WO1998055085A1 - Compositions pharmaceutiques et procedes - Google Patents

Compositions pharmaceutiques et procedes Download PDF

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Publication number
WO1998055085A1
WO1998055085A1 PCT/US1998/005346 US9805346W WO9855085A1 WO 1998055085 A1 WO1998055085 A1 WO 1998055085A1 US 9805346 W US9805346 W US 9805346W WO 9855085 A1 WO9855085 A1 WO 9855085A1
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WIPO (PCT)
Prior art keywords
diol
cis
trans
exo
butanediol
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PCT/US1998/005346
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English (en)
Inventor
David A. Brown
Alexander A. Khorlin
Krystyna Lesiak
Wu Yun Ren
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Codon Pharmaceuticals, Inc.
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Publication date
Priority claimed from PCT/US1997/016642 external-priority patent/WO1998011882A1/fr
Application filed by Codon Pharmaceuticals, Inc. filed Critical Codon Pharmaceuticals, Inc.
Priority to AU65659/98A priority Critical patent/AU6565998A/en
Priority to US09/086,547 priority patent/US6214888B1/en
Priority to US09/085,917 priority patent/US6623724B2/en
Publication of WO1998055085A1 publication Critical patent/WO1998055085A1/fr
Priority to US10/667,630 priority patent/US6955804B2/en
Priority to US11/251,217 priority patent/US7250157B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol

Definitions

  • the present invention relates to regulating the melanin content of mammalian melanocytes ; regulating pigmentation in mammalian skin, hair, wool or fur; treating or preventing various skin and proliferative disorders; increasing the differentiation of mammalian neuronal cells for purposes of treating neurodegenerative diseases or nerve damage; and stimulating cellular nitric oxide (NO) synthesis, cyclic guanosine monophosphate levels (cGMP) , and protein kinase G (PKG) activity for purposes of treating diseases mediated by deficiencies in the NO/cGMP/PKG signal transduction pathway; by administration of various compounds, including alcohols, diols and/or triols and their analogues .
  • NO nitric oxide
  • cGMP cyclic guanosine monophosphate levels
  • PKG protein kinase G
  • U.S. Patent 5,352,440 is directed to increasing melanin synthesis in melanocytes and increasing pigmentation by administration of certain diacylglycerol compounds .
  • U.S. Patent 5,532,001 is directed to increasing pigmentation in mammalian skin via administration of certain DNA fragments .
  • U.S. Patent 5,554,359 is directed to increasing levels of melanin in melanocytes by administration of lysosomotropic agents .
  • the present invention provides a method for increasing the melanin content of mammalian melanocytes, which comprises administering to said melanocytes an effective amount of a C 3 -C 50 diol, which may be aliphatic or aromatic, linear, branched, mono-, bi- or polyclicic, saturated or unsaturated, unsubstituted, mono- or polysubstituted.
  • a C 3 -C 50 diol which may be aliphatic or aromatic, linear, branched, mono-, bi- or polyclicic, saturated or unsaturated, unsubstituted, mono- or polysubstituted.
  • Another aspect of the present invention concerns a method for increasing pigmentation in mammalian skin, hair or wool, which comprises administering to said mammal an effective amount of one or more compounds described above.
  • Another aspect of the present invention concerns a method for treating a skin proliferative disorder or a disorder of keratinization in a mammal, which comprises administering to a mammal in need of such treatment an effective amount of one or more compounds described above.
  • a further aspect of the present invention concerns a method for preventing a skin proliferative disorder or a disorder of keratinization in a mammal, which comprises administering to a mammal in need of such preventive treatment an effective amount of one or more compounds described above.
  • An additional aspect of the present invention concerns a method for treating a tumorous or cancerous disorder whereby application of one or more of the compounds described above results in reversal of said disorder by virtue of induction of differentiation of cancerous or tumorous cells to a less- or non-proliferative phenotype.
  • cancerous or tumorous disorders include, but are not limited to, proliferative disorders of a dermatological nature .
  • the present invention provides a composition for increasing the melanin content of mammalian melanocytes, which comprises: a) an effective amount of one or more compounds described above; and b) a suitable carrier.
  • the present invention provides a composition for treating a skin proliferative disorder or a disorder of keratinization, which comprises: a) an effective amount of one or more compounds described above; and b) a suitable carrier.
  • the present invention provides a composition for preventing a skin proliferative disorder, which comprises : a) an effective amount of one or more compounds described above; and b) a suitable carrier.
  • the present invention additionally provides a method for increasing the melanin content of mammalian melanocytes, which comprises administering to said melanocytes an effective amount of one or more compounds having the following structure:
  • each X is independently selected from a single or double bond; or a group containing from one atom to twenty atoms, at least one of which is carbon, nitrogen, oxygen or sulfur; each R ⁇ is independently selected from hydrogen; halogen; an acyl or amino acyl group containing from one atom to twenty atoms, at least one of which is carbon, nitrogen, oxygen, or sulfur; or a group containing from one atom to twenty atoms, one of which is carbon, nitrogen, oxygen, or sulfur;
  • R 2 is a linear, branched or unbranched, cyclic, bicyclic or polycyclic group containing from one atom to fifty atoms, at least one of which is carbon, nitrogen, oxygen, or sulfur, and each R is independently selected from R ⁇ ; R 2 ; hydroxyl, methyl , hydroxy ethyl, - (CH 2 ) n CH 3 - , - (CH 2 ) n OH, -
  • n is independently an integer from 0-25 ; and pharmaceutically acceptable salts or prodrugs thereof, with the proviso that with reference to the first listed structure only, when the X to which R x is attached is a single bond and each R is acyl and one of R x is hydroxymethyl (HOCH 2 -) , then the sum of carbon atoms in R., ⁇ is greater than one.
  • Another aspect of the present invention concerns a method for increasing pigmentation in mammalian skin, hair or wool, which comprises administering to said mammal an effective amount of one or more compounds described above.
  • Another aspect of the present invention concerns a method for treating a skin proliferative disorder or a disorder of keratinization in a mammal, which comprises administering to a mammal in need of such treatment an effective amount of one or more compounds described above.
  • a further aspect of the present invention concerns a method for preventing a skin proliferative disorder or a disorder of keratinization in a mammal, which comprises administering to a mammal in need of such preventive treatment an effective amount of one or more compounds described above.
  • An additional aspect of the present invention concerns a method for treating a tumorous or cancerous disorder whereby application of one or more of the compounds described above results in reversal of said disorder by virtue of induction of differentiation of cancerous or tumorous cells to a less- or non-proliferative phenotype.
  • cancerous or tumorous disorders include, but are not limited to, proliferative disorders of a dermatological nature .
  • the present invention provides a composition for increasing the melanin content of mammalian melanocytes, which comprises: a) an effective amount of one or more compounds described above; and b) a suitable carrier.
  • the present invention provides a composition for treating a skin proliferative disorder or a disorder of keratinization, which comprises: a) an effective amount of one or more compounds described above; and b) a suitable carrier.
  • the present invention provides a composition for preventing a skin proliferative disorder, which comprises : a) an effective amount of one or more compounds described above; and b) a suitable carrier.
  • the present invention provides a method of altering pigmentation in mammalian skin, hair, wool or fur, which comprises administering to a mammal an effective amount of a compound which alters cellular production of nitric oxide, wherein an increase in nitric oxide production results in increased pigmentation, and a decrease in nitric oxide production results in decreased pigmentation.
  • the present invention provides a method of altering pigmentation in mammalian skin, hair, wool or fur, which comprises administering to a mammal an effective amount of a compound which alters cellular production of cyclic guanosine monophosphate, wherein an increase in cyclic guanosine monophosphate production results in increased pigmentation, and a decrease in cyclic guanosine monophosphate production results in decreased pigmentation.
  • the present invention provides a method of altering pigmentation in mammalian skin, hair, wool or fur, which comprises administering to a mammal an effective amount of a compound which alters cellular activity of protein kinase G, wherein an increase in protein kinase G activity results in increased pigmentation, and a decrease in protein kinase G activity results in decreased pigmentation.
  • the present invention provides a method of identifying a substance which alters pigmentation in mammalian melanocytes, which comprises evaluating the effect the substance has on cellular production of nitric oxide, wherein if such production is altered, then the pigmentation in mammalian melanocytes is altered.
  • the present invention provides a method of identifying a substance which alters pigmentation in mammalian melanocytes, which comprises evaluating the effect the substance has on cellular production of cyclic guanosine monophosphate, wherein if such production is altered, then the pigmentation in mammalian epidermal melanocytes is altered.
  • the present invention provides a method of identifying a substance which alters pigmentation in mammalian melanocytes, which comprises evaluating the effect the substance has on cellular activity of protein kinase G, wherein if such activity is altered, then the pigmentation in mammalian epidermal melanocytes is altered.
  • the present invention provides a method for increasing the differentiation of mammalian neuronal cells, which comprises administering to a mammal in need of such increase an effective amount of a C 3 -C 50 diol, which may be aliphatic or aromatic, linear, branched, mono-, bi- or polyclicic, saturated or unsaturated, unsubstituted, mono- or polysubstit ted.
  • a C 3 -C 50 diol which may be aliphatic or aromatic, linear, branched, mono-, bi- or polyclicic, saturated or unsaturated, unsubstituted, mono- or polysubstit ted.
  • the present invention provides a method for increasing the differentiation of mammalian neuronal cells, which comprises administering to a mammal in need of such increase an effective amount of one or more compounds having the following structure:
  • R 2 is a linear, branched or unbranched, cyclic, bicyclic or polycyclic group containing from one atom to fifty atoms, at least one of which is carbon, nitrogen, oxygen, or sulfur, and each R is independently selected from R ⁇ ; R 2 ; hydroxyl , methyl , hydroxymethyl , - (CH 2 ) n CH 3 - , - (CH 2 ) n OH, -
  • n is independently an integer from 0-25; and pharmaceutically acceptable salts or prodrugs thereof .
  • the present invention provides a composition for increasing the differentiation of mammalian neuronal cells, which comprises: a) an effective amount of one or more compounds described just above; and b) a suitable carrier.
  • the present invention provides a method for stimulating cellular synthesis of nitric oxide (NO) , which comprises administering to mammalian cells in need of such stimulation an effective amount of a C 3 -C 50 diol, which may be aliphatic or aromatic, linear, branched, mono-, bi- or polyclicic, saturated or unsaturated, unsubstituted, mono- or polysubstituted.
  • a C 3 -C 50 diol which may be aliphatic or aromatic, linear, branched, mono-, bi- or polyclicic, saturated or unsaturated, unsubstituted, mono- or polysubstituted.
  • the present invention provides a method for stimulating cellular synthesis of nitric oxide (NO) , which comprises administering to mammalian cells in need of such stimulation an effective amount of a compound having the structure:
  • each X is independently selected from a single or double bond; or a group containing from one atom to twenty atoms, at least one of which is carbon, nitrogen, oxygen or sulfur; each R ⁇ is independently selected from hydrogen; halogen; an acyl or amino acyl group containing from one atom to twenty atoms, at least one of which is carbon, nitrogen, oxygen, or sulfur; or a group containing from one atom to twenty atoms, one of which is carbon, nitrogen, oxygen, or sulfur;
  • R 2 is a linear, branched or unbranched, cyclic, bicyclic or polycyclic group containing from one atom to fifty atoms, at least one of which is carbon, nitrogen, oxygen, or sulfur, and each R is independently selected from R 1( - R 2 ; hydroxyl , methyl , hydroxymethyl , - (CH 2 ) n CH 3 - , - (CH 2 ) n OH, -
  • the present invention provides a composition for stimulating cellular synthesis of nitric oxide (NO), which comprises: a) an effective amount of one or more compounds described just above; and b) a suitable carrier.
  • a composition for stimulating cellular synthesis of nitric oxide (NO) which comprises: a) an effective amount of one or more compounds described just above; and b) a suitable carrier.
  • Figures 1A-1D are printouts from an Oncor Imaging SystemTM of Fontana-Masson stained guinea pig skin biopsy samples as described in Example 5.
  • Figure 2 is a series of bar graphs depicting the structure activity results obtained in Example 7.
  • Figures 3A-3D are printouts of normal human epidermal melanocytes and melanosomes as described in Example 8.
  • Figures 4A-4B are printouts as described in Example 10.
  • Figure 5 is a series of bar graphs depicting the structure activity results obtained in Example 13.
  • Figures 6A-6B are photographs of treated guinea pig skin as described in Example 14.
  • Figures 7A-7D are printouts as described in Example Figures 8A-8D are printouts as described in Example 16.
  • Figures 9A-9D are printouts as described in Example 17.
  • the present invention is based on the unique observation that certain compounds effectively and efficiently induce melanogenesis in mammalian cells, which has several consequences.
  • increasing melanogenesis leads to increasing the melanin content of melanocytes, and hence results in increased pigmentation or darkened color of the skin, hair wool or fur.
  • the present invention is useful in the treatment of hypopigmentation disorders, such as albinism, vitiligo, etc. It is also believed that increasing the pigmentation of skin according to the present invention will protect such skin from subsequent UV light damage, sunburn, photoaging and development of skin cancers.
  • the present invention may be used to treat hyperproliferative disorders such as actinic keratosis, basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma, dermatofibrosarcoma protuberans, hemangioma, nevus flammeus, xanothoma, Kaposi ' s sarcoma, mastocytosis, mycosis fungoides, lentigo, nevocellular nevus, lentigo maligna, malignant melanoma, and metastatic carcinoma.
  • hyperproliferative disorders such as actinic keratosis, basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma, dermatofibrosarcoma protuberans, hemangioma, nevus flammeus, xanothoma, Kaposi ' s sarcoma, mastocytosis, mycosis fungoides, lentigo,
  • the present methods and compositions are also useful in the treatment of diseases characterized by inflammation and disturbance of keratinization, including psoriasis vulgaris, psoriasis eosinophilia, acne vulgaris, acne conglobata, acne fulminans, osteoma cutis, nodulocystic acne, cystic acne and benign and premalignant dermatoses .
  • the compounds also effectively and efficiently increase differentiation of neuronal cells, including
  • the present invention is useful for treating diseases or disorders marked by reduction of neuronal dendricity and function, including but not limited to Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, or any other neurodegenerative disease, or physical or toxic damage to brain, spinal or peripheral nerve cells. Further, the present invention is useful for restoring or optimizing neuronal communication, function or performance.
  • the present invention is particularly useful for treating Parkinson's disease which is specifically marked by depletion of dopamine synthesis .
  • the present invention is useful for treating neuronal proliferative, tumorous, or cancerous disorders, or said disorders in any other cell type that might be similarly affected.
  • the present invention is useful for treating additional neurodegenerative disorders or neuropathies including but not limited to diffuse cerebral cortical atrophy, Lewy-body dementia, Pick disease, mesolimbocortical dementia, thalamic degeneration, Huntington chorea, cortical-striatal-spinal degeneration, cortical-basal ganglionic degeneration, cerebrocerebellar degeneration, familial dementia with spastic paraparesis, polyglucosan body disease, Shy-Drager syndrome, olivopontocerebellar atrophy, progressive supranuclear palsy, dystonia musculorum deformans, Hallervorden-Spatz disease, Meige syndrome, familial tremors, Gilles de la Tourette syndrome, acanthocytic chorea, Friedreich ataxia, Holmes familial cortical cerebellar atrophy, Gerstmann-
  • NO/cGMP/PKG signal transduction pathway Unlike previous compounds like nitroglycerin and isosorbide dinitrate that stimulate this pathway by releasing NO upon reaction with intracellular sulfhydryl groups (Smith and Reynard, 1992, Pharmacology, W. B. Saunders Co., Philadelphia, PA, pp.
  • the compounds of this invention appear to act by direct stimulation of nitric oxide synthase (NOS) activity, thus generating NO de novo .
  • NOS nitric oxide synthase
  • the compounds of the present invention will provide a preferred alternative method of treatment for conditions presently treated by NO donors .
  • NO/cGMP/PKG pathway mediates melanogenesis induced by ultraviolet light (Romero-Graillet, et al . , 1996, J. Biol . Chem.
  • X is independently selected from a single bond; or C x -C 10 alkylene, C 2 -C 10 alkenylene, or C 2 -C 10 alkynylene, each of which may contain one or more different heteroatoms or heteroatoms of the same type. More preferably each R x is independently selected from hydrogen; fluoro; chloro; or C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, C 7 -C 20 aralkyl,
  • C 8 -C 20 aralkenyl, C 8 -C 20 aralkinyl, or C 6 -C 20 aryl each of which may contain one or more different heteroatoms or heteroatoms of the same type, or carboxyl, carboxamido, carbalkoxy, sulfamido, sulfonamido; hydroxyl, or amino. More preferably R 2 contains from two to twenty carbon atoms, each may contain one or more different heteroatoms or heteroatoms of the same type.
  • Particularly preferred compounds of this invention are 2 , 3-cis/exo-pinanediol ( [IR, 2R, 3S, 5R] -[-] -pinanediol and [IS, 2S, 3R, 5S] -[+] -pinanediol] ; 2, 3-cis/exo-bornanediol; 5- norbornene-2 , 2-dimethanol; norbornane-2 , 2-dimethanol; 2- hydroxy-2-norbornanemethanol; 1- (exo-2-norbornyl-) -propan- 1, 2-diol; and 1- (endo-2-norbornyl-) -propan-1, 2-diol.
  • compositions of the present invention contemplate the use of one or more of the above-mentioned compounds as an active ingredient for various uses .
  • the active ingredient (s) is combined with an acceptable carrier to form a topical formulation which may be placed on the skin for dermatological uses .
  • Topical formulations may include ointments, lotions, pastes, creams, gels, drops, suppositories, sprays, liquids, shampoos, powders and transdermal patches. Thickeners, diluents, emulsifiers, dispersing aids or binders may be used as needed.
  • one function of the carrier is to enhance skin penetration of the active ingredient (s) , and should be capable of delivering the active ingredient (s) to melanocytes under in vivo conditions .
  • Suitable carriers are well known to one of ordinary skill, and include liposomes, ethanol, dimethylsulfoxide (DMSO) , petroleum jelly (petrolatum) , mineral oil (liquid petrolatum) , water, dimethylformamide, dekaoxyethylene-oleylether, oleic acid, 2-pyrrolidone and Azone® brand penetration enhancer (Upjohn) .
  • a particularly preferred composition includes an active ingredient ( s) as described above, with one of 2-pyrrolidone, oleic acid and/or Azone® as penetration enhancer, solubilized in a base of water, ethanol, propanol and/or propylene glycol (the latter component having properties of a carrier, penetration enhancer and an active ingredient as described herein) .
  • the compositions of the present invention may also include other active ingredients, as well as inert or inactive ingredients .
  • the dose regimen will depend on a number of factors which may readily be determined, such as severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with a course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved, or a cosmetically desired degree of melanogenesis (tanning) is achieved, depending on the application.
  • topical formulations such as creams, lotions, solutions, etc.
  • unit dosage form compositions according to the present invention will contain from about 0.01 mg to about 100 mg of active ingredient, preferably about 0.1 mg to about 10 mg of active ingredient.
  • compositions of the present invention also contemplate the use of one or more of the above-mentioned compounds as an active ingredient to stimulate neuronal differentiation, dendricity, and/or tyrosine hydroxylase activity (with resultant increased dopamine synthesis) and/or to treat disease conditions related to the NO/cGMP/PKG pathway.
  • the active ingredient (s) is given orally, intravenously, or transdermally in an acceptable formulation.
  • a particularly preferred carrier for some formulations is 1, 2-propylene glycol since it is an excellent solvent for certain compounds in this invention including but not limited to 5-norbornene-2 , 2-dimethanol, 5-norbornane-2 , 2-dimethanol and 3 , 3-dimethyl-l, 2- butanediol. Additionally, 1, 2-propylene glycol as carrier has itself, as described in this invention, similar but lesser activity than the preferred active ingredient (s) . Depending on the specific application, the compositions of the present invention may also include other active ingredients, as well as inert or inactive ingredients.
  • the dose regimen will depend on a number of factors which may readily be determined, such as severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with a course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved.
  • One of ordinary skill may readily determine optimum dosages, dosing methodologies and repetition rates.
  • unit dosage form compositions according to the present invention will contain from about 0.01 mg to about 100 mg of active ingredient, preferably about 0.1 mg to about 10 mg of active ingredient.
  • Topical formulations (such as creams, lotions, solutions, etc.) may have a concentration of active ingredient of from about 0.01% to about 50%, preferably from about 0.1% to about 10%.
  • Another aspect of the present invention is based on the observation that the subject compounds which stimulate melanin production act via the Nitric Oxide/cyclic Guanosine monophosphate/Protein Kinase G ("NO/cGMP/PKG”) pathway.
  • NO/cGMP/PKG Nitric Oxide/cyclic Guanosine monophosphate/Protein Kinase G
  • the present invention includes not only the compounds described above, but any compound which acts via the NO/cGMP/PKG pathway to stimulate melanin synthesis by increasing cellular production of NO, cGMP or PKG.
  • agents which decrease cellular production of NO, cGMP or PKG will decrease or suppress melanin production and pigmentation in mammalian skin, hair, fur or wool, and the present invention is also directed to those compositions and methods.
  • Such is useful in, for example, the lightening of skin, hair, wool or fur for cosmetic purposes, or the treatment of hyperpigmentation or uneven pigmentation disorders such as vitiligo, dermal melanocytosis, Franceschetti-Jadassohn Syndrome, etc.
  • the formulation and dosing would be as described above with respect to pigmentation applications.
  • Discovery of the pathway through which the present compounds act also leads to methods for screening compounds for melanogenic activity and potency, or for their ability to reduce or suppress melanogenesis, based on measurement of generation of nitric oxide (NO) or measurement of nitric oxide synthesis (NOS) activity.
  • Methods for measurement of NO or NOS include but are not limited to the following well known methods. Measurement of NO is usually based on the fact that NO rapidly decomposes to nitrate and nitrite in aqueous solution. Nitrate reductase is added to culture media or cell extracts to ensure complete conversion of nitrate to nitrite.
  • NOS activity is measured by adding [ 3 H] -arginine to intact tissues or protein extracts, and measuring release of 3 H resulting from the conversion of arginine to citrulline during the enzymatic formation of NO by NOS (Baudouin and Tachon, 1996, J. Invest . Dermatol . 106:428-431) .
  • the production of cGMP or activity of PKG can be used as a screening tool.
  • cGMP may be measured by commercially available immunoassay (see Romero-Graillet, et al . , 1996, J. Biol . Chem. 271:28052-28056) .
  • PKG may be measured by cyclic GMP dependent kination of a primary histone target (see Hidaka, et al . , Biochemistry 1984, 23, 5036-5041)
  • Example 1 The Cloudman S91 mouse melanoma cell line was obtained from American Type Culture Collection (ATCC) . Cells were cultured in Dulbecco ' s Modified Eagles Medium (DMEM) containing 10% calf serum, 2 mM L-glutamine, 10 U Penicillin/ml and 10 ug Streptomycin/ml according to a previously published protocol (Eller, et al . , Proc . Natl.
  • DMEM Dulbecco ' s Modified Eagles Medium
  • Tables 1 and 2 below show the results obtained when testing formulations containing various concentrations of 1, 2-propanediol as the active ingredient. In the control, no test compound was added to the medium.
  • Example 2 The same procedure as in Example 1 was followed, except that ethanol, and isomers of propanediol and butanediol were used as test compounds .
  • the results are set forth in Tables 3 and 4.
  • the data demonstrate that several isomers of propanediol and butanediol induce melanogenesis and differentiation of S91 melanoma cells. Both 50 mM propanediol (PG) or butanediol (BD) resulted in an approximate 1.5-fold increase of melanogenesis, while 150 mM resulted in about a 2-fold increase following a single treatment.
  • PG propanediol
  • BD butanediol
  • Ethanol had no effect on cells at 340 mM but was toxic at 850 mM as indicated by low cell survival. Ethanol did not induce melanogenesis at any concentration tested. Glycerol (G) had only a slight effect on melanogenesis and differentiation at the concentrations tested in this experiment, indicating that triols may be less effective inducers of these phenotypes than diols.
  • Melanogenesis is the most characteristic feature of melanocyte differentiation (J. Cell Sci . 107:1095-1103, 1994) , and, is inversely correlated with rate of proliferation in melanoma cell lines (Neoplasia 31:545-9, 1984; Biochem. Biophys . Res . Commun. 177:545-50, 1991; Exp. Dermatol . 4:192-198, 1995).
  • increased proliferation commensurate with dedifferentiation are hallmarks of rapid tumor progression and a poor prognosis, while decreased proliferation and differentiation are indicative of more long-term survival ( Introduction to the Cellular and Molecular Biology of Cancer, L. M. Franks and N. Teich, 1987, Oxford University Press) .
  • the ability of the present compounds to induce melanogenesis and slow cell growth is indicative of their ability to act as chemotherapeutic agents .
  • Induction of melanogenesis combined with a reduced rate of cellular proliferation is indicative of induction of differentiation in S91 cells.
  • the change of cellular morphology from a rounded, spindly appearance to a flattened, cuboidal appearance is further indication of differentiation in S91 cells (Exp. Cell Res. 191:209-218, 1990).
  • the compounds of the present invention are not only tanning agents, but also chemotherapeutic agents capable of delaying tumor progression and increasing long-term survival .
  • Examples 1 and 2 were followed to examine the effect of additional compounds on melanogenesis in S91 cells.
  • Table 5 show the concentration of a number of compounds required to induce 2-fold or greater melanization in S91 cells. Many compounds are more potent than those described in Examples 1 and 2.
  • 2, 3-pyridinediol was potent at 100 uM; 1, 4-dioxane-2 , 3-diol and ⁇ -estradiol at 500 uM; 5-norbornene-2, 2-dimethanol at 5 mM; 3 , 3-dimethyl-l, 2- butanediol and 1, 2-cis-cyclopentanediol at 10 M; and
  • the PKC inhibitors H7 (1- [5-isoquinolinyl-sulfonyl] -2- methyl-piperazine) and D-sphingosine also induced melanogenesis in S91 cells.
  • these PKC inhibitors enhanced melanogenesis induced by propylene glycol in S91 cells.
  • EXAMPLE 4 Normal human epidermal melanocytes (NHEMs) were examined for induction of melanogenesis using cells and media from Clonetics Corporation (San Diego, California) . Cells were cultured exactly as specified by the supplier. Based on induction of a 1.5-fold increase of melanin in NHEMs, the most potent compound examined was 2 , 3-pyridinediol at 200 uM, followed by 5-norbornene-2, 2-dimethanol at ⁇ 5 mM, 3 , 3-dimethyl-l, 2-butanediol at 12.5 mM, and 2, 3 -dimethyl-2, 3-butanediol and 1, 2-cis-cyclopentanediol at 50 mM (Table 6) . D-Ribose was inactive in NHEMs when tested over a range of concentrations up to a toxic dose. These results show that compounds of the present invention that exhibit activity in S91 cells, also exhibit activity in normal human melanocytes
  • EXAMPLE 5 Compounds were tested for melanogenic activity in vivo by application to American short-haired guinea pigs .
  • Treatment sites were created by removal of fur using Nair® brand depilatory. Compounds were applied in 25 ⁇ l volumes twice per day for 5 days to each treatment spot as indicated in Table 7.
  • the numbers presented are the relative melanogenesis rating (mean ⁇ SE) , and are arranged according to the relative location on the animal, with the head being to the left and the tail being to the right.
  • Tyrosinase is the rate limiting enzyme in the melanogenic pathway. Its measurement provides a highly specific and sensitive indication of degree of induction of melanogenesis by test compounds. All cell culture conditions and treatments were as described above in Examples 1-3. Following treatments, cells were trypsinized, counted by Coulter, pelleted by centrifugation at 1000 X g, and analyzed for tyrosinase activity using modifications of previously described procedures (Pomerantz, S. H., 1966, J. Biol. Chem. 241:161-168; Jara, et al., 1988, Pigment Cell Res. 1:332-339.). Briefly, cell pellets were solubilized by sonicating for 5 seconds in 600 ul 50 mM phosphate buffer pH 6.8 containing 0.5%
  • 3-dimethyl-1,2-butanediol (3 , 3-M-l, 2-BD) and 5-norbornene-2, 2-dimethanol (5-NBene-2, 2-DM) result in the greatest induction of tyrosinase on both a cellular and protein basis.
  • 100 uM 2, 3-pyridinediol (2,3-Pyd) induced 2-fold increases of melanin (Example 3, Table 5)
  • 500 uM 2,3-Pyd induced only low levels of tyrosinase relative to that induced by 5 mM 5-NBene-2, 2-DM or 3 , 3-M-l, 2-BD, and, higher levels of 2,3-Pyd were toxic.
  • 5-NBene-2,2-DM and 3, 3-M-l, 2-BD are nontoxic at concentrations that induce much higher levels of tyrosinase, and thus are preferred agents for induction of melanogenesis in this embodiment. Since 5-NBene-2, 2-DM induces nearly equivalent levels of tyrosinase at 5-fold lower concentrations than 3 , 3-M-l, 2-BD, it is particularly preferred.
  • IBMX (3-isobutyl-l-methylxanthine) is well known to those in the art as potent inducer of melanogenesis and tyrosinase, and is provided as a positive control .
  • any saturated or unsaturated compound derived from or related to norbornane is included as a component of this invention, including but not limited to compounds derived from bornane, pinane, camphene and camphor.
  • PKA highly specific protein kinase A
  • H-89 N- [2- (p-bromocinnamylamino) -ethyl] -5- isoquinolinesulfinamide»2HCl; Chijiwa, et al . , 1990, J. Biol . Chem. 265:5267-5272
  • PLC highly specific protein kinase C
  • GF109203X Bisindolylmaleimide; Toullec, et al., 1991, J. Biol . Chem. 266:15771-15781
  • GF109203X Bisindolylmaleimide; Toullec, et al., 1991, J. Biol . Chem. 266:15771-15781
  • GF109203X Bisindolylmaleimide; Toullec, et al., 1991, J. Biol . Chem. 266:15771-15781
  • Tyrosinase was measured in normal human epidermal melanocytes (NHEM) using procedures identical to those described for S91 cells (Example 6) , except that media from 5 day treatment periods was retained and centrifuged at 200 X g, 1600 X g, or 17,300 X g for analysis of tyrosinase activity in the extracellular exported melanosomal particulate fraction, and in the resultant supernatant media fraction.
  • tyrosinase was also measured by an in si tu assay wherein radiolabelled tyrosine was added directly to freshly replaced media of NHEM for a period of 24 hrs following a 5 day treatment period (Abdel-Malek, et al . , 1992, J " . Cell . Physiol . 150:416-425). Results showed that 5 mM 5-NBene-2, 2-DM induced tyrosinase to a greater extent in the in situ assay, in cells, in extracellular particulate melanosomal fractions, and in the media of NHEM than did 25 mM
  • Example 9 Highly specific inhibitors of the cAMP/PKA (protein kinase A) or PKC (protein kinase C) pathways do not inhibit induction of melanogenesis by 5-NBene-2, 2-DM in S91 cells (Example 6, Table 10) .
  • each of the nitric oxide (NO) scavenger PTIO (2-phenyl-4, 4, 5, 5-tetramethyl- imidazoline-l-oxyl-3-oxide) , the cyclic guanosine monophosphate (cGMP) inhibitor LY83583 (6-anilino-5, 8- quinolinequinone) , and the PKG (protein kinase G) inhibitor KT58223 reduce induction of melanogenesis by 5-NBene-2 , 2-DM in S91 cells (Table 13) .
  • results are similar to those obtained for ultraviolet radiation wherein induction of melanogenesis did not occur via either the cAMP/PKA or PKC pathways (Friedmann and Gilchrest, 1987, J " . Cell . Physiol 133:88-94; Carsberg, et al . , J. Cell . Sci .
  • PTIO Nitric oxide scavenger 3
  • KT5823 PKG inhibitor
  • the PC12 rat pheochromocytoma cell line was obtained from American Type Culture Collection (ATCC) . Cells were cultured in 85% RPMI 1640 medium, 10% horse serum (heat inactivated at 56°C for 30 minutes) , 5% fetal bovine serum, 25 U/ml penicillin, and 25 ug/ml streptomycin (Greene, et al . , 1991, "Methodologies for the culture and experimental use of the rat PC12 rat pheochromocytoma cells line", pp. 207-225, In: Culturing Nerve Cells, The MIT Press, Cambridge, Massachusetts) .
  • PC12 rat pheochromocytoma cells are considered to be an excellent model for neuronal cells because they respond to treatment with nerve growth factor (NGF) by acquisition of a number of properties of neurons including cessation of proliferation, extension of neurons, acquisition of electrical excitability, and increased neurotransmitter synthesis (Greene, et al . , 1991 and references therein).
  • NGF nerve growth factor
  • PC12 cells are used as a model for studies of prevention or cure of neurodegenerative diseases since they provide a robust screen for agents that maintain neuron survival and prevent neuron cell death in serum-free media (Rukenstein, et al . , 1991, J " . Neurosci .
  • Agents are considered to be potentially useful for treatment of neurodegenerative disorders if they not only promote PC12 cell survival, but also increase neurite outgrowth (Rukenstein, et al . , 1991). Agents are considered to be particularly useful for treatment of neurodegenerative disorders if they promote PC12 cell survival and neurite outgrowth in the absence of "priming" with NGF (Rukenstein, et al . , 1991).
  • PC12 cells are considered to be an especially good model for studies of Parkinson's disease (Michel, et al . , 1994, Europ . J. Neurosci . Assoc .
  • PC12 cells have been used as a model to study aspects of Alzheimer's disease (Shen, et al . , 1995, Brain Res . 671:282-292), amyotrophic lateral sclerosis (Durham, et al . , 1995, Clin . Exp. Pharmacol . Physiol .
  • cells were plated at 15,000 cells/35 mm dish. Two days following plating, cell culture media was replaced with that containing treatments. One week later, media and treatments were replaced with fresh media and treatments. Two weeks following the initial treatments, cells were examined microscopically, and the portion of cells exhibiting dendricity was estimated. Cells were harvested by trypsinization and counted by Coulter Counter.
  • Cells were pelleted by centrifugation at 200 X g, and cell pellets were lysed in 600 ul 50 mM Tris/Acetate pH 6.0/0.2% Triton X-100 by vortexing, sonicating 5 seconds, incubating on ice for 30 minutes, followed by revortexing. Protein was determined on aliquots of cell lysate by the Bradford Coomassie Blue method (Bradford, 1967, Anal . Biochem. 72:248-254) using Bio-Rad Protein Assay Kit I.
  • Tyrosine hydroxylase activity was determined by incubating 100 ul of PC12 cell lysate with 100 ul of the following reaction mixture at 37°C for 15 min: 200 mM sodium acetate pH 6.0, 50 uM L-tyrosine, 2000 U Cat/ml, 50 mU dihydropteridine reductase/ml, 0.1 mM NADH final, 200,000 cpm 3H L- tyrosine/100 ul, 0.1 mM NSD1015 (3-hydroxybenzylhydrazine) , and 100 uM tetrahydrobiopterin (BH4) (Nagatsu, et al . ,
  • Ethanol used as a solvent for 3, 3-M-l, 2-BD and 5-NBene-2, 2-DM, and IBMX (3-isobutyl-l-methylxanthine) , which increases cellular cAMP levels, resulted in only minor effects relative to the agents of this invention.
  • the reduced cell numbers resulting from treatment with 1,2-PG, 3, 3-M-l, 2-BD or 5-NBene-2 , 2-DM are in part indicative of the differentiation process induced by treatments.
  • some cells detached concomitantly with the acquisition of dendricity that occurred earlier than for other treatments .
  • This detachment phenomenon has been noticed previously for PC12 cells induced to differentiate with NGF, and can be avoided by coating treatment dishes with collagen (reviewed in Greene, et al . , 1991).
  • the compounds of this invention induced dendricity and tyrosine hydroxylase activity in the absence of priming with NGF, a prerequisite for induction of neurite extension by many other agents tested on PC12 cells (Steiner, et al . 1991 , Nature Medicine 3:421-428; Rukenstein, et al . 1991, J " . Neurosci . 11:2552-2563).
  • Several agents under consideration as treatments for neurodegenerative diseases do not promote neurite extension even in NGF-primed PC12 cells (e.g.,
  • IGF-I and IGF-II are neurotrophic peptides that cannot cross the blood-brain barrier and therefore require gene therapy implantation at the site of action (Haase, et al . 1997, Nature Medicine 3:429-436).
  • L-Dopa which is presently used for treatment of Parkinson's disease is toxic (Yahr, M. D. 1993, Adv. Neurol . 60:11-17), in part, by generation of peripherally formed dopamine (Riederer, et al .
  • the agents of the present invention (i) act directly without a requirement for NGF; (ii) induce neuronal differentiation thereby setting into motion cellular reprogramming to the desired phenotype; (iii) induce tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis; (iv) are small molecule drugs that are likely to cross the blood brain barrier; and (v) have no known ability to form semiquinone, quinone or other toxic intermediates, it is contemplated that the agents of this invention will be particularly advantageous for treatment of neurodegenerative diseases including but not limited to Parkinson ' s disease.
  • Example 13 Further studies using S91 cells and the methods described in Example 6 showed that 2 , 3-cis/exo-pinanediol ( [IR, 2R, 3S, 5R] -[-] -pinanediol) had greater melanogenic activity than 5 -norbornene-2, 2-dimethanol when tested over a range of concentrations (Figure 5). 2, 3-cis/exo- pinanediol induced 2.6-fold more tyrosinase activity than 5-norbornene-2 , 2-dimethanol when tested at 500 uM, 5.2-fold more at 1 mM, and 7.3-fold more at 2.5 mM (calculated from data in Figure 5) .
  • nitric oxide was measured in cell-free media from S91 cells following treatment with a range of concentrations of 2, 3-cis/exo-pinanediol or 5- norbornene-2, 2-dimethanol for 4 days.
  • nitric oxide is converted into nitrite and nitrate with seconds of production. Therefore, nitric oxide is measured by first converting nitrate to nitrite using nitrate reductase, followed by addition of Greiss reagent to detect nitrite as optical density at 550 nm (Moshage, et al . , 1995, Clin. Chem. 41:892-896; Schmidt, et al .
  • induction of melanogenesis by diols occurs via the nitric oxide pathway. It follows that measurement of induction of nitric oxide, cGMP or PKG may provide biochemically relevant screening assays for compounds that may be melanogenic. Thus, the utilization of these assays to screen compounds for melanogenic activity is claimed in the present invention.
  • ETOH ethanol solvent control for 5 mM 5-NBene-2 , 2-DM and 5 mM 2,3-cs/ex-PD (lower treatment concentrations received proportionally less ETOH)
  • Example 14 In studies using the guinea pig model identical to that described in Example 5, 2 , 3-cis/exo-pinanediol ( [IR, 2R,3S, 5R]- [-] -pinanediol) exhibited 2- to 4-fold more melanogenic activity than equivalent concentrations of 5- norbornene-2, 2-dimethanol when compared using treatment spots in the posterior half of animals (c and d in Table 19 and Figure 6) . In Figure 6, a, b, c and d indicate treatment spots that transverse the anterior-posterior axis along the backs of guinea pigs.
  • Figure 6A top row, shows spots a-d treated with 50% ETOH
  • Figure 6A bottom row, shows spots a-d treated with 8.7M 1, 2-cis-cyclopentanediol in 20% ETOH
  • Figure 6B top row, shows spots a-d treated with 1M 5-norbornene-2, 2-dimethanol in 8.5M propylene glycol, 20% ETOH, and 2% 2-pyrrolidone
  • Figure 6B bottom row, shows spots a-d treated with 1M 2, 3-cis/exo- pinanediol .
  • Example 15 As a continuance of structure activity studies, a variety of pinanediol derivatives and related monocyclic derivatives were examined for melanogenic activity using the S91 cell line and procedures for analysis of tyrosinase described in Example 6. All of the compounds examined herein were either bicyclic- or monocyclic-monoterpenes (Table 20) . In general, bicyclic-monoterpenes were more potent inducers of melanogenesis than monocyclic- monoterpenes , and within each of these groups, diols were more potent than alcohols, while non-hydroxylated compounds exhibited little or no activity (Table 20) .
  • (-) -Isopinochampheol an alcohol closely related to (IR, 2R, 3S, 5R) -(-) -pinanediol (also known as [-]-2- hydroxyisopinocampheol) , possessed considerably less melanogenic activity (Table 20).
  • (-)- isopinochampheol resulted in detachment of cells from culture dishes at concentrations where (IR, 2R, 3S, 5R) - (-) - pinanediol was a highly efficacious inducer of melanogenesis, indicating that the alcohol was more toxic than the diol.
  • Cis-p-menthane-3 , 8-diol and trans-p-menthane-3 , 8-diol were the most potent monocyclic monoterpenes examined (Table 20) . However, these possessed much less melanogenic activity than any of the bicyclic-monoterpene diols examined (Table 20) . Similar to results for the bicyclic- monoterpenes , the alcohols exhibited only low levels of melanogenic activity, and were toxic at the higher concentrations tested (Table 20). Moreover, R-(+)- limonene, a non-hydroxylated monocyclic-monterpene exhibited little or no melanogenic activity.
  • trans- p-menthane-2, 8-diol exhibited much less melanogenic activity than cis-p-menthane-3 , 8-diol or trans-p-menthane-
  • cis-p-menthane-3, 8-diol and trans-p-menthane-3, 8-diol possess six member rings.
  • cis-p-menthane-3 , 8-diol and trans-p-menthane-3 , 8- diol are markedly more potent than either monocyclic hexanediol or pentanediol (Example 3 and Table 5) .
  • a range of substituents including but not limited to methyl groups may increase melanogenic activity of monocyclic compounds .
  • sobrerol 4 1.8X 2. OX 2.3X 3.5X
  • PC12 cells were grown and treated with 5-norbornene- 2 , 2-dimethanol as described in Example 10, except that cells were analyzed one week after treatments, rather than two weeks after treatments. Furthermore, in addition to the analysis described in Example 10, the media of cells was also collected for analysis of nitric oxide (NO) . For measurement of NO, media was centrifuged at 200 X g for 5 min to remove cells and debris . Nitric oxide was measured using a CalBiochem (San Diego, CA) Colorimetric Nitric Oxide Assay Kit .
  • nitric oxide is converted into nitrite and nitrate within seconds of production in biological fluids, nitric oxide is measured by first converting nitrate to nitrite using nitrate reductase, followed by addition of Greiss reagent to detect nitrite as optical density at 550 nm (Moshage, et al . , 1995, Clin . Chem. 41:892-896; Schmidt, et al . , 1995, Biochemica 2:22)).
  • Results show that the degree of neurite outgrowth (% with neurites of length exceeding cell body diameter) , the amount of nitric oxide (NO) released into the media, the amount of NO generated per cell, and tyrosine hydroxylase (Tyr Hydrox) activity all increased following treatment with 5-norbornene-2, 2-dimethanol (5-NBene-2, 2-DM) (Table 21) . Induction of NO and tyrosine hydroxylase were apparent at 1 mM 5-NBene-2, 2-DM, and appeared to reach a maximum at 2.5 mM 5-NBene-2 , 2-DM, since no further increases were observed at 5 mM 5-NBene-2 , 2-DM (Table 21).
  • FIG. 8 shows photographs of cultured unstained PC12 cells.
  • PC12 cells are untreated.
  • PC12 cells in Figure 8B were treated for one week with 2.5 mM 5-NBene- 2, 2-DM, while cells in Figures 8C and 8D were treated with 5 mM 5-NBene-2, 2-DM.
  • PC12 cells treated with 5 mM 5-NBene- 2, 2-DM exhibited both longer and more branched neurites than those treated with 2.5 mM 5-NBene-2 , 2-DM.
  • Example 17 To further elucidate and substantiate the role of NO in differentiation of PC12 cells, additional studies were done using the guanylyl cyclase inhibitor LY83583. This inhibitor blocks differentiation of PC12 cells otherwise induced by NO donors, indicating they act via cGMP. Similarly it was contemplated that if 5-NBene-2, 2-DM was inducing NO within PC12 cells, and if this endogenously generated NO was responsible for inducing differentiation of PC12 cells by the cGMP pathway, then LY83583 should block the effects of 5-NBene-2 , 2-DM. All PC12 culture and treatment methods were the same as in Examples 10 and 16.
  • Co-treatment of PC12 cells with 5-NBene-2, 2-DM and the guanylyl cyclase inhibitor LY83583 shows that blockage of the NO/cGMP/PKG pathway completely blocks neurite outgrowth otherwise induced by 5-NBene-2 , 2-DM.
  • Figure 9A shows photographs of unstained, untreated, cultured PC12 cells.
  • Figure 9B shows induction of extensive neurite outgrowth in PC12 cells treated with 5 mM 5-NBene-2, 2-DM for two weeks.
  • Figure 9D shows that PC12 cells cotreated with both 5 mM 5- NBene-2,2-DM and 0.1 uM LY83583 for two weeks exhibit no neurite outgrowth, similar to untreated PC12 cells (Figure 9A) or PC12 cells treated with 0.1 uM LY83583 alone for two weeks ( Figure 9C) .
  • Results shown here and in Example 16 show that 5-
  • NBene-2,2-DM induces nitric oxide activity, that induction of nitric oxide activity is associated with induction of neurite outgrowth and tyrosine hydroxylase activity, and that LY83583 can block neurite outgrowth induced by 5- NBene-2,2-DM.
  • 5-NBene-2 ,2-DM stimulates PC12 cell differentiation by stimulating the NO/cGMP/PKC signal transduction pathway. Since 5-NBene- 2, 2-DM cannot be a source of NO, 5-NBene-2 , 2-DM must stimulate NO production within PC12 cells. Furthermore, this endogenously generated NO must act via stimulating cGMP production, since the effects of 5-NBene-2, 2-DM on PC12 cells differentiation are blocked by LY83583.
  • the compounds of the present invention are distinguished from NO donors not only by the fact that they induce synthesis of NO within treated cells, but also because in contrast to NO donors (Hindley, et al . , 1997, J. Neurosci . Res . 47:427-439), the compounds of the present invention induce neurite outgrowth from PC12 cells in the absence of co-treatment with NGF. Thus, unlike NO donors which induce no neurite outgrowth in the absence of NGF (Hindley, et al . , 1997, J " . Neurosci . Res . 47:427-439), the compounds of the present invention induce neurite outgrowth in the complete absence of added NGF.
  • diols including but not limited to 5-NBene-2 , 2-DM and 2 , 3-cis/exo-pinanediol will induce NO production and differentiation of PC12 cells . Since many different types of diols, and some related alcohols and triols, similarly induce differentiation of melanoma cells and melanocytes, it is contemplated that many or all of these will be active as inducers of PC12 and neuronal cell differentiation. It is further contemplated that similar to 5-NBene-2 , 2-DM, many of these diols will stimulate PC12 cell and neuronal cell differentiation, at least in part, via the NO/cGMP/PKG pathway.
  • Example 18 As discussed in Example 10, the compounds of the present invention appear to be particularly efficacious relative to other compounds proposed for treatment of neurodegenerative diseases (Steiner, et al . , 1997, Nature Med. 3:421-428; Rukenstein, et al . , 1991, J " . Neurosci . 11:2552-2563) in that they act without co-treatment with nerve growth factor ( ⁇ GF) . Furthermore, as discussed in Examples 16 and 17, although the compounds of the present invention induce PC12 differentiation at least in part via the ⁇ O/cGMP/PKG pathway, NO donors are ineffective without the presence of NGF. The purpose of this study was to determine if addition of NGF would markedly improve the ability of the compounds of this invention to stimulate PC12 differentiation.
  • ⁇ GF nerve growth factor
  • PC12 cells were treated with the bicyclic diol 5-norbornene-2, 2-dimethanol (5-NBene-2, 2-DM) , the bicyclic diol 2, 3-cis/exo-pinanediol (2,3-cs/ex-PD), or the bicyclic alcohol (S) -cis-verbenol (S-cs-VBol) .
  • concentration of NGF used for co- treatments (0.5 ng/ml) was identical to that shown to be essential for induction of PC12 cell differentiation by immunophilins (Steiner, et al . , 1997, Nature Med. 3:421- 428) .
  • NGF 50 ng/ml NGF was used as a positive control (Steiner, et al . , 1997, Nature Med. 3:421-428). Cells were plated, treated and neurite outgrowth evaluated as described in Example 16, Table 21, except that cells were evaluated 7 and 21 days after initiation of treatments.
  • Results showed that supplementary NGF resulted in a slight stimulation of neurite outgrowth when PC12 cells were examined 7 days after the initiation of treatments, but mixed results were obtained when cells were examined 21 days after the initiation of treatments (Table 22) .
  • the highest levels of neurite outgrowth were seen following 7 days treatment with 50 ng/ml NGF, following 7 days treatment with 5 mM 2,3-cs/ex-PD, and following 21 days treatment with 5 mM 5-NBene-2 , 2-DM (Table 22); highest levels of neurite outgrowth were apparently unaffected by inclusion of 0.5 ng/ml NGF.
  • Results show that the compounds of this invention can induce significant levels of neurite outgrowth (similar to the 50 ng/ml NGF positive control) without inclusion of supplementary NGF.
  • results show that although the bicyclic alcohol S-cis-Verbenol (S-cs-VBol) induced some neurite outgrowth, it was much less effective than the bicyclic diols examined here.
  • Values are estimates wherein neurites were counted as extended if their length exceeded the cell body's diameter. Evaluations include single cells and cells on periphery of clumps, but exclude cells in middle of clumps.
  • 5-norbornene-2 , 2-dimethanol (5-NBene-2, 2-DM) induced NO oxide in several different cell types in addition to melanoma cells (Example 13, Table 18) and PC12 cells (Example 16, Table 21) .
  • These include normal human epidermal melanocytes (NHEM) and normal human umbilical vein endothelial cells (HUVEC) .
  • NHEM and HUVEC were obtained from Clonetics Corporation, San Diego, and cultured exactly as described by the supplier. Nitric oxide measurements were done as described above (Example 13), on cell-free media collected seven days following the initiation of treatments .
  • the compounds of this invention will also induce NO production in NHEM and other cell types . Additionally, the compounds of this invention induce NO synthesis in endothelial cells (HUVEC in Table 23) . Induction of NO production in bodily endothelial cells is well known to be associated with stimulation of vasodilation and relief of vasoconstrictive disorders including but not limited to heart disease, hypertension, stroke, and obstructive pulmonary disease (described above in the Detailed Description of the Invention) . Thus, it is contemplated that the compounds of this invention will be useful for treating a number of disorders related to impairment of circulation.
  • the finding that representatives of the compounds of this invention act to stimulate NO production from a diverse array of cells types indicates that these compounds may be universal inducers of the NO/cGMP/PKG pathway.
  • the compounds of this invention are contemplated to provide an alternative treatment for many different types of disorders presently treated by administration of NO donors (including but not limited to those described above in the Detailed Description of the Invention) .
  • the compounds of this invention are contemplated to be useful for treating any disorder or condition in which clinical applications or research show to be amenable to remediation by stimulation of some or all components of the NO/cGMP/PKG pathway (including but not limited to those described above in Detailed Description of the Invention) .

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Abstract

L'invention porte sur des procédés et des compositions permettant de réguler la teneur en mélanine dans les mélanocytes mammaliens, de réguler également la pigmentation de la peau, des poils, de la laine ou de la fourrure chez les mammifères; de traiter ou prévenir diverses dermatoses et maladies prolifératives; d'accroître la différentiation des cellules neuronales mammaliennes en vue de traiter des maladies neurodégénératives ou des atteintes nerveuses; et de stimuler la synthèse de l'oxyde nitrique (NO) cellulaire, les taux cycliques de l'acide guanylique (cGMP) et l'activité de la protéine kinase G (PKG) en vue de traiter des maladies induites par des déficiences dans le processus de transduction du signal NO/cGMP/PKG. Ce procédé consiste à administrer divers composés tels que des alcools, des diols et/ou des triols et leurs analogues.
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EP1261572A1 (fr) * 2000-03-10 2002-12-04 Applied Genetics Incorporated Dermatics Composes dermatologiques
US6623724B2 (en) 1996-09-18 2003-09-23 Applied Genetics Incorporated Dermatics Dermatological compositions and methods
CN102497777A (zh) * 2009-08-26 2012-06-13 巴斯夫欧洲公司 脂环族二醇作为生物杀伤剂的用途
WO2017043935A1 (fr) * 2015-09-09 2017-03-16 한국생명공학연구원 Composition pour prévenir ou traiter des maladies liées à une faiblesse musculaire comprenant du sobrerol
WO2022026381A1 (fr) * 2020-07-27 2022-02-03 Board Of Regents, The University Of Texas System Agents de contraste pour la détection d'activités enzymatiques en fonction de la synthèse de la mélanine
WO2022045671A1 (fr) * 2020-08-26 2022-03-03 주식회사 뉴롤메드 Composition pour prévenir ou traiter un accident vasculaire cérébral ischémique, contenant du sobrerol en tant que principe actif

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EP0957903A1 (fr) * 1996-09-18 1999-11-24 Codon Pharmaceuticals, Inc. Compositions pharmaceutiques et procedes
EP0957903A4 (fr) * 1996-09-18 2002-10-09 Codon Pharmaceuticals Inc Compositions pharmaceutiques et procedes
US6623724B2 (en) 1996-09-18 2003-09-23 Applied Genetics Incorporated Dermatics Dermatological compositions and methods
US6955804B2 (en) 1996-09-18 2005-10-18 Applied Genetics Incorporated Dematics Dermatological compositions and methods
US7250157B2 (en) 1996-09-18 2007-07-31 Applied Genetics Incorporated Dermatics Dermatological compositions and methods
EP1261572A1 (fr) * 2000-03-10 2002-12-04 Applied Genetics Incorporated Dermatics Composes dermatologiques
EP1261572A4 (fr) * 2000-03-10 2004-05-26 Applied Genetics Inc Dermatics Composes dermatologiques
CN102497777A (zh) * 2009-08-26 2012-06-13 巴斯夫欧洲公司 脂环族二醇作为生物杀伤剂的用途
WO2017043935A1 (fr) * 2015-09-09 2017-03-16 한국생명공학연구원 Composition pour prévenir ou traiter des maladies liées à une faiblesse musculaire comprenant du sobrerol
KR101810651B1 (ko) * 2015-09-09 2017-12-20 한국생명공학연구원 소브레롤을 포함하는 근력 약화 관련 질환의 예방 또는 치료용 조성물
CN108601755A (zh) * 2015-09-09 2018-09-28 韩国生命工学研究院 用于预防或治疗肌无力相关疾病的、包含水合蒎醇的组合物
US10765642B2 (en) 2015-09-09 2020-09-08 Korea Research Institute Of Bioscience And Biotechnology Composition for preventing or treating muscle weakness-related diseases comprising sobrerol
CN108601755B (zh) * 2015-09-09 2021-08-10 韩国生命工学研究院 用于预防或治疗肌无力相关疾病的、包含水合蒎醇的组合物
WO2022026381A1 (fr) * 2020-07-27 2022-02-03 Board Of Regents, The University Of Texas System Agents de contraste pour la détection d'activités enzymatiques en fonction de la synthèse de la mélanine
WO2022045671A1 (fr) * 2020-08-26 2022-03-03 주식회사 뉴롤메드 Composition pour prévenir ou traiter un accident vasculaire cérébral ischémique, contenant du sobrerol en tant que principe actif
KR20220026806A (ko) * 2020-08-26 2022-03-07 주식회사 뉴롤메드 소브레롤을 유효성분으로 함유하는 허혈성 뇌졸중 예방 또는 치료용 조성물

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