WO1997032839A9 - 9-cis-retinoic acid esters and amides and uses thereof - Google Patents

9-cis-retinoic acid esters and amides and uses thereof

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Publication number
WO1997032839A9
WO1997032839A9 PCT/US1996/002979 US9602979W WO9732839A9 WO 1997032839 A9 WO1997032839 A9 WO 1997032839A9 US 9602979 W US9602979 W US 9602979W WO 9732839 A9 WO9732839 A9 WO 9732839A9
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compound
wherem
ret
noyloxy
cis
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PCT/US1996/002979
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French (fr)
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WO1997032839A1 (en
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Priority claimed from PCT/US1996/002979 external-priority patent/WO1997032839A1/en
Publication of WO1997032839A1 publication Critical patent/WO1997032839A1/en
Publication of WO1997032839A9 publication Critical patent/WO1997032839A9/en

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  • the present invention relates to the held of 9-c ⁇ s retinoic acid and its esters and amides, and particularly to their uses for a variety of therapeutic and prophylactic treatments of the skin.
  • the present invention particularly relates to 9-c ⁇ s-ret ⁇ no ⁇ c acid esters which are effective m the treatment of acne and other skin disorders when administered either topically or orally and which show few if any side effects.
  • retinoids include retinol (Vitamin A), retinal, all-lrans-retinoic acid, and 13-c ⁇ s-ret ⁇ no ⁇ c acid, as well as a variety of esters and similar derivatives.
  • Vitamin A has long been employed for dermal treatments, particularly for the treatment of acne in a variety of its manifestations.
  • the use of Vitamin A itself has been limited because of the toxic character of the compound when administered in excess.
  • Vitamin A esters such as Vitamin A palmitate, for example, are considered safer, although these materials too have substantial levels of toxi ⁇ ty that limits the concentrations at which the compounds can be administered.
  • b-Carotene as a Vitamin A precursor has also been explored, with the expectation of greater safety. The precursor is less effective, however, since it is itself largely inactive and must be cleaved to the active Vitamin form before the desired effects are produced, and the cleavage is difficult to manage, predict and control.
  • Retinal has not achieved any significant acceptance for dermal uses because of the instability of the compound under exposure to heat, oxygen and ultraviolet light. The instability of the compound is unacceptable for most candidate uses.
  • All-trans-rehnoic acid is, to date, the most commonly used dermal retmoid, m topical form (RETIN A®, Ortho Pharmaceuticals, Inc., a subsidiary of Johnson & Johnson). It has been approved for use in the treatment of acne vulgaris and related forms of acne. A substantial level of administration for other indications has not yet been approved, including anti-w ⁇ nkling and antiactinic treatments of the skin. All-trans-retinoic acid has been demonstrated to be irritating to the sk , producing inflammation m a substantial proportion of users In severe cases of cystic acne, oral doses of 13- ⁇ s retinoic a ⁇ d have proved quite effective (ACCUTANE®, Roche Dermatologies, a Division of Hoffmann-LaRoche Inc.).
  • the compound is, however, highly teratogemc and mutagenic, and is strictly contraindicated m women of child- bearing potential.
  • a number of retinoids have been identified with antiaging and antiactmic properties, including esters and amides of 13-c ⁇ s-ret ⁇ no ⁇ c a ⁇ d and all-trans-retinoic a ⁇ d. In many cases these compounds have activities comparable to the parent a ⁇ d and comparable inflammatory and irritating characteristics, although some are known to be safer and less irritating than others (sometimes at the expense of reduced effectiveness).
  • Such retinoids have also been shown to be of benefit in the reduction of skm cancers and precancerous lesions of the skm, although to date use for such indications have not been approved by regulatory authorities.
  • Retinol vitamin A
  • retinoic acid vitamin A acid
  • certain of its analogs are known to have beneficial effects in the treatment of acne and keratiruzing skin disorders
  • Acne affects large patient populations and is a common inflammatory skm disorder which usually localizes on the face Fortunately, the disease usually disappears and in the mterval of months or years between onset and resolution, therapy, although not curative, can satisfactorily suppress the disease in the majority of patients.
  • retinoids therapies for acne mvolve local and systemic administration of vitamin compounds, collectively know as retinoids.
  • Topical application of all-trans-retmoic acid has been t ⁇ ed with some success, particularly against comedones or blackheads, but this condition frequently returns when the treatment is withdrawn.
  • AU-trans-retinoic a ⁇ d is also known as trehnoin. These terms are used interchangeably throughout this specification.
  • retmoic a ⁇ d applied topically can be highly irritating and its use can be painful for the patient depending on the concentration used and the frequency of application.
  • a number of side effects complicates the administration of large doses of vitamin A Among the many symptoms of hypervitarrunosis A are weight loss, desquamation of the skm, hair loss, irritation of the oral and pharyngeal mucosa, and nose bleeds, headaches, bone pain, hver toxi ⁇ ty due to storage of vitamin A in the hver, papilledena, pseudotumor cereb ⁇ , de ⁇ uneralization, and pe ⁇ osteal thickemng of the bones. Because of these and other side effects of oral treatment with vitamin A and all-trans-re noic a ⁇ d, which produces similar side effects, they are rarely recommended for dermatopathic conditions.
  • the daily dosage of this compound ranged from 30-300 mg taken over from 2 to 8 weeks.
  • Other drugs presently used in the treatment of acne include benzoyl peroxide, tretinoin (a ⁇ - trans-retinoic acid, Retin-A Ortho), clindamy ⁇ n, tetracyline, erythromycin, minocycline, and estrogens (for females).
  • Benzoyl peroxide is considered safe and effective m mild and moderate acne treatment.
  • Tretmom is effective but has the previously mentioned deletenous side effects, as well as acceleratmg photocarcinogenesis.
  • the antibiotics are reasonably effective but have side effects such as gastrointestinal problems including reports of pseudomembranous colitis.
  • Estrogens are sometimes effective in treatmg acne, but the side effects of these drugs make them less than desirable.
  • retinoids In general terms, there is a balancing of safety related issues against effectiveness of the retinoids.
  • the most common indications for retinoids are not related to mortality or severe morbidity issues. Acne vulgans, wrinkles and skin photo-damage are largely cosmetic concerns. While the psychodynamics of such conditions and their effective treatment can be quite compelling, significant nsks of side effects are not j ustifiable and safer and more effective modalities of treatment are needed.
  • Other indications for the use of retinoids in dermal therapeutic indications are more significant, and higher levels of adverse side effects are more justifiable, but even m those contexts, greater safety is needed.
  • Another object of the mvention is the treatment of a vanety of skin conditions and diseases with formulations of novel esters and amides of 9- ⁇ s-ret ⁇ no ⁇ c acid.
  • Still another object of the present mvention is to provide a method for altering the structure and appearanc e of the skin through the use of novel esters and amides of 9- ⁇ s-ret ⁇ no ⁇ c acid.
  • 9-c ⁇ s-retmo ⁇ c a ⁇ d and its esters and amides are synthesized and formulated for administration for the treatment of skm conditions and diseases without the mducement of dermatitis wherem there is apphed topically to the epidermis of the skin a non- untatmg retmoid compnsed of the esters and amides of 9-c ⁇ s-ret ⁇ no ⁇ c a ⁇ d, the retmoids havmg the formulae:
  • n is a number from 1 to 5; wherein R' is H or any of the lower alkyls ranging from Ci to C ⁇ ; wherein R" is
  • R"' is the hydrocarbon backbone of fatty a ⁇ ds; wherein R"" is R" or the hydrocarbon backbone of fatty acids; wherem R""' is the lower alkyls rangmg from Ci to C ⁇ ; and further, when there are two or more R', R", R"', R"", or R"' groups attached to the same carbon, each R", R", R"', R"", or R"' group may be the same as or different from the other R', R", R"', R"", or R""' groups attached to that carbon.
  • This invention is directed to novel derivatives of 9-cis-retinoic acid which are useful in the treatment of acne and the like, but which minimize the irritating side-effects associated with 13-cis- retinoic acid and all-trans-retinoic acid and many of their esters and amides as used in treatments of acne and related dermal indications.
  • the derivatives have the formulae:
  • n is a number from 1 to 5; wherem R' is H or any of the lower alkyls ranging from Ci to C ⁇ ; wherem R" is o o
  • R"' is the hydrocarbon backbone of fatty acids; wherem R"" is R" or the hydrocarbon backbone of fatty a ⁇ ds; wherem R""' is the lower alkyls rangmg from Ci to C ⁇ ; and further, when there are two or more R', R", R"', R"", or R"' groups attached to the same carbon, each R', R", R"', R"", or R"' group may be the same as or different from the other R', R", R"', R"", or R""' groups attached to that carbon.
  • esters and amides of 9- ⁇ s-retmo ⁇ c are not per se known to the art.
  • Illustrative compounds include: l-(9- ⁇ s-ret ⁇ noyloxy)-2-propanone, l-(9- ⁇ s-retmoyloxy)-3-decanoyloxy-2-propanone, l,3-b ⁇ s-(9- ⁇ s-ret ⁇ noyloxy)-2-propanone, l-(9- ⁇ s-retmoyloxy)-2-pmacolone,
  • Each test compound and a vehicle control is applied topically to the dorsal trunk of the rhino mouse.
  • the utnculus diameters are measured with a ocular micrometer.
  • the assay is based upon the work of Khgman, et al (1979) and Van Scott (1972).
  • Khgman, et al. The Effect on Rhino Mouse Skin of Agents which Influence Keratiruzation and Exfohation, J. Invest. Derm. 73: 354-358 (1979).
  • Van Scott Experimental Animal Integumental Models for Screenmg Potential Dermatologic Drugs, In Pharmacology of the Skm, eds. Montagna et al, New York, Appleton-Century-Crofts, 1972, pp.
  • the dorsal trunk of the rhino mouse is the test site.
  • Each test compound is dissolved in alcohol.propylene glycol (70:30, v:v) or other suitable vehicle and topically applied (0.1 ml) to the dorsal trunk once daily, five consecutive days/week for two weeks.
  • administration may be oral (p.o.) in a suitable vehicle.
  • the animals are sacrificed by cervical dislocation.
  • the treated dorsal trunk skin is removed from the animal and placed mto 0.5% acetic acid for up to 18 hours at approximately 4 °C.
  • the epidermis with the acne cysts is separated from the underlying dermis.
  • the sheets of epidermis are processed by rout e methods to permanent whole mounts for microscopic examination.
  • full-thickness samples may be taken, stamed (H&E), and examined by light microscopy.
  • the utnculus diameters are measured with an ocular micrometer to compare effects of test compounds to vehicle control and/or reference compound on cyst reduction. Light microscopy is used to determine effects on cell differentiation. The results are summanzed in Table 1.
  • All-trans-retinoic a ⁇ d is used as a control.
  • the compounds of the present mvention are synthesized from 9- ⁇ s-ret ⁇ no ⁇ c a ⁇ d.
  • the a ⁇ d is itself synthesized by the method of Boehm, et al., supra.
  • denvatives can be apphed topically or orally without causing imtation or with less imtation than found with state of the art retmoid based treatments, and are an effective and safe treatment for a wide diversity of dermal conditions, i.e., acne vulgans, cystic acne, hyperpigmentation, hypo-pigmentation, psonasis, dermal and epidermal hypoplasia and keratoses, the reduction of wrinkling of the skm as an incident of aging and actinic damage, normalization of the production of sebum, the reduction of enlarged pores, promotmg the rate of wound healing, limiting of scar tissue formation during healing and the like.
  • dermal conditions i.e., acne vulgans, cystic acne, hyperpigmentation, hypo-pigmentation, psonasis, dermal and epidermal hypoplasia and keratoses, the reduction of wrinkling of the skm as an incident of aging and actinic damage, normalization of
  • lchthyoses e.g., ichthyosis hystnx, epidermolytic hyperkeratosis, and lamellar ichthyosis
  • folhcular disorders e.g., pseudofolhcuhtes, senile comedones, nevus comidonicas, and tnchostatis spinulosa
  • benign epithelial tumors e.g., flat warts, tnchoepithehoma, and molluscum contagiosum
  • perforated dematoses e.g., elastosis perforans senpigmosa and Kyrles disease
  • disorders of keratinization e.g., Daners disease, keratoderma, hyperkeratosis planta ⁇ s, pitynas
  • retinoids can mcrease the rate of wound healing.
  • Retinoids have sometimes been defined narrowly as compnsing simply vitamin A (rebnol) and its denvatives such as vitamin A aldehyde (retmal), vitamm A a ⁇ d (retinoic a ⁇ d), compnsing the so-called natural retmoids.
  • retmoids have been defined as a much larger class of chemical compounds that have physiochemical similanties to vitamin A and its denvatives.
  • wound-heahng retmoid is all-trans retmoic acid which is also known as tretmom.
  • Tretmom showed a rate of healing of 6% when the skm of pigs was pre-treated with a 0.05% tretmom cream for 10 days pnor to partial-thickness skm wounding. Applymg tretmom after wounding had a deletenous effect on healing. Hung et al., Arch. Dermatol. 125:65-69 (1989) believe that this deletenous effect is due to the inflammation that is caused by tretinoin. Khgman, J. Am. Acad. Dermatol., 15779-85 (1986) studied hairless mice by irradiation with a sun lamp. The mice were then treated topically with vanous concentrations of tretmom for several weeks. The subepidermal repair zone in the treated mice was significantly wider than that in the untreated control group. The collagen was histochemically and ultrastructurally normal; fibroblasts were numerous and morphologically hyper-active.
  • the venous ulcer is the most common leg ulcer. Lewis, Custis 44:123-24 (1989), states that venous ulcers are not a skin problem, but a problem of disordered circulation. From his clinical experience, the best treatment is the apphcation of external pressure to counteract the high pressure transmitted through mcompetent perforatmg veins from the deep venous system to the venules of the skin.
  • Retmoids affect the differentiation, maintenance and proliferation of many cell types whether they are of ectodermal, endodermal or mesodermal ongin; or whether they are epithelial, fibroblastic or mesenchymal.
  • Retmoids affect the differentiation, maintenance and proliferation of many cell types whether they are of ectodermal, endodermal or mesodermal ongin; or whether they are epithelial, fibroblastic or mesenchymal.
  • Brown et al New Eng. J. Med., 321:76-79 (1989) demonstrated that the topical application of epidermal growth factor accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns.
  • Paired donor sites were c reated in patients who required skin grafting either for burns or reconstructive surgery.
  • One donor site from each patient was treated topically with silver sulfadiazine cream and one was treated topically with silver sulfadiazine containing epidermal growth factor.
  • Total healing time m these 12 patients was 9-21 days with an average of 12 days.
  • the healing time of the donor sites that received the epidermal growth factor was accelerated by an average 1.5 days.
  • Hunt and La Van, New Eng. J Med., 321:111-112 (1989) commenting on the work of Brown et al, state that a 15% acceleration of the healing time of a patient with burns may save many days of pam or hospitahzation.
  • Sheffield, W. et al. in EPO 339,905-AZ discloses a wound healing composition compnsed of at least one polypeptide growth factor havmg human metagenic or angiogenic activity at least one retmoid.
  • the composition is pondered to have a synorgistic effect with respect to growth factors and retmoids alone.
  • compositions of the present mvention may be topically apphed to the wound site in any suitable pharmaceutically acceptable vehicle, for example, a liquid earner such as propylene glycol ethanol, propylene glycol ethanol chloroform, and the like.
  • a liquid earner such as propylene glycol ethanol, propylene glycol ethanol chloroform, and the like.
  • a preferred hquid composition is a solution of a small amount of at least one of the compounds m combmation with from about 25 to about 75% by volume of 95% ethanol and from about 75 to about 25% by volume of hquid glycol.
  • a typical solvent earner of this type compnses 75% by volume 95% ethyl alcohol and 30% by volume propylene glycol
  • the preferred concentration of the active compound in these compositions is at least 0.01% by weight, most preferably from about 0.1 % to about 0.5% by weight and most preferably from about 0.05% to about 0.2% by weight, but any therapeutically effective concentration may be used.
  • compositions of the present mvention may also be formulated in any number of other ways, depending on whether an aqueous solution, cream or ointment is desired and whether it would be used/and its site of use set as on the surface of the skin or in the eye.
  • compositions formulated as a cream may contain a cream stabilizer such an xanthen gum, an emulsifier preferably a non-iomc emulsifier, at least one hquid and one sohd hydrophobic matenal selected from the hquid and sohd fatty a ⁇ ds, fatty alcohols, fatty a ⁇ d esters, pharmaceutical grades of waxes and hydrocarbons, the latter rangmg from liquids through semi- hquids such as petrolatum, to sohds and the likes, preservative, an antioxidant, and water
  • compositions of the present mvention are useful m eye drop formulations, eye gels, eye creams, lyposome or micelle formulations, acquest vehicles for soaking soaked gauze dressmgs, burn dressmgs, artifi ⁇ al skins, sutures and staple coatmgs, ointments, lotions or creams, gel formulation, foams and the like. Additional matenals such as buffers, preservatives, adjusting agents, antioxidants, polymers for adjusting viscosity or for use as extenders and ex ⁇ pients may be used m the compositions. Methods for mcreasmg the rate of healing a wound compnses applymg or contacting the compositions of the present mvention directly to the wound.
  • composition is permitted to remain in contact with the wound for a penod of time sufficient to mcrease the rate of cell growth at the wound site.
  • Such methods mclude incorporating any composition of the present mvention mto a cream formulation or soaking a gauze dressing with an acquest solution of the composition and then applymg the cream or soaked gauze to a wound site such as a burn, donor site wound, ulcer or any type of cutaneous wound. Additionally, sutures or staples may be coated or soaked with the acquest composition and used to close an open wound.
  • the type of wounds that may be healed usmg the composition of the present invention are those which result from any medical or ac ⁇ dental injury which causes epithelial damage such asophthalmic wounds, such as those which result from corneal ulcers, cutaneous wounds, such as burn wounds, donor site wounds from skm transplants and ulcers. Additionally, dermatological c onditions in which the skm has been damaged may be treated with the compositions of the present mvention. Leg and foot ulcers may also be treated with compositions of the present mvention. Any wound that does not result in total skm loss but retains a portion of the dermis may be treated using the compositions of the present mvention.
  • Trie causes and mechanisms of psonasis and psonahc conditions are not fully known.
  • the disease is genetic, and has been asso ⁇ ated with increased levels of certain histocompatibilitv antigens. It has been reported that persons with elevated HLA-Cw6 are 9 to 15 times more likely to develop psonasis than others. No cure is known, and persons with the disease expenence lifelong, penodic eruptions of scaly plaques, papules and, in some persons, pustules, which may appear on any dermal surface. The disease may be exacerbated by some important drugs, including lithium, Beta-blockers, and antimalenals.
  • Treatment with systemic steroids provide rapid clearing of psoriasis, but often cause a worsenmg manifestation of the condition when the medication is withdrawn, m a rebound phenomenon, which has led to the abandonment of corticostenods as a routme treatment.
  • Clinically diagnosed psonasis is treated with a vanety of procedures and agents. Most commonly, topical applications of steroids, anthralm, coal tar formulations, intralesional injections of steroids, occlusive dressmgs employed with topical formulations, and the administration of ultraviolet hght (both UVB and, less commonly, UVA are employed m combination with anthral , coal tar formulations, and photosensitizers, such as psoralens) are employed.
  • ultraviolet hght both UVB and, less commonly, UVA are employed m combination with anthral , coal tar formulations, and photosensitizers, such as psoralens
  • vanety of systemic treatments employed as well, including the administration of methotrexate, hydrea, etretinate and cyclosporm.
  • the treatment of psonasis involves a balancing of short term palliation and limited benefits of the more benign treatments agamst the greater potency and more senous side effects and consequences of the more efficacious systemic treatments.
  • Ultraviolet hght treatments are generally effective and mvolve a minimum of side effects, but require a large number of office visits; unsupervised self-admmistration of UVB and UVA with photosensitizers is extremely unsafe and is not prescnbed.
  • the mode of action of the compounds of the present mvention is not known.
  • the compounds of the present mvention are beheved to be potent inhibitors of hpoxidase,
  • the mouse ear edema model is based on the induction of edema by the topical administration of arachadonic acid to the dermis of the ear of the specimen.
  • a more recently developed model which has achieved less acceptance as a predictive screen in the evaluation of potential antipsonatic agents, is the hamster model. The procedure of the hamster model is discussed m detail, infra.
  • the compounds of the present mvention are readily formulated with conventional pharmaceutical earners, and may be convemently administered by any convement route, mcludmg un, lv, lp, subcutaneous and intralesional (local) injections, oral administration, and topical (dermal) application, with or without occlusion.
  • LD50 values by oral administration in rats illustrates that the compounds of the present mvention do not represent a substantial threat of acute toxi ⁇ ty.
  • esters and amides of 9- ⁇ s retmoic a ⁇ d are topically apphed to the skm site exhibiting charactenstics to be treated m any suitable pharmaceutically-acceptable vehicle, as for example, a hquid earner such as propylene glycol-ethanol.
  • a prefened hquid composition is a solution of a small amount of at least one of the compounds of the mvention m a combination of
  • composition from about 75% to 25% by volume of a hquid glycol.
  • a typical solvent earner of this type compnses 70% by volume 95% ethyl alcohol and 30% by volume propylene glycol.
  • a small but effective amount of an antioxidant such as butylated hydroxytoluene may also be included m the composition.
  • a typical solvent carrier of this type compnses 70% by volume 95% ethyl alcohol and 30% by volume propylene glycol.
  • An antioxidant at a concentration of 0.01 to about 0.1 % by weight may be incorporated m the earner.
  • the preferred concentration of the active compound m these compositions is at least about
  • 0 01 % by weight more preferably from about 0.01% to about 0.5% by weight and most preferably from about 0.05% to about 0.2% by weight, but any therapeutically effective concentration may be used. Concentrations less than 2.5% by weight will normally be employed.
  • Such topical formulations will generally be made up to contam from about 0.025 to about 2.5 weight percent of the active 9- ⁇ s-ret ⁇ no ⁇ d, preferably from about 0.05 to 0.5 weight percent, although m most circumstances the concentration is not narrowly cntical.
  • the compounds are both highly potent and highly safe, and an exceptionally wide range of concentrations may be employed as indicated.
  • the concentrations to be employed may range widely, from 0.5 to 50 milligrams per kilogram of body weight.
  • the compounds are sensitive to ultraviolet hght, and are oxidized when exposed to air and heat or other oxidizing conditions.
  • the compounds of the present mvention are highly hydrophobic in character, and formulation of the compounds requires that these charactenstics be taken mto account.
  • a further advantage of the compounds is their non-irntating charactenstic when apphed topically. This highly desirable charactenstic is not seen when all-trans-retinoic a ⁇ d is used.
  • Boehm, et al J Med Chem., Vol. 37, No 3, pp. 408-414 (1994). Boehm et al demonstrate that 9-c ⁇ s-ret ⁇ no ⁇ c a ⁇ d is more effi ⁇ ent at binding the two known sub-famihes of lntracellular receptors and each of the three defined sub-types within each of the subfamilies, and predict that 9- ⁇ s-ret ⁇ no ⁇ c acid will provide improved therapeutic indices when compared to other retmoic acids (i.e., all-trans-retinoic a ⁇ d and 13- ⁇ s-ret ⁇ no ⁇ c a ⁇ d).
  • retmoic acids i.e., all-trans-retinoic a ⁇ d and 13- ⁇ s-ret ⁇ no ⁇ c a ⁇ d.
  • the activation of the retmoid receptors has been associated with induced or accelerated mitosis of normal skm cells, and it is the induced cell growth, particularly epithehal cell growth, which is most often associated with the improved skm properties occurring with use of retmoids to treat the skm
  • Abnormal skm cells i.e., those charactenzing photodamaged or age damaged skm, neoplasia and keratoses, cells m hyperpigmented skm regions, psonatic skm cells, and the like, have been associated with defective or compromised retmoid receptors. It is thus hypothesized that treatment of the skin with retmoids functions to stimulate mitosis and proliferation of normal cells, while the abnormal cells are not stimulated and do not parti ⁇ pate m the increased levels of mitosis.
  • the abnormal cells associated with the foregoing conditions are preferentially replaced by normal cells.
  • the enhanced bmdmg of all the types of receptors of the 9- ⁇ s-ret ⁇ no ⁇ c acid is thus a direct basis for predicting a high therapeutic mdex compared to other retmoid acids.
  • esters and amides of the 9- ⁇ s-ret ⁇ no ⁇ c a ⁇ d do not show any detectable imtation or inflammation of the skm among users.
  • the activity of the esters and amides is, however, comparable in magnitude with the 9-c ⁇ s-ret ⁇ no ⁇ c a ⁇ d itself and substantially increased m comparison with all-trans-retmoic a ⁇ d and 13-c ⁇ s-ret ⁇ no ⁇ c acid.
  • the solution is stirred for 24 hours at room temperature with a magnetic stirrer.
  • the sample is concentrated lo about 5 ml and chromatographed on a neutral aluminum oxide (Aldrich #19, 997- 4) column (14X1.8 cm).
  • the alumina is deactivated with 20 ml of water per 1.0 kg of alumina.
  • the sample is eluted stepwise with 100 ml of 20% dichloromethane in hexane, 100 ml of 50% dichloromethane m hexane, and finally with 250 ml of dichloromethane.
  • the sample elutes quickly and the vast majonty of the prties remain on the column.
  • Fractions of 25 ml are collected and evaluated by thin layer chromatography (TLC) on silic gel (EM Reagents #5775) develop with ethyl 0 acetate:heptane (1:3).
  • TLC thin layer chromatography
  • silic gel EM Reagents #5775
  • the fractions containmg the product are combmed and concentrated to give an orange oil which solidifies on cooling to give 0.55 g of sohd.
  • the NMR (CDC13) spectrum of Compound 1 is identical to the spectrum of 9- ⁇ s-retmo ⁇ c a ⁇ d except for two additional peaks and the lack of a carboxyhc acid peak.
  • the two additional peaks are (singlet, 2 protons, --OCH2 CO--) and (singlet, 3 protonsCOCH3).
  • the structure is confirmed by NMR. 0 Elemental analysis for the compound gives a theoretical value for C23 H32 Ch of 77.49% C,
  • NMR (CDC13) spectrum of Compound 2 is identical to the spectrum of 9- ⁇ s-ret ⁇ no ⁇ c acid except for three additional peaks and the lack of a carboxyhc acid peak. The structure is confirmed by NMR.
  • the sohd is collected, dissolved m dichloromethane and dried (Na 2 S0 4 ).
  • the sohd contamed large quantities of water which had to be removed.
  • the dichloromethane is removed under vacuum and the oil is dissolved m acetone and placed m the freezer (-10 °C) overnight.
  • the disubstituted dihydroxyaeetone greatlyty separated and is removed.
  • Suffi ⁇ ent water is added to make the solution about 25% water by volume and after standing overnight m the freezer, the product separated and is recrystalhzed from dichloromethane/hexane to give 5.2 g, mp 91-93 °C
  • Compound 8 is similar to Compound 1 with a terminal methyl hydroxyl group instead of a methyl group. This modification should make Compound 8 more glycende-hke and more hvdrophihc.
  • Compound 10 is synthesized by coupling 9- ⁇ s-ret ⁇ no ⁇ c acid with N-hydroxysuc ⁇ nimide (Zimmerman, et al, The Effect of Active Ester Components on Racemization m the Synthesis of Reptiles by the Dicyclohexyl-carbodiimide Method, J. Am. Chem. Soc, 89: 7151-7152 (1967)). The structure is confirmed by NMR.
  • Compound 10 is the most easdy hydrolyzed prodrug.
  • the N-hydroxy-succinimide group gives an activated ester linkage and is used in peptide synthesis because of this property.
  • EXAMPLE 11 TOPICAL ASSAY FOR THE INHIBITION OF SKIN CANCERS
  • the usefulness of the retmoid compounds of the present mvention for the inhibition of skm cancers is demonstrated by testmg in the ornithine decarboxylase (ODC) assay an ester Compound 1, l-(9- ⁇ s-ret ⁇ noyloxy)-2- ⁇ nacolone and Compound 2, 2-(9- ⁇ s-ret ⁇ noyloxy)-4- methoxyaeetophenone).
  • ODC/Retinoid Bioassay is based on the method of Verma, A. K. and Boutwell, R. K.,
  • the ODC assay measures a compounds effect on the prevention of the induction of ODC, namely the effect of the retmoid compound on the inhibition of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) induced ODC activity.
  • TPA tumor promoter 12-0-tetradecanoylphorbol-13-acetate
  • the assay is earned out usmg CD-I mice (aged 7 to 9 weeks) The dorsal hair of the mice is shaved 3-4 days before testing. Four mice are used for each pomt.
  • the test retmoids, at one of two dose levels (1.7 and 17 nmoles) dissolved in 0.2 ml of acetone is apphed topically to the back of each shaved mouse.
  • TPA TPA 17 nM
  • Control groups are treated with either acetone alone, TPA, or tretmom.
  • the mice are sacrificed by cervical dislocation 5 hours after TPA treatment.
  • the dorsal skm encompassing the shaved and TPA exposed area is ex ⁇ sed and placed in a
  • the epidermal layers from the 4 mice are pooled and placed in a homogemzahon tube with 2 ml of ODA buffer (10 nM tns-HCl with 0.050 nM pyndoxal phosphate, 0.050 nM ethylenediammetetraacetic a ⁇ d (EDTA), 1 mM dithiothreitol, pH 7.5).
  • ODA buffer 10 nM tns-HCl with 0.050 nM pyndoxal phosphate, 0.050 nM ethylenediammetetraacetic a ⁇ d (EDTA), 1 mM dithiothreitol, pH 7.5.
  • the pooled epidermal layers are homogenized for 15 seconds at 0 °C. usmg a Polytron homogemzer at a setting of 7.5.
  • the homogenate is centnfuged at 30,000 x g and the supernatant fraction is pipetted mto
  • the homogenate is assayed for ODC activity as descnbed by Verma and Boutwell to measure the release of 14 C ⁇ 2 from labeled DL(1-14C) o ⁇ uthine.
  • Incubations are carried out m disposable centrifuge tubes with center well holders containing filter paper impregnated with sodium hydroxide to absorb 14 C ⁇ 2 .
  • the mcubation mixture consisted of 90 ml of L-orruthine, 350 ml of ODC buffer, 100 ml of 14C-orn ⁇ th ⁇ ne (1.32 nm, Sp. Act:4.4 pCi/pM) and 10 ml of test sample. After mcubation at 37 °C.
  • Hamsters are dosed orally with the test compound and vehicle alone. The reduction in sebaceous gland size is estimated microscopically in relation to the control.
  • the assay is unique and propnetary to Ortho Pharmaceutical Corp. and is based upon the work of Plewig et al (1977) and Gomex et al. (1980). Plewig, et al, Hamster Ear Model for Sebaceous Glands, J. Invest. Derm. 68: 171- 176 (1977). Gomez, et al, Effect of 13-c ⁇ s-Ret ⁇ no ⁇ c A ⁇ d on the Hamster Flank Organ, J. Invest. Derm. 74: 392-397 (1980).
  • the compounds of the mvention are as effective as 13- ⁇ s- retinoic acid m both topical and oral applications
  • the data presented is raw data which does not take mto account the differences in molecular weight between the compounds of the mvention and 13- ⁇ s- or all-trans-retinoic acid. If one does take this mto account, as must be done to accurately compare the activity of the vanous compounds, it can be seen that many of the compounds of this mvention are equally or more effective than either 13- ⁇ s- or all-trans-retinoic acid.
  • the animals are in the weight range between 2.0 and 3.0 kilograms, and are approximately 11 weeks of age. Animals selected for the test are not subjected to any previous experimental procedures, and their skm is free from imtation, trauma and disease.
  • a dose of 0.5 ml of a test solution composed of 0.025 g of 2 ⁇ (9-cis-retmoyloxy)-4- methoxyacetophenone in a hquid solution composed of 75 ml of ethyl alcohol, 25 ml of propylene glycol 400, and 0.025 g by weight of butylated hydroxytoluene is apphed to one mtact and one abraded skm site per animal. Six animals are treated m this manner.
  • a control group of six animals is treated m an identical manner except that 2-(9- ⁇ s- ret ⁇ noyloxy)-4-methoxyacetophenone is absent from the control solution.
  • the apphcation sites are prepared by clipping the skm of the trunk free of hair approximately 24 hours before apphcation of the dose.
  • One apphcation site on each animal is abraded by making minor incisions through the stratum corneum, but not sufficient to disturb the derma (that is, not suffi ⁇ ently deep to produce bleedmg).
  • the second apphcation site is mtact skm.
  • the dose is apphed to a small area (approximately 6 cm 2 ) of sk n and covered with a gauze patch which is held m place with Vetrap bandaging.
  • the patches are apphed to one mtact site and one abraded site per animal.
  • the test substance is kept in contact with the skin for 24 hours.
  • the skm is not nnsed following the 24 hour exposure penod.
  • 2-(9-c ⁇ s-ret ⁇ noyloxy)-4-methoxyacetophenone is evaluated m a study of its potential to produce dermal imtation. Comparisons are made of tretmom, isotretmom, 2-(9- ⁇ s-ret ⁇ noyloxy)-4- methoxyacetophenone, and the vehicle solution. In the first test, four solutions are used. The control consists of vehicle solution, namely a solution of 60% by volume ethanol and 40% by volume polyethylene glycol.
  • the other three solutions are 0.025% solutions of tretmom, isotretinoin, or 2-(9- ⁇ s-retmoyloxy)-4- methoxyacetophenone m 60% by volume ethanol and 40% by volume polyethylene glycol.
  • Four patients paint two saturated cotton swabs of each of the four solutions on four different areas of the inner forearm, twice daily for ten days. No l tant reactions occur.
  • the control consists of vehicle solution, namely a solution of 90% by volume ethanol and 10% by volume polyethylene glycol.
  • the other three solutions are 0.075% solutions of tretmom, isotretmom, or 2-(9-c ⁇ s-ret ⁇ noyloxy)-4- methoxyacetophenone in 90% by volume ethanol and 10% by volume polyethylene glycol.
  • Four patients paint two saturated cotton swabs of each of the four solutions on four different areas of the inner forearm, twice daily for ten days. Only one subject experiences an irritant reaction. On day two, the tretmom area begins reacting with redness and peeling. On day seven, the lsotretinoin area begins reacting with redness and peeling. By day nine, both areas are still reacting, the tretinom area more mtensely than the isotretmom area. There is no reaction in either the 2-(9- ⁇ s-ret ⁇ noyloxy) ⁇ l- methoxyacetophenone or the control areas.
  • the three solutions are 0075% solutions of tretmom, isotretmom, or 2-(9- ⁇ s-ret ⁇ noyloxy)-4-m-ethoxyacetophenone m 90% by volume ethanol and 10% by volume polyethylene glycol.
  • the tests are earned out m double-blind fashion, that is, neither the subjects nor the mvestigator know the contents of the solutions during the study
  • the four subjects are provided with and apphed to their entire faces (omitting the eyehds) and dorsal surface of the nght forearm, once daily for 12 to 16 weeks, 0.1% concentration of 2-(9-c ⁇ s-ret ⁇ noyloxy)-4-methoxyacetophenone m a hydrophihc cream vehicle.
  • the left forearm of each patient is treated daily with a non-medicated moisturizer of the patients choice. All subjects are evaluated every 4 weeks through the study for redness, peeling, skm surface texture and wrinkling.
  • Biopsies (using a 4 mm punch) are taken from the dorsal surface of the right upper forearm at the beginning of the study and agam from the same area at the end of the study.
  • biopsies are stained with H & E, Al ⁇ an Blue and collagen/ elastic stains and compared by a qualified dematopathologist.
  • TISSUE CULTURE ASSAYS It has been shown while investigating the effect of tretinoin on keratmocytes that tretmom has the capa ⁇ ty to stimulate proliferation of quiescent keratmocytes m vitro (Vararut, et al, 1989). Varanit, et al , conclude that tretmom stimulates keratmocyte growth, in part, by increasing epidermal growth factor receptor RNA and transforming growth factor-alpha production (Mitra cl al, 1989).
  • the esters and amides of 9- ⁇ s-ret ⁇ no ⁇ c acid are evaluated usmg several assays developed by Nickoloff.
  • One of the major thrusts is the delineation of the effect of vanous kerat ocyte growth modulating factors on cultured human keratmocytes.
  • the tissue culture assays used are given m full detad m Nickoloff, B J , Mitra, R.S., Riser, B.L., Dixit, V.M., and Vararu, J., Am. J. Pathol., 132:543-551
  • One to two-microhter droplets of the cell suspension are dehvered with a micropipette into the wells of a microtiter ⁇ ilture dish. After cooling, the agarose droplets are covered with 0.2 ml of the overlay medium After mcubation, migration of the cells is examined daily for 1 to 3 days by phase contrast microscopy usmg an Olympus phase contrast microscope. The distance to the leadmg edge of migrating cells from the edge of the agarose droplet is determined on four sides of each droplet.
  • Motility Assay Micropore Filter Assay Nitrocellulose filters 12m (pore diameter) are used to separate a modified Boyden chamber mto two fluid-filled (KGM) compartments. Keratmocytes are placed m the upper compartment and allowed to migrate mto the filters. After 20 hours, the filters are stamed with hematoxyhn and eosin and the number of migrating cells is determined microscopically. Enzyme-Linked Immunosorbent Assay (ELISA) ELISAs are used to quantify the amount of lmmunoreactive FN (fibronectin) TSP
  • a standard bmding assay 2 x 105 keratmocytes in 24 well plates are washed twice with KBM. The cells are washed once with Earl's balanced salt solution (EBSS) containing 0.2% bovine serum albumm. The cells are chilled and then incubated with medium composed of EBSS with 0.2 bovine serum albumm (bmdmg medium). After 6 hours the assay is terminated by washing the monolayers with cold bmding medium, solubilization of cells in O l N sodium hydroxide containing 1 % SDS and countmg m a gamma counter. Non-specific bmdmg was determined by adding an excess of unlabeled epidermal growth factor (EGF) to parallel samples well and was no greater than 5% of the total amount bound under any treatment condition.
  • EGF epidermal growth factor
  • the bmdmg data are analyzed by a Scratchard plot Measure of Transforming Growth
  • TGF-alpha TGF-alpha
  • TGF-alpha produced cultured keratmocytes
  • semiconfluent keratmocytes containmg KGM are maintained for 48 hours at 37 °C, and the conditioned medium is assayed for TGF-alpha usmg radio immunoassay kit. Duplicate ahquots are removed and immediately reduced and denatured. The samples are run with at least five different known TGF- alpha standards.
  • TGF-alpha To determine the amount of TGF-alpha that may be bound to the keratmocyte cell surface, thoroughly washed keratmocytes are exposed to a cold acid wash m 50 mM glycme, 100 mM NaCl; pH 3.0 for 4 mm at 4 °C. Measurement of TGF-alpha mRNA
  • RNA is isolated by centnfugation. RNA concentration is determined by absorbance at 260 ran and confirmed by nondenatunng agarose gel electrophoresis. RNA are size-fractionated by electrophoresis on 1 % formaldehyde-agarose gel and transferred to denvatized nylon membrane. Filters are hybndized against 2 P labeled probes prepared by random priming. Specific hybndization is estimated by subjecting the autoradiographs of the blots to laser scanning densitometry. EXAMPLE 18
  • the cells After two days and change of the low calcium serum-free medium (keratmocyte growth medium, KGM-Clonetics Corp., San Diego, Calif.) containing EGF, insulin, and bovine pituitary extract, the cells are allowed to proliferate in the dark at 37 °C for an additional 3 days in the presence and absence of the 9- ⁇ s-ret ⁇ no ⁇ d of Example 1.
  • the cell counts after three days are as follows (these cell counts are +/- a 10% standard error):
  • a topical assay to test for pseudocomedone (utnculus) reduction m the rhmo mouse is conducted. Each test compound and a vehicle control was apphed topically to the dorsal trunk of the rhmo mouse. The utnculus diameters are measured with a ocular micrometer.
  • the assay is based on the work of Khgman, et al (1979) and Van Scott (1972).
  • Van Scott Pharmacology of the Skin, eds. Montagna et al, New York, Appelton-Century- Crofts, 1972, pp. 523-533. Mann, Anat.
  • the dorsal trunk of the rhino mouse is the test site. Each test compound is dissolved m alcohohpropylene glycol (70:30, v:v) or other suitable vehicle and topically apphed (0.1 ml) to the dorsal trunk once daily, five consecutive days/week for two weeks. Following treatment, the animals are sacrificed by cervical dislocation. The treated dorsal trunk skm is removed from the animal and placed mto 0.5% acetic acid for up to 18 hours at approximately 4 °C. After this, the epidermis with the "acne cysts" is separated from the underlymg dermis. The sheets of epidermis are processed by routine methods to permanent whole mounts for microscopic examination. Also, full- thickness samples are taken, stamed (H&E) and examined by hght microscope.
  • H&E stamed
  • the utnculus diameters are measured with an ocular micrometer to compare effects of test compounds to vehicle control and/or reference compound on cyst reduction. Light microscopy is used to determine effects on cell differentiation. The results are summanzed m Table 7.
  • Example 2 The compound of Example 2 is 2.5 times less irntatmg than all-trans-RA m the rhino mouse and 3.3 times less irntatmg than all-trans-RA m rabbit dermal imtation.
  • EXAMPLE 21 - 30 The compound of Example 2 is 2.5 times less irntatmg than all-trans-RA m the rhino mouse and 3.3 times less irntatmg than all-trans-RA m rabbit dermal imtation.
  • Epithehal regeneration is the rate limiting factor m the healing of donor sites and partial thickness wounds.
  • An mcrease in heahng rate would allow donor sites to be reharvested more frequently and thus allow closure of the burn wound in a more timely fashion.
  • the more rapid heahng of partial thickness injunes would mean less pain for the patient and a more timely return to so ⁇ ety as a functioning member.
  • the objectives of this study are to:
  • the performance of this study requires pigs be used for a penod of 14 days The study lasts for 10 weeks using a total of 10 pigs.
  • the pigs are anesthetized usmg 10 mg/kg ketamine and 2 mg/kg xylazme mtramuscular injection.
  • Lidocaine is used as a local subcutaneous mjection.
  • Rows of surgically created 2 cm X 2 cm wounds are created on the dorsum of the animal. The wounds are formed usmg a Padgett electnc dermatome (Padgett Company, Kansas City, MO) set at 0.016 inch.
  • Each pig is used to test a different concentration of 9- ⁇ s-retmo ⁇ ds; and, different wounds are used to test varying numbers of apphcations per day, one act g as a control receiving no 9- ⁇ s-ret ⁇ no ⁇ d.
  • the number of apphcations per day vanes from once a day to four times a day. Kaltostat is used as a delivery mechanism.
  • the animals are anesthetized four times daily and appropnate 9- ⁇ s-retmo ⁇ d apphcations are made lo designated sites.
  • Wound sites are covered by Compressor Gnp ® tubular elastic bandage #12 (36-48" circumference).
  • Wound biopsies are taken from designated sites numbered 1 through 5 on each wound, chosen by a random method, on days 3, 5, 7, 9 and 12 for histological exammation and companson. Photographic documentation are obtained during the course of the study.
  • the pigs are anesthetized as usual and then euthanized usmg 0.14 ml/lb. intravenous T61.
  • Each wound is a partial thickness wound created by a Padgett electnc dermatome. 2)Each pig is used to test vanous numbers of applications per day on one concentration of topical 9- ⁇ s-ret ⁇ no ⁇ d.
  • Biopsies are taken from randomly chosen biopsy sites numbered 1 through 5
  • Biopsies are taken on 5 days of the study, on days 3, 5, 7, 9, 12. Biopsies do not contact the edge of the wound.
  • the fdtrates were combmed and diluted with additional water and then extracted three tunes with ether.
  • the combmed organic extracts were dned over MgS ⁇ 4 and concentrated under reduced pressure.
  • Purification by sihca gel flash column chroma lography usmg 20:1 hexane.ethyl acetate as eluent gave the pure methyl ester of 9- ⁇ s-ret ⁇ no ⁇ c acid (II) m the amount of 530 mg, a yield of 80% .
  • the structure was confirmed by NMR.
  • Human neonatal foreskin keratmocytes and human neonatal foreskin frbroblasts were isolated and grown in monlayer culture.
  • Culture medium for keratmocytes was Keratmcyte Growth Medium (KGM) from Clonetics, Inc., San Diego, CA.
  • the culture medium for fibroblasts was Dulbecco's Modified Eagle's Medium (DMEM) supplemented with non-essential ammo a ⁇ ds and 10% fetal bovine serum.
  • the culture medium was obtained from GIBCO, Grand Island, NY and the FBS was from Hyclone Labs, Inc., Logan, UT
  • Cells at passage 2 - 4 were plated m wells of a 24 well dish at approximately 5 X 10 4 cells per well m their respective culture medium. Incubation was at 37 °C with 5% CO2. After the cells attached and spread overmght, they were washed two times in serum-free keratmcyte basal medium (KBM) and incubated m 1 ml of KBM or m 1 ml of KGM. Compound IV was added m varying concentrations to the wells. All-trans-retinoic a ⁇ d (or no treatment) was added to control wells. The cells were incubated for an additional two days for fibroblasts and three days for keratmocytes, and were then harvested and counted. The effect of TV and of the controls are shown for both cell types in Tables 9 and 10:
  • Tables 9 and 10 demonstrate that Compound IV is effective to stimulate both fibroblast and keratinocyte viabihty and growth, but in a fashion more selective than retinoic acid. By comparison of the effects of retinoic acid and IV in these tests, it is possible to demonstrate that IV shows a potent "retinoid" effect on keratinocytes and a less active result with fibroblasts, consistent with the separation of the therapeutic activity from the well known and limiting side effects of retinoids.

Abstract

Esters and amides of 9-cis-retinoic acid are synthesized, formulated into pharmaceutically acceptable carriers and administered for the treatment of acne vulgaris, cystic acne, hyperpigmentation, hypopigmentation, psoriasis, dermal and epidermal hypoplasia and kerotoses, the reduction of wrinkling of the skin as an incident of aging and actinic damage, normalization of the production of sebum, the reduction of enlarged pores, promoting the rate of wound healing, limiting of scar tissue formation during healing and the like. They are additionally useful for treatment or amelioration of the same additional classes of skin disorders as is retinoic acid itself and other retinoids. These disorders include ichthyoses (e.g., ichthyosis hystrix, epidermolytic hyperkeratosis, and lamellar ichthyosis), follicular disorders (e.g., pseudofolliculites, senile comedones, nevus comidonicas, and trichostatis spinulosa), benign epithelial tumors (e.g., flat warts, trichoepithelioma, and molluscum contagiosum), perforated dematoses (e.g., elastosis perforans seripiginosa and Kyrles disease), and disorders of keratinization (e.g., Dariers disease, keratoderma, hyperkeratosis plantaris, pityriasis rubra pilaris, lichen planus acanthosis nigricans, and psoriasis). The esters and amides of 9-cis-retinoic acid are also effective for the non-irritating treatment of effects attributable to aging and particularly to photodamage and photoaging. The use of these compounds extends to non-irritating treatments involving the retardation and reversal of additional dermal and cosmetic conditions which are ameliorated by tretinoin such as the effacement of wrinkles, improvement in appearance, namely color and condition of the skin, spots caused from exposure to the sun as well as other skin disorders. The esters and amides of 9-cis-retinoic acid are exceptionally active when compared to other retinoids employed for such indications, and are also exceptionally safe in effective therapeutic doses in contrast to other retinoids.

Description

9-CIS RETINOIC ACID ESTERS AND AMIDES AND USES THEREOF
BACKGROUND OF THE INVENTION TECHNICAL FIELD:
The present invention relates to the held of 9-cιs retinoic acid and its esters and amides, and particularly to their uses for a variety of therapeutic and prophylactic treatments of the skin. The present invention particularly relates to 9-cιs-retιnoιc acid esters which are effective m the treatment of acne and other skin disorders when administered either topically or orally and which show few if any side effects.
RELATED CASES The mventor of the present invention is one of the inventors of prior patents, 4,677,120,
4,885,311, 4,994,491, 5,049,584; 5,124,356; and Re. 34,075.
STATE OF THE ART
A number of dermal uses have been developed for a wide diversity of retinoids, including retinol (Vitamin A), retinal, all-lrans-retinoic acid, and 13-cιs-retιnoιc acid, as well as a variety of esters and similar derivatives.
Vitamin A has long been employed for dermal treatments, particularly for the treatment of acne in a variety of its manifestations. The use of Vitamin A itself has been limited because of the toxic character of the compound when administered in excess.
Vitamin A esters, such as Vitamin A palmitate, for example, are considered safer, although these materials too have substantial levels of toxiαty that limits the concentrations at which the compounds can be administered. b-Carotene, as a Vitamin A precursor has also been explored, with the expectation of greater safety. The precursor is less effective, however, since it is itself largely inactive and must be cleaved to the active Vitamin form before the desired effects are produced, and the cleavage is difficult to manage, predict and control.
Retinal has not achieved any significant acceptance for dermal uses because of the instability of the compound under exposure to heat, oxygen and ultraviolet light. The instability of the compound is unacceptable for most candidate uses.
All-trans-rehnoic acid is, to date, the most commonly used dermal retmoid, m topical form (RETIN A®, Ortho Pharmaceuticals, Inc., a subsidiary of Johnson & Johnson). It has been approved for use in the treatment of acne vulgaris and related forms of acne. A substantial level of administration for other indications has not yet been approved, including anti-wπnkling and antiactinic treatments of the skin. All-trans-retinoic acid has been demonstrated to be irritating to the sk , producing inflammation m a substantial proportion of users In severe cases of cystic acne, oral doses of 13-αs retinoic aαd have proved quite effective (ACCUTANE®, Roche Dermatologies, a Division of Hoffmann-LaRoche Inc.). The compound is, however, highly teratogemc and mutagenic, and is strictly contraindicated m women of child- bearing potential. A number of retinoids have been identified with antiaging and antiactmic properties, including esters and amides of 13-cιs-retιnoιc aαd and all-trans-retinoic aαd. In many cases these compounds have activities comparable to the parent aαd and comparable inflammatory and irritating characteristics, although some are known to be safer and less irritating than others (sometimes at the expense of reduced effectiveness). Such retinoids have also been shown to be of benefit in the reduction of skm cancers and precancerous lesions of the skm, although to date use for such indications have not been approved by regulatory authorities.
Retinol (vitamin A) and retinoic acid (vitamin A acid), its isomers, and certain of its analogs are known to have beneficial effects in the treatment of acne and keratiruzing skin disorders Acne affects large patient populations and is a common inflammatory skm disorder which usually localizes on the face Fortunately, the disease usually disappears and in the mterval of months or years between onset and resolution, therapy, although not curative, can satisfactorily suppress the disease in the majority of patients.
A small number of acne patients with severe disease show little or no response to intensive therapeutic efforts mcludmg the use of high doses of oral tetracyclme, dapsone, predmsone, and, m women, estrogen. In many cases, these drugs afford only a modest degree of control while the side effects of these agents severely restrict their usefulness. Patients with nodulocystic acne suffer from large, inflammatory, suppurative nodules appearing on the face, and frequently the back and chest In addition to their appearance, the lesions are tender and often purulently exudative and hemorrhagic. Disfiguring scars are frequently inevitable.
Therapies for acne mvolve local and systemic administration of vitamin compounds, collectively know as retinoids. Topical application of all-trans-retmoic acid has been tπed with some success, particularly against comedones or blackheads, but this condition frequently returns when the treatment is withdrawn. (AU-trans-retinoic aαd is also known as trehnoin. These terms are used interchangeably throughout this specification.) Additionally, retmoic aαd applied topically can be highly irritating and its use can be painful for the patient depending on the concentration used and the frequency of application.
A number of side effects complicates the administration of large doses of vitamin A Among the many symptoms of hypervitarrunosis A are weight loss, desquamation of the skm, hair loss, irritation of the oral and pharyngeal mucosa, and nose bleeds, headaches, bone pain, hver toxiαty due to storage of vitamin A in the hver, papilledena, pseudotumor cerebπ, deπuneralization, and peπosteal thickemng of the bones. Because of these and other side effects of oral treatment with vitamin A and all-trans-re noic aαd, which produces similar side effects, they are rarely recommended for dermatopathic conditions.
Chrome sun exposure has been determined to create a number of skm disorders mcludmg skm cancer which is usually discernible by the presence of lesions known as keratoses as well as photoaging (or dermatohehosis) of the skm which is characterized by wrinkling, sallowness, roughness and mottled pigmentation. In an article entitled, Topical Tretmom Improves Photoaged Skm, JAMA 259, vol. 4, pgs. 527-532, Jan. 22/29, 1988, the authors Webb et al. report that photoaging of the skin of middle-aged and elderly Caucasians could be improved within a 16-week period by daily topical application of a cream containing 0.1 % tretinoin (all-trans-retinoic acid)
A side effect which complicates the administration of tretmom, is that the therapy is irritating to the skin and induces dermatitis of several weeks duration in almost all of the subjects undergomg the tretmom therapy. Redness, peeling, stinging, burning and dryness were consistently expeπenced by nearly all subjects. Eleven of fifteen subjects experienced dermatitis severe enough to require the use of topical steroids to control the dermatitis Three of fifteen withdrew from the tretmom therapy due to the severity of the tretinoin-induced dermatitis. Also effects on the histology of the epidermal and stratum corneum layers of the dorsal forearm skm were noted in the tretmom treated areas. Because of these side effects, recommendation for use of the therapy is inhibited and is not used to full advantage. A method of dermal therapy that would retain the effectiveness of tretmom but which would be essentially non-irπtatmg would provide a much needed solution to the treatment of photoagmg. Further, non-irπta ng effective treatment of other skm disorder such as skin cancer would meet a long felt need m dermal therapy.
The Handbook of Nonprescπption Drugs, 5* ed., 1977, A.P.A. pub , pp. 140, 319, 320, discloses the use of vitamin A and retinoic acid in the treatment of acne (unspecified). However, the disclosure of this publication is opposite to that of the subject mvention, m that it states, The systemic use of vitamin A for the treatment of acne, ... is not warranted by clinical evidence at p. 140; and that, Treatments that have been abandoned or have not been proved effective include oral vitamin A at p. 320.
J V. Straumford reported a systemic usage of large oral doses of retinol, the alcohol form of vitamin A, over a long period of time for the treatment of acne. (Straumford, J. V., Vitamin A: Its Effect on Acne, Northwest Med., 42: 219-255, August, 1943). These results, however, have been disputed and systemic therapy of acne utilizing retinol has been challenged by other investigators. (Anderson, J. A. D., et al., Vitamin A m Acne Vulgans, Bπt. Med. J., 2:294-296, August, 1963; Lynch, F. W., et al , Acne Vulgans Treated with Vitamin A, Arch. Derm. 55:355, 357, March, 1947; and Mitchell, G. H., et al, Results of Treatment of Acne Vulgans by Intramuscular Injections of Vitamin A, Arch. Derm. 64:428-434. October, 1951.) Topical administration of retinoic acid for the treatment of acne was reported by Khgman, et al , (Arch. Derm. 99:469-476, 1969, U.S. Pat. No. 3,729,568). The effectiveness of this treatment as disclosed by Khgman is often associated with a noticeable irritating effect of topically applied retinoic acid. Esters and amides of trans-retmoic acid which are useful for the treatment of acne are claimed m U.S. Pat. Nos. 4,055, 659 (all-trans-retinoyloxyacetamide), 4,126,697 (4-(all-trans- retmoyloxyacetyl)-catechol), 4,126,698 (2-hydroxyethyl all-trans-retinoate), and 4,304,787 (benzyl all- trans-retinoate). All four of these patents to Gander, et al. also disclose mixed 2-hydroxy-l-propyl and l-hydroxy-2-ρroρyl all-trans-retinoates, N-(3,4-methylene-dιoxyphenyl methyl) all-trans- retinamide, and 4-rutrobenzyl all-trans-retinoate. The effectiveness of all these compounds was shown through testing which measured mcrease m DNA synthesis in epidermal cells. This ability has been associated with the effectiveness of retinoic aαd m the treatment of acne. See, for example, Chnstophers and Braun-Falco, Stimulation of Epidermal DNA-Synthesis with Vitamm A-Acid, Arch. K n. Exp. Derm. 232: 427-433 (1968) and Wolfe, et al, Changes m Epidermal Differentiation After Vitamin A Acid, Arch. Khn. Exp. Derm. 237: 744-795 (1970). No claim is made and no testing is disclosed in the Gander, et al. patents which indicates that the esters or amides show fewer or greater side effects than trans-retmoic acid.
The process for treating acne vulgans topically utilizing retinal, the aldehyde form of vitamin A, is disclosed in U.S. Pat. No. 3,932,665. The aldehyde form, unhke the acid form of vitamm A, exerts its therapeutic effect without producmg imtation, inflammation, erythema, or peeling of the skin. This patent also discloses the topical use of 13-αs-retιnal m the treatment of acne vulgans.
The method of treating acne with C-20 and C-22 vinylogs of desmethyl retmoic acid is disclosed in U.S. Pat. No. 3,882,244. These vinylogs as disclosed m the patent are applied topically to the site of the acne infection as a solution, ointment, or powder. The treatment of acne vulgaris with retmoic aαd analogs, particularly ll-(2,6,6-tnmethylcyclohex-l-enyl-l)-5,9-dιmethylundeca- 2,4,6,8,10-pentenoιc aαd is disclosed m U.S. Pat. No. 3,934,028. This compound can be used either internally or topically. When taken orally, the daily dosage of this compound ranged from 30-300 mg taken over from 2 to 8 weeks. However, there is no indication that the compound leads to remission from the disease after administration of the compound is withdrawn. Other drugs presently used in the treatment of acne include benzoyl peroxide, tretinoin (aϋ- trans-retinoic acid, Retin-A Ortho), clindamyαn, tetracyline, erythromycin, minocycline, and estrogens (for females).
Benzoyl peroxide is considered safe and effective m mild and moderate acne treatment.
Tretmom is effective but has the previously mentioned deletenous side effects, as well as acceleratmg photocarcinogenesis. The antibiotics are reasonably effective but have side effects such as gastrointestinal problems including reports of pseudomembranous colitis. Estrogens are sometimes effective in treatmg acne, but the side effects of these drugs make them less than desirable.
The use of 13-cis-retinoic acid derivatives for the treatment of acne and other skin diseases is disclosed in U.S. Pat. No. 4,677,120 of Parish et al. All-trans analogs are disclosed in U.S. Pat. No. 4,885,311. The derivatives are claimed for use in either oral or topical treatment of the disease. These derivatives have been found to minimize the toxic side-effects associated with the use of all-trans and 13-cis-retinoic acid in the treatment of acne.
See also the following prior art cited in the record of our additional related prior patents, 4,677,120, 4,885,3 1, 4,994,491; 5,049,584; 5,124,356; and Re. 34,075:
U.S. PATENT DOCUMENTS
2,424,994 8/1947 Milas 260-410.9V 2,576,103 11/1951 Cawley et al. 260-410.9V 2,917,523 12/1959 Pommer et al. 260-410.9V 2,951,853 9/1960 Matsui 260-410.9V 3,287,382 11/1966 van Leeu en 260/410.9V 3,928,400 12/1975 Olson et al. 260/410.9V 3,931,257 01/1976 Pawson 260/408 3,984,544 10/1976 Casmer et al. 514-177 4,055,659 10/1977 Gander et al. 514-552 4,108,880 8/1978 Gander et al. 260-410 4,190,594 2/1980 Gander et al. 260-404 4,216,224 8/1980 Yu et al. 514-561 4,529,600 7/1985 Dawson et al. 514-529 4,677,120 6/1987 Parish et al. 514-549 4,877,805 10/1989 Khgman 514-381 4,885,311 12/1989 Khgman 514/549 4,888,342 12/1989 Khgman 514/419
NON-U.S. PATENT DOCUMENTS
106926 5/1984 EP
2050658 5/1972 DE
2081478 12/1971 FR
PROBLEMS IN THE ART
In general terms, there is a balancing of safety related issues against effectiveness of the retinoids. The most common indications for retinoids are not related to mortality or severe morbidity issues. Acne vulgans, wrinkles and skin photo-damage are largely cosmetic concerns. While the psychodynamics of such conditions and their effective treatment can be quite compelling, significant nsks of side effects are not justifiable and safer and more effective modalities of treatment are needed. Other indications for the use of retinoids in dermal therapeutic indications are more significant, and higher levels of adverse side effects are more justifiable, but even m those contexts, greater safety is needed.
While a vast array of modifications of the existing compounds have been mvestigated, there is a considerable need to provide greater safety at the existing levels of effectiveness or greater levels of effectiveness at the existing level of safety. It would, of course, be most beneficial to increase both safety and effectiveness of the retinoids
OBJECTS OF THE INVENTION
It is an object of the present invention to provide novel esters and amides of 9-αs-retιnoιc
It is another object of the present invention to provide dermal formulations of novel esters and amides of 9-cιs-retιnoιc acid.
Another object of the mvention is the treatment of a vanety of skin conditions and diseases with formulations of novel esters and amides of 9-αs-retιnoιc acid.
Still another object of the present mvention is to provide a method for altering the structure and appearanc e of the skin through the use of novel esters and amides of 9-αs-retιnoιc acid.
BRIEF SUMMARY OF THE INVENTION
In the present mvention, 9-cιs-retmoιc aαd and its esters and amides are synthesized and formulated for administration for the treatment of skm conditions and diseases without the mducement of dermatitis wherem there is apphed topically to the epidermis of the skin a non- untatmg retmoid compnsed of the esters and amides of 9-cιs-retιnoιc aαd, the retmoids havmg the formulae:
Figure imgf000008_0001
wherem R is o O O
II o o
II II
CR'"2 CR' — CR' '. CCHjOCK
- CR'
Figure imgf000009_0001
Figure imgf000009_0002
OCR'"
II o
wherem X is H, F, Cl, Br, • I, OH,
O O O II
OR, OR', • OCR', CR'. CH, CN,
Figure imgf000009_0003
wherein n is a number from 1 to 5; wherein R' is H or any of the lower alkyls ranging from Ci to Cβ; wherein R" is
O O
II II COR', CR', CR', and R'
wherein R"' is the hydrocarbon backbone of fatty aαds; wherein R"" is R" or the hydrocarbon backbone of fatty acids; wherem R""' is the lower alkyls rangmg from Ci to Cβ ; and further, when there are two or more R', R", R"', R"", or R""' groups attached to the same carbon, each R", R", R"', R"", or R""' group may be the same as or different from the other R', R", R"', R"", or R""' groups attached to that carbon.
DETAILED DESCRIPTION
This invention is directed to novel derivatives of 9-cis-retinoic acid which are useful in the treatment of acne and the like, but which minimize the irritating side-effects associated with 13-cis- retinoic acid and all-trans-retinoic acid and many of their esters and amides as used in treatments of acne and related dermal indications. The derivatives have the formulae:
Figure imgf000010_0001
wherein R is o O O
II O o o
II II
CR'"2 CR' — CR'"2 CCHjOCR'" II CR'"2 CNHR
-CR'
Figure imgf000010_0002
Figure imgf000010_0003
Figure imgf000010_0004
wherem X is
H, F, Cl, ' Br, ■ I, OH,
O O O II II II
OR, OR', - OCR', CR' CH, CN,
NO 2 NH, - NHR', NR'2 and ° κR
O R
wherem n is a number from 1 to 5; wherem R' is H or any of the lower alkyls ranging from Ci to Cβ; wherem R" is o o
II II COR', CR', CR', and R'
or R'; wherem R"' is the hydrocarbon backbone of fatty acids; wherem R"" is R" or the hydrocarbon backbone of fatty aαds; wherem R""' is the lower alkyls rangmg from Ci to Cβ; and further, when there are two or more R', R", R"', R"", or R""' groups attached to the same carbon, each R', R", R"', R"", or R""' group may be the same as or different from the other R', R", R"', R"", or R""' groups attached to that carbon.
The esters and amides of 9-αs-retmoιc are not per se known to the art. Illustrative compounds include: l-(9-αs-retιnoyloxy)-2-propanone, l-(9-αs-retmoyloxy)-3-decanoyloxy-2-propanone, l,3-bιs-(9-αs-retιnoyloxy)-2-propanone, l-(9-αs-retmoyloxy)-2-pmacolone,
2-(9-αs-retmoyloxy)-acetophenone,
9-αs-retιnoyloxy methyl 2,2-dιmethyl propanoate,
2-(9-αs-retmoyloxy)-n-methyl-acetamιde, l-(9-αs-rehnoyloxy)-3-hydroxy-2-propanone, l-(9-cιs-retιnoyloxy)-2,3-dιoleoylproρanone, and sucαmmidyl 9-αs-retmoate.
The structure of 9-αs-retmoιc aαd is shown m Formula 1:
Figure imgf000012_0001
9-cιs-rθtιπoιc acid Formula 1
For comparison, the structure of all-trans-ret oic acid and 13-αs-retιnoιc acid are shown in Formulae 2 and 3, respectively.
al rans-rβtinoic aαd
Figure imgf000012_0002
Formula 2
13-cιs retinoic acid
Figure imgf000012_0003
Formula 3
TOPICAL ASSAY
A topical assay to test for pseudocomedone (utnculus) reduction m the rhino mouse is conducted.
Each test compound and a vehicle control is applied topically to the dorsal trunk of the rhino mouse. The utnculus diameters are measured with a ocular micrometer. The assay is based upon the work of Khgman, et al (1979) and Van Scott (1972). Khgman, et al., The Effect on Rhino Mouse Skin of Agents which Influence Keratiruzation and Exfohation, J. Invest. Derm. 73: 354-358 (1979). Van Scott, Experimental Animal Integumental Models for Screenmg Potential Dermatologic Drugs, In Pharmacology of the Skm, eds. Montagna et al, New York, Appleton-Century-Crofts, 1972, pp. 523- 533. Mann, Hair Loss and Cyst Formation m Hairless and Rhino Mutant Mice, Anat. Rec. 170: 485- 500 (1971). Mezick, et al , Topical and Systemic Effects of Retmoids on Horn-Filled Utnculus Size m the Rhino Mouse. A Model to Quantify Antikeratiruzing Effects of Retmoids, J. Invest Derm. 83. 110-113 (1984). Mezick, et al, Anti-Acne Activity of Retmoids in the Rhino Mouse, In Models in Dermatology, eds. Maibach, et al , Basel, Karger, 1985.
The dorsal trunk of the rhino mouse is the test site. Each test compound is dissolved in alcohol.propylene glycol (70:30, v:v) or other suitable vehicle and topically applied (0.1 ml) to the dorsal trunk once daily, five consecutive days/week for two weeks. Also, administration may be oral (p.o.) in a suitable vehicle. Following treatment, the animals are sacrificed by cervical dislocation. The treated dorsal trunk skin is removed from the animal and placed mto 0.5% acetic acid for up to 18 hours at approximately 4 °C. After this, the epidermis with the acne cysts is separated from the underlying dermis. The sheets of epidermis are processed by rout e methods to permanent whole mounts for microscopic examination. Also, full-thickness samples may be taken, stamed (H&E), and examined by light microscopy.
The utnculus diameters are measured with an ocular micrometer to compare effects of test compounds to vehicle control and/or reference compound on cyst reduction. Light microscopy is used to determine effects on cell differentiation. The results are summanzed in Table 1.
TABLE 1
TOPICAL RHINO MOUSE ASSAY
RETINOID % CONCENTRATION UTRICULUS REDUCTION
Compound 1 0.1 +++
Compound 2 0.1 +++ all-trans-retmoic aαd 0.1 ++
All-trans-retinoic aαd is used as a control.
Compound 1 and Compound 2 have the formulas identified n Examples 1 and 2 below.
+=shght activity
++=moderate activity
+++=substantιal activity
From the results it can be seen that the compounds of the mvention are effective m topical applications. The data presented is raw data which does not take mto account the differences in molecular weight between the compounds of the mvention and all-trans-retinoic aαd.
The compounds of the present mvention are synthesized from 9-αs-retιnoιc aαd. The aαd is itself synthesized by the method of Boehm, et al., supra.
The preparation of the compounds of the present mvention is illustrated by the examples.
Melting points are determined on a Thomas-Hooever capillary pomt apparatus, 1H-NMR spectra are taken with a Vanan EM-360-A spectrometer, and elemental analysis is done by Atlantic Microlab, Inc., of Atlanta, Ga. These denvatives can be apphed topically or orally without causing imtation or with less imtation than found with state of the art retmoid based treatments, and are an effective and safe treatment for a wide diversity of dermal conditions, i.e., acne vulgans, cystic acne, hyperpigmentation, hypo-pigmentation, psonasis, dermal and epidermal hypoplasia and keratoses, the reduction of wrinkling of the skm as an incident of aging and actinic damage, normalization of the production of sebum, the reduction of enlarged pores, promotmg the rate of wound healing, limiting of scar tissue formation during healing and the like. They are additionally useful for treatment or amelioration of the same additional classes of skin disorders as is retinoic acid itself and other retmoids. These disorders mclude lchthyoses (e.g., ichthyosis hystnx, epidermolytic hyperkeratosis, and lamellar ichthyosis), folhcular disorders (e.g., pseudofolhcuhtes, senile comedones, nevus comidonicas, and tnchostatis spinulosa), benign epithelial tumors (e.g., flat warts, tnchoepithehoma, and molluscum contagiosum), perforated dematoses (e.g., elastosis perforans senpigmosa and Kyrles disease), and disorders of keratinization (e.g., Daners disease, keratoderma, hyperkeratosis plantaπs, pitynasis rubra pilaπs, lichen planus acanthosis nign ans, and psonasis) The esters and amides of 9-αs-retιnoιc acid are also effective for the non-irntating treatment of effects attributable to aging and particularly to photodamage and photoaging. The use of these compounds extends to non-untating treatments involving the retardaUon and reversal of additional dermal and cosmetic conditions which are amehorated by tret om such as the effacement of wrinkles, improvement in appearance, namely color and condition of the skm, spots caused from exposure to the sun as well as other skm disorders.
For many years the medical commumty has devoted a considerable amount of research time and effort to discovering new and better ways of promotmg healing of wounds. Of particular concern are wounds such as burns, ulcers and corneal wounds which are difficult to treat and have a long time for complete healing. Research efforts have been devoted to discovering new ways of creasing the rate of healing wounds to minimize the nsk of infection and reduce the painful penod of recovery. There is a need n the medical community for pharmaceutical compositions that can be easily apphed to wounds and mcrease their rate of healing.
It is known that retinoids can mcrease the rate of wound healing.
Retinoids have sometimes been defined narrowly as compnsing simply vitamin A (rebnol) and its denvatives such as vitamin A aldehyde (retmal), vitamm A aαd (retinoic aαd), compnsing the so-called natural retmoids. However, recently the retmoids have been defined as a much larger class of chemical compounds that have physiochemical similanties to vitamin A and its denvatives.
An example of a wound-heahng retmoid is all-trans retmoic acid which is also known as tretmom.
Tretmom showed a rate of healing of 6% when the skm of pigs was pre-treated with a 0.05% tretmom cream for 10 days pnor to partial-thickness skm wounding. Applymg tretmom after wounding had a deletenous effect on healing. Hung et al., Arch. Dermatol. 125:65-69 (1989) believe that this deletenous effect is due to the inflammation that is caused by tretinoin. Khgman, J. Am. Acad. Dermatol., 15779-85 (1986) studied hairless mice by irradiation with a sun lamp. The mice were then treated topically with vanous concentrations of tretmom for several weeks. The subepidermal repair zone in the treated mice was significantly wider than that in the untreated control group. The collagen was histochemically and ultrastructurally normal; fibroblasts were numerous and morphologically hyper-active.
Klein, Acta. Dermatovener (Stockholm) 74:171-74 (1975) found that the skm of rats pre- treated with 1 % tretmom for 3 days before and 0.1 % after the wound was made accelerated healing by 18% when compared to the control group. The skm excision included the panniculus cornices down to the fascia of the spinal musculature. Only 0.1% tretmom was used postoperatively to prevent a worsening of dermatitis which always occurred after the preliminary treatment with tretinoin.
Ubels et al., Am. J. Ophthalmol., 95:353-58 (1983) treated experimental corneal epithelial wounds in rabbits. Treatment with 0.1 % tretmom three times per day resulted in a 21 % mcrease in the healing rate compared to the control eyes. Treatment five times a day resulted in a 35% mcrease in the healing rate.
Hunt, J. Am. Acad. Dermatol. 15:817-21 (1986) reviewed vitamin A and wound heahng. He states that, although few comparative studies have been done, tretinom appears to bo the most wound-active retmoid.
The effectiveness of topical tretmom has been known for some time, but its chief drawback has been local primary irritation. It is the cutaneous safety of tretinom rather than the systemic toxicity potential that is the cause for concern (See Papa, C, Acta Dermatovener (Stockholm) 74:128- 32 (1975)). Advances m the development of newer dosage forms have increased the number of patients who can tolerate topical tretinom, but the dermatitis caused by tretinoin is still an unwanted side reaction. Weiss et al, JAMA 259:527-32 (1988), m their study on treatment, found that 92% of the patients expenenced dermatitis. To mitigate this inflammation, some patients required potent topical steroids (flucinonide or desoximatasome).
The venous ulcer is the most common leg ulcer. Lewis, Custis 44:123-24 (1989), states that venous ulcers are not a skin problem, but a problem of disordered circulation. From his clinical experience, the best treatment is the apphcation of external pressure to counteract the high pressure transmitted through mcompetent perforatmg veins from the deep venous system to the venules of the skin.
Rust et al, Brit. J. Dermatol. 120:101-105 (1989), found that a vasodilator, Ketanserin, healed the lesions in a patient with recalcitrant ulceration of both lower legs diagnosed as being due to live-diploid vascuhtis. As discussed above, tretinoin can accelerate skin healing, but what might be more important for some leg ulcers is Klingman's supra (1986) findings that topical tretinom augmented the repair of ultraviolet damaged tissue m the hairless mouse with new blood vessel formation. A retmoid may have utility in treating leg ulcers by stimulating new blood vessel formation and inducing skm growth.
Retmoids affect the differentiation, maintenance and proliferation of many cell types whether they are of ectodermal, endodermal or mesodermal ongin; or whether they are epithelial, fibroblastic or mesenchymal. For a review of pnor developments in retmoid therapy, see Pawson, A.A., et al, J.Med. Chem., 25:1269-1277 (1982). A more recent discussion of retmoids m research and clinical medicine can be found in the publication of a symposium held in Geneva: J. H. Saurat, Editor, Retmoids, New Trends m Research and Therapy , Karger publishing Co. (1985).
Several researchers have presented experimental results which suggest that retinoic aαd increases the mitogenic activity of epidermal growth factor and its b dmg to its cell surface receptors in vitro. For example, see Jeten, A.M., J. Cell Physiol., 110(3).235-40 (1982); Harper, R.A., et al, Endocrinology, 107(6):2113-2114 (1980); and Roberts, A.B., et al, Cancer Res., 44:1635-1641 (1984). One mvestigator has suggested that the effect of retmoids on the binding of EGF to its receptor is that the retmoids mcrease the number of EGF receptor sites and therefore enables the binding of a greater number of EGF molecules to the receptors. See, Jetten, A.M., Nature, 284:626-629 (1980); and Jetten, A.M., Fed. Proc, 43(1):134-139 (1984).
Brown et al , New Eng. J. Med., 321:76-79 (1989) demonstrated that the topical application of epidermal growth factor accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns. Paired donor sites were c reated in patients who required skin grafting either for burns or reconstructive surgery. One donor site from each patient was treated topically with silver sulfadiazine cream and one was treated topically with silver sulfadiazine containing epidermal growth factor. Total healing time m these 12 patients was 9-21 days with an average of 12 days. The healing time of the donor sites that received the epidermal growth factor was accelerated by an average 1.5 days. Hunt and La Van, New Eng. J Med., 321:111-112 (1989), commenting on the work of Brown et al, state that a 15% acceleration of the healing time of a patient with burns may save many days of pam or hospitahzation.
Sheffield, W. et al. in EPO 339,905-AZ, discloses a wound healing composition compnsed of at least one polypeptide growth factor havmg human metagenic or angiogenic activity at least one retmoid. The composition is reputed to have a synorgistic effect with respect to growth factors and retmoids alone.
However, growth factors exist as natural molecules on very small quantities and require expensive recombmant DNA technologies to produce quantities m pharmaceutically useful amounts. Although tretinom can be produced using suffiαently inexpensive technology to be commercially viable, it is limited by its local primary imtation The compositions of the present mvention may be topically apphed to the wound site in any suitable pharmaceutically acceptable vehicle, for example, a liquid earner such as propylene glycol ethanol, propylene glycol ethanol chloroform, and the like. A preferred hquid composition is a solution of a small amount of at least one of the compounds m combmation with from about 25 to about 75% by volume of 95% ethanol and from about 75 to about 25% by volume of hquid glycol. A typical solvent earner of this type compnses 75% by volume 95% ethyl alcohol and 30% by volume propylene glycol The preferred concentration of the active compound in these compositions is at least 0.01% by weight, most preferably from about 0.1 % to about 0.5% by weight and most preferably from about 0.05% to about 0.2% by weight, but any therapeutically effective concentration may be used.
The compositions of the present mvention may also be formulated in any number of other ways, depending on whether an aqueous solution, cream or ointment is desired and whether it would be used/and its site of use set as on the surface of the skin or in the eye.
Compositions formulated as a cream may contain a cream stabilizer such an xanthen gum, an emulsifier preferably a non-iomc emulsifier, at least one hquid and one sohd hydrophobic matenal selected from the hquid and sohd fatty aαds, fatty alcohols, fatty aαd esters, pharmaceutical grades of waxes and hydrocarbons, the latter rangmg from liquids through semi- hquids such as petrolatum, to sohds and the likes, preservative, an antioxidant, and water
The compositions of the present mvention are useful m eye drop formulations, eye gels, eye creams, lyposome or micelle formulations, acquest vehicles for soaking soaked gauze dressmgs, burn dressmgs, artifiαal skins, sutures and staple coatmgs, ointments, lotions or creams, gel formulation, foams and the like. Additional matenals such as buffers, preservatives, adjusting agents, antioxidants, polymers for adjusting viscosity or for use as extenders and exαpients may be used m the compositions. Methods for mcreasmg the rate of healing a wound compnses applymg or contacting the compositions of the present mvention directly to the wound. The composition is permitted to remain in contact with the wound for a penod of time sufficient to mcrease the rate of cell growth at the wound site. Such methods mclude incorporating any composition of the present mvention mto a cream formulation or soaking a gauze dressing with an acquest solution of the composition and then applymg the cream or soaked gauze to a wound site such as a burn, donor site wound, ulcer or any type of cutaneous wound. Additionally, sutures or staples may be coated or soaked with the acquest composition and used to close an open wound.
The type of wounds that may be healed usmg the composition of the present invention are those which result from any medical or acαdental injury which causes epithelial damage such asophthalmic wounds, such as those which result from corneal ulcers, cutaneous wounds, such as burn wounds, donor site wounds from skm transplants and ulcers. Additionally, dermatological c onditions in which the skm has been damaged may be treated with the compositions of the present mvention. Leg and foot ulcers may also be treated with compositions of the present mvention. Any wound that does not result in total skm loss but retains a portion of the dermis may be treated using the compositions of the present mvention.
Trie causes and mechanisms of psonasis and psonahc conditions are not fully known. The disease is genetic, and has been assoαated with increased levels of certain histocompatibilitv antigens. It has been reported that persons with elevated HLA-Cw6 are 9 to 15 times more likely to develop psonasis than others. No cure is known, and persons with the disease expenence lifelong, penodic eruptions of scaly plaques, papules and, in some persons, pustules, which may appear on any dermal surface. The disease may be exacerbated by some important drugs, including lithium, Beta-blockers, and antimalenals. Treatment with systemic steroids provide rapid clearing of psoriasis, but often cause a worsenmg manifestation of the condition when the medication is withdrawn, m a rebound phenomenon, which has led to the abandonment of corticostenods as a routme treatment.
For many individuals, the clinical manifestations are associated with emotional components, which often result m attempts at concealment, self-consciousness and the avoidance of diagnosis and treatment.
By vanous estimates, psonasis occurs m from 1 to 3 percent of the population worldwide. In the United States, it has been estimated that five hundred to six hundred thousand patients consult physiαans each year for psonatic conditions.
Clinically diagnosed psonasis is treated with a vanety of procedures and agents. Most commonly, topical applications of steroids, anthralm, coal tar formulations, intralesional injections of steroids, occlusive dressmgs employed with topical formulations, and the administration of ultraviolet hght (both UVB and, less commonly, UVA are employed m combination with anthral , coal tar formulations, and photosensitizers, such as psoralens) are employed.
There are a vanety of systemic treatments employed as well, including the administration of methotrexate, hydrea, etretinate and cyclosporm.
Typically, the treatment of psonasis involves a balancing of short term palliation and limited benefits of the more benign treatments agamst the greater potency and more senous side effects and consequences of the more efficacious systemic treatments.
Ultraviolet hght treatments are generally effective and mvolve a minimum of side effects, but require a large number of office visits; unsupervised self-admmistration of UVB and UVA with photosensitizers is extremely unsafe and is not prescnbed.
As with all antipsonatics, the mode of action is not certainly known, but is believed be through the binding of one or more enzymes, or through the inhibition of bmdmg of one or more enzymes (competitive bmdmg). A vanety of enzyme mediated pathways are under investigation as the basis for psonasis. Thus far, the evidence m the art is suggestive, but not fully probative, of several mechanisms, as discussed infra.
As m the case of other therapeutic modalities, the mode of action of the compounds of the present mvention is not known. The compounds of the present mvention are beheved to be potent inhibitors of hpoxidase,
Each of the compounds synthesized m the present mvention has been screened for topical activity. While the data obtained are set out m detail below, m general terms, all the claims evaluated as having substantial topical activity, at least comparable to the best results observed with the systemic therapies of the pnor art.
There are two animal models in common use m the art at the present tune. These are the ustomary mouse ear edema test and the less common hamster model
The mouse ear edema model is based on the induction of edema by the topical administration of arachadonic acid to the dermis of the ear of the specimen. A more recently developed model, which has achieved less acceptance as a predictive screen in the evaluation of potential antipsonatic agents, is the hamster model. The procedure of the hamster model is discussed m detail, infra.
All the compounds synthesized m the present mvention have been evaluated m the mouse ear edema model, and have shown substantial levels of activity. No adverse reactions have been observed.
A few of the compounds of the present invention have also been evaluated m the hamster model. The activity has been confirmatory of that observed m the mouse ear edema screen. Again, no toxic or adverse effects have been observed.
The compounds of the present mvention are readily formulated with conventional pharmaceutical earners, and may be convemently administered by any convement route, mcludmg un, lv, lp, subcutaneous and intralesional (local) injections, oral administration, and topical (dermal) application, with or without occlusion.
Administration in known aiumal models commonly recognized m the art as reasonably predictive of antipsonatic activity have shown potent anti-inflammatory action The most widely recognized and employed antipsonatic screen, the mouse ear edema test, described above, shows activity which equals or exceeds that of most other therapeutic modahties which can be screened in this model.
At the same time, no adverse side reactions, toxiαties or adverse indications have been observed to date. LD50 values by oral administration in rats illustrates that the compounds of the present mvention do not represent a substantial threat of acute toxiαty.
Thus m practicing the treatment of skin m accordance with the practice of the present invention, the esters and amides of 9-αs retmoic aαd are topically apphed to the skm site exhibiting charactenstics to be treated m any suitable pharmaceutically-acceptable vehicle, as for example, a hquid earner such as propylene glycol-ethanol. A prefened hquid composition is a solution of a small amount of at least one of the compounds of the mvention m a combination of
(A) from about 25% to about 75% by volume of 95% ethanol and
(B) from about 75% to 25% by volume of a hquid glycol. A typical solvent earner of this type compnses 70% by volume 95% ethyl alcohol and 30% by volume propylene glycol. A small but effective amount of an antioxidant such as butylated hydroxytoluene may also be included m the composition. A typical solvent carrier of this type compnses 70% by volume 95% ethyl alcohol and 30% by volume propylene glycol. An antioxidant at a concentration of 0.01 to about 0.1 % by weight may be incorporated m the earner. The preferred concentration of the active compound m these compositions is at least about
0 01 % by weight, more preferably from about 0.01% to about 0.5% by weight and most preferably from about 0.05% to about 0.2% by weight, but any therapeutically effective concentration may be used. Concentrations less than 2.5% by weight will normally be employed.
Such topical formulations will generally be made up to contam from about 0.025 to about 2.5 weight percent of the active 9-αs-retιnoιd, preferably from about 0.05 to 0.5 weight percent, although m most circumstances the concentration is not narrowly cntical. The compounds are both highly potent and highly safe, and an exceptionally wide range of concentrations may be employed as indicated.
It is also possible, although generally less preferred to administer the retmoids of the present mvention orally, or even parenterally, subcutaneously or, if desired, mtravenously. Such systemic administration produces systemic effects which are not generally preferred, as higher dosages may be required to achieve the required therapeutic levels to produce the desired action. When admmistered systemically, the concentrations to be employed may range widely, from 0.5 to 50 milligrams per kilogram of body weight. The compounds are sensitive to ultraviolet hght, and are oxidized when exposed to air and heat or other oxidizing conditions.
Like most retmoids, the compounds of the present mvention are highly hydrophobic in character, and formulation of the compounds requires that these charactenstics be taken mto account. A further advantage of the compounds is their non-irntating charactenstic when apphed topically. This highly desirable charactenstic is not seen when all-trans-retinoic aαd is used.
The 9-αs-retιnoιc acid on which the present mvention is based is per se known. Boehm, et al, J Med Chem., Vol. 37, No 3, pp. 408-414 (1994). Boehm et al demonstrate that 9-cιs-retιnoιc aαd is more effiαent at binding the two known sub-famihes of lntracellular receptors and each of the three defined sub-types within each of the subfamilies, and predict that 9-αs-retιnoιc acid will provide improved therapeutic indices when compared to other retmoic acids (i.e., all-trans-retinoic aαd and 13-αs-retιnoιc aαd).
Competitive binding studies show 9-cιs-retιnoιc acid has the lowest equihbnum constants of all retmoids for all six subfamilies of retmoid receptors
Boehm et al hypothesize that 9-cιs-retmoιc aαd is an endogenous isomer of the retmoid family of biologically active retmoids occurring in vivo
The activation of the retmoid receptors has been associated with induced or accelerated mitosis of normal skm cells, and it is the induced cell growth, particularly epithehal cell growth, which is most often associated with the improved skm properties occurring with use of retmoids to treat the skm
Abnormal skm cells, i.e., those charactenzing photodamaged or age damaged skm, neoplasia and keratoses, cells m hyperpigmented skm regions, psonatic skm cells, and the like, have been associated with defective or compromised retmoid receptors. It is thus hypothesized that treatment of the skin with retmoids functions to stimulate mitosis and proliferation of normal cells, while the abnormal cells are not stimulated and do not partiαpate m the increased levels of mitosis. As the process of stimulated mitosis continues over time, accompanied by the normal death and sloughing off of cells at the surface of the skin, the abnormal cells associated with the foregoing conditions are preferentially replaced by normal cells. The enhanced bmdmg of all the types of receptors of the 9-αs-retιnoιc acid is thus a direct basis for predicting a high therapeutic mdex compared to other retmoid acids.
Along with the heightened binding activity of the 9-cιs-retιnoιc aαd comes an equally mflammatory and irritating qualities of retinoids. Thus, by the data shown by Boehm et al , increased potency is expected to be accompanied by comparably or equally heightened side effects which limit the safety of the retmoids. The prediction of a higher therapeutic mdex is also a prediction of higher safety problems.
We have found that esters and amides of the 9-αs-retιnoιc aαd do not show any detectable imtation or inflammation of the skm among users. The activity of the esters and amides is, however, comparable in magnitude with the 9-cιs-retιnoιc aαd itself and substantially increased m comparison with all-trans-retmoic aαd and 13-cιs-retιnoιc acid. We achieve increased effectiveness, similar to that projected by Boehm, et al. for the 9-αs-retιnoιc acid, but without the conelative mcrease m hazards. As a consequence, a greatly improved therapeutic profile is achieved without safety problems.
EXAMPLES rj EXAMPLE 1
SYNTHESIS OF COMPOUND 1: l-(9-CIS-RETINOYLOXY)-2-PINACOLONE
Into a 100 ml round bottom flask is added 1.0 g (0.0033 moles) 9-c ls-retmoic acid, 25 ml of anhydrous methanol, and 0.2 g (0.0035 moles) of KOH. Trie solution is stirred at room temperature 0 until the 9-αs-retιnoιc aαd dissolves. After the solvent is removed under vacuum, 25 ml of acetonitnle is added and the solution is again concentrated to a semisohd under vacuum. Chloroacetone, (2.0 g, 0.032 moles), 0 1 g 18-crown-6 (0.00038 mole), and 100 ml of acetonitnle are added. The solution is stirred for 24 hours at room temperature with a magnetic stirrer. The sample is concentrated lo about 5 ml and chromatographed on a neutral aluminum oxide (Aldrich #19, 997- 4) column (14X1.8 cm). The alumina is deactivated with 20 ml of water per 1.0 kg of alumina.
The sample is eluted stepwise with 100 ml of 20% dichloromethane in hexane, 100 ml of 50% dichloromethane m hexane, and finally with 250 ml of dichloromethane. The sample elutes quickly and the vast majonty of the impunties remain on the column. Fractions of 25 ml are collected and evaluated by thin layer chromatography (TLC) on silic gel (EM Reagents #5775) develop with ethyl 0 acetate:heptane (1:3). The fractions containmg the product are combmed and concentrated to give an orange oil which solidifies on cooling to give 0.55 g of sohd.
Tnturating the sample with 10 ml of cold 95% ethanol produces a sharp melt g pomt.
TLC on silica gel (EM Reagents #5735) develop with 1.3 ethyl acetate:heptane shows one spot, Rf =0.41. TLC on alummum oxide (EM Reagents #5581) develop with 1:3 ethyl acetate:heptane 5 shows one spot, Rf =0.73.
The NMR (CDC13) spectrum of Compound 1 is identical to the spectrum of 9-αs-retmoιc aαd except for two additional peaks and the lack of a carboxyhc acid peak. The two additional peaks are (singlet, 2 protons, --OCH2 CO--) and (singlet, 3 protonsCOCH3). The structure is confirmed by NMR. 0 Elemental analysis for the compound gives a theoretical value for C23 H32 Ch of 77.49% C,
9.05% H; the found values are 77.52% C and 9.17% H. EXAMPLE 2
SYNTHESIS OF COMPOUND 2: 2-(9-CIS-RETINOYLOXY)-4'-METHOXYACETOPHENONE 5 The procedure used in Example 1 is followed with minor modifications. The reaction is earned out in a 250 ml round bottomed flask with 1.0 g of 9-αs-retιnoιc aαd and a 20% molar excess of 2-chloro-4-methoxyacetophenone. (2-chloro-4-methoxyacetophenone is prepared from the Fnedel-Crafts aeylation of anisole with chloroacetic anhydnde.) After completion of the reaction, the product is isolated by column chromatography under the same conditions as in Example 1 except that a larger column (11 cmX4 cm diameter) is used. The product at this pomt, however, contains unreacted 2-chloro-4-methoxyacetophenone. A homogeneous product is obtained by recrystal zation form 100 ml of 95% ethanol to give 0.88 g of a yellow sohd.
TLC on sihca gel (EM Reagents #5735) develop with 1:3 ethyl acetate:heptane shows one spot, Rf =0.45. TLC on aluminum oxide (EM Reagents #5581) develop with 1:3 ethyl acetate:heptane shows one spot, Rf =0.69.
The NMR (CDC13) spectrum of Compound 2 is identical to the spectrum of 9-αs-retιnoιc acid except for three additional peaks and the lack of a carboxyhc acid peak. The structure is confirmed by NMR.
Elemental analysis for the compound gives a theoretical value for C29 H3β O4 of 77.64% C, 8.09% H; the found values are 77.58% C and 8.10% H. EXAMPLE 3
SYNTHESIS OF COMPOUND 3 l-(9-CIS-RETINOYLOXY)-3-DECANOYLYOXY-2-PROPANONE
The following synthetic scheme is used for the synthesis of Compound 3. Into a 500 ml round bottom flask fitted with a reflux condenser and magnetic stirrer is added 20 g (0.22 mole) of dihydroxyaeetone dimer, 300 ml of acetone, 30 ml of DMF, and 30 ml of pyndine and 14 g (0.078 mole) of decanoyl chlonde. The dihydroxyaeetone dimer dissolved as the acid chlonde is added. The solution is refluxed for 30 mm. and then stirred for 1 hr. The reaction is poured mto 2 1 of cracked ice and allowed to stand for 2 hrs. as the product crystallized. The sohd is collected, dissolved m dichloromethane and dried (Na2S04). The sohd contamed large quantities of water which had to be removed. The dichloromethane is removed under vacuum and the oil is dissolved m acetone and placed m the freezer (-10 °C) overnight. The disubstituted dihydroxyaeetone impunty separated and is removed. Suffiαent water is added to make the solution about 25% water by volume and after standing overnight m the freezer, the product separated and is recrystalhzed from dichloromethane/hexane to give 5.2 g, mp 91-93 °C
TLC on sihca gel developed with 4/1 heptane/ethyl acetate, visualized with iodine, showed the product to be homogeneous. NMR (CDC13/DMSO-D6) showed the conect ratio forOCH2COCH2 — protons to C9H9 protons. The NMR spectrum is complex as is the NMR spectrum of dihydroacetone. Into a 100 ml round bottom flask equipped with a reflux condenser and a magnetic stirrer is added 2.5 g (0.01 mole) of the above alcohol and 15 ml of thionyl chlonde. The solution is refluxed for 70 mrn and the unreacted thionyl chlonde is removed under vacuum. Toluene, 25 ml, is added and removed under vacuum to remove the last traces of thionyl chlonde. The remaimng oil solidified on cooling and is recrystalhzed from 15 ml of hexane to give 1.45 g (54% yield) mp 40-41 rC. The structure is confirmed by NMR. The coupling of the alkyl chlonde to 9-αs-retιnoιc aαd is earned out as described for
Compound 1. Us g equal molar concentration of the above chloro compound and 9-cιs-retιnoιc aαd a yield of 41% can be obtamed TLC indicates the sample to be homogenous. Us g the same conditions as descnbed for Compound 1, on sihca gel
Figure imgf000024_0001
and on aluminum oxide Rf=0.37. The NMR spectrum of the product is essentially the combmed spectra of the two reagents, 9-αs-retmoιc acid and the substituted chloromethyl ketone, which are coupled to give Compound 2. There is one mmor change, however. The peak for (COCH Cl) disappears and the methylene hydrogens (ofCOCH2θ) had shifted where 2 singlets appeared and are separated by about 0.02 ppm (4 protons, -CO2CH2COCH2OCO--). The structure is confirmed by NMR. EXAMPLE 4 SYNTHESIS OF COMPOUND 4 l BIS-(9-CIS-RETINOYLOXY)-2-PROPANONE
The procedure for Compound 1 is used for Compound 4 with minor changes. Instead of chloroacetone, 1,3-dιchloroacetone (0.2 g, 0.0016 mole) is used. The solution is stirred for 24 hrs. before chromatography. The reaction is worked up as described for Compound 1 to give 1.41 g (38% yield). After tnturatmg with 2X2 ml hexane the meltmg pomt mcreased and sharpened. TLC indicated sample to be homogenous. Usmg the same conditions as described for Compound 1, on sihca gel Rf=0.67 and on aluminum oxide Rf=0.42. NMR (CDC13) spectrum of Compound 3 is identical to the spectrum of 9-cιs-retιnoιc acid except for the additional peak (smglet, 2 protons, — OCH2CO— ). The structure is confirmed by NMR. EXAMPLE 5
SYNTHESIS OF COMPOUND 5 9-CIS-RETINOYLOXY METHYL PHENYL KETONE
The procedure for Compound 1 is used for Compound 5 with minor changes. Instead of chloroacetone, alpha-chloroacetophenone is used. The structure is confirmed by NMR. Compound 5 is similar to Compound 1, except that the terminal methyl group has been replaced with a phenyl group. This will allow molecular modification by phenyl group substitution in order to spread the physicochemical properties of the denvatives for Quantitative Structure- Activity Relationship (QSAR) studies (Purcell et al, Strategy of Drug Design: A Molecular Guide to Biological Activity, Wiley, New York, 1973). EXAMPLE 6
SYNTHESIS OF COMPOUND 6 9-CIS-RETINOYLOXYMETHYL 2,2-DIMETHYLPROPANOATE
The procedure for Compound 1 is used for Compound 6 with minor changes. Instead of chloroacetone, chloromethyl pivilate is used. The structure is confirmed by NMR. Compound 6 uses the same ester denvative as the antibiotic prodrug pivampiαlhn m which the acyloxy-methyl ester is hydrolyzed by non-specific esterases to generate ampicilhn (Sinkula, Apphcation of the Pro-Drug Approach to Antibiotics, m Pro-drugs as Novel Drug Delivery Systems, ACS Symposium Senes (1974), p. 116-153). EXAMPLE 7
SYNTHESIS OF COMPOUND 7 2-(9-CIS-RETINOYLOXY)-N-METHYL-ACETAMIDE
The procedure for Compound 1 is used for Compound 7 with minor changes. Instead of chloroacetone, N-methyl chloroacetamide is used. The structure is confirmed by NMR. Compound 7 is designed to explore hydrophihαty: the N-methyl acetamide group is very hvdrophihc (Wolfenden, Waterlogged Molecules, Sαence, 222. 1087-1093 (1983)). EXAMPLE 8
SYNTHESIS OF COMPOUND 8 l-(9-CIS-RETINOYLOXY)-3-HYDROXY-2-PROPANONE Compound 8 is prepared by reacting the aαd chlonde of 9-cιs-retιnoιc acid with an excess of dihydroxyaeetone. State of the art synthetic schemes are available (Haslam, Recent Developments m Methods for the Estenfication and Protection of the Carboxyl Group, Tetrahedron, 36: 2409-2433 (1980)) and can also be used as an alternative. The structure is confirmed by NMR.
Compound 8 is similar to Compound 1 with a terminal methyl hydroxyl group instead of a methyl group. This modification should make Compound 8 more glycende-hke and more hvdrophihc. EXAMPLE 9
SYNTHESIS OF COMPOUND 9 l-(9-CIS-RETINOYLOXY)-2,3-DIOLEOYLPROPANE Compound 9 is prepared by reacting oleoyl chlonde with glyceraldehyde followed by reduction of the aldehyde group with sodium borohydnde. This gives a 1,2-dιsubstιtuted glycende with an available hydroxyl for coupling. The coupling of 9-αs-retmoιc aαd is earned out as for Compound 8. This scheme is based on the synthetic procedure for glycende denvatives of aspirin (Paris, et al , Glycendes as Prodrugs. 2. l,3-Dιalkanoyl-2-(2-methyl-4-oxo-l,3-benzodιoxan-2-yl) glycendes (Cychc Aspirin Tnglycendes) as Antiinflammatory Agents, J. Med Chem., 23: 79-82 (1980)). The structure is confirmed by NMR. Compound 9 is based on evidence that topical application of glycendes is an effective means of incorporating essential fatty aαds mto the skm (Prottery, et al., The Repair of Impaired Epidermal Barner Function m Rats by the Cutaneous Application of Linoleic Aαd, Bntish J. of Derm., 94: 13-21, (1976)). EXAMPLE 10
SYNTHESIS OF COMPOUND 10 SUCCINIMIDYL 9-CIS-RETINOATE
Compound 10 is synthesized by coupling 9-αs-retιnoιc acid with N-hydroxysucαnimide (Zimmerman, et al, The Effect of Active Ester Components on Racemization m the Synthesis of Reptiles by the Dicyclohexyl-carbodiimide Method, J. Am. Chem. Soc, 89: 7151-7152 (1967)). The structure is confirmed by NMR.
Compound 10 is the most easdy hydrolyzed prodrug. The N-hydroxy-succinimide group gives an activated ester linkage and is used in peptide synthesis because of this property EXAMPLE 11 TOPICAL ASSAY FOR THE INHIBITION OF SKIN CANCERS
The usefulness of the retmoid compounds of the present mvention for the inhibition of skm cancers is demonstrated by testmg in the ornithine decarboxylase (ODC) assay an ester Compound 1, l-(9-αs-retιnoyloxy)-2-ριnacolone and Compound 2, 2-(9-αs-retιnoyloxy)-4- methoxyaeetophenone). The ODC/Retinoid Bioassay is based on the method of Verma, A. K. and Boutwell, R. K.,
Cancer Res. (1977) 37.2196-2201. The ODC assay measures a compounds effect on the prevention of the induction of ODC, namely the effect of the retmoid compound on the inhibition of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) induced ODC activity. The assay is earned out usmg CD-I mice (aged 7 to 9 weeks) The dorsal hair of the mice is shaved 3-4 days before testing. Four mice are used for each pomt. The test retmoids, at one of two dose levels (1.7 and 17 nmoles) dissolved in 0.2 ml of acetone is apphed topically to the back of each shaved mouse. A smgle dose of TPA (17 nM) is apphed to the back of each treated mouse 30 minutes later. Control groups are treated with either acetone alone, TPA, or tretmom. The mice are sacrificed by cervical dislocation 5 hours after TPA treatment. The dorsal skm encompassing the shaved and TPA exposed area is exαsed and placed in a
100 ml beaker containing distilled water maintained at 51 °C - 57 °C. The skm is soaked for 50-70 seconds at this temperature with intermittent stirring. The skm is placed epidermis side up in a chilled (0 ° -5 °C.) stainless steel plate and the epidermal layer is scraped off with a razor blade. The epidermal layers from the 4 mice are pooled and placed in a homogemzahon tube with 2 ml of ODA buffer (10 nM tns-HCl with 0.050 nM pyndoxal phosphate, 0.050 nM ethylenediammetetraacetic aαd (EDTA), 1 mM dithiothreitol, pH 7.5). The pooled epidermal layers are homogenized for 15 seconds at 0 °C. usmg a Polytron homogemzer at a setting of 7.5. The homogenate is centnfuged at 30,000 x g and the supernatant fraction is pipetted mto a storage tube and frozen for about 72 hours.
The homogenate is assayed for ODC activity as descnbed by Verma and Boutwell to measure the release of 14Cθ2 from labeled DL(1-14C) oπuthine. Incubations are carried out m disposable centrifuge tubes with center well holders containing filter paper impregnated with sodium hydroxide to absorb 14Cθ2. The mcubation mixture consisted of 90 ml of L-orruthine, 350 ml of ODC buffer, 100 ml of 14C-ornιthιne (1.32 nm, Sp. Act:4.4 pCi/pM) and 10 ml of test sample. After mcubation at 37 °C. for 45 minutes, 0.5 ml of 2M chilled αtnc acid (4 °C.) is added and incubation is contmue for an additional 30 minutes to msure complete absorption of 14Cθ2. The filter paper is removed from the center well holders and set m 1 ml of water m capped scintillation vials for at least 1 hour before addmg RBI 3820 scintillation cocktail. Radioactivity is measured in a Tri Carb Scintillation Counter. Results are expressed as pmol of 14Cθ2 released m 30 mmutes per milligram of protem based on the specific activity of DL-14C-ornιthιne. The results are expressed m Table 2 below as the % reduction in ODC activity as compared to the control, for the compounds of each of the preceding Examples:
TABLE 2
Compound Concentration (nM) ODC Activity (nM 14C02 /30 m/mg Protem) Reduction
1 17.0 +++
1 1.7 +++
2 17.0 +++
2 1.7 ++
3 1.7 +++
4 1 7 +++
5 1.7 ++
6 1.7 +++
8 1.7 +++
9 1.7 ++
10 1.7 +++
Acetone 0.0 NA*
TPA 17.0 0
Tretinom 17.0 +++
*NA=not apphcable +=shght activity ++=moderate activity +++=substantιal activity The results recorded in the Table mdicate that the ret oid compounds of the present mvention possess biological activity that inhibits TPA mduced ODC activity rendering these compounds useful for treatmg malignant skin disorders. EXAMPLE 12
TOPICAL ASSAY FOR THE INHIBITION OF SKIN CANCERS
Another topical assay is conducted following the procedure descnbed above. All-trans- retmoic acid is used as a control. Compounds 1, 2, 5, 6, and 10 are tested The results are summanzed m Table 3:
TABLE 3
TOPICAL RHINO MOUSE ASSAY
RETINOID CONCENTRATION UTRICULUS REDUCTION Compound 1 0.1 +++ Compound 2 0.1 +++ Compound 5 0.1 ++ Compound 6 0.1 +++ Compound 10 0.1 ++ all-trans-retinoic acid 0.1 ++
+=shght activity ++=moderate activity +++=substantιal activity EXAMPLE 13
ORAL ASSAY
An oral assay for hamster sebaceous gland reduction is conducted.
Hamsters are dosed orally with the test compound and vehicle alone. The reduction in sebaceous gland size is estimated microscopically in relation to the control. The assay is unique and propnetary to Ortho Pharmaceutical Corp. and is based upon the work of Plewig et al (1977) and Gomex et al. (1980). Plewig, et al, Hamster Ear Model for Sebaceous Glands, J. Invest. Derm. 68: 171- 176 (1977). Gomez, et al, Effect of 13-cιs-Retιnoιc Aαd on the Hamster Flank Organ, J. Invest. Derm. 74: 392-397 (1980).
For oral (p.o.) studies, male Synan golden hamsters are dosed at 5 ml/kg, once daily, five consecutive days/week for two or three weeks. Modified but similar dosmg schedules could be used. Control hamsters are dosed with vehicle alone at 5 ml/kg. Following final treatment (up to 72 hours), the hamsters are sacrificed m a CO2 atmosphere. Samples of each test site are placed mto 10% buffered formalin and histologicaϋy prepared. Senal sections of each sample are stained (H&E) and examined microscopically.
The reduction m sebaceous gland size is estimated microscopically in relation to the control treated sites To quantify sebaceous gland size, cross-sectional areas are measured with an image analyzer system. The results are summarized m Table 4:
TABLE 4
SEBACEOU S GLAND ASSAY
RETINOID DOSE (mg/kg) SEBACEOUS GLAND
REDUCTION
Compound 1 32 +++ Compound 2 32 +++ Compound 3 32 +++ 13-αs-retιnoιc aαd 32 ++ all-trans-retinoic acid 32 +++
+=shght activity ++=moderate activity
+++=substantιal activity
From the results it can be seen that the compounds of the mvention are as effective as 13-αs- retinoic acid m both topical and oral applications The data presented is raw data which does not take mto account the differences in molecular weight between the compounds of the mvention and 13-αs- or all-trans-retinoic acid. If one does take this mto account, as must be done to accurately compare the activity of the vanous compounds, it can be seen that many of the compounds of this mvention are equally or more effective than either 13-αs- or all-trans-retinoic acid. A further advantage of the compounds of the invention over 13-cιs-retιnoιc aαd, is their non-irntating charactenstics when apphed topically. This highly desirable charactenstic is not seen when 13-αs- retmoic acid is used. EXAMPLE 14
TOPICAL ASSAY FOR PRIMARY ACUTE DERMAL IRRITATION
Compound 2 of Example 2, 2-(9-αs-retιnoyloxy)-4-methoxy-acetophenone is evaluated for its potential to produce primary dermal imtation after a single topical application to the skin tissue of rabbits.
Twelve healthy, young, adult, female New Zealand White rabbits (Orycetolagus cuniculus), are used m the study. The animals are purchased from a registered commercial breedmg laboratory.
At the start of the study, the animals are in the weight range between 2.0 and 3.0 kilograms, and are approximately 11 weeks of age. Animals selected for the test are not subjected to any previous experimental procedures, and their skm is free from imtation, trauma and disease.
A dose of 0.5 ml of a test solution composed of 0.025 g of 2~(9-cis-retmoyloxy)-4- methoxyacetophenone in a hquid solution composed of 75 ml of ethyl alcohol, 25 ml of propylene glycol 400, and 0.025 g by weight of butylated hydroxytoluene is apphed to one mtact and one abraded skm site per animal. Six animals are treated m this manner.
A control group of six animals is treated m an identical manner except that 2-(9-αs- retιnoyloxy)-4-methoxyacetophenone is absent from the control solution.
The apphcation sites are prepared by clipping the skm of the trunk free of hair approximately 24 hours before apphcation of the dose. One apphcation site on each animal is abraded by making minor incisions through the stratum corneum, but not sufficient to disturb the derma (that is, not suffiαently deep to produce bleedmg). The second apphcation site is mtact skm.
The dose is apphed to a small area (approximately 6 cm2) of sk n and covered with a gauze patch which is held m place with Vetrap bandaging. The patches are apphed to one mtact site and one abraded site per animal. The test substance is kept in contact with the skin for 24 hours. The skm is not nnsed following the 24 hour exposure penod.
Animals are observed for signs of erythma and edema 24 and 72 hours after apphcation of the test matenal. Observations are scored accordmg to the Draize Scale for Scoring Skm Reactions as m Draize, J. H., Dermal Toxicity, Appraisal of the Safety of Chemicals m Foods, Drugs and Cosmetics Dermal Toxicity, pp. 46-59, Association of Food and Drug Officials of the U.S., Topeka, Kans., 1965. Observations at the different scheduled times indicates that no signs of erythema or edema formation are evident in any of the 12 test animals at any observation time penod. Animals are weighed at the beginning and at the end of the observation penod. All 12 animals exhibited a gam m body weight. No overt signs of toxiαty are evident during the course of the study. EXAMPLE 15
TOPICAL ASSAY FOR COMPARATIVE DERMAL IRRITATION
2-(9-cιs-retιnoyloxy)-4-methoxyacetophenone is evaluated m a study of its potential to produce dermal imtation. Comparisons are made of tretmom, isotretmom, 2-(9-αs-retιnoyloxy)-4- methoxyacetophenone, and the vehicle solution. In the first test, four solutions are used. The control consists of vehicle solution, namely a solution of 60% by volume ethanol and 40% by volume polyethylene glycol. The other three solutions are 0.025% solutions of tretmom, isotretinoin, or 2-(9-αs-retmoyloxy)-4- methoxyacetophenone m 60% by volume ethanol and 40% by volume polyethylene glycol. Four patients paint two saturated cotton swabs of each of the four solutions on four different areas of the inner forearm, twice daily for ten days. No l tant reactions occur. In the second test, four other solutions are used. The control consists of vehicle solution, namely a solution of 90% by volume ethanol and 10% by volume polyethylene glycol. The other three solutions are 0.075% solutions of tretmom, isotretmom, or 2-(9-cιs-retιnoyloxy)-4- methoxyacetophenone in 90% by volume ethanol and 10% by volume polyethylene glycol. Four patients paint two saturated cotton swabs of each of the four solutions on four different areas of the inner forearm, twice daily for ten days. Only one subject experiences an irritant reaction. On day two, the tretmom area begins reacting with redness and peeling. On day seven, the lsotretinoin area begins reacting with redness and peeling. By day nine, both areas are still reacting, the tretinom area more mtensely than the isotretmom area. There is no reaction in either the 2-(9-αs-retιnoyloxy)^l- methoxyacetophenone or the control areas.
In the third test, three solutions are used. The three solutions are 0075% solutions of tretmom, isotretmom, or 2-(9-αs-retιnoyloxy)-4-m-ethoxyacetophenone m 90% by volume ethanol and 10% by volume polyethylene glycol. Four patients pamt two saturated cotton swabs of the 2-(9- cιs-retιnoyloxy)-4-methoxyacetophenone solution twice daily on one cheek of their faces. To the other c heek they apply two saturated cotton swabs of either tretmom or isotretmom.
The tests are earned out m double-blind fashion, that is, neither the subjects nor the mvestigator know the contents of the solutions during the study
Clinical assessments are made daily of the subjects cheeks. All subjects develop lrntant reactions by the third or fourth day of the study. Cheeks of subjects painted with solutions containing 2-(9-αs-retιnoyloxy)-4'-methoxy-acetophenone are found to be shghtly lintated or not irntated at all during the six days the study is conducted. By way of contrast, the cheeks of subjects pamted with solutions containmg tretmom or isotretmom develop reactions which are so mtense with redness and peeling that all subjects discontinue apphcation on or before the sixth day of the study. EXAMPLE 16
TOPICAL ASSAY FOR ANTIAGING AND ANTIACTINIC EFFECTS
Four subjects aged 49 to 73, three females and one male havmg significant, easily observe, sun-damaged, wrinkled, aged skin of the face and forearms are subjects of a study to determine the effect of topical apphcation of 2-(9-αs-retιnoyloxy)-4-methoxyacetophenone m the treatment of dermatohehosis. The three females had moderately severe sun-damaged skm and wrinkles of the forearms, hands and face. The one male (aged 73) has extremely severe sun damage m these areas as well as multiple actinic keratoses. The four subjects are provided with and apphed to their entire faces (omitting the eyehds) and dorsal surface of the nght forearm, once daily for 12 to 16 weeks, 0.1% concentration of 2-(9-cιs-retιnoyloxy)-4-methoxyacetophenone m a hydrophihc cream vehicle. The left forearm of each patient is treated daily with a non-medicated moisturizer of the patients choice. All subjects are evaluated every 4 weeks through the study for redness, peeling, skm surface texture and wrinkling. Biopsies (using a 4 mm punch) are taken from the dorsal surface of the right upper forearm at the beginning of the study and agam from the same area at the end of the study.
The biopsies are stained with H & E, Alαan Blue and collagen/ elastic stains and compared by a qualified dematopathologist.
Facial assessment of the patients mdicates that all show an improvement m their dermatohehosis. Two of the four patients show very significant improvement m faαal smoothness, dryness and fine wrinkling. Moderate improvement of these parameters are observed m the other two patients. The improvements begin at about two months mto the study and contmue throughout the remainder of the study.
All the patients mvolved in the study are pleased by the improved appearance of their skin and note that they feel their facial skin is fresher, clearer and more attractive during the study.
Assessment of the forearms of the patients mdicates that three show improvement in surface texture (smoothness), surface dryness and f e wrinkling withm two months after apphcation of the ream containmg 2-(9-αs-retιnoyloxy)-4-methoxy-acetophenone is initiated. This improvement is mamtamed throughout the remainder of the study and is readily apparent when right and left forearms are compared. The one patient who does not show improvement is found to be non- comphant with the instructions for use and uses only spanng apphcation of the cream containmg 2- (9-αs-retιnoyloxy)-4-methoxyacetophenone and limits treatment to one small spot on the forearm. No significant imtation is expenenced by any patient. Very shght pmkness and a feeling of sbght tightness m facial skin develops m two patients after more hberal use of the cream is encouraged.
Companson of the biopsies taken at the onset of the study with those taken after the treatment period mdicates no significant differences m before treatment and after treatment biopsies. While this test does not have a control, the results are compared with the results obtained from a similar study conducted by Weiss et al m which 0.1 % concentration of retmoic aαd m a hydrophihc cream vehicle or vehicle alone is apphed to faαal skm and dorsal forearm skm. In the Weiss study, it is observed that vehicle alone had no clinical or histological effect but that retmoic acid cream, after 16 weeks of use had some positive effects on the surface texture and wrinkling of sun damaged faαal skm, and on the histology of the epidermal and stratum corneum layers of dorsal forearm skm. Also noted m this study is the occurrence of a moderately severe irntancy level from usmg retmoic aαd cream. EXAMPLE 17
TISSUE CULTURE ASSAYS It has been shown while investigating the effect of tretinoin on keratmocytes that tretmom has the capaαty to stimulate proliferation of quiescent keratmocytes m vitro (Vararut, et al, 1989). Varanit, et al , conclude that tretmom stimulates keratmocyte growth, in part, by increasing epidermal growth factor receptor RNA and transforming growth factor-alpha production (Mitra cl al, 1989).
The esters and amides of 9-αs-retιnoιc acid are evaluated usmg several assays developed by Nickoloff. One of the major thrusts is the delineation of the effect of vanous kerat ocyte growth modulating factors on cultured human keratmocytes. The tissue culture assays used are given m full detad m Nickoloff, B J , Mitra, R.S., Riser, B.L., Dixit, V.M., and Vararu, J., Am. J. Pathol., 132:543-551
(1988); Nickoloff, B.J., Mitra, R.S , Elder, J.T , Fisher, G.J., and Voorhees, J.J., Bnt. J. Dermatol., 121,
161174 (1989); Nickoloff, B.J. and Mitra, R.S., J. Invest. Dermatol., 1989. In the tissue culture work, the primary cultures of keratmocytes are obtamed from normal appearmg skm from volunteers, normal adult face-hft skin or neonatal foreskin.
Cell Proliferation Tissue Culture
When the primary cultures of keratmocytes are subcofluent (10-14 days), thev are passed mto plastic Peln dishes usmg 0.03% trypsin, 001% EDTA The KGM (Keratmocyte growth medium) is replaced every 2-3 days and these cells are considered to be passage No. 1. When the keratmocytes were subcofluent, they are removed and seeded. The day of adding IFN-gamma (gamma mterferon) is designated as day 0. On day 4, the medium is removed and the cells are washed once with fresh KGM and the keratmocytes are detached usmg 0.03% trypsin, 0.01 % EDTA and an ahquot is placed into a hemocytometer for manual counting using a phase contrast microscope. Motility Assay Agarose Drop Explant Technique
Keratmocytes removed from cultures dishes by usmg 0.03% trypsin, 0.01% EDTA; 106 keratmocytes are centnfuged mto a 0.1 ml cell pellet and resuspended in 0.3 ml of KGM containing 0.2% agarose. One to two-microhter droplets of the cell suspension are dehvered with a micropipette into the wells of a microtiter αilture dish. After cooling, the agarose droplets are covered with 0.2 ml of the overlay medium After mcubation, migration of the cells is examined daily for 1 to 3 days by phase contrast microscopy usmg an Olympus phase contrast microscope. The distance to the leadmg edge of migrating cells from the edge of the agarose droplet is determined on four sides of each droplet.
Motility Assay - Micropore Filter Assay Nitrocellulose filters 12m (pore diameter) are used to separate a modified Boyden chamber mto two fluid-filled (KGM) compartments. Keratmocytes are placed m the upper compartment and allowed to migrate mto the filters. After 20 hours, the filters are stamed with hematoxyhn and eosin and the number of migrating cells is determined microscopically. Enzyme-Linked Immunosorbent Assay (ELISA) ELISAs are used to quantify the amount of lmmunoreactive FN (fibronectin) TSP
(thrombospondin) and LN (lamimn) produced by keratmocytes and secreted into the αdture medium. Bnefly, cells grown in cultures dishes are washed and then incubated in KGM or KBM (growth factor-depnved keratmocyte basal medium), the culture fluids are harvested, clarified by low-speed centnfugation and added to wells of a 96-well plate. Purified FN, LN or TSP are added to each assay plate to serve as a standard. After 4-hour mcubation, the culture medium from the cells, the control culture medium and the standards are removed from the wells and the ELISAs are performed. Ligand Binding Assay
In a standard bmding assay, 2 x 105 keratmocytes in 24 well plates are washed twice with KBM. The cells are washed once with Earl's balanced salt solution (EBSS) containing 0.2% bovine serum albumm. The cells are chilled and then incubated with medium composed of EBSS with 0.2 bovine serum albumm (bmdmg medium). After 6 hours the assay is terminated by washing the monolayers with cold bmding medium, solubilization of cells in O l N sodium hydroxide containing 1 % SDS and countmg m a gamma counter. Non-specific bmdmg was determined by adding an excess of unlabeled epidermal growth factor (EGF) to parallel samples well and was no greater than 5% of the total amount bound under any treatment condition.
The bmdmg data are analyzed by a Scratchard plot Measure of Transforming Growth
Factor-alpha Protein (TGF-alpha)
To determine the amount of TGF-alpha produced cultured keratmocytes, semiconfluent keratmocytes containmg KGM are maintained for 48 hours at 37 °C, and the conditioned medium is assayed for TGF-alpha usmg radio immunoassay kit. Duplicate ahquots are removed and immediately reduced and denatured. The samples are run with at least five different known TGF- alpha standards.
To determine the amount of TGF-alpha that may be bound to the keratmocyte cell surface, thoroughly washed keratmocytes are exposed to a cold acid wash m 50 mM glycme, 100 mM NaCl; pH 3.0 for 4 mm at 4 °C. Measurement of TGF-alpha mRNA
Keratmocytes (3 x 106) are lysed and RNA is isolated by centnfugation. RNA concentration is determined by absorbance at 260 ran and confirmed by nondenatunng agarose gel electrophoresis. RNA are size-fractionated by electrophoresis on 1 % formaldehyde-agarose gel and transferred to denvatized nylon membrane. Filters are hybndized against 2P labeled probes prepared by random priming. Specific hybndization is estimated by subjecting the autoradiographs of the blots to laser scanning densitometry. EXAMPLE 18
HUMAN KERATINOCYTE GROWTH ASSAY
Three separate experiments are designed to determine whether the compounds of the present mvention would influence the growth of cultured human keratmocytes. The effects of all- trans-retinoic acid are established as a control. Each run utilizes a similar protocol. The procedure is as follows: The 2nd passage adult human keratmocytes are seeded onto 35 mm plastic Petn dishes with 0.32 x 106 cells/plate present at the beginning of the experiment. After two days and change of the low calcium serum-free medium (keratmocyte growth medium, KGM-Clonetics Corp., San Diego, Calif.) containing EGF, insulin, and bovine pituitary extract, the cells are allowed to proliferate in the dark at 37 °C for an additional 3 days in the presence and absence of the 9-αs-retιnoιd of Example 1. The cell counts after three days are as follows (these cell counts are +/- a 10% standard error):
TABLE 6
Cells/Plate (duphcatc dishes, 4 separate counts pooled
KGM Alone 1.98x10* Retmoid Concentratιon(mg/ml)
0.01 2.63xl06
0.05 2.10x10s
0.10 1.64x106
0.50 1.49xl06
1.0 1.36x10^
5.0 0.72x10*
The lowest concentration (.01 and .05 g/ml) of the 9-cιs-retιnoιd stimulates keratmocyte growth, whereas the higher concentration (05, 1.0 and 5.0 g/ml) inhibits growth These dose- dependent pro-prohferative and anti-prohferative results are exactly the same as observed with all- trans-retinoic acid, except the present retmoid compound is more potent, as there is no growth promotmg effects of all-trans-retinoic acid until l.Og/ml concentration is reached (approximately 100 times higher concentration). There is no obvious effect on the differentiation of the keratmocytes.
EXAMPLE 19
TOPICAL ASSAY
A topical assay to test for pseudocomedone (utnculus) reduction m the rhmo mouse is conducted. Each test compound and a vehicle control was apphed topically to the dorsal trunk of the rhmo mouse. The utnculus diameters are measured with a ocular micrometer. The assay is based on the work of Khgman, et al (1979) and Van Scott (1972). Khgman, et al, J. Invest. Derm., 73:354-358 (1979). Van Scott, Pharmacology of the Skin, eds. Montagna et al, New York, Appelton-Century- Crofts, 1972, pp. 523-533. Mann, Anat. Rec, 170:485-500 (1971). Mezick, et al, J. Invest. Derm., 83:110-113 (1984). Mezick et al, Models m Dermatology, eds. Maibach, et al, Basel, Karger, 1985.
The dorsal trunk of the rhino mouse is the test site. Each test compound is dissolved m alcohohpropylene glycol (70:30, v:v) or other suitable vehicle and topically apphed (0.1 ml) to the dorsal trunk once daily, five consecutive days/week for two weeks. Following treatment, the animals are sacrificed by cervical dislocation. The treated dorsal trunk skm is removed from the animal and placed mto 0.5% acetic acid for up to 18 hours at approximately 4 °C. After this, the epidermis with the "acne cysts" is separated from the underlymg dermis. The sheets of epidermis are processed by routine methods to permanent whole mounts for microscopic examination. Also, full- thickness samples are taken, stamed (H&E) and examined by hght microscope.
The utnculus diameters are measured with an ocular micrometer to compare effects of test compounds to vehicle control and/or reference compound on cyst reduction. Light microscopy is used to determine effects on cell differentiation. The results are summanzed m Table 7.
TABLE 7
RHINO MOUSE UTRICULUS REDUCTION
Retmoid Dose (%) Utnculus ED^ (%) Relative Potency
Reduction f95% Conf. Inter.) all-trans-Retinoic 0.0005 34.1
Aαd
0.005 49.9 0.004
0.05 72.8
Example 2 0.0005 25.2
0.005 45.7 0.01 0.4 (0.3, 0.5)
0.05 - 61.1
EXAMPLE 20
THE DRAIZE TEST FOR IRRITATION
The results of testmg done on rabbits to determine dermal i tation are shown m Table 8. Table 8
Rabbit Dermal Irritation
Retmoid Dose (%) Erythema Grade Relative Potency (95% Conf. Inter.) all-trans-Retmoic 0.001 0.4
Acid
0.01 0.8 0.01
0.1 2.8
Example 1 0.001 0.0
0.01 0.42 0.08 0.3 (0.1, 0.5)
0.1 1.75
The compound of Example 2 is 2.5 times less irntatmg than all-trans-RA m the rhino mouse and 3.3 times less irntatmg than all-trans-RA m rabbit dermal imtation. EXAMPLE 21 - 30
THE EPITHELIAL WOUND HEALING MODEL
Wounds that do not result in total skm loss but retain a portion of their dermis heal primarily by epidermal regeneration. It is known that tretmom will accelerate skm healing but it is irntatmg and makes it unattractive for this use. In previous work molecular modification of isotretmom observed the therapeutic benefits of isotretmom without imtation. See Pansh et al, U.S. Patent Nos 4,677,120 and 4,885,311. It was hypothesized that the new 9-αs-rehnoιds might retain the therapeutic benefit as wound healing chemicals without the associated i tation. Several of the 9-cιs- retmoids were synthesized and evaluated usmg a battery of assays designed to measure the potential for epidermal regeneration and skm imtation. Ten 9-αs-retιnoιds were synthesized. Compounds were selected that have sufftαent solubility m the vehicles commonly used for topical apphcations. Therefore, relatively low molecular weight denvatives were prefened. The 10 c ompounds selected for synthesis are shown m Examples 21 - 30
Epithehal regeneration is the rate limiting factor m the healing of donor sites and partial thickness wounds. An mcrease in heahng rate would allow donor sites to be reharvested more frequently and thus allow closure of the burn wound in a more timely fashion. In addition, the more rapid heahng of partial thickness injunes would mean less pain for the patient and a more timely return to soαety as a functioning member. The objectives of this study are to:
1) study the rate of re-epithe zation of a partial thickness injury in the pig model treated with topical tretmom denvatives compared to control wounds, 2) develop a dose response determination for such a topical apphcation,
3) review the heahng process via histological exammation
The performance of this study requires pigs be used for a penod of 14 days The study lasts for 10 weeks using a total of 10 pigs. The pigs are anesthetized usmg 10 mg/kg ketamine and 2 mg/kg xylazme mtramuscular injection. Lidocaine is used as a local subcutaneous mjection. Rows of surgically created 2 cm X 2 cm wounds are created on the dorsum of the animal. The wounds are formed usmg a Padgett electnc dermatome (Padgett Company, Kansas City, MO) set at 0.016 inch. Each pig is used to test a different concentration of 9-αs-retmoιds; and, different wounds are used to test varying numbers of apphcations per day, one act g as a control receiving no 9-αs-retιnoιd. The number of apphcations per day vanes from once a day to four times a day. Kaltostat is used as a delivery mechanism.
The animals are anesthetized four times daily and appropnate 9-αs-retmoιd apphcations are made lo designated sites. Wound sites are covered by Compressor Gnp ® tubular elastic bandage #12 (36-48" circumference). Wound biopsies are taken from designated sites numbered 1 through 5 on each wound, chosen by a random method, on days 3, 5, 7, 9 and 12 for histological exammation and companson. Photographic documentation are obtained during the course of the study. After the 14 days of study, the pigs are anesthetized as usual and then euthanized usmg 0.14 ml/lb. intravenous T61. l)Each wound is a partial thickness wound created by a Padgett electnc dermatome. 2)Each pig is used to test vanous numbers of applications per day on one concentration of topical 9-αs-retιnoιd.
Concentrations:
2 pigs-standardization of technique 2 pιgs-2 mg/% 2 pιgs-4 mg/%
2 pιgs-8 mg/ % 2 pιgs-12 mg/% 3)Bιopsιes are taken for histological examination of wound sites to determine amount epithelial regeneration. Row 1 is used as the biopsy site.
Biopsies are taken from randomly chosen biopsy sites numbered 1 through 5
Biopsies are taken on 5 days of the study, on days 3, 5, 7, 9, 12. Biopsies do not contact the edge of the wound.
4)Vιsual healing is observed and recorded using photographic documentation with a digital camera, computer analysis and an analyst program. 5)Test rows receive topical 9-αs-retιnoιds.
6)Control rows do not receive topical 9-αs-retιnoιds.
7)Test sites are covered by Compressognp tubular elastic bandage.
The results show an mcrease m the rate of wound heahng of about 25 to about 30 % for the 9-cιs-retιnoιc aαd esters and amides of the present mvention, compared with untreated control sites, compared to about 15 to about 18% for all-trans-retinoic acid treated sites. Control sites on test animals showed an increase m wound heahng rate of about 5% compared to untreated animals, indicating a slight systemic effect of the treatments. EXAMPLE 31
A mixture was formed of 2.42 g (2789 mmol) of Mnθ2, 0.35 g (7.2 mmol) of NaCN, 0.12 ml (2.1 mmol) glacial acetic acid m 20 ml of CH3OH at 0 °C. A separate solution was formed of 600 mg (2.1 mmol) of 9-αs-retιnal (I) m 9 ml of CH30H, also at 0 °C. The solution of 9-αs-retιnal was added dropwise to the mixture and stirred for 2.5 h at constant temperature and the reaction mixture was fdtered through Cehte. The filter cake was washed with a 1:1 mixture of CH^OH and water. The fdtrates were combmed and diluted with additional water and then extracted three tunes with ether. The combmed organic extracts were dned over MgSθ4 and concentrated under reduced pressure. Purification by sihca gel flash column chroma lography usmg 20:1 hexane.ethyl acetate as eluent gave the pure methyl ester of 9-αs-retιnoιc acid (II) m the amount of 530 mg, a yield of 80% . The structure was confirmed by NMR.
Figure imgf000039_0001
9-cιs retinal (I)
The methyl ester of 9-cιs-retιnoιc acid (II), 530 mg, 1.68 mmol, was dissolved in 3 ml of THF, 8 ml CH30H and 3 ml of water. A solution of 0.52 g (9.3 mmol) KOH m 8 ml CH3OH was added dropwise at ambient temperature and stirred for 24 hours. The bulk of the solvents, THF and CH3OH, were removed under reduced pressure. The resulting aqueous phase was washed twice with ether and adjusted to pH 3 with IN HQ. The reaction product was extracted with ether (three times). The organic extracts were combmed, dned over MgS04 and concentrated under reduced pressure to afford pure 9-αs-retιnoιc acid (III), 0.446 g, at a yield of 88% The structure was confirmed by NMR.
Figure imgf000040_0001
A solution of 0.450 g (1.50 mmol) of 9-αs-retιnoιc acid (IΙI)was formed m 17 ml of DMF at 0 °C. In sequence, 0.97 g (3.0 mmol) cesium carbonate and then 0.25 ml (1.9 mmol) 1-chloropιnacolone Here added to the solution. The reaction mixture was stirred at ambient temperature for 2 h, and the reaction was then quenched with the addition of 20 ml water The reaction product was extracted with methylene chlonde four times The organic extracts were combmed and dned over MgSθ4 and then concentrated under reduced pressure Sihca gel column purification usmg 5 1 hexane. ethyl acetate produced 480 g of product IV with a very small amount of 1-chloropιnacolone. Repeated recrystalhzation with ethanol at low temperature gave 320 mg of pure IV, havmg a meltmg pomt of 81 °C The structure was confirmed by NMR.
Figure imgf000040_0002
Human neonatal foreskin keratmocytes and human neonatal foreskin frbroblasts were isolated and grown in monlayer culture. Culture medium for keratmocytes was Keratmcyte Growth Medium (KGM) from Clonetics, Inc., San Diego, CA. The culture medium for fibroblasts was Dulbecco's Modified Eagle's Medium (DMEM) supplemented with non-essential ammo aαds and 10% fetal bovine serum. The culture medium was obtained from GIBCO, Grand Island, NY and the FBS was from Hyclone Labs, Inc., Logan, UT
Cells at passage 2 - 4 were plated m wells of a 24 well dish at approximately 5 X 104 cells per well m their respective culture medium. Incubation was at 37 °C with 5% CO2. After the cells attached and spread overmght, they were washed two times in serum-free keratmcyte basal medium (KBM) and incubated m 1 ml of KBM or m 1 ml of KGM. Compound IV was added m varying concentrations to the wells. All-trans-retinoic aαd (or no treatment) was added to control wells. The cells were incubated for an additional two days for fibroblasts and three days for keratmocytes, and were then harvested and counted. The effect of TV and of the controls are shown for both cell types in Tables 9 and 10:
TABLE 9
HUMAN FIBROBLASTS
Treatment Concentration, mg/ml Number of Cells X 104 None 5.7 ± 0.6 Retinoic Acid
0.25 10.1 ± 0.2 0.5 10.1 ± 0.1 1.0 9.6 ± 0.1
Compound IV
0.1 6.4 + 0.6 0.25 5.8 ± 0.1 0.5 5.5 ± 0.8 1.0 7.9 ± 0.1 5.0 8.5 ± 0.6 10.0 7.5 ± 0.9
TABLE 10
HUMAN KERATINCYTES
Concentration Number of Cells X 104
Treatment mg/ml KMB KGM None 8.4 ± 0.3 28.6 ± 3.4 Retinoic Acid
0.25 13.7 ± 1.6 24.7 ± 1.7
0.5 15.2 ± 0.1 17.7 ± 0.8
1.0 12.3 ± 1.0 10.9 ± 1.8
Compound IV
0.10 13.1 ± 0.5 27.4 ± 0.1
0.25 13.1 ± 1.0 25.9 ± 0.4
0.5 13.8 ± 2.4 22.5 ± 0.1
1.0 14.0 ± 0.9 21.2 ± 0.8
5.0 11.2 ± 1.2 6.4 ± 0.1 10.0 8.4 ± 0.2 4.5 ± 0.2
Tables 9 and 10 demonstrate that Compound IV is effective to stimulate both fibroblast and keratinocyte viabihty and growth, but in a fashion more selective than retinoic acid. By comparison of the effects of retinoic acid and IV in these tests, it is possible to demonstrate that IV shows a potent "retinoid" effect on keratinocytes and a less active result with fibroblasts, consistent with the separation of the therapeutic activity from the well known and limiting side effects of retinoids.

Claims

1. A compound of the formula selected from the group consisting of:
Figure imgf000043_0001
wherein R is a member selected from the group consisting of: o o o O O O
II II
-CR'"2 CCHjOCR'" II
-CR'", CNHR'
- CR'
Figure imgf000043_0002
Figure imgf000043_0003
Figure imgf000043_0004
OCR'"
II o
wherein X is H, F, Cl, Br, I, OH, o O o
OR, OR', OCR', CR'. CH, CN,
Figure imgf000043_0005
wherein n is a number from 1 to 5; wherein R' is H or any of the lower alkyls ranging from lo C(,; wherem R" is a member selected from the group consistmg of o o
II II COR', CR', CR', and R'
wherem R"' is the hydrocarbon backbone of fatty a╬▒ds; wherem R"" is R" or the hydrocarbon backbone of fatty a╬▒ds; wherem R""' is the lower alkyls rangmg from to C(, , and further, when there are two or more R', R", R"', R"", or R""' groups attached to the same carbon, each R", R", R"', R"", or R""' group may be the same as or different from the other R', R", R"', R"", or R""' groups attached to that carbon
2 The compound of claim 1, having the formula:
Figure imgf000044_0001
10
3 The compound of claim 1 wherem said compound is l-(9-╬▒s-retmoyloxy)-3-decanoyloxy- 2-propanone.
4. The compound of claim 1 wherem said compound is l,3-b╬╣s-(9-╬▒s-ret╬╣noyloxy)-2- propanone.
I i' 5. The compound of claim 1 wherem said compound is l-(9-╬▒s-ret╬╣noyloxy) 2-p╬╣nacolone.
6 The compound of claim 1 wherem said compound is 2-(9-╬▒s-ret╬╣noyloxy)-acetophenone
7 The compound of claim 1 wherein said compound is 9-╬▒s-ret╬╣noyloxy methyl 2,2- dimethyl propanoate.
8. The compound of claim 1 wherem said compound is 2-(9-c╬╣s-ret╬╣noyloxy)-n-methyl- 20 acetamide.
9. The compound of claim 1 wherein said compound is l-(9-╬▒s-ret╬╣noyloxy)-3-hydroxy-2- propanone.
10. The compound of claim 1 wherem said compound is l-(9-╬▒s-ret╬╣noyloxy)-2,3- dioleoylpropanone.
25 11. The compound of claim 1 wherem said compound is suc╬▒mmidyl 9-╬▒s-ret╬╣noate.
12. A pharmaceutical composition for the treatment of the skin for a condition responsive to retinoids which compnses an effective amount of a 9-cis-retino╬╣d compound of the formula:
Figure imgf000045_0001
wherem R is a member selected from the group consistmg of: o O O O
-CR'". CR' -CR'" CCHjOCR'" II II
-CR'"2 CHOCOR'
- C
Figure imgf000045_0002
Figure imgf000045_0003
Figure imgf000045_0004
wherein X is H, F, Cl, Br, I, OH, O O O OR, OR', OCR', CR'. CH, CN,
NO 2 NHj , NHR', NR'ΓÇ₧ and
Figure imgf000045_0005
wherem n is a number from 1 to 5; wherein R' is H or any of the lower alkyls ranging from to ; wherem R" is a member selected from the group consisting of: o o
II II COR', CR', CR', and R'
wherein R"' is the hydrocarbon backbone of fatty a╬▒ds; wherem R"" is R" or the hydrocarbon backbone of fatty acids; wherem R""' is the lower alkyls rangmg from O to C╬▓ ; and further, when there are two or more R', R", R"', R"", or R""' groups attached to the same carbon, each R", R", R"', R"", or R""' group may be the same as or different from the other R', R", R'", R"", or R"'" groups attached to that carbon. said compound admixed with a pharmaceutically-acceptable vehicle.
13. The composition of claim 12, wherem said pharmaceutically-acceptable vehicle is a topical, oral or lnjectable vehicle.
14. The composition of claim 12, wherem said 9-c╬╣s-ret╬╣no╬╣d compound compnses from about 0.01 % to about 0.5% by weight of said composition.
15. The composition of claim 12, wherem said 9-╬▒s-ret╬╣no╬╣d compound compnses from about 0.05% to about 0.2% by weight of said composition.
16. The composition of claim 12, wherem said vehicle is a mixture selected from the group consisting of propylene glycol-ethanol and propylene glycol-ethanol chloroform.
17. The composition of claim 12, wherem said 9-╬▒s-ret╬╣no╬╣d compound compnses a compound havmg the formula:
Figure imgf000046_0001
18. The composition of claim 12, wherem said 9-c╬╣s-ret╬╣no╬╣d compound compnses l-(9-╬▒s- ret╬╣noyloxy)-2-propanone.
19. The composition of claim 12, wherem said 9-c╬╣s-ret╬╣no╬╣d compound comprises l-(9-╬▒s- ret╬╣noyloxy)-3-decanoyloxy-2-propanone.
20. The composition of claim 12, wherem said 9-c╬╣s-ret╬╣no╬╣d compound compnses l,3-b╬╣s-(9- us-ret╬╣noyloxy)-2-propanone.
21. The composition of claim 12, wherein said 9-╬▒s-ret╬╣no╬╣d compound compnses l-(9-╬▒s- retmoyloxy)-2-pmacolone.
22. The composition of claim 12, wherem said 9-c╬╣s-ret╬╣no╬╣d compound compnses 2-(9-╬▒s- ret╬╣noyloxy)-acetophenone.
23. The composition of claim 12, wherem said 9-╬▒s-ret╬╣no╬╣d compound comprises 9-c╬╣s- retmoyloxv methyl 2,2-dunethyl propanoate.
24. The composition of claim 12, wherem said 9-cis-retmoid compound compnses 2-(9-c╬╣s- retmoyloxy)-n-methyl-acetam╬╣de.
25. The composition of claim 12, wherem said 9-c╬╣s-ret╬╣no╬╣d compound comprises l-(9-╬▒s- ret╬╣noyloxy)-3-hydroxy-2-propanone.
26. The composition of claim 12, wherem said 9-c╬╣s-ret╬╣no╬╣d compound compnses l-(9-╬▒s- ret╬╣noyloxy)-2,3-d╬╣oleoylpropanone.
27. The composition of claim 12, wherem said 9-c╬╣s-ret╬╣no╬╣d compound compnses succinimidyl 9-c╬╣s-ret oate.
28. A method for treatmg the skm for a condition responsive to retmoids which compnses: topical apphcation to the site of said subject of a pharmaceutical composition which compnses an effective amount of a 9-╬▒s-ret╬╣no╬╣d compound of the formula:
Figure imgf000047_0001
wherein R is a member selected from the group consisting of:
O O
II II
-CR"'. CR' -CR"1 CCHjOCR"'
-CR'
Figure imgf000048_0001
Figure imgf000048_0002
Figure imgf000048_0003
OCR'"
II o
wherein X is H, ' F, Cl, Br, Γûá L OH, O O O
II OR, OR', ΓÇó OCR', CR'. CH, CN,
NO? ΓÇó NH, , NHR', NR'╬╖ and
O R'
wherein n is a number from 1 to 5; wherein R' is H or any of the lower alkyls ranging from O to C╬▓; wherein R" is a member selected from the group consisting of: o O
II II
- COR', - CR', -CR', and
wherein R"' is the hydrocarbon backbone of fatty acids; wherein R"" is R" or the hydrocarbon backbone of fatty acids; wherein R""' is the lower alkyls ranging from to C╬▓ ; and further, when there are two or more R', R", R"\ R"", or R""' groups attached to the same carbon, each R", R", R"', R"", or R""' group may be the same as or different from the other R', R", R"', R"", or R""' groups attached to that carbon. said compound admixed with a pharmaceutically-acceptable topical vehicle
5 29. The method of claim 28, wherem said 9-╬▒s-ret╬╣no╬╣d compound compnses from about
0.01 % to about 0.5 % by weight of said composition
30. The method of claim 28, wherem said 9-c╬╣s-ret╬╣no╬╣d compound compnses from about 0.05% to about 0.2% by weight of said composition.
31. The method of claim 28, wherem said condition is a member selected from the group n c consisting of acne vulgans, cystic acne, hyperpigmentation, hypopigmentation, psonasis, dermal and epidermal hypoplasia and kerotoses, normalization of the production of sebum, the reduction of enlarged pores, promotmg the rate of wound heahng, limiting of scar tissue formation dunng heahng, ichthyosis hystrix, epidermolytic hyperkeratosis, and lamellar ichthyosis, pseudofolhcuhtis, senile comedones, nevus comidonicas, and tnchostatis spinulosa, benign epithehal tumors 5 (mcludmg flat warts, tnchoepithehoma, and molluscum contagiosum), perforated dematoses (mcludmg elastosis perforans senpigmosa and Kyrles disease), and disorders of keratinization (mcludmg Daners disease, keratoderma, hyperkeratosis plantans, pitynasis rubra pdans, lichen planus acanthosis mgncans, and psoriasis, effects attnbutable to aging, photodamage photoaging, the effacement of wrinkles, and the normalization of color and condition of the skin and spots 0 caused from exposure to the sun.
32. The method of claim 28, wherem said pharmaceutically-acceptable vehicle is a topical, oral or injectable vehicle.
33. The method of claim 28, wherein said pharmaceutically-acceptable vehicle is a topical vehicle. 5
34. The method of claim 28 wherem said compound is l-(9-╬▒s-ret╬╣noyloxy)-2-propanone.
35. The method of claim 28 wherem said compound is l-(9-╬▒s-ret╬╣noyloxy)-3-decanoyloxy- 2-propanone.
36. The method of claim 28 wherem said compound is l,3-b╬╣s-(9-╬▒s-ret╬╣noyloxy)-2- propanone. 0
37. The method of claim 28 wherem said compound is l-(9-╬▒s-ret╬╣noyloxy)-2-p╬╣nacolone.
38. The method of claim 28 wherem said compound is 2-(9-╬▒s-retrnoyloxy)-acetophenone.
39. The method of claim 28 wherem said compound is 9-╬▒s-ret╬╣noyloxy methyl 2,2-d╬╣methyl propanoate.
40. The method of claim 28 wherem said compound is 2-(9-╬▒s-ret╬╣noyloxy)-n-methyl- acetamide.
41. The method of claim 28 wherem said compound is l-(9-c╬╣s-ret╬╣noyloxy)-3-hydroxy-2- propanone.
42. The method of claim 28 wherein said compound is l-(9-cis-retinoyloxy)-2,3- dioleoylpropanone.
43. The method of claim 28 wherem said compound is succirumidyl 9-c╬╣s-retmoate.
44. A method for treatmg the skin of a subject requiring such treatment for a condition responsive to retmoids which compnses: oral administration to said subject of an effective amount of a 9-cis-retmoid compound of the formula:
Figure imgf000050_0001
wherem R is a member selected from the group consistmg of:
O O O O
II II
-CR'". CR' II II
-CR"'., CCHjOCR'" -CR'", CHOCOR'
- CR'
Figure imgf000050_0002
Figure imgf000050_0003
Figure imgf000050_0004
OCR'"
II o wherein X is H, F, Cl, Br, I, OH,
O O O
II I I II OR, OR', OCR', CR'. CH, CN,
Figure imgf000051_0001
wherein n is a number from 1 to 5; wherein R' is H or any of the lower alkyls ranging from to C╬▓; wherein R" is a member selected from the group consisting of: o o
II II COR', CK', CR', and R'
wherein R"' is the hydrocarbon backbone of fatty acids; wherein R"" is R" or the hydrocarbon backbone of fatty acids; wherein R""' is the lower alkyls ranging from Q to C╬▓ ; and further, when there are two or more R', R", R"', R"", or R""' groups attached to the same carbon, each R", R", R"', R"", or R""' group may be the same as or different from the other R', R", R"', R"", or R""' groups attached to that carbon.
45. The method of claim 44 wherein said compound has the formula:
Figure imgf000051_0002
46. The method of claim 44 wherein said compound is l-(9-cis-retinoyloxy)-2-ρropanone.
47. The method of claim 44 wherein said compound is l-(9-cis-retinoyloxy)-3-decanoyloxy- 2-propanone.
48. The method of claim 44 wherein said compound is l,3-bis-(9-tis-retinoyloxy)-2- propanone.
49. The method of claim 44 wherein said compound is l-(9-cis-retinoyloxy)-2-pinacolone.
50. The method of claim 44 wherein said compound is 2-(9-cis-retinoyloxy)-acetophenone.
51. The method of claim 44 wherein said compound is 9-cis-retinoyloxy methyl 2,2-dimethyl propanoate.
52. The method of claim 44 wherein said compound is 2-(9-cis-retinoyloxy)-n-methyl- acetamide.
53. The method of claim 44 wherein said compound is l-(9-cis-retinoyloxy)-3-hydroxy-2- propanone.
54. The method of claim 44 wherein said compound is l-(9-cis-retinoyloxy)-2,3- dioleoylpropanone.
55. The method of claim 44 wherein said compound is succuiimidyl 9-ris-retinoate.
56. A pharmaceutical composition for the treatment of wounds which comprises an effective cell growth stimulating amount of a wound-treating compound of the formula:
Figure imgf000052_0001
wherein R is o O O
II O O O
II II
CR'"2 CR' ΓÇö CR'"2 CCHjOCR'" II II II CR'"2 CNHR' CR'"2 CHOCOR
O o O O
II II II
CR'"OCCR' - CR'"OCNR' CR'OCCHiOH ΓÇö CR'"2 CΓÇö O
Figure imgf000052_0002
wherein X is H, F, Cl, Br, I, OH, p o o II II II OR, OR', OCR', CR'. CH, CN,
Figure imgf000053_0001
wherein n is a number from 1 to 5; wherein R' is H or any of the lower alkyls ranging from C\ to C╬▓; wherein R" is o o
II II COR', CR', CR', and R'
wherem R"' is the hydrocarbon backbone of fatty acids; wherein R"" is R" or the hydrocarbon backbone of fatty acids; wherein R""' is the lower alkyls rangmg from Ci to ╬▓ ; and further, when there are two or more R', R", R"', R"", or R""' groups attached to the same carbon, each R", R", R'", R"", or R""' group may be the same as or different from the other R', R", R"', R"", or R""' groups attached to that carbon, admixed with a pharmaceutically acceptable earner.
57. The pharmaceutical composition of claim 56 wherein said compound is l-(9-cis- retinoyloxy)-2-propanone.
58. The pharmaceutical composition of claim 56 wherem said compound is l-(9-c╬╣s- retinoyloxy)-3-decanoyloxy-2-propanone.
59. The pharmaceutical composition of claim 56 wherein said compound is l,3-bis-(9-cis- retinoyloxy)-2-propanone.
60. The pharmaceutical composition of claim 56 wherein said compound is l-(9-cis- ret╬╣noyloxy)-2-p╬╣nacolone.
61. The pharmaceutical composition of claim 56 wherein said compound is 2-(9-cis- ret╬╣noyloxy)-acetophenone.
62. The pharmaceutical composition of claim 56 wherem said compound is 9-╬▒s-retinoyloxy methyl 2,2-dimethyl propanoate.
63. The pharmaceutical composition of claim 56 wherein said compound is 2-(9-cis- retinoyloxy)-n-methyl-acetamide.
64. The pharmaceutical composition of claim 56 wherein said compound is l-(9-cis- retinoyloxy)-3-hydroxy-2-proρanone.
65. The pharmaceutical composition of claim 56 wherein said compound is l-(9-ds- retinoyloxy)-2,3-dioleoylpropanone.
66. The pharmaceutical composition of claim 56 wherein said compound is succinimidyl 9- cis-retinoate.
67. The pharmaceutical composition of claim 56 wherein said wound-healing compound comprises from about 0.005% to about 0.1% by weight of said composition.
68. The pharmaceutical composition of claim 56, wherein said wound-healing compound comprises from about 0.01% to about 0.05% by weight of said composition.
69. The pharmaceutical composition of claim 56, wherein said vehicle is a mixture selected from the group consisting of propylene glycol-ethanol and propylene glycol-ethanol chloroform.
70. A method for treating wounds in a subject requiring such treatment which comprises: topical apphcation to the wound of said subject of a pharmaceutical composition which comprises an effective wound healing amount of a compound of the formula:
Figure imgf000054_0001
wherein R is o O O
II II II CR"', CR' ΓÇö CR'"2 CCH,OCR -CR'"2 CNHR' -CR"' CHOCOR'
-CR'O
Figure imgf000055_0001
Figure imgf000055_0002
Figure imgf000055_0003
OCR'"
II o
wherein X is H, F, Cl, Br, Γûá I, OH,
O O O II
OR, OR', ΓÇó OCR', CR'. CH, CN,
O
II
NO - Γûá NH,, - NHR', NRV and -N ,
O
wherein n is a number from 1 to 5; wherein R' is H or any of the lower alkyls ranging from to C╬▓; wherein R" is o O
II II
- COR', - CR', -CR', and
wherein R"' is the hydrocarbon backbone of fatty acids; wherein R"" is R" or the hydrocarbon backbone of fatty adds; wherein R""' is the lower alkyls ranging from to C╬▓ ; and further, when there are two or more R', R", R"', R"", or R""' groups attached to the same carbon, each R", R", R"', R"", or R""' group may be the same as or different from the other R', R", R"', R"", or R""' groups attached to that carbon, admixed with a pharmaceutically acceptable earner.
71. The method of claim 70 wherem said compound is l-(9-╬▒s-ret╬╣noyloxy)-2-propanone.
72. The method of claim 70 wherem said compound is l-(9-αs-retιnoyloxy)-3-decanoyloxy- 2-proρanone.
73. The method of claim 70 wherem said compound is l,3-b╬╣s-(9-╬▒s-ret╬╣noyloxy)-2- propanone.
74. The method of daim 70 wherein said compound is l-(9-╬▒s-ret╬╣noyloxy)-2-p╬╣nacolone.
75. The method of claim 70 wherem said compound is 2-(9-c╬╣s-ret╬╣noyloxy)-acetophenone.
76. The method of claim 70 wherem said compound is 9-╬▒s-ret╬╣noyloxy methyl 2,2-dimethyl propanoate.
77. The method of claim 70 wherein said compound is 2-(9-╬▒s-ret╬╣noyloxy)-n-methyl- acetamide.
78. The method of claim 70 wherem said compound is l-(9-╬▒s-retmoyloxy)-3-hydroxy-2- propanone.
79. The method of claim 70 wherem said compound is l-(9-╬▒s-ret╬╣noyloxy)-2,3- dioleoylpropanone.
80. The method of claim 70 wherem said compound is succimmidyl 9-╬▒s-ret╬╣noate.
81. The method of claim 70, wherein said wound-heahng compound compnses about 0.005% to about 0.1 % by weight of said composition.
82. The method of claim 70, wherem said wound healmg compound compnses from about 0.01% to about 0.05% by weight of said composition.
83. The method of claim 70, wherein said vehicle is a mixture selected from the group consistmg of propylene glycol-ethanol, propylene glycol-ethanol-chloroform.
84. The 9-cis-retinoid having the structure:
Figure imgf000057_0001
85. The 9-ds-retinoid having the structure:
Figure imgf000057_0002
86. The 9-cis-retinoid having the structure:
Figure imgf000057_0003
87. The 9-cis-retinoid having the structure:
Figure imgf000058_0001
88. The 9-cis-retinoid having the structure:
Figure imgf000058_0002
89. The 9-cis-retinoid having the structure:
Figure imgf000058_0003
j.
Figure imgf000059_0001
hav 'CH«Cj 7 fo cfa.
Figure imgf000059_0002
S" >.
Figure imgf000059_0003
8 TϊtA<?-< i-'le6it~<f k* f«?) fc* rf tc/uKL
Figure imgf000060_0001
PCT/US1996/002979 1996-03-04 1996-03-04 9-cis-retinoic acid esters and amides and uses thereof WO1997032839A1 (en)

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