimmune/inflammatory component, in which antigen-antibody complexes cause infiltration of lymphocytes and other ~ 25 inflammatory cells (leukocytes) into the stratum = corneum. (Beutner, et al, in Beutner, et al, eds., Immunopathology of the Skin. New York: John Wiley = and Sons, 1987, pp 255-60). Once this leukocyte infiltration occurs, neutrophils release inflammatory mediators such as serine proteases and oxygen radicals which contribute to tissue damage.
In fact, abnormal protease activity has been found in psoriatic skin (Franki, JE., Hopsu-Havu, V., Arch Derm Res. 1976, 256, 113-26; Glinski, W. et al, J. Invest. Dermatol, 1980: 75(6) 481-7;

O95/00136 2C . "~ PCT~S94/06949 Dubertret, L., et al, Br. ~. Dermatol, 1982, 107, 499-504; Ohtani, O., et al, J. Invest Dermatol., 1980: 78(4) 280-4).

I:C. Diseases Mediated by Proteases There are a number of diseases that affect the skin and mucosal membranes which have been found to be mediated by proteases. Examples of 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 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. l, McGraw-Hill, Inc., New York, pp. 967-973).
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, WO95/00~6 PCT~S94/069~

2~

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 &eneral 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). Topical tretinoin, cyclosporine, and systemic antifungal agents, such as griseofulvin, have been reported to be somewhat effective in treating severely symptomatic oral lichen planus. No large, well designed studies, however, have proven the efficacy of these therapies. The use of potent topical steroids, particularly on mucosal surfaces, can result in dangerous side effects.
Bullous Diseases A number of the protease mediated diseases are classified as bullous disorders. Bullae, more commonly known as blisters, are circumscribed, fluid contA;n;ng elevated lesions which are usually more than 5 mm in diameter. Diseases exhibiting ~ 30 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 ~woss/nol36 ~ S S ~CT~594/~6949 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. Pemphigus vulgaris and pemphigus vegetans exhibit blisters above the basal layer of the skin (i.e., the first layer of keratinocytes in the epidermis). In pemphigus foliaceus and pemphigus erythematosus, 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 hi.stologically resemble a row of tombstones at the base of the blister.
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 WO95/00~6 i2~ 6 g9 ~ PCT~S941069 ~

commonly 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.
Because of the severity of symptoms and the high morbidity and mortality associated with pemphigus, hospitalization is often necessary.
Untreated or unresponsive pemphigus patients can develop sepsis, cachexia, and major fluid and electrolyte imbalances similar to those observed in burn patients.
Current treatment of pemphigus involves the = use of corticosteroids, including high dosages of oral prednisone or prednisolone. Accordingly, these patients must be closely monitored for adrenocorticoid side effects. It has also been reported that immunosuppressive agents such as cyclophosphamide, azathioprine, methotrexate and cyclosporine-A, or a combination of immunosuppressive agents with high doses of prednisone may be useful in the symptomatic treatment of pemphigus (Lever, J. Am. Acad.
= Dermatol. 1979, Vol. 1, pp. 2-31). As with treatment with prednisone or prednisolone alone, ~W095/00~6 ~2~a~9~ PCT~S94/06949 patients undergoing immunosuppressive treatment must be closely monitored for adverse side effects.
Treatment of pemphigus with gold compounds alone or in combination with prednisone has also been reported (Lever, ~. Am. Acad. Dermatol. 1979, Vol.
1, pp. 2-31).
Bullous Pemphigoid 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.
As with pemphigus, 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, 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.

W095/00~6 ~ PCT~S94/069 ~
~6~ 12-54). 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.
Current treatments for this disorder are less than satisfactory (see Bleicher, supra; Arndt, K.
in Fitzpatrick, Eisen, Wolff, Freedberg and Austen, Dermatology in General Medicine, 1987, Vol. 1, 15 McGraw-Hill, Inc., New York, pp. 582-584). As with pemphigus, treatment of cicatricial pemphigoid often requires high doses of systemic corticosteroids and immunosuppressive agents.
Because of the scarring associated with cicatricial pemphigoid, long term systemic steroids have been used in these patients despite the side effects.
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. For example, in one study, cytotoxic proteases were identified in the blister fluid of pemphigus and pemphigoid patients (Grando, Glukhenky, Drannik, Kostromin and Chernyavsky, Int.
35 ~. Tissue React. 1989, Vol. 11, pp. 195-201).
Similar observations have been reported by other investigators (Lauharanta, Salonen and Vaheri, Acta ~ W095/00~6 21 6 ~ 9 S S PCT~S94/06949 ?~

Derm. Venereol. 1989, Vol. 69, pp. 527-9; Morioka, Naito and Ogawa J. Invest. Dermatol. 1981, Vol. 76, pp. 337-41; Singer, Sawka, Samowitz and Lazarus, J.
rnveSt . Dermatol. 1980, Vol. 74, pp. 363-7).
~nflammatory responses, such as those seen in lichen planus, result in the local production and/or elaboration of proteases and tissue injury at the disease site. (Barnhart, Quintana, Lenon, Bluhm and Riddle, Ann. N. Y. Acad. Sci. 1968, Vol.
146, pp. 527-39; Janoff and Carp, Monogr. Pathol.
1982, Vol. 23, pp. 62-82; Grando, Glukhenky, Drannik, Epshein, Kostromin and Korostash, Arch.
Dermatol. 1989, Vol. 125, pp. 925-30; Forster, J.
Dent. Res. 1972, Vol. 51, pp. 257-63). Finally, in the case of pemphigus, there is evidence that direct induction of proteinase activity by autoantibodies significantly contributes to the pathogenesis of the disease (Singer, Hashimoto and Lazarus, Springer Semin. Immunopathol. 1981, Vol.
4, pp. 17-32).
The 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). By contrast, the natural plasma proteinase inhibitor, alpha-l-proteinase inhibitor, completely inhibited acantholysis formation in the mouse model. Based on this work, it appears that only certain proteinase inhibitors are effective in the treatment of pemphigus.
C~n~er Sores (Aphthous Ulcers) Aphthous ulcers are inflammatory lesions of unknown etiology that can effect any mucosal surface, but occur most often in the mouth W095l00~6 ;~ PCT~S94/069 ~
~6~9 -14-(Cropley, T.G. in Manual of Clinical Problems in Dermatology, Olbricht, S.M., Bigby, M.e., Arndt, K.A., eds. Little, Brown, and Co. Boston, 1992, pp.
312). The presence of an extensive inflammatory infiltrate in these lesions often result in tissue injury, which reflects the actions of a host of soluble mediators such as proteases and tumor necrosis factor. Current treatments include hygienic measures, topical anesthetics and various unproven therapies such as oral suspensions of tetracyclines and systemic and topical corticosteroids. Patients are frequently instructed to avoid trauma to the oral cavity (such as sharp bread crusts or hard toothbrushes) which may precipitate new ulcers.
In light of the seriousness of the symptoms associated with hyperkeratosis and disorders mediated by proteases, there clearly r~m~; n-~ a need for effective, safe topical and systemic methods for their treatment.
Therefore, it is an object of the present invention to provide a method for the topical treatment of disorders mediated by proteases.
It is another object of the present invention to provide a method for the systemic treatment of the disorders mediated by proteases.
It is a further object of the present invention to provide a method for the topical treatment of diseases characterized by hyperkeratosis.
It is another object of this invention to provide compositions for the treatment of disorders characterized by hyperkeratosis or mediated by proteases.

~ 095/00~6 21 6 q 955 PCT~S94/06949 Summary of the Invention It has been discovered that cutaneous conditions characterized by hyperkeratosis or mediated by proteases can be treated with 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.
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. Once the lipid soluble NAC thioester traverses the stratum corneum, simple hydrolysis of the thioester, by thioesterases or other mech~ni smC, 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 Z5 conditions, including those which are discussed herein. The 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. In an alternative embodiment, the thioester or thioether residue that is cleaved in vivo exhibits an independent beneficial biological activity.
In one embodiment of the invention, a method and composition is provided for the treatment of a WO95/00~6 ~6 4 PCT~S94/069 cutaneous condition characterized by hyperkeratosis, including ichthyosis, keratoderma, lichen planus, and psoriasis, in a human or other m~mmAl. 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.
In a preferred method of a~mi ni ctration, the active compounds are a~m; n i.ctered 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 mi n i m~ 1 systemic effects.
It has been discovered that the active compounds, and specifically the NAC thioesters, are not only useful to treat hyperkeratotic disorders, but may decrease inflam.mation associated with the disorder. The compounds can also be used as immunosuppressants.
Preferred 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-1-cyclohexen-1-yl)-2,4,6, 8-nonatetraenoyl-N-acetyl-L-cysteine), lactic acid (S-lactoyl-N-acetyl-L-cysteine) and the thioesters of other a-hydroxy acids, including but not limited to glycolic acid and a-hydroxybutyric acid.
In another embodiment, a method for the ~ W095/00~6 ~ ~21 ~ ~ 5 ~ PCT~S94/06949 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), is provided wherein 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. The active compound or its derivative is a~m; n; stered 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. When treating lesions of the oral mucosa, the taste and odor of NAC can be masked with an additive such as lemon or peppermint oil.
Skin or mucosal lesions on non-oral membranes (for example, ocular, vaginal, nasal, or 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 a~m; n; ctered in an effective dosage range to cause suppression of the symptoms. In one embodiment, 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. In another WOg5/00~6 Z1~ 4 9 ~ ~ PCT~S94106~

f embodiment, 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.
In an alternative embodiment, a method for the treatment of disorders mediated by proteases is provided that includes a~m; n istering 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.

Detailed Description of the Invention I. N-Acetylcysteine and its Derivatives The term alkyl, as used herein, refers to a saturated straight, branched, or cyclic (or a combination thereof) hydrocarbon of Cl to C22, 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.

The term aryl, as used herein, refers to phenyl, or substituted phenyl, wherein the substituent is halo, alkyl, alkoxy, alkylthio, = haloalkyl, hydroxyalkyl, alkoxyalkyl, methylenedioxy, cyano, C(O)(alkyl), carboxylic acid, C02alkyl, amide, amino, alkylamino or dialkylamino, and wherein the aryl group can have ~ WO95/00136 21 i ' ' PCT~S94/06949 up to 3 substituents.
The term aralkyl refers to an aryl group with an alkyl substituent.
The term 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.
As used herein the term fatty acid refers to a long chain (C6 to C22) aliphatic carboxylic acid The term "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.
The term 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 NR4+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, aluminum, or magnesium, zinc, bismuth, copper, cobalt, nickel, cadmium, and the like, or a cation of a nitrogenous base including but not limited to N,N-dibenzylethylene-diamine, D-glucosamine, or ethylenediamine.

W09~/00~6 ~ PCT~S94/0694~
æl6~955 II. N-Acetylcysteine and its Derivatives 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.
N-acetylcysteine (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 Denantiomers. 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. In a preferred embodiment, 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 . ~.
Med . 1988, Vol. 319, pp. 1557-62). A large overdose of acetaminophen results in a larger portion of the drug being metabolized via a free radical (cytochrome P-450) pathway which results in hepatic cellular necrosis. N-acetylcysteine, when administered within the first few hours of overdose, protects the liver by acting as an alternate substrate for coniugation with and ~ 095/00~6 21 6~ 9SS PCT~S94/06949 ~ f detoxification of the reactive metabolite.
In addition to its mucolytic and free radical scavenging activities, NAC has been reported to be an effective collagenase inhibitor (Lemp and Roddy, Ann. Ophthalmol. 1974, Vol. 6, pp. 893-5) and an antioxidant in vivo (Knight, K.R., MacPhadyen, K., Lepore, D.A., Kuwata, N., Eadie, P.A., 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). It has also been reported that 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.
In yet another capacity, N-acetylcysteine can act as an inhibitor of tumor necrosis factor production in vivo ( Peristeris, P. et al, Cel l .
Immunol . 1992, Vol. 140, pp. 390-99).
As used herein, the term pharmaceutically acceptable derivative or salt of N-acetylcysteine refers to either:
(i) any compound that upon ~m; n; stration to the recipient, is capable of providing directly or indirectly, the compounds disclosed herein;
including, or alternatively, (ii) a compound of the formula:
~ CH~ ~COOR

\C/

WO95/00~6 ~ 9 5~ PCT~S94/069 wherein Rl 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, aspartoyl, glutaroyl, lysinyl, argininyl, and histidinyl; and R2 is hydrogen, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, C(O or S)alkyl, C(O or S)aryl, C(O or S)alkaryl, C(O or S)aralkyl, C(O or S)alkyloxyalkyl, C(O or S)acyloxyalkyl, or phosphate. R2 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. Alternatively, R2 can be the residue of lactic acid, retinoic acid, or ascorbic acid (to form the thioester). or other a-hydroxy acid (including but not limited to glycolic a~ d), or the residue of a dicarboxylic ~WO9~/00~6 `21 6 ~ 9 ~ ~ PCT~S94/06949 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. In yet another embodiment, R2 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-O,O-diacetic acid, 1,4-bis-carboxymethyl benzene, 2,2-bis-(4-hydroxyphenyl)propane-O,O-diacetic acid, 1,4-phenylene-dipropionic acid, and cyclohexane dicarboxylic acid (wherein N-acetylcysteine is bound through one or more carboxylic acid groups to form thioesters). In one embodiment, at least one of Rl or R2 is not hydrogen.
Non-limiting examples of amine-cont~;~ing 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.
In another embodiment, a dimer or oligomer of NAC or its derivative is provided. The dimer can be formed by joining two -SR2 moieties to form a disulfide bridge (with the el;m;n~tion of the R2 groups), or by joining two carboxylate moieties to for.m an anhydride. Alternatively, the dimer can be formed by combining an -SR2 moiety of NAC or its derivative with a CO2RI moiety of another NAC
molecule or derivative to form a thioester dimer.

WO95/00~6 ~ PCT~S94/069j~
'`'~9S~ _ In another embodiment, two or more NAC molecules or derivatives are combined through thioester linkages.

III. Phanmaceutical Compositions of NAC
Humans, equine, canine, bovine, feline and other animals, and in particular, mAmmAl5, suffering from hyperkeratosis or diseases mediated by proteases, can be treated by administering to the patient or ~n;m~l 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 a~m; n; stered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid, cream, gel or solid form.
As used herein, the term pharmaceutically acceptable salts or complexes refers to salts or complexes that retain the desired biological activity of the above-identified compounds and exhibit m;n;mAl 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 am.monia, N,N-dibenzylethylene-diamine, D-glucosamine, or ethylene~; ~mi ne.
In general, 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 ~Wo9~/00~6 PCT~S94/06949 ~ ~SS

~he bioavailability and rate of metabolism of the active species, thus providing control over the delivery of the active species. For example, modifications of the active molecule, such as alteration of charge, can effect water and lipid solubility and thus alter the potential for percutaneous absorption. For example, increasing the 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). Further, the 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.
In one embodiment, N-acetylcysteine or its derivative or salt is applied in the form of a topical composition. The 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. In one embodiment, 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).
In another embodiment, 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.

W095/00~6 ~ PCT~S94/069 ~
` 21~495~

Alternatively, 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.
The vehicle, or carrier, can be modified to enhance cutaneous absorption, enhance the reservoir effect, or m; nim; ze potential irritancy or neuropharmacological effects of the composition.
Examples of 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., M~enh~ 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 ph~rm~ceutically 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 ~095/00~6 PCT~S94tO6949 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 a~min;~tered in any suitable unit dosage form, including but not limited to pills, tablets, troches, and caplets cont~;n;ng 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 ~m; n; stered 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.
The 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 W095/00~6 PCT~S94/069~
216495~ ~

according to the individual need and the professional judgment of the person a~mi n; stering or supervising the administration of the compositions. The 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 a~m; ni stered 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. For the purpose of oral therapeutic administration, 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 = Z5 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 (phenylalanine); a flavoring agent such as peppermint, lemon, cinnamon, methyl salicylate, or ~W095/00~6 2 1 4. 4 9 S S PCT~S94/06949 orange flavoring. 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.
In one embodiment, 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.
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.

W095/00~6 ~ ' , PCT~S94/069 2~ 6;~'S5 _30-IV. Methods for the Evaluation of Effectiveness of NAC in the Treatment of Disorders Characterized by Hyperkeratosis.
The effectiveness of 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. 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.

~ W095/00~6 21 ~9 S~ PCT~S94/06949 ~ .~ C
V. Methods for the Evaluation of Effectiveness of NAC in the Treatment of Pemphigus in Model Systems.

The effectiveness of 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.
1. Experimental pro~ e for purification of pemphigus antibodies from human donors.

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, ollowed by ion exchange chromatography. IgG
fractions prepared in this manner are free of significant protease contamination when assayed.
~urther or alternate purification regimens can include protein-A or protein-G binding and elution, and various chromatography schemes that exploit differences in the size and charge of the IgG, as known to those skilled in the art. The fractions are concentrated and sterilized via filtration.
The pemphigus anti-body titer in the serum is then measured.

W095/00~6 PCT~S94/069 ~
2~64g~i5 2. Organ Culture Model for Pemphigus Production of 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. Complement is not required for the in vitro production of acantholysis since heating the pemphigus sera at 56 C for thirty minutes does not prevent acantholysis (Lever, J. Am. Acad. Dermatol. 1979, Vol. 1, pp. 2-31).
The ability of 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 cont~i n; ng C02 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 e~m; ned by routine histologic (hemotoxylin and eosin st~;n;ng) 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 _~W095/00~6 PCT~S94/06949 6~9SS

(1-24 hours) with NAC, its derivative or salt prior to addition of pemphigus lgG. Acantholysis is evaluated on a scale of (-), (+), (++), or (+++)~
where (-) is no acantholysis, (+) is positive on ].0-30%, (++) is positive on 30-70%, and (+++) is positive on 70-100% of the epidermis in the histologic section.

3. Neonatal Mouse Model for Pemphigus The ability of NAC or its derivative or salt to reduce the symptoms of pemphigus in vivo can be evaluated in a neonatal mice model (Anhalt, Labib, ~oorhees, Beals and Diaz, N. Engl. ~. 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 ~n;m~l S 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 ~or direct immuno-fluorescence. Human pemphigus antibodies are also monitored in the ~nim~ls' 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.
Specifically, within 30 minutes of pemphigus lgG injection, the neonatal mice receive injections of NAC, its salt, or its derivative prepared in PBS. The a~m; n; stered 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 WO9S/00136 PCT~S941069 ~
216495~

the solutions to be injected are sterilized by filtration through an 0.45 ~m millipore filter.
Effects of inhibitors on epidermal acantholysis by pemphigus IgG in neonatal mice are evaluated visually (positive if the presence of Nikolsky sign is observed; i.e., apparently normal epidermis can be separated at the basal layer and rubbed off when pressed with a sliding motion on any part of the skin surface) as well as histologically (acantholytic changes are examined at five sites) 24 hours after pemphigus IgG is injected. To carry out biochemical analysis 24 hours after pemphigus IgG injection the mice are sacrificed and the whole skin of each ~ n; m~ I removed. At least five different sites from each removed skin are then examined for histologic analysis.
In addition to acantholysis, the effect of NAC, its derivatives and its salts on the level of the protease, plasminogen activator, in the neonatal mouse epidermis is determined. Skin samples are removed as described above at 3 and 24 hours after injection of pemphigus IgG with preinjection of the test compound. The skin is isolated by heating the skin at 56C for 30 seconds and putting it through 2 freeze thaw cycles. It is then homogenized and spun at 4C for 2 hours in = 0.01 M sodium monophosphate, pH 7.0 and centrifuged at 750 g for 10 min. The pellet is extracted with 2 M potassium thiocyanate (KSCN) with 0.01% Triton X-100 4C for 2 hours. 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.
Invest Dermatol, 1989, vol. 93, 173-177).

~095/00136 PCT~S94/06949 ~ 6,~,~,S~

VI. Methods for the Evaluation N-acetylcysteine in Humans with oral lesions.
The effectiveness of treatment of patients with oral lesions resulting from lichen planus, bullous pemphigoid, cicatricial pemphigoid, pemphigus or canker sores (aphthous ulcers) with NAC or its derivatives or salts thereof can be evaluated as described generally for treatment of lichen planus by Eisen, Ellis, Duell, Griffiths and Voorhees, in N. ~ngl. J. Med. 1990, Vol. 323, pp.
290-4. For example, patients with symptomatic oral lichen planus are given either placebo or a topical N- acetylcysteine solution, gel, or ointment containing 1 to 50% NAC or other test compound.
The solutions are swished for several minutes and expectorated or swallowed several times daily.
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 m; n i m~ 1 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.
Analogously, 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.

VII. Evaluation of the Effectiveness of NAC
Derivatives or Salts as I.~,.unosuppressant or Antiinflammatory Agent.

In another embodiment, this invention is a method for the treatment of cutaneous, ocular, or mucosal hypersensitivity and/or hyperproliferative W095/00~6 ~ - PCT~S94/069~
,.," ~,,~,64~S~

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 apharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier.
The thioester and thioether derivative of N-acetylcysteine, or the salt thereof, isadministered 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, iritis, 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.
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 r~
~37--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. For example, 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 VVB) light, for example, as described in Arch.
Biochem. Biophys., 298:87-90 (1992); ~. Invest.
Dermatol., 89:1410-1414 (1987); Arch. Dermatol.
Ref., 278:445-448 (1986).
Specifically, healthy 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 mA~;mize ear swelling. The W light source should be located 24.25 inches from the mice. The WB
radiation can range between 150 and 250 mJ/cm2. The mice are subjected to radiation for 3 to 5 minutes during which period they are contained in a plastic box with no cover. At an appropriate time after or during ultraviolet radiation, the test compound is applied to the ~n im~ls' W-light treated skin.
Alternatively, 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 W095/00~6 r PCT~S941069j~

` ~i6 ~g5$ -38-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.) Systemic Treatment Sensitization and challenge for contact hypersensitivity can be measured as follows. The abdomens of mice are shaved with electric clippers, and then 50 yl of a 4% (w/w) solution of oxazolone in 4:1 (v:v) acetone:olive oil is applied to the shaved abdomen, and 5 yl of the same solution is applied to each hind footpad. Five to seven days later, the mice are challenged for contact hypersensitivity by applying 10 yl 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. 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.

O95/00~6 ~ PCT~S94/06949 The right ears of control mice are similarly treated, but with vehicle alone. In the case of experiments designed to evaluate the topical effect of the thioester or thioether derivative of N-5 acetylcysteine, its derivatives or salts on the r sensitization phase, only the right ear is to be challenged. Evaluation of Ear Swellinq Response - Immediately before and 24 or 48 hours after application of oxazolone, the ear thicknesses are 10 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 CO2 15 inhalation after the last experimental time point, and the ears are then processed for histologic eXAm; n~tion .
Quantification of Leukocyte Infiltration -Both ears of each mouse are fixed in 4.0~ buffered 20 formalin and then processed routinely and embedded in paraffin for preparation of 6-7 ~m-thick hematoxylin and eosin-stained sections. The sections are coded and exAm;ned with an ocular grid at 400x under light microscopy by an observer 25 unaware of the identity of the individual slides.
The number of leukocytes/mm2 of dermis is calculated by counting all of the leukocyte cells in an area of at least 0.14 mm2 of dermis.
Statistical Analysis - Differences between 30 groups can be assessed by the 2-tailed Student's t test (paired for comparisons of left and right ears in the same mice, unpaired for comparisons between diferent groups of mice).
Effect of topically administered Active 35 Compound on tissue swelling associated with oxazolone-induced contact hypersensitivity reactions/Effect of time of administration. To test W095/00~6 PCT~S94/069~

2 1 ~ 4 9 5 ~ 40 the effect of the active compounds on the development of the contact hypersensitivity reactions, or on the magnitude or time course of reactions which have already been elicited, 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) ~nim~l s . 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.
In a separate experiment, twenty-four hours after oxazolone challenge, 100 mg/ml active drug in a suitable vehicle is applied to both surfaces of the right ears of some mice, whereas vehicle alone is applied to both surfaces of the ears of control (0~ test drug) mice. The change in ear thickness is then determined 24 hours after treatment with the active compound, i.e. at 48 hours after challenge with oxazolone. This experiment tests the ability of the active compound to suppress contact hypersensitivity reactions which already have developed VIII. Evaluation of Inhibition of Inflammatory Response When applied topically, phorbol-12-myristate-13-acetate (PMA) induces edema (tissue swelling), leukocyte infiltration, and eventually, epidermal hyperplasia in the ears of mice. This model of 095/00~6 PCT~S94/069 2 16 ~9~ 41-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.
1.) Elicitation and Analysis of Acute Inflammatory Response. 10 ~1 of a solution of phorbol-12-myristate-13-acetate (PMA) (1 mg/ml in acetone) is applied to the inner surface of the left (challenged) mouse ear and 10 ~1 of acetone is applied to the right (control) ear.
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.
In methods analogous to the procedures described above, 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.
Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description of the invention. Such modifications and variations are intended to fall within the scope of the appended claims.

Claims (79)

We claim:

1. A method for the treatment of disorders mediated by proteases in mammals that result in skin or mucosal lesions, comprising the step of:
topically applying to the skin or mucosal lesion an effective amount of N-acetylcysteine or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier for topical administration.

2. A method for the treatment of disorders mediated by proteases in mammals that result in skin or mucosal lesions, comprising the step of:
systemically administering an effective amount of N-acetylcysteine or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier for systemic administration.

3. A method for the treatment of disorders mediated by proteases in mammals that result in skin or mucosal lesions, comprising the step of:
topically applying to the skin or mucosal lesion an effective amount of a derivative of N-acetylcysteine of the formula:

wherein R1 is H, 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; 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 R2 is H, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, C(O or S)alkyl, C(O or S)aryl, C(O or S)alkaryl, C(O or S)aralkyl, C(O or S)alkyloxyalkyl, C(O or S)acyloxyalkyl, phosphate, and or an inorganic cation; the residue of a saturated or unsaturated fatty acid; the residue of lactic acid, retinoic acid, or ascorbic acid; or the residue of an alkyl or aromatic dicarboxylic acid;
or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier for topical administration.

4. A method for the treatment of disorders mediated by proteases in mammals that result in skin or mucosal lesions, comprising the step of:
systemically administering an effective amount of a derivative of N-acetylcysteine of the formula:

wherein R1 is H, 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; 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 R2 is H, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, C(O or S)alkyl, C(O or S)aryl, C(O or S)alkaryl, C(O or S)aralkyl, C(O or S)alkyloxyalkyl, C(O or S)acyloxyalkyl, phosphate, and or an inorganic cation; the residue of a saturated or unsaturated fatty acid; the residue of lactic acid, retinoic acid, or ascorbic acid; or the residue of an alkyl or aromatic dicarboxylic acid;
or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier for systemic administration.

5. The method of claim 1 wherein the compound is applied in the form of a 10 or 20 percent aqueous solution.

6. The method of claim 3 wherein the compound is applied in the form of a 10 or 20 percent aqueous solution.

7. The method of claim 1 wherein the compound is applied in the form of a 1 to 100% topical solution, gel, ointment, cream, lotion or foam.

8. The method of claim 3 wherein the compound is applied in the form of a 1 to 100% topical solution, gel, ointment, cream, lotion or foam.

9. The method of claim 1 wherein the compound is applied several times a day.

10. The method of claim 3 wherein the compound is applied several times a day.

11. The method of claim 1 wherein the compound is applied with gauze bandages soaked in the compound.

12. The method of claim 3 wherein the compound is applied with gauze bandages soaked in the compound.

13. The method of claim 1 wherein the compound is applied orally and swished and expectorated or swallowed.

14. The method of claim 3 wherein the compound is applied orally and swished and expectorated or swallowed.

15. The method of claim 3 wherein R1 is an amino acid.

16. The method of claim 4 wherein R1 is an amino acid.

17. The method of claim 15 wherein the amino acid is selected from the group consisting of lysine and arginine.

18. The method of claim 16 wherein the amino acid is selected from the group consisting of lysine and arginine.

19. The method according to claim 3 wherein R1 is an amine-containing antibiotic.

20. The method according to claim 4 wherein R1 is an amine-containing antibiotic.

21. The method according to claim 19 wherein the antibiotic is selected from the group consisting of erythromycin, propionylerythromycin, neomycin, gentomycin, tobramycin, and mechlocycline.

22. The method according to claim 20 wherein the antibiotic is selected from the group consisting of erythromycin, propionylerythromycin, neomycin, gentomycin, tobramycin, and mechlocycline.

23. The method according to claim 1 wherein N-acetylcysteine is administered as the sodium salt.

24. The method according to claim 2 wherein N-acetylcysteine is administered as the sodium salt.

25. The method of claim 1 wherein the disease is pemphigus.

26. The method of claim 1 wherein the disease is bullous pemphigoid.

27. The method of claim 1 wherein the disease is cicatricial pemphigoid.

28. The method of claim 1 wherein the disease is lichen planus.

29. The method of claim 1 wherein the disease is canker sores (aphthous ulcers).

30. The method of claim 2 wherein the disease is pemphigus.

31. The method of claim 2 wherein the disease is bullous pemphigoid.

32. The method of claim 2 wherein the disease is cicatricial pemphigoid.

33. The method of claim 2 wherein the disease is lichen planus.

34. The method of claim 2 wherein the disease is canker sores (aphthous ulcers).

35. The method of claim 3 wherein the disease is pemphigus.

36. The method of claim 3 wherein the disease is bullous pemphigoid.

37. The method of claim 3 wherein the disease is cicatricial pemphigoid.

38. The method of claim 3 wherein the disease is lichen planus.

39. The method of claim 3 wherein the disease is canker sores (aphthous ulcers).

40. The method of claim 4 wherein the disease is pemphigus.

41. The method of claim 4 wherein the disease is bullous pemphigoid.

42. The method of claim 4 wherein the disease is cicatricial pemphigoid.

43. The method of claim 4 wherein the disease is lichen planus.

44. The method of claim 4 wherein the disease is canker sores (aphthous ulcers).

45. A pharmaceutical composition for topical delivery comprising an effective amount of a derivative of N-acetylcysteine as described in claim 3 to treat a disorder mediated by proteases in mammals that results in skin or mucosal lesions.

46. A pharmaceutical composition for topical delivery comprising an effective amount of a derivative of N-acetylcysteine as described in claim 4 to treat a disorder mediated by proteases in mammals that results in skin or mucosal lesions.

47. A pharmaceutical composition for systemic delivery comprising an effective amount of a derivative of N-acetylcysteine as described in claim 2 to treat a disorder mediated by proteases in mammals that results in skin or mucosal lesions.

48. A pharmaceutical composition for systemic delivery comprising an effective amount of a derivative of N-acetylcysteine as described in claim 4 to treat a disorder mediated by proteases in mammals that results in skin or mucosal lesions.

49. The method of claim 3 or 4 wherein R2 is selected from the group consisting of the residue of 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.

50. The method of claim 3 or 4 wherein R2 is selected from the group consisting of the residue of cromolyn, nedocrimil, or other mast cell stabilizers, azelaic acid, or methotrexate.

51. The pharmaceutical composition of claim 47 or 48 wherein R2 is selected from the group consisting of the residue of 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, neodecanoic, neononanoic, palmitoleic or petriselinic acid.

52. The pharmaceutical composition of claim 47 or 48 wherein R2 is selected from the group consisting of the residue of cromolyn, nedocrimil, or other mast cell stabilizers, azelaic acid, or methotrexate.

53. A method for the treatment of a hyperkeratotic disorder comprising topically applying to the affected area an effective amount of a compound of the formula:

wherein R1 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; 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 R2 is hydrogen, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, C(O or S)alkyl, C(O or S)aryl, C(O or S)alkaryl, C(O or S)aralkyl, C(O or S)alkyloxyalkyl, C(O or S)acyloxyalkyl, phosphate, or an inorganic cation; the thioester formed between N-acetylcysteine and the residue of a saturated or unsaturated fatty acid, the residue of lactic acid or other .alpha.-hydroxy acids, retinoic acid, or ascorbic acid; or the residue of an alkyl or aromatic dicarboxylic acid;
or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier for topical administration.

54. The method of claim 53 wherein the compound is applied in the form of a topical solution, gel, ointment, cream, lotion or foam.

55. The method of claim 53 wherein the compound is applied with gauze bandages soaked in the compound.

56. The method of claim 53, wherein R2 is a fatty acid residue.

57. The method according to claim 56 wherein the fatty acid is selected from the group lauric, oleic, caproic, linoleic, linolenic, caprylic, capric, myristic, palmitic, stearic, arachidic, behenic, lignoceric, heptanoic, nonanoic, undecanoic, tridecanoic, pentadecanoic, heptadecanoic, nonadecanoic, heneicosanoic, tricosanoic, arachidonic, docosahexanoic, elaidic, erucic, nervonic, palmitoleic or petriselinic acid.

58. The method according to claim 53 wherein R2 is selected from the group consisting of the residue of cromolyn, nedocrimil, azelaic acid, methotrexate and other mast cell stabilizers.

59. The method of claim 53 wherein the mammal is a human.

60. The method of claim 53, wherein the compound is selected from the group consisting of (S-oloeyl-N-acetyl-L-cysteine), (S-lauryl-N-acetyl-L-cysteine), (S-myristoyl-N-acetyl-L-cysteine), (S-caprolyl-N-acetyl-L-cysteine), (S-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoyl-N-acetyl-L-cysteine), and (S-lactoyl-N-acetyl-L-cysteine).

61. A topical composition for the treatment of a hyperkeratotic disorder comprising an effective amount of a compound of the formula:

wherein R1 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; 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 R2 is hydrogen, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, C(O or S)alkyl, C(O or S)aryl, C(O or S)alkaryl, C(O or S)aralkyl, C(O or S)alkyloxyalkyl, C(O or S)acyloxyalkyl, phosphate, or an inorganic cation; the thioester formed between N-acetylcysteine and the residue of a saturated or unsaturated fatty acid, the residue of lactic acid or other .alpha.-hydroxy acids, retinoic acid, or ascorbic acid; or the residue of an alkyl or aromatic dicarboxylic acid, and wherein at least one of R1 and R2 is not hydrogen;
or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier for topical administration.

62. The composition of claim 61 in the form of a topical solution, gel, ointment, cream, lotion or foam.

63. The composition of claim 61 in the form of a soaked gauze bandage.

64. The composition of claim 61, wherein R2 is a fatty acid residue.

65. The composition of claim 64 wherein the fatty acid is selected from the group lauric, oleic, caproic, linoleic, linolenic, caprylic, capric, myristic, palmitic, stearic, arachidic, behenic, lignoceric, heptanoic, nonanoic, undecanoic, tridecanoic, pentadecanoic, heptadecanoic, nonadecanoic, heneicosanoic, tricosanoic, arachidonic, docosahexanoic, elaidic, erucic, nervonic, palmitoleic or petriselinic acid.

66. The composition of claim 61, wherein the compound is selected from the group consisting of (S-oloeyl-N-acetyl-L-cysteine), (S-lauryl-N-acetyl-L-cysteine), (S-myristoyl-N-acetyl-L-cysteine), (S-caprolyl-N-acetyl-L-cysteine), (S-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6, 8-nonatetraenoyl-N-acetyl-L-cysteine), and (S-lactoyl-N-acetyl-L-cysteine).

67. A method for the treatment of pathological conditions associated with immune responses or inflammatory conditions in mammals, comprising the step of:
topically applying to the skin or mucosal lesion an effective amount of a lipid soluble derivative of N-acetylcysteine of the formula:

wherein R1 is hydrogen, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl, 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; an amine salt formed by the reaction of an amine-containing antibiotic with the carboxylic acid group of the N-acetylcysteine, or a pharmaceutically acceptable cation, and R2 is alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, C(O or S)alkyl, C(O or S)aryl, C(O or S)alkaryl, C(O or S)aralkyl, C(O or S)alkyloxyalkyl, C(O or S)acyloxyalkyl, phosphate, and or an inorganic cation; the residue of a saturated or unsaturated fatty acid; the residue of lactic acid or other .alpha.-hydroxy acids, retinoic acid, or ascorbic acid; or the residue of an alkyl or aromatic dicarboxylic acid;
or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier for topical administration.

68. The method of claim 67, wherein the pathology associated with an immune response is contact hypersensitivity.

69. The method of claim 67 wherein the compound in combination with an ophthalmic carrier is topically applied to the eye.

70. The method of claim 67, wherein the compound is applied cutaneously.

71. The method of claim 67, wherein the compound is applied to mucosal membranes.

72. The method of claim 67, wherein the daily topical dose of compound is between 0.01 and 60 grams.

73. The method of claim 67, wherein the compound is applied in a concentration between 0.01 and 100%.

74. A compound of the formula:

wherein R1 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; an amine salt formed by the reaction of an amine-containing antibiotic with the carboxylic acid group of the N-acetylcysteine, or a pharmaceutically acceptable cation, and R2 is the residue of a saturated or unsaturated fatty acid.

75. The compound of claim 74, wherein R2 is selected from the group consisting of lauric, oleic, caproic, linoleic, linolenic, caprylic, capric, myristic, palmitic, stearic, arachidic, behenic, lignoceric, heptanoic, nonanoic, undecanoic, tridecanoic, pentadecanoic, heptadecanoic, nonadecanoic, heneicosanoic, tricosanoic, docosahexanoic, elaidic, erucic, nervonic, palmitoleic or petriselinic acid.

76. A compound selected from the group consisting of S-oloeyl-N-acetyl-L-cysteine; S-lauryl-N-acetyl-L-cysteine; S-myristoyl-N-acetyl-L-cysteine; S-caprolyl-N-acetyl-L-cysteine; and S-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoyl-N-acetyl-L-cysteine).

77. A compound of the formula:

wherein R1 is H, 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; 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 R2 is H, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, C(O or S)alkyl, C(O or S)aryl, C(O or S)alkaryl, C(O or S)aralkyl, C(O or S)alkyloxyalkyl, C(O or S)acyloxyalkyl, phosphate, and or an inorganic cation; the residue of a saturated or unsaturated fatty acid; the residue of lactic acid, retinoic acid, or ascorbic acid; or the residue of an alkyl or aromatic dicarboxylic acid;
or a pharmaceutically acceptable salt thereof, for use in medical therapy, for example, for the treatment or prophylaxis of hyperkeratosis or diseases mediated by proteases.

78. Use of a compound of the formula:

wherein R1 is H, 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; 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 R2 is H, alkyl, aryl, alkaryl, aralkyl, alkyloxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl, C(O or S)alkyl, C(O or S)aryl, C(O or S)alkaryl, C(O or S)aralkyl, C(O or S)alkyloxyalkyl, C(O or S)acyloxyalkyl, phosphate, and or an inorganic cation; the residue of a saturated or unsaturated fatty acid; the residue of lactic acid, retinoic acid, or ascorbic acid; or the residue of an alkyl or aromatic dicarboxylic acid; or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of hyperkeratosis or a disease mediated by proteases.

79. A compound of the formula:

wherein R1 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; an amine salt formed by the reaction of an amine-containing antibiotic with the carboxylic acid group of the N-acetylcysteine, or a pharmaceutically acceptable cation, and R2 is the residue of a saturated or unsaturated fatty acid, for use in medical therapy, for example, for the treatment or prophylaxis of inflammatory conditions.
--> 80. A compound of the formula:

wherein R1 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; an amine salt formed by the reaction of an amine-containing antibiotic with the carboxylic acid group of the N-acetylcysteine, or a pharmaceutically acceptable cation, and R2 is the residue of a saturated or unsaturated fatty acid, for use in medical therapy, for example, for use as an immunosuppressant.