Abstract:
Disclosed are methods for increasing the differentiation of mammalian neuronal cells for purposes of treating neurodegenerative diseases or nerve damage by administration of various compounds including alcohols, diols and/or triols and their analogues.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of application Ser. No. 60/026,577 filed Sep. 18, 1996, of application Ser. No. 60/035,947 filed Jan. 21, 1997, of application Ser. No. 60/036,863 filed Feb. 4, 1997, and of application Ser. No. 60/048,597 filed Jun. 4, 1997. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     Not Applicable 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to increasing the differentiation of mammalian neuronal cells for purposes of treating neurodegenerative diseases or nerve damage by administration of various compounds including alcohols, diols and/or triols and their analogues. 
     2. Description of Related Art 
     The compositions which are the subject of the present invention have been found to increase the melanin content of mammalian melanocytes, increase pigmentation in the epidermis of a mammal, and treat or prevent various skin and proliferative disorders. See U.S. application Ser. No. 60/026,577 filed Sep. 18, 1996; application Ser. No. 60/035,947 filed Jan. 21, 1997; application Ser. No. 60/036,863 filed Feb. 4, 1997, and application Ser. No. 60/048,597 filed Jun. 4, 1997. It has now been found that the present compositions may be used for treating neurodegenerative diseases or nerve damage. 
     SUMMARY OF THE INVENTION 
     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:                           
     wherein 
     X 1 , X 2 , and X 3  are independently selected from a single bond; or a group containing from one atom to twenty atoms, at least one of which is carbon, nitrogen, oxygen or sulfur; 
     each of R 1  and R 2  is independently selected from hydrogen; halogen; or a group containing from one atom to twenty atoms, one of which is carbon, nitrogen, oxygen, or sulfur; 
     each of R 3  and R 4  is independently selected from hydrogen or an acyl or amino acyl group containing from one atom to twenty atoms, at least one of which is carbon, nitrogen, oxygen, or sulfur; 
     R 5  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 6  is independently selected from hydrogen; halogen; or a group containing from one atom to twenty atoms, one of which is carbon, nitrogen, oxygen, or sulfur; hydroxyl, hydroxymethyl, —(CH 2 ) n OH, —(CH 2 ) n OR 1 , —(CH 2 ) n —CH(OH)—CHOH, —(CH 2 ) n —CH(OH)—CH(OH)R 1 , —(CH 2 ) n —CH(OH)—(CH 2 ) n —CH 2 (OH), —(CH 2 ) n —CH(OH)—(CH 2 ) n —CH(OH)R 1  or —CH 2 OR 3 , wherein each n is independently an integer from 0-25; 
     and pharmaceutically acceptable salts thereof In another aspect, 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 above; and 
     b) a suitable carrier. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS. 1A-1B are photographs as described in Example 1. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is based on the unique observation that certain compounds effectively and efficiently increase differentiation of neuronal cells, including increased neuronal dendricity and neuronal tyrosine hydroxylase activity, which has several consequences. First, increasing dendricity leads to increased neuronal communication, thereby increasing neuronal function and performance. Thus, the present invention is useful for treating diseases or disorders marked by reduction of neuronal dendricity and function, including but not limited to Parkinson&#39;s disease, amyotrophic lateral sclerosis, Alzheimer&#39;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. 
     Second, increasing tyrosine hydroxylase activity directly increases dopamine synthesis. Thus, the present invention is particularly useful for treating Parkinson&#39;s disease which is specifically marked by depletion of dopamine synthesis. 
     Third, induction of neuronal differentiation reverses neuronal proliferative disorders. Thus, 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. 
     Finally, since the methods and compositions described herein induce differentiation, dendricity and tyrosine hydroxylase in a neuronal cell model, 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-Straussler-Scheinker disease, progressive spinal muscular atrophy, progressive balbar palsy, primary lateral sclerosis, hereditary muscular atrophy, spastic paraplegia, peroneal muscular atrophy, hypertrophic interstitial polyneuropathy, heredopathia atactica polyneuritiformis, optic neuropathy, and ophthalmoplegia. 
     The active compounds according to the present invention have the structures described above. More preferably, each X is independently selected from a single bond; or C 1 -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 of R 1  and R 2  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  aralkynyl, 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 each of R 3  or R 4  is independently selected from hydrogen or C 1 -C 18  acyl, which may contain one or more different heteroatoms or heteroatoms of the same type. More preferably R 5  contains from two to twenty carbon atoms, each may contain one or more different heteroatoms or heteroatoms of the same type. 
     The preparation of the present compounds would be apparent to one of ordinary skill, and many of them are commercially available. Representative preferred compounds include, but are not limited to: 
     1,2-Ethanediol 
     1,2-Propanediol (Propylene Glycol) 
     (S)-(+)-1,2-Propanediol [(S)-(+)-1,2-Propylene Glycol] 
     1,3-Propanediol 
     2,3-Dimethyl-2,3-Butanediol 
     2,3-Dimethyl-1,2-Butanediol 
     1-Phenyl-1,2-Propanediol 
     2-Methyl-1,3-Propanediol 
     1,2-Butanediol 
     1,3-Butanediol 
     1,4-Butanediol 
     2,3-Butanediol 
     (2R,3R)-(−)-2,3-Butanediol 
     (2S,3S)-(+)-2,3-Butanediol 
     2,3-meso-Butanediol 
     1,2-Pentanediol 
     1,4-Pentanediol 
     1,5-Pentanediol 
     2,4-Pentanediol 
     1,2-cis-cyclopentanediol 
     1,2-trans-cyclopentanediol 
     1,2-cis-cyclohexaneanediol 
     1,2-trans-cyclohexanediol 
     1,2-dihydroxy-4,5-cyclohexanediol carbonate 
     1,2,4,5-tetrahydroxycyclohexane 
     1,2-Hexanediol 
     1,5-Hexanediol 
     1,6-Hexanediol 
     2,5-Hexanediol 
     1,2-Heptanediol 
     1,7-Heptanediol 
     7-Octene-1,2-diol 
     1,2-Octanediol 
     1,8-Octanediol 
     1,2-Nonanediol 
     1,9-Nonanediol 
     1,2-Decanediol 
     1,10-Decanediol 
     1,2-Dodecanediol 
     1,12-Dodecanediol 
     1,2-Tetradecanediol 
     1,14-Tetradecanediol 
     1,2-Hexadecanediol 
     1,16-Hexadecanediol 
     Glycerol 
     1,2,4-Butanetriol 
     1,2,3-Trihydroxyhexane 
     1,2,6-Trihydroxyhexane 
     1,2,3-Heptanetriol 
     β-estradiol 
     azabicyclo-(2,2,1)-heptanediol-3-one 
     1,4-dioxane-2,3-diol 
     5-norbornene-2,2-dimethanol 
     norbornane-2,2-dimethanol 
     2,3-norbornanediol (exo or endo or cis or trans) 
     2,3-cis-exo-norbornanediol 
     α-norborneol 
     2-norbornanemethanol 
     norbornane 
     borneol 
     camphor 
     camphene 
     camphane 
     norbornane acetic acid 
     norbornane-carboxylic acid 
     norbornane-dicarboxylic acid 
     2-endo-hexadecylamino-5-norbornene-2-exo-methanol 
     2-endo-hexadecylamino-5-norbornene-2,3-exo-dimethanol 
     2-(propyl-1,2-diol)-norbornane 
     1,2-dithiane-trans-4,5-diol 
     2,3-pyridinediol 
     2,3-pyridinediol hydrogen chloride 
     2,3-pyridinediol glycolic acid 
     2,3-dipyridyl-2,3-butanediol 
     2,2,4,4-tetramethyl-1,3-cyclobutanediol 
     Particularly preferred compounds of this invention are 5-norbornene-2,2-dimethanol; norbornane-2,2-dimethanol; 2-norbornanemethanol; 1,2-cis-cyclopentanediol; 2,3-cis-exo-norbornanediol, 2-(propyl-1,2-diol)-norbornane and 3,3-dimethyl-1,2-butanediol. Other preferred compounds are 1,2-trans-cyclopentanediol; 2,3-dimethyl-2,3-butanediol; 2-methyl-1,3-propanediol; 2,3-butanediol; and propylene glycol. 
     The methods and compositions of the present invention 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). In a preferred embodiment, 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-1,2-butanediol. Additionally, 1,2-propylene glycol as carrier has itself, as described in this invention, similar but lessor 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. In general, it is contemplated that 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%. 
     The use of and useful and novel features of the present methods and compositions will be further understood in view of the following non-limiting examples. 
     EXAMPLE 1 
     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, Mass.). Cells were cultured directly on plastic dishes at 37° C. in 5% CO 2  in a humidified incubator. 
     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). In addition, 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.  11:255-2563). 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). By virtue of their ability to express tyrosine hydroxylase and thereby synthesize dopamine, PC12 cells are considered to be an especially good model for studies of Parkinson&#39;s disease (Michel, et al., 1994,  Europ. J. Neurosci. Assoc.  6:577-586 and references therein). In addition, neurite outgrowth in PC12 cells has been used to identify agents that stimulate the regeneration of severed neuronal axons in the peripheral nerves of adult mammals (Sandrock, A. W. and Matthew, W. D., 1987,  Proc. Natl. Acad. Sci. U.S.A.  84:6934-6938). Moreover, PC12 cells have been used as a model to study aspects of Alzheimer&#39;s disease (Shen, et al., 1995,  Brain Res.  671:282-292), amyotrophic lateral sclerosis (Durham, et al., 1995,  Clin. Exp. Pharmacol. Physiol.  22:366-67), Down&#39;s syndrome (Groner, et al., 1994,  Biomed. Pharmacother.  48:231-240), and age-related neurodegeneration (Taglialatela, et al., 1996,  J. Neurochem.  66:1826-1835). 
     For testing compounds for induction of dendricity (neurite outgrowth) and tyrosine hydroxylase activity in this invention, 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×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 tyrosine, 2000 U Cat/ml, 50 mU dihydropteridine reductase/ml, 0.1 mM NADH final, 200,000 cpm 3H tyrosine/100 ul, 0.1 mM NSD1015 (3-hydroxybenzylhydrazine), and 100 uM tetrahydrobiopterin (BH4) (Nagatsu, et al., 1969,  Anal. Biochem.  9:122-126; Ribeiro, et al. 1991,  J. Biol. Chem.  16207-16211). Reactions were stopped by addition of 200 ul 10% activated charcoal in 0.1N HCl and incubation on ice for 15 min. This mixture was centrifuged at 17,300×g for 5 min, and 200 ul supernatant was then filtered through a 0.22 uM GV Durapore centrifugal filter unit (Millipore) by centrifuging at 17,300×g for 5 min. Filtrate was added to 4 ml Fisher Plus scintillation fluid and counted on a Hewlett Packard scintillation counter. Tyrosine hydroxylase activity was measured as tritium release and was calculated as dpm/ug protein and dpm/10 3  cells per hour. 
     Microscopic examination showed that a large portion of PC12 cells treated with 5 mM 5-norbornene-2,2-dimethanol (5-NBene-2,2-DM) acquired dendritic processes (Table 1, and compare untreated PC12 cells in FIG. 1A with 5-NBene-2,2-DM treated PC12 cells in FIG.  1 B). Lesser increases of dendritic processes were noted following treatment with 3,3-dimethyl-1,2-butandiol (3,3-M-1,2-BD) or 1,2-propylene glycol (1,2-PG) (Table 1). The most notable increases of tyrosine hydroxylase activity resulted from treatment with 25 mM 3,3-M-1,2-BD and 5 mM 5-NBene-2,2-DM (Table 1). Treatment with 1,2-PG, 3,3-M-1,2-BD and 5-NBene-2,2-DM increased the amount of protein per cells, a feature often associated with induction of differentiation. Increases of protein per cells were manifested morphologically as an increase in cell size (compare untreated PC12 cells in FIG. 1A with 5-NBene-2,2-DM treated PC12 cells in FIG.  1 B). Examination of the data in Table 1 shows that increases of tyrosine hydroxylase per cell as a result of treatment with 1,2-PG, 3,3-M-1,2-BD or 5-NBene-2,2-DM, were in part, a result of increases of the amount of protein per cell. Ethanol (ETOH), used as a solvent for 3,3-M-1,2-BD and 5-NBene-2,2-DM, and IBMX (3-isobutly-1-methylxanthine), which increases cellular cAMP levels, resulted in only minor effects relative to the agents of this invention. 
     
       
         
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 dpm/hr 
               
             
          
           
               
                   
                   
                   
                 ug 
                 Tyrosine 
                 Tyrosine 
               
               
                   
                 Cells/Dish 
                 % Den- 
                 Protein/ 
                 Hydroxylase 
                 Hydroxylase 
               
               
                   
                 (×10 3 ) 
                 dritic 
                 10 3  Cells 
                 /10 3  Cells 
                 /ug Protein 
               
               
                   
                   
               
             
          
           
               
                 Untreated 
                 0.728 
                  1% 
                 0.47 
                 3708 
                 7888 
               
               
                 Untreated 
                 0.490 
                  1% 
                 0.61 
                 4812 
                 7888 
               
               
                 Mean Untreated 
                 0.609 
                  1% 
                 0.54 
                 4260 (1.00×) 
                 7888 (1.00×) 
               
               
                 17 mM ETOH (0.1%) 
                 0.410 
                  2% 
                 0.78 
                     7344 (1.72×) 1   
                 9416 (1.19×) 
               
               
                 85 mM ETOH (0.5%) 
                 0.367 
                  5% 
                 0.82 
                 7308 (1.72×) 
                 8912 (1.13×) 
               
               
                 100 mM 1,2-PG 
                 0.180 
                 10% 
                 1.66 
                 12988 (3.05×)  
                 7824 (0.99×) 
               
               
                 300 mM 1,2-PG 
                 0.197 
                  2% 
                 1.57 
                 16152 (3.79×)  
                 10288 (1.30×)  
               
               
                 10 mM 3,3-M-1,2-BD 
                 0.214 
                 25% 
                 1.11 
                 8828 (2.07×) 
                 7952 (1.01×) 
               
               
                 25 mM 3,3-M-1,2-BD 
                 0.044 
                  5% 
                 2.22 
                 37148 (8.72×)  
                 16732 (2.12×)  
               
               
                 5 mM 5-NBene-2,2-DM 
                 0.155 
                 50% 
                 1.64 
                 28956 (6.80×)  
                 17656 (2.23×)  
               
               
                 10 mM 5-NBene-2,2-DM 
                 0.010 
                 25% 
                 2.33 
                 12732 (3.00×)  
                 5464 (0.69×) 
               
               
                 0.1 mM IBM× 
                 0.346 
                  2% 
                 1.20 
                 9148 (2.15×) 
                 7624 (0.97×) 
               
               
                   
               
               
                   1 Fold increase relative to mean untreated control value.  
               
             
          
         
       
     
     The reduced cell numbers resulting from treatment with 1,2-PG, 3,3-M-1,2-BD or 5-NBene-2,2-DM are in part indicative of the differentiation process induced by treatments. However, in the case of treatment with 25 mM 3,3-M-1,2-BD and 10 mM 5-NBene-2,2-DM, 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). Treatment with collagen also shortens the time required for dendrite formation and greatly increases the extent of dendrite formation in response to treatment with NGF (reviewed in Greene, et al., 1991). Thus, it is contemplated that the compounds of this invention will prove to exhibit more activity when tested on collagen-coated dishes. 
     Induction of differentiation as indicated by induction of dendricity, induction of tyrosine hydroxylase activity, increased cellular protein levels and induction of cell cycle arrest as indicated by reduced growth, indicate that the compounds of this invention can act as chemotherapeutic agents for treatment of neural tumorous and cancerous disorders and additional neural proliferative disorders. In addition, it is contemplated that the compounds of this invention will treat tumorous, cancerous and proliferative disorders arising from additional cell types. 
     It should be particularly noted that 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. 1997,  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; Rukenstein, et al., 1991 and references therein). Moreover, many agents under consideration for treatment of neurodegenerative diseases including GDNF (glial cell-derived neurotrophic factor) being developed for treatment of Parkinson&#39;s disease 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). Furthermore, L-Dopa which is presently used for treatment of Parkinson&#39;s disease is toxic (Yahr, M. D. 1993,  Adv. Neurol.  60:11-17), in part, by generation of peripherally formed dopamine (Riederer, et al. 1993,  Adv. Neurol.  60:626-635), and in part, by virtue of its ability to form highly reactive semiquinone and quinones via autooxidation (Karg, et al. 1989,  Acta Derm. Venereol.  69:521-524). Given that 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&#39;s disease.