METHODS AND PHARMACEUTICAL COMPOSITIONS EMPLOYING DESMETHYLSELEGILINE

Abstract: Methods and pharmaceutical compositions for using the selegiline metabolite desmethylselegiline and stereoisomeric forms thereof. In particular, the present invention provides novel compositions and methods for using desmethylselegiline, and/or ent-desmethylselegiline, for selegiline-responsive diseases and conditions. Diseases and conditions responsive to selegiline include those produced by neuronal degeneration or neural trauma and those due to immune system dysfunction. Desmethylselegiline, and/or ent-desmethylselegiline, is either administered orally or by a route of administration which does not rely upon gastrointestinal absorption. Desmethylselegiline is the R-(-) enantiomer of N-methyl-N-(prop-2-ynyl)-2-aminophenylpropane and ent-desmethylselegiline is the S(+) enantiomer. Claimed compositions include both the R-(-) and S-(+) isomers as well as mixtures thereof. Pharmaceutically acceptable acid addition salts may also be used. Effective dosages are a daily dose of at least about 0.0015 mg/kg of body weight.

Description: Methods and Pharmaceutical Compositions Employing Desmethylselegiline Field of the Invention The present invention pertains to methods and pharmaceutical compositions for using the selegiline metabolite desmethylselegiline and its enantiomer, ent-desmethyl¬ selegiline. In particular, the present invention provides compositions and methods for using these agents for selegiline-responsive diseases and conditions. Background of the Invention Two distinct monoamine oxidase enzymes are known in the art: monoamine oxidase A (MAO- A) and monoamine oxidase B (MAO-B). The cDNAs encoding these enzymes show different promoter regions and distinct exon portions, indicating they are encoded independently at different gene positions. In addition, analysis of the two proteins has shown differences in their respective amino acid sequences. The first compound found to selectively inhibit MAO-B was R-(-)-N-methyl-N- (prop-2-ynyl)-2-aminophenylpropane, also known as L-(-)-deprenyl, R-(-)-deprenyl, or selegiline. Selegiline has the following structural formula: The selectivity of selegiline in the inhibition of MAO-B is important to its safety profile following oral administration. Tranylcypromine (an MAO inhibitor introduced some thirty years ago but subsequently withdrawn due to its severe hypertensive side effects), in contrast to selegiline, is a non-selective inhibitor of MAO. The acute toxicity of tranylcypromine arises from inhibition of MAO- A, which interferes with the metabolism of tyramine. Tyramine is normally metabolized in the gastrointestinal tract by MAO- A but when MAO- A is inhibited, tyramine absorption is increased following consumption of tyraιτι-u e-contøiιιing foods such as cheese, beer, herring, etc. This results in the release of catecholamines which can precipitate a hypertensive crisis, producing the "cheese effect. " This effect is characterized by Goodman and Gilman as the most serious toxic effect associated with MAO- A inhibitors. One of the metabolites of selegiline is its N-desmethyl analog. Structurally, the desmethylselegiline metabolite is the R (-) enantiomeric form of a secondary amine of the formula: Heretofore, desmethylselegiline was not known to have pharmaceutically useful MAO-related effects, i.e. , potent and selective inhibitory effects on MAO-B. In the course of determining the usefulness of desmethylselegiline for the purposes of the present invention, the MAO-related effects of desmethylselegiline were more completely characterized. This characterization has established that desmethylselegiline has exceedingly weak MAO-B inhibitory effects and no advantages in selectivity with respect to MAO-B compared to selegiline. For example, the present characterization established that selegiline has an IC50 value against MAO-B in human platelets of 5 x 10 9 M whereas desmethylselegiline 's IC 50 value is 4 x 10 7 M, indicating the latter is approximately 80 times less potent as an MAO- B inhibitor than the former. Similar characteristics can be seen in the following data measuring inhibition of MAO-B and MAO-A in rat cortex mitochondrial-rich fractions: Table 1 : Inhibition of MAO by Selegiline and Desmethylselegiline Percent Inhibition Cone. selegiline desmethylselegiline MAO-B MAO-A MAO-B MAO-A 0.003μM 16.70 - 3.40 - O.OlOμM 40.20 - 7.50 - 0.030μM 64.70 - 4.60 - O.lOOμM 91.80 - 6.70 - 0.300μM 94.55 9.75 26.15 0.0 l.OOOμM 95.65 32.55 54.73 0.70 3.000μM 98.10 65.50 86.27 4.10 lO.OOOμM - 97.75 95.15 11.75 30.000μM - - 97.05 - lOO.OOOμM - - 1 56.10 | As is apparent from the above table, selegiline is approximately 128 times more potent as an inhibitor of MAO-B relative to MAO-A, whereas desmethylselegiline is only 97 times more potent as an inhibitor of MAO-B relative to MAO-A. Accordingly, desmethylselegiline appears to have an approximately equal selectivity for MAO-B compared to MAO-A as selegiline, albeit with a substantially reduced potency. Analogous results are obtained in rat brain tissue. Selegiline exhibits an ICso for MAO-B of 0.11 x 10 "7 M whereas desmethylselegiline' s IC50 value is 7.3 x 10 " 7 M, indicating desmethylselegiline is approximately 70 times less potent as an MAO-B inhibitor than selegiline. Both compounds exhibit low potency in inhibiting MAO-A in rat brain tissue, 0.18 x 10 "5 for selegiline, 7.0 x 10 * 5 for desmethylselegiline. Thus, desmethyl¬ selegiline is approximately 39 times less potent than selegiline in inhibiting MAO-A. Based on its pharmacological profile as set forth above, desmethylselegiline as an MAO-B inhibitor provides no advantages in either potency or selectivity compared to selegiline. To the contrary, the above in vitro data suggest that use of desmethylselegiline as an MAO-B inhibitor requires on the order of 70 times the amount of selegiline. The potency of desmethylselegiline as an MAO-B inhibitor in vivo has been reported by Heinonen, E. H., et al. ("Desmethylselegiline, a metabolite of selegiline, is an irreversible inhibitor of MAO-B in human subjects," referenced in Academic Dissertation "Selegiline in the Treatment of Parkinson's Disease," from Research Reports from the Department of Neurology, University of Turku, Turku, Finland, No. 33 (1995), pp. 59- 61). According to Heinonen, desmethylselegiline in vivo has only about one-fifth the MAO-B inhibitory effect as selegiline, i.e., a dose of 10 mg of desmethylselegiline would be required for the same MAO-B effect as 1.8 mg of selegiline. The various diseases and conditions for which selegiline is known to be useful include: depression (U.S. patent 4,861,800); Alzheimer's disease and Parkinson's disease, particularly through the use of transdermal dosage forms, including ointments, creams and patches; macular degeneration (U.S. patent 5,242,950); age-dependent degeneracies, including renal function and cognitive function as evidenced by spatial learning ability (U.S. patent 5,151 ,449) ; pituitary-dependent Cushing ' s disease (U.S. patent 5 , 192 , 808) ; immune system dysfunction (U.S. patent 5,276,057); and schizophrenia (U.S. patent 5,151,419). PCT Published Application WO 92/17169 discloses the use of selegiline in the treatment of neuromuscular and neurodegenerative disease and in the treatment of CNS injury due to hypoxia, hypoglycemia, ischemic stroke or trauma. Although selegiline is known to be effective in treating the foregoing conditions, neither the precise number or nature of its mechanism or mechanisms of action are known. However, there is evidence that selegiline provides neuroprotection or neuronal rescue, possibly by reducing oxidative neuronal damage, increasing the amount of the enzyme superoxide dismutase, and/or reducing dopamine catabolism. For example, PCT Published Application WO 92/17169 reports that selegiline acts by directly maintaining, preventing loss of, and/or assisting in, the nerve function of animals. The biochemical effects of selegiline on neuronal cells has been extensively studied. For example, see Tatton, et al. , "Selegiline Can Mediate Neuronal Rescue Rather than Neuronal Protection," Movement Disorders 8 (Supp 1):S20-S30 (1993); Tatton, et a/., "Rescue of Dying Neurons," J. Neurosci. Res. 30:666-672 (1991); and Tatton, et al., "(-)- Deprenyl Prevents Mitochondrial Depolarization and Reduces Cell Death in Trophically- Deprived Cells," 11th Int'l Symp. on Parkinson 's Disease, Rome, Italy, March 26-30, 1994. Selegiline is known to be useful when administered to a subject through a wide variety of routes of administration and dosage forms. For example U.S. patent 4,812,481 (Degussa AG) discloses the use of concomitant selegiline-amantadine in oral, peroral, enteral, pulmonary, rectal, nasal, vaginal, lingual, intravenous, intraarterial, intracardial, intramuscular, intraperitoneal, intracutaneous, and subcutaneous formulations. U.S. patent 5,192,550 (Alza Corporation) describes a dosage form comprising an outer wall impermeable to selegiline but permeable to external fluids. This dosage form may have applicability for the oral, sublingual or buccal administration of selegiline. Similarly, U.S. patent 5,387,615 discloses a variety of selegiline compositions, including tablets, pills, capsules, powders, aerosols, suppositories, skin patches, parenterals, and oral liquids, including oil-aqueous suspensions, solutions, and emulsions. Also disclosed are selegiline- containing sustained release (long acting) formulations and devices. In addition to desmethylselegiline, selegiline produces one other principal direct metabolite, memamphetamine. Both desmethylselegiline and methamphetamine are further metabolized to amphetamine. The last two metabolites, amphetamine and methamphetamine, are known to have the potential to exert neurotoxic effects on dopamine neurons, and are undesirable by-products. Unlike selegiline, desmethylselegiline does not produce methamphetamine as a metabolite, only amphetamine. Accordingly, although a highly potent and selective MAO-B inhibitor, selegiline's practical use is circumscribed by its dose-dependent specificity for MAO-B, and the pharmacology of selegiline metabolites generated after administration. Summary of the Invention The present invention relates to the surprising discovery that both desmethyl¬ selegiline ("DMS" or "R(-)DMS") and its enantiomer (ent-desmethylselegiline, abbreviated as "Ent-DMS" or "S(+)DMS") are useful in providing selegiline-like effects in subjects, notwithstanding dramatically reduced MAO-B inhibitory activity and an apparent lack of enhanced selectivity for MAO-B compared to selegiline. In particular, the present invention relates to the surprising discovery that desmethylselegiline, ent-desmethylselegiline and their isomeric mixtures provide a more advantageous way of obtaining selegiline-like therapeutic effects in selegiline -responsive diseases or conditions. Thus, the present invention provides novel pharmaceutical compositions in which desmethylselegiline and/or ent-desmethylselegiline are employed as the active ingredients and novel therapeutic methods involving the administration of desmethylselegiline and ent-desmethylselegiline. Specifically, the present invention provides: (1) an improved method for obtaining selegiline-like therapeutic effects in a subject suffering from a selegiline-responsive disease or condition, which comprises: administering to said subject desmethylselegiline, ent-desmethylselegiline, or a mixture thereof, in an amount sufficient to produce a selegiline-like therapeutic effect. ; and (2) a pharmaceutical composition comprising, in addition to one or more optional pharmaceutically acceptable excipients or carriers, an amount of desmethylselegiline, ent- desmethylselegiline, or mixtures thereof, such that one or more unit doses of said composition administered on a periodic basis is effective to treat one or more selegiline- responsive diseases or conditions in a subject to whom said unit dose or unit doses are administered. As used herein the term "selegiline-responsive disease or condition" refers to any of the various diseases or conditions in mammals, including humans, for which selegiline is known to be useful. In particular, a "selegiline-responsive disease or condition" refers to the various diseases and conditions described above, e.g., Alzheimer's disease, cognitive dysfunction, neuronal rescue, and the like. Similarly, the term "selegiline-like therapeutic effect" refers to one or more of the salutary effects exerted by selegiline in a subject being treated for a selegiline-responsive disease or condition. The selegiline-responsive diseases or conditions related to neuronal degeneration or trauma which respond to the present methods include Parkinson's disease, Alzheimer's disease, depression, glaucoma, macular degeneration, ischemia, diabetic neuropathy, attention deficit disorder, post polio syndrome, multiple sclerosis, impotence, narcolepsy, chronic fatigue syndrome, alopecia, senile dementia, hypoxia, cognitive dysfunction, negative symptomatology of schizophrenia, amyotrophic lateral sclerosis, Tourette's syndrome, tardive dyskinesia, and toxic neurodegeneration. The present invention also encompasses the restoration or improvement of immune system function by R(-)DMS, S(+)DMS or mixtures thereof. Such improvement or restoration has been reported to occur when selegiline is administered to animals. The conditions or diseases treatable include age-dependent immune system dysfunction, AIDS, cancer and infectious diseases. Depending upon the particular route employed, either desmethyselegiline or ent- desmethylselegiline are administered in the form of a free base or as a physiologically acceptable non-toxic acid addition salt. Such salts include those derived from organic and inorganic acids such as, without limitation, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinic acid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid, salicylic acid, phthalic acid, embonic acid, enanthic acid, and the like. The use of salts, especially the hydrochloride, is particularly desirable when the route of administration employs aqueous solutions, as for example parenteral administration; use of delivered desmethylselegiline, or ent-desmethylselegiline, in the form of the free base is especially useful for transdermal administration. Accordingly, reference herein to the administration of DMS or ent-DMS or to mixtures thereof encompasses both the free base and acid addition salt forms. The optimal daily dose of desmethylselegiline and/or ent-desmethylselegiline useful for the purposes of the present invention is determined by methods known in the art, e.g., based on the severity of the disease or condition being treated, the condition of the subject to whom treatment is being given, the desired degree of therapeutic response, and the concomitant therapies being administered to the patient or animal. Ordinarily, however, the attending physician or veterinarian will administer an initial dose of at least about 0.0015 mg/kg, calculated on the basis of the free secondary amine, with progressively higher doses being employed depending upon the response to the therapy. Typically the daily dose will be about 0.01 mg/kg and may extend to about 0.5 mg/kg of the patient's body weight (all such doses again being calculated on the basis of the free secondary amine). These guidelines further require that the actual dose be carefully titrated by the attending physician or veterinarian depending on the age, weight, clinical condition, and observed response of the individual patient or animal. The daily dose can be administered in a single or multiple dosage regimen. The dosage form and regimen may permit, for example, a continuous release of relatively small amounts of the active ingredient from a single dosage unit, such as a transdermal patch, over the course of one or more days. This is particularly desirable in the treatment of chronic conditions such as Parkinson's disease, Alzheimer's disease, and depression. Alternatively, it can be desirable in conditions such as ischemia or neural damage to administer one or more discrete doses by a more direct systemic route such as intravenously or by inhalation. In still other instances such as glaucoma and macular degeneration, localized administration, such as via the intraocular route, can be indicated. In the case of oral administration, the present invention encompasses the unexpected discovery that the oral use of desmethylselegiline, and/or ent-desmethylselegiline, is more effective than oral selegiline for use in a variety of conditions and diseases. Accordingly, desmethylselegiline and its enantiomer are employed orally in subjects where the use of selegiline itself would be contraindicated due to side effects. Pharmaceutical compositions containing desmethylselegiline and/or ent- desmethylselegiline can be prepared according to conventional techniques. For example, preparations for parenteral routes of administration for desmethylselegiline, e.g., intramuscular, intravenous and intraarterial routes, can employ sterile isotonic saline solutions. Sterile isotonic solutions can also be employed for intraocular administration. Transdermal dosage unit forms of desmethylselegiline and/or ent- desmethylselegiline can be prepared utilizing a variety of previously described techniques (see e.g. , U.S. Patent Nos. 4,861,800; 4,868,218; 5,128,145; 5,190,763; and 5,242,950; and EP-A 404807, EP-A 509761, and EP-A 593807). For example, a monolithic patch structure can be utilized in which desmethylselegiline is directly incorporated into the adhesive and this mixture is cast onto a backing sheet. Alternatively desmethylselegiline, and/or ent- desmethylselegiline, can be incorporated as an acid addition salt into a multilayer patch which effects a conversion of the salt to the free base, as described for example in EP-A 593807. Desmethylselegiline and/or ent-desmethylselegiline can also be administered by a device employing a lyotropic liquid crystalline composition in which, for example, 5 to 15% of desmethylselegiline is combined with a mixture of liquid and solid polyethylene glycols, a polymer, and a nonionic surfactant, optionally with the addition of propylene glycol and an emulsifying agent. For further details on the preparation of such transdermal preparations, reference can be made to EP-A 5509761. Since the term "ent-desmethylselegiline" refers to the S(+)-isomeric form of desmethylselegiline, reference above to mixtures of selegiline and ent-desmethylselegiline includes both racemic and non-racemic mixtures of optical isomers. Subjects treatable by the present preparations and methods includes both human and non-human subjects for which selegiline-like therapeutic effects are known to be useful. Accordingly, the compositions and methods above provide especially useful therapies for mammals, especially domesticated mammals. Thus, the present methods and compositions are used in treating selegiline-responsive diseases or conditions in canine and feline species. Successful use of the compositions and methods above requires employment of an effective amount of desmethylselegiline, ent-desmethylselegiline or a mixture thereof. Although both desmethylselegiline and ent-desmethylselegiline are dramatically less potent than selegiline as inhibitors of MAO, employment of these agents, or a mixture of these agents, does not require a commensurately increased dosage to obtain a selegiline-like therapeutic response. Surprisingly, dosages necessary to attain a selegiline-like therapeutic effect are on the same order as the known doses of selegiline. Accordingly, because both desmethylselegiline and ent-desmethylselegiline exhibit a much lower inhibition of MAO-A at such dosages, desmethylselegiline and ent-desmethylselegiline provide a substantially wider margin of safety with respect to MAO-A associated toxicity compared to selegiline. In particular, the risk of the adverse effects of MAO-A inhibition, e.g., hypertensive crisis, are minimized due to the 40-70 fold reduced potency for MAO-A inhibition. As described above and notwithstanding its demonstrably inferior inhibitory proper- ties with respect to MAO-B inhibition, desmethylselegiline and its enantiomer are signifi¬ cantly more effective than selegiline in treating selegiline-responsive conditions, e.g., conditions resulting from neuronal degeneration or neuronal trauma. In this regard desmethylselegiline and/or its enantiomer, like selegiline itself, are particularly useful when administered by a route which does not rely upon upper GI tract or other gastrointestinal absorption. Preferred routes include the parenteral, topical, transdermal, intraocular, buccal, sublingual, intranasal, inhalation, vaginal, and rectal routes. As noted above, the present invention encompasses the additional discovery that desmethylselegiline can be employed in both optically active forms and in racemic form, i.e., as mixtures of desmethylselegiline and ent-desmethylselegiline. Desmethylselegiline, its enantiomer and mixtures thereof are conveniently prepared by methods known in the art, as described below in Example 1. Brief Description of the Figures Figure 1: Effect of Selegiline on Neuron Survival. Mesencephalic cultures were prepared from embryonic 14 day rats. Cultures were used at about 1.5 million cells per plate and were maintained either in growth medium alone (control cultures) or in growth medium supplemented with selegiline. On day 1, 8 and 15, cells were immunostained for the presence of tyrosine hydroxylase ("TH"). Solid bars represent results obtained for cultures maintained in the presence of 50 μM selegiline and open bars represent results for control cultures. In all cases, results are expressed as a percentage of TH positive cells present in control cultures on day 1. The abbreviation "DIV" refers to "days in vitro. " Asterisks or stars above bars both in Figure 1 and the figures discussed below indicates a result that differs from controls in an amount that is statistically significant, i.e. P<0.05 Figure 2: [ 3 H]-Dopamine Uptake in Mesencephalic Cells. Cells, cultured as described above for Figure 1 , were tested for their uptake of labeled dopamine and results are shown in Figure 2. Solid bars represent uptake in cells maintained in the presence of 50 μM selegiline and open bars represent uptake in control cultures Figure 3: Effect of Selegiline on Glutamate Receptor Dependent Neuronal Cell Death. Rat embryonic mesencephalic cells were cultured as described above. After allowing cultures to stabilize, the culture medium was changed daily for a period of 4 days to induce glutamate receptor-dependent cell death. Depending on the culture, medium contained either 0.5, 5.0 or 50 μM selegiline. After the final medium change, cultured cells were immunostained for the presence of tyrosine hydroxylase. From left to right, bars represent results for controls, 0.5, 5.0 and 50 μM selegiline Figure 4: Effect of Selegiline on Dopamine Uptake in Neuronal Cultures. Rat mesencephalic cells were cultured and medium was changed on a daily basis as discussed for Figure 3. Uptake of tritiated dopamine by cells was measured and results are shown in the figure. From left to right, bars are in the same order as for Figure 3. Figure 5: Effect of R(-)Desmethylselegiline on Glutamate Receptor Dependent Neuronal Cell Death. Rat embryonic mesencephalic cultures were prepared as described above except that R(-)DMS was used instead of selegiline. On day 9, the number of TH positive cells in cultures was determined. Results are expressed as a percentage of control. From left to right, bars show results for controls, 0.5, 5 and 50 μM R(-)DMS. Figure 6: Effect of R(-)Desmethylselegiline on Dopamine Uptake in Neuronal Cultures. Cell cultures were prepared as described above for Figure 5 and then tested for uptake of tritiated dopamine. Results for controls and for cells maintained in the presence of 0.5 μM, 5 μM and 50 μM desmethylselegiline are shown from left to right in the figure. Figure 7: Comparison of Dopamine Uptake in Mesencephalic Cells Incubated in the Presence of Different Monoamine Oxidase Inhibitors. Rat embryonic mesencephalic cells were prepared as described for Figures 3-6 and incubated in the presence of a variety of monoamine oxidase inhibitors. The inhibitors examined were selegiline; R(-) desmethylselegiline; pargyline; and clorgyline, all at concentrations of 0.5, 5 and 50 μM. In addition, cells were incubated in the presence of the glutamate receptor blocker MK-801 at a concentration of 10 μM. Cultures were tested for uptake of tritiated dopamine. Figure 8: Inhibition of Neuronal Dopamine Neuronal Re-Uptake by Deprenyl and the Enantiomers of Desmethylselegiline. An in vitro nerve terminal preparation (synaptosome preparation) was prepared using fresh rat neostriatal tissue. This was examined for its ability to take up tritiated dopamine in buffer alone or in buffer supplemented with various concentrations of selegiline, R(-)desmethylselegiline or S(+)desmethylselegiline. Uptake in the presence of each of the MAO inhibitors was expressed as a percent inhibition relative to uptake in the presence of buffer alone and results are shown in Figure 8. As indicated in the figure, the plots were used to determine the ID 50 for each test agent. The ID 50 for S(+)DMS was 20 μM; for selegiline, 80 μM; and for R(-)DMS about 100 μM. Figure 9: In Vivo MAO-B Inhibition in Guinea Pig Hippocampus. Various doses of selegiline, R(-)desmethylselegiline, and S(+)desmethylselegiline were injected daily into guinea pigs for a period of 5 days. Animals were then sacrificed and the MAO-B activity in the hippocampus portion of the brain was determined. Results were expressed as a percent inhibition relative to hippocampus MAO-B activity in control animals and are shown in Figure 9. The plots were used to determine the ID 50 dosage for each agent. The ID50 for selegiline was about 0.03 mg/kg; and for both enantiomers of DMS, about 0.3 mg/kg. Figure 10: In Vitro Interferon ("IFN") Production in Spleen Cells from Rats Injected with Selegiline, R(-)DMS or S(+)DMS. F344 rats were injected ip, daily for 60 days with saline, selegiline, R(-)DMS or S(+)DMS. All injected rats were old, i.e., between 18 and 20 months of age. After allowing a 10 day "wash out" period in which no injections were made, rats were killed and their spleens removed. The in vitro production of interferon-γ by the stimulated spleen cells (lymphocytes) was determined and compared with production by spleen cells (lymphocytes) from non-injected old rats and from young rats (3 months old). From left to right, the bars reflect results for spleen cells from young rats; from old rats, from old rats injected with saline; from old rats injected with 0.25 mg/kg of selegiline; from old rats injected with 1.0 mg/kg of selegiline; from rats injected with 0.025 mg/kg of R(-)DMS; from rats injected with 0.25 mg/kg of R(-)DMS; from rats injected with 1.0 mg/kg of R(-)DMS; and from rats injected with 1.0 mg/kg of S(+)DMS. The horizontal line indicates the point at which IFN levels differ significantly (P< .05) from the levels seen using young rat spleen cells. Results are expressed as units oer ml. Figure 11 : In Vitro IFN Production in Spleen Cells From Old Rats: The same results shown in Figure 10 are repeated 11 but without the inclusion of the results for young rat spleen cells. "#" indicates a result significantly different (P<0.05) from that obtained for spleens from old rats injects with saline. "*" indicates a result significantly diferent from all groups except old rats injected with 1.0 mg/kg of deprenyl. Figure 12: In Vitro Interleukin-2 Production by Spleen Cells from Old Rats. Rats were injected as described above (see Figure 10) and the production of interleukin-2 by stimulated spleen cells was determined. The order of bars from left to right is the same as for Figure 10. Figure 13: Percentage of Rat Spleen Cells that are IgM Positive. Rats were injected with saline, selegiline, R(-)DMS or S(+)DMS as described above (see Figure 10). The spleens from the rats were assayed to determine the percentage of cells that were IgM positive and results are shown in the figure. The horizontal line in the figure corresponds to the decrease in IgM percentage that is statistically significant (P<0.05) relative to the percentage seen in spleens obtained from young rats. The bars are in the same order from left to right as in Figures 10 and 12. Figure 14: Percentage of Rat Spleen Cells that are CD5 Positive. The experiment of Figure 13 was repeated but instead of measuring the percentage of cells that are IgM positive, the percentage that are CD5 positive was determined. Detailed Description of the Invention The surprising utility of desmethylselegiline and ent-desmethylselegiline in treating selegiline-responsive diseases or conditions is attributable in part to their powerful action in preventing loss of dopaminergic neurons by promoting repair and recovery. Hence, at doses as low as 0.01 mg/kg, at which little or no MAO-B inhibition is generally observed, a reversal in neuronal damage and/or death can be observed. Because desmethylselegiline and ent-desmethylselegiline prevent loss and facilitate recovery of nerve cell function, they are of value in a wide variety of neurodegenerative and neuromuscular diseases. In this regard, desmethylselegiline and ent-desmethylselegiline are substantially more potent than selegiline, as described more empirically in the examples below. The Examples are for illustrative purposes only and are not intended to limit the scope of the invention. Examples Example 1 : Preparation of Desmethylselegiline and Ent-desmethylselegiline A. Desmethylselegiline Desmethylselegiline (designated below as "R (-) DMS") is prepared by methods known in the art. For example, desmethylselegiline is a known chemical intermediate for the preparation of selegiline as described in U.S. Patent No. 4,925,878. Desmethyl¬ selegiline can be prepared by treating a solution of R(+)-2-aminophenylpropane (levoamphetamine) : in an inert organic solvent such as toluene with an equimolar amount of a reactive propargyl halide such as propargyl bromide, Br-CH 2 -C≡CH, at slightly elevated tem¬ peratures (70°-90°C). Optionally the reaction can be conducted in the presence of an acid acceptor such as potassium carbonate. The reaction mixture is then extracted with aqueous acid, for example 5% hydrochloric acid, and the extracts are rendered alkaline. The nonaqueous layer which forms is separated, for example by extraction with benzene, dried, and distilled under reduced pressure. Alternatively the propargylation can be conducted in a two-phase system of a water- immiscible solvent and aqueous alkali, utilizing a salt of R(+)-2-aminophenylpropane with a weak acid such as the tartrate, analogously to the preparation of selegiline as described in U.S. Patent No. 4,564,706. B. Ent-Desmethylselegiline Ent-desmethylselegiline (designated below as "S(+)DMS") is conveniently prepared from the enantiomeric S(-)-2-aminophenylpropane (dextroamphetamine), i.e., following the procedures set forth above for desmethylselegiline. C. Mixtures of Enantiomers Mixtures of enantiomeric forms of desmethylselegiline, including racemic desmethylselegiline, are conveniently prepared from enantiomeric mixtures, including racemic mixtures of the above aminophenylpropane starting material. D. Conversion Into Acid Addition Salts N-(prop-2-ynyl)-2-aminophenylpropane in either optically active or racemic form can be converted to a physiologically acceptable non-toxic acid addition salt by conventional techniques such as treatment with a mineral acid. For example, hydrogen chloride in isopropanol is employed in the preparation of desmethylselegiline hydrochloride. Either the free base or salt can be further purified, again by conventional techniques such as recrystallization or chromatography. Example 2: Neuronal Survival as Measured Using Tyrosine Hydroxylase The effect of desmethylselegiline on neuron survival can be correlated to tyrosine hydroxylase, the rate limiting enzyme in dopamine biosynthesis. Assays are performed by determining the number of tyrosine hydroxylase positive cells in cultured E-14 embryonic mesencephalic cells over a period of 7 to 14 days. Protection in this system has been seen with a variety of trophic factors including BDNF, GDNF, EGF, and β-FGF. A. Test Methods Timed pregnant Sprague-Dawley rats are used to establish neuronal cultures from embryonic rat brain on the 14th day of gestation. Mesencephalon is dissected out without the membrane coverings and collected in Ca ++ and Mg ++ free balanced salt solution at 4°C. Tissue fragments are dissociated in chemically defined medium by mild trituration with a small bore pasteur pipette. Cell suspension is plated in polyornithine-coated 35 mm Falcon plastic dishes (0.1 mg/ml, Sigma) at a density of 1.5X10 * cells/dish. Cultures are maintained at 37°C in an atmosphere of 10% CO 2 /90% air and 100% relative humidity, and fed twice weekly with chemically defined medium consisting of MEM/F12 (1 :1, Gibco), glucose (33 mM), HEPES (15 mM), NaHCO 3 (44.6 raM), transferrin (100 mg/ml), insulin (25 mg/ml), putrescine (60 nM), sodium selenite (30 nM), progesterone (20 nM), and glutamine (2 mM). Control cells receive no further additions. The medium used for other cells also included test substance, e.g. selegiline, at one or more concentrations. Cultures are fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 minutes at room temperature, permeabilized with 0.2% Triton X-100 for 30 minutes and incubated with an antibody against tyrosine hydroxylase (1 :1000; Eugene Tech) for 48 hours at 4°C in the presence of a blocking serum. They are then stained using a peroxidase-coupled avidin-biotin staining kit (Vectastain ABC kit; Vector Labs) with 3\3'- diaminobenzidine as a chromagen. The number of dopaminergic neurons in cultures is determined by counting the cells positively immunostained with TH antibodies. 100 fields (0.5 mm X 0.5 mm) in two transverse strips across the diameter of the dish, representing 2.5% of the total area, are counted using a Nikon inverted microscope at 200X magnification. B. Results Using the procedures described above, the following results were obtained: Table 2: Effect of Selegiline and DMS on the Survival of TH Positive Cells Control Selegiline Desmethylselegiline Mean Mean %Cont Mean %Cont Cone. 0.5μM 108.55 201.70 ± 25.01 185.81 246.00 ± 22.76 226.62 5 μM 237.00 ± 12.59 218.33 357.95 ± 25.76 329.76 50 μM 292.28 ± 17.41 269.25 391.60 ± 34.93 360.76 Example 3: Neuronal Survival as Measured Using Dopamine Uptake In addition to determining the number of TH positive cells in culture (see Example 2) the protective effect of desmethylselegiline on neuronal cells also can be determined by directly measuring dopamine uptake. The amount of uptake by the cultured brain cells corresponds to axonal growth. A. Test Methods Cell cultures, established in the manner discussed above, are incubated with [^H]dopamine (0.5 mCi/ml; 37 Ci/mmol; New England Nuclear) for 15 minutes in the presence of ascorbic acid (0.2 mg/ml) in PBS (pH 7.3), supplemented with 0.9 mM CaCl 2 and 0.5 mM MgCl at 37 °C. After two rinses and a 5 minute incubation with fresh buffer, H]dopamine accumulated within the cells is released by incubating the cultures with 95% ethanol for 30 minutes at 37 °C. Preparations are then added to 10 ml Ecoscint (National Diagnostics) and counted in a scintillation spectrometer. Nonspecific uptake values are obtained by blocking dopaminergic neuronal uptake with 10 mM mazindol. B. Results Using the above procedure, the results shown in Table 3 were obtained. Table 3: Effect of Selegiline and DMS on 3 H-Dopamine Uptake Cont. Selegiline Desmethylselegiline Mean Mean %Cont Mean %Cont Cone. 0.5μM 11982 14452 ± 212 120.6 24020 ± 800 200.4 5 μM - 16468 ± 576 137.5 34936 ± 2119 291.5 50 μM - 33018 ± 1317 275.5 56826 ± 2656 474.3 | C. Conclusions from Examples 2 and 3 The results described in Examples 2 and 3 indicate that desmethylselegiline is superior to selegiline as a neuroprotective agent. This is true notwithstanding the fact that desmethylselegiline in much less potent than selegiline as an inhibitor of MAO-B. Example 4: Neuroprotective Action of Desmethylselegiline Enantiomers in Cultured Doparmne-Containing Mesencephalic Neurons In Vitro The survival of mesencephalic, dopamine-containing neuronal cultures of rat brain tissue was used in these experiments to examine neuroprotective properties of selegiline and R(-) desmethylselegiline. The number of TH positive neurons is directly proportional to the survival of dopaminergic neurons and 3 H-dopamine uptake is a measure of axonal growth in these neurons A. Effect of Selegiline on the Survival of Dopaminergic Neurons. Mesencephalic cultures prepared from embryonic day 14 rats were treated with 0.5, 5 or 50 μM selegiline for 15 days, beginning on the day of plating. (For a more detailed discussion of the culturing of cells and other methods used in these experiments see Mytilineou et al., J. Neurochem.61 : 1470-1478 (1993).) Survival and growth of dopamine neurons was evaluated by tyrosine hydroxylase (TH) immunocytochemistry and [ 3 H]dopamine uptake and results are shown in figures 1 and 2. No effect was observed on neuron survival of when selegiline was tested at concentrations of 0.5 and 5 μM. At 50 μM, selegiline reduced the loss of TH-positive neurons at 8 and 15 days after plating (Figure 1) and increased dopamine uptake at 15 days (Figure 2). In separate experiments it was determined that pre-treatment with 0.5, 5, 50 or 100 μM selegiline did not inhibit the uptake of dopamine under the assay conditions used in the experiments. The results indicate that the uptake values obtained reflect dopamine neuron survival and process outgrowth and that selegiline has a neuroprotective effect. B. Effect of Selegiline on Glutamate Receptor Dependent Cell Death. The neuroprotective effect of selegiline was also examined using an experimental paradigm that causes neuronal cell death that can be blocked by inhibition of glutamate receptors. In these experiments cells were plated and allowed to stabilize for several days. The growth medium of the cells was then changed on a daily basis to induce cell death that can be prevented by blocking glutamate receptors, e.g. using MK-801. After 4 days of daily medium changes cultures were stained for tyrosine hydroxylase and assayed for uptake of tritiated dopamine. The results shown in Figures 3 and 4 further support the conclusion that selegiline promotes the survival of dopaminergic neurons. C. Effect of Desmethylselegiline on the Survival of Dopamine Neurons. Using the glutamate receptor dependent model of neuron death, an even more potent protection of dopaminergic neurons was provided when desmethylselegiline was used in place of selegiline. Even at the lowest dose tested (0.5 μM), desmethylselegiline caused a significant reduction in the loss of TH positive neurons (Figure 5) and a significant increase in dopamine uptake (Figure 6) relative to control cultures in which medium was sued without supplementation with either selegiline or desmethylselegiline. D. Comparison With Other MAO Inhibitors. Using the glutamate receptor dependent paradigm of neurotoxicity, the effects of selegiline and desmethylselegiline were compared with two other MO A inhibitors, pargyline and clorgyline (Figure 7). In agreement with previous results, measurement of dopamine uptake indicated neuron protection by 50 μM deprenyl and 5 and 50 μM desmethylselegiline. Pargyline did not appear to offer any protection at the concentrations used, while clorgyline protected at 50 μM. As expected protection was also obtained by the NMDA receptor blocker MK-801 (10 μM). E. Effect of DMS Enantiomers on 3 H-Dopamine Uptake The data summarized in Table 4 suggests that both (R-)DMS and S(+)DMS are effective as neuroprotectants in mesencephalic dopamine-containing neurons in culture. Table 4: Effect of DMS Enantiomers on Dopamine Uptake 3 H-Dopamine uptake Treatment as a percentage + SEM Control 100 ± 14.14% R(-) DMS (lOnM) 140.82 ± 26.20% S(+) DMS (lOnM) 234 ± 38.36% These results demonstrate that, compared to untreated control cells, there was 40% and 134% more axonal growth and terminal axonal survival after treatment with R(-) DMS and S(+) DMS, respectively, Thus, S(+) DMS may be an even more potent and/or efficacious neuroprotectant than R(-) DMS. Example 5: Desmethylselegiline and Ent-Desmethylselegiline as Inhibitors of Dopamine Re-Uptake The biological actions of the brain neurotransmitter dopamine are terminated at the synapse by a high-affinity, sodium and energy -dependent transport system (neuronal re- uptake) present within the limiting membrane of the presynaptic dopamine - containing nerve terminal. Inhibition of this transport mechanism would extend the actions of dopamine at the synapse and therefore enhance dopamine synaptic transmission. A. Method of Testing The R(-) and S(+) enantiomers of desmethylselegiline (DMS) were tested for their ability to inhibit the dopamine re-uptake system and compared to selegiline. Inhibitory activity in this assay is indicative of agents of value in the treatment of diseases which require enhanced synaptic dopamine activity. Presently this would include Parkinson's Disease, Alzheimer's Disease and Attention Deficit Hyperactivity Disorder (ADHD). The assay system used was essentially that described by Fang et al. (Neuro- pharmacology 55:763-768 (1994)). An in vitro nerce-temiinal preparation (synaptosome preparation) was obtained form fresh rat neostriatal brain tissue. Transport by dopamine nerve-terminals was estimated by measuring the uptake of tritiated dopamine. B. Results As seen in the data presented in Table 5, selegiline, R(-) DMS and S(+) DMS all inhibited dopamine re-uptake by dopamine-containing nerve terminals. Selegiline and R(-) DMS were approximately equipotent. In contrast, S(+) DMS was 4-5 times more potent than either selegiline or R(-) DMS. Table 5: 3 H-Dopamine Uptake By Rat Neostriatal Brain Tissue Agent Concentration % Reduction x±SEM Dopamine lμM 52.0 ± 4.9 lOμM 80.9 ± 0.4 Selegiline lOOnM 7.0 ± 3.6 lμM 13.9 ±4.7 lOμM 16.3 ±3.8 lOOμM 59.8 ±1.0 R(-) DMS lOOnM 11.5±1.0 lμM 10.7 ±2.8 lOμM 20.1 ±3.1 lOOμM 51.3±2.6 S(+) DMS lOOnM 15.3 ±7.7 lμM 24.1 ±11.7 lOμM 47.0 ±3.1 lOOμM 76.9 ±1.8 Relative potency can be expressed in terms of the concentration required to inhibit dopamine re-uptake by 50% (ID 50 ). The ID 50 values were determined graphically (see Figure 8) and are shown below in Table 6. Table 6: Concentrations Needed to Inhibit Dopamine Uptake by 50% Agent lUso Selegiline ~ 80 μm R(-)DMS =100μm S(+)DMS =20μm C. Conclusions The results demonstrate that, at the appropriate concentration, selegiline and each of the enantiomers of DMS inhibit transport of released dopamine at the neuronal synapse and enhance the relative activity of this neurotransmitter at the synapse. In this regard, S(+) DMS is more potent than selegiline which, in turn, is more potent than R(-) DMS. This effect is indicative of agents beneficial in the treatment of Parkinson's Disease, Alzheimer's Disease and Attention Deficit Hyperactivity Disorder (ADHD). Of the agents tested, S(+) DMS appears to be the most preferred with regard to the treatment of ADHD. Example 6: Actions of the R(-) and S(+) Enantiomers of Desmethylselegiline (DMS) on Human Platelet MAO-B and Guinea Pig Brain MAO-B and MAO-A Activity Human platelet MAO is comprised exclusively of the type -B isoform of the enzyme. In the present study, the inhibition of this enzyme by the two enantiomers of DMS was determined and compared with inhibition due to selegiline. In addition, the present study examined the two enantiomers of DMS for inhibitory activity with respect to the MAO-A and MAO-B in guinea pig hippocampal tissue. Guinea pig brain tissue is the best animal model for studying brain dopamine metabolism, the enzyme kinetics of the multiple forms of MAO and the inhibitory properties of novel agents that interact with these enzymes. The multiple forms of MAO in this animal species show identical kinetic properties to those found in human brain tissue. Finally, agents were injected into guinea pigs to determine the extent to which they might act as inhibitors of brain MAO in vivo. B. Method of Testing: The test system utilized the in vitro conversion of specific substrates of MAO-A ( 14 C-serotonin) and MAO-B ( 14 C-phenylemylamine) by human platelets and/or guinea pig hippocampal homogenates. The rate of conversion of each substrate was measured in the presence of S(-t-) DMS, R(-) DMS or selegiline and compared to the isozyme activity in the absence of these agents. A percent inhibition was calculated from these values. Potency was evaluated by comparing the concentration of each agent which caused a 50% inhibition (IC 50 value). In some experiments, R(-) DMS, S(-f-) DMS or selegiline was administered in vivo subcutaneously (s.c), once a day for 5 days prior to sacrifice, preparation of enzyme hippocampal homogenates, and the in vitro assay of MAO-A and MAO-B activity. These experiments were performed to demonstrate that the DMS enantiomers were capable of entering brain tissue and inhibiting MAO activity. C. Results MAO-B Inhibitory Activity In Vitro Results for MAO-B inhibition are shown in Tables 7 and 8. IC S0 values for MAO-B inhibition and potency as compared to selegiline is shown in Table 9. Table 7: MAO-B Inhibition in Human Platelets Agent Concentration % Inhibition x ± SEM Selegiline 0.3nM 8.3 ±3.4 5nM 50.3 ± 8.7 lOnM 69.0 ±5.5 30nM 91.0±1.4 lOOnM 96.0 ±1.6 300nM 96.0 ±1.6 lμM 96.6 ±1.6 R(-) DMS lOOnM 14.3 ±3.6 300nM 42.1 ±4.0 lμM 76.9 ±1.47 3μM 94.4 ± 1.4 lOμM 95.8 ±1.4 3μM 95.7 ±2.3 S(+) DMS 300nM 6.4 ±2.8 lμM 11.1 ±1.0 3μM 26.6 ±1.9 lOμM 42.3 ±2.3 30μM 68.2 ±2.34 lOOμM 83.7 ±0.77 ImM 94.2 ±1.36 Table 8: MAO-B Inhibition in Guinea Pig Hippocampus Agent Concentration % Inhibition x ±SEM Selegiline 0.3nM 28.3±8.7 5nM 81.2±2.6 lOnM 95.6 ±1.3 30nM 98.5 ±0.5 lOOnM 98.8 ±0.5 300nM 98.8 ±0.5 lμM 99.1 ±0.45 R(-) DMS lOOnM 59.4 ±9.6 300nM 86.2 ±4.7 lμM 98.2 ±0.7 3μM 98.4 ±0.95 lOμM 99.1 ±0.45 30μM 99.3 ±0.40 S(+) DMS 300nM 18.7±2.1 lμM 44.4 ±6.4 3μM 77.1 ±6.0 lOμM 94.2 ±1.9 30μM 98.3 ±0.6 lOOμM 99.3 ± 0.2 lmM 99.9 ±0.1 Table 9: IC, n Values for the Inhibition of MAO-B Guinea Pig Treatment Human Platelets Hippocampal Cortex Selegiline 5nM(l) lnM(l) R(-) DMS 400 nM (80) 60 nM (60) S(-l-) DMS 1400 nM (2800) 1200 nM (1200) ( ) = potency compared to selegiline As observed, R(-) DMS was 20-35 times more potent than S(+) DMS as an MAO- B inhibitor and both enantiomers were less potent than selegiline. MAO-A Inhibitory Activity Results obtained from experiments examining the inhibition of MAO-A in guinea pig hippocampus are summarized in Table 10 and IC 50 values for the two enantiomers of DMS and for selegiline are shown in Table 11. Table 10: MAO-A Inhibition in Guinea Pig Hippocampus Agent Concentration % Reduction x ± SEM Selegiline 300nM 11.95 ±2.4 lμM 22.1 ±1.2 3μM 53.5 ±2.7 lOμM 91.2 ±1.16 lOOμM 98.1 ±1.4 lmM 99.8 ±0.2 R(-) DMS 300nM 4.8 ±2.1 lμM 4.2 ±1.5 3μM 10.5 ±2.0 lOμM 19.0 ±1.3 lOOμM 64.2 ±1.5 lmM 96.5 ±1.2 S(+) DMS lμM 3.3 ±1.5 3μM 4.3 ±1.0 lOμM 10.5 ±1.47 100 μM 48.4 ±1.8 lmM 92.7 ±2.5 lOmM 99.6± 0.35 Table 11 : IC 50 Values for the Inhibition of MAO-A ICso for MAQ-A in Guinea Pig Treatment Hippocampal Cortex Selegiline 2.5 μM (1) R(-) DMS 50.0 μM (20) S(+) DMS 100.0 μM (40) ( ) = potency compared to selegiline R(-) DMS was twice as potent as S(+) DMS as an MAO-A inhibitor and both were 20-40 times less potent than selegiline. Moreover, each of these agents were 2-3 orders of magnitude, i.e., 100 to 1000 times, less potent as inhibitors of MAO-A than inhibitors of MAO-B in hippocampal brain tissue. Therefore, selegiline and each enantiomer of DMS can be classified as selective MAO-B inhibitors in brain tissue. Results of In Vivo Experiments Each enantiomer of DMS was administered in vivo by subcutaneous injection once a day for five consecutive days, and inhibition of brain MAO-B activity was then deteπnined. Under these conditions, R(-) DMS and S(+) DMS were equipotent as MAO- B inhibitors but ten times less potent than selegiline. Results are shown in Figure 9 and IC S0 values are summarized in Table 12. Table 12: IC 50 Values for Brain MAO-B When Agents are Injected Prior to Assay ICso for MAQ-B in Guinea Pig Treatment Hippocampal Cortex Selegiline 0.03 mg/kg R(-) DMS 0.30 mg/kg S(+) DMS 0.30 mg/kg This experiment demonstrates that each of the enantiomers of DMS will penetrate the blood brain-barrier and inhibit brain MAO-B after parenteral in vivo administration. It also demonstrates that the potency differences as an MAO-B inhibitor observed in vitro between each of the DMS enantiomers and selegiline, are reduced under in vivo conditions. The dosages administered in this experiment, were not sufficient to inhibit MAO-A activity. D. Conclusions: R(-) DMS and S(+) DMS both demonstrate activity as an MAO-B and MAO-A inhibitors. Each enantiomer was selective for MAO-B. S(+) DMS was generally less potent than R(-) DMS and both enantiomers of DMS were less potent than selegiline in inhibiting both MAO-A and MAO-B. Both enantiomers demonstrated activity after in vivo administration, indicating that these enantiomers are able to enter brain tissue after parenteral administration. The ability of these agents to inhibit MAO-A or MAO-B suggests that these agents are of value as therapeutics for Parkinson's disease, Alzheimer's disease or depression. Example 7: In Vivo Neuroprotection by the Enantiomers of Desmethylselegiline The ability of the enantiomers of DMS to prevent neurological deterioration was examined by administering the agents to the wobbler mouse, an animal model of motor neuron diseases, particularly amyotrophic lateral sclerosis (ALS). Wobbler mice exhibit progressively worsening forelimb weakness, gait disturbances, and flexion contractions of the forelimb muscles. B. Test Method R(-)DMS, S( + )DMS or placebo was administered to wobbler mice by daily intra- peritoneal injection for a period of 30 days in a randomized, double-blind study. At the end of this time mice were examined for grip strength, running time, resting locomotive activity and graded for semiquantitative paw posture abnormalities, and semiquantitative walking abnormalities. The investigators who prepared and injected the solutions to the animals and who analyzed behavioral changes were different. Assays and grading were performed essentially as described in Mitsumoto et al., Ann. Neurol. 56:142-148 (1994). Grip strength of the front paws of a mouse was determined by allowing the animal to grasp a wire with both paws. The wire was connected to a gram dynamometer and traction is applied to the tail of the mouse until the animal is forced to release the wire. The reading on the dynamometer at the point of release is taken as a measure of grip strength. Running time is defined as the shortest time necessary to traverse a specified distance, e.g. 2.5 feet and the best time of several trials is recorded. Paw posture abnormalities are graded on a scale based upon the degree of contraction and walking abnormalities are graded on a scale ranging from normal walking to an inability to support the body using the paws. Locomotive activity is determined by transferring animals to an examination area in which the floor is covered with a a square grid.. Activity is measured by the number of squares traversed by a mouse in a set time interval, e.g. 9 minutes. C. Results At the beginning of the study, none of the groups were different in any variables, indicating that the three groups were comparative at the baseline. Weight gain was identical in all three groups, suggesting that no major side effects occurred in any animals. Table 13 summarizes differences that were observed in the mean grip strength of the test animals: Table 13: Mean Grip Strength in Wobbler Mice Treated with R(-) or S(+) DMS Treatment N Grip Strength (ς ) Control (placebo) 10 9(0-15) R(-)DMS 9 20(0 - 63) S(+)DMS 9 14(7 - 20) N = number of animals analyzed Grip strength dropped markedly at the end of the first week in all animals. At the end of the study, grip strength was the least in control animals, ranging from 0 to 15 g, with a mean of 9 g. The R(-) group had a mean grip strength of 20 g with values ranging from 0 to 63 g. The third group, injected with S(+) DMS, had a mean grip strength of 14 g, with individual values ranging from 7 to 20 g. While the variability in grip strength in the treated animal groups prevented a meaningful statistical analysis of this data, the mean grip strength measured in the DMS-treated animals was greater than for the controls. Running time, resting locomotive activity, semiquantitative paw posture abnormality grading, and semiquantitative walking abnormality grading were also tested. None of these tests, however, showed data suggesting that any one of these three groups differ from the other. Example 8: Immune System Restoration by R(-)DMS and S(+)DMS There is an age-related decline in immunological function that occurs in animals and humans which makes older individuals more susceptible to infectious disease and cancer. U.S. patents 5,276,057 and 5,387,615 suggest that selegiline is useful in the treatment of immune system dysfunction. The present experiments were undertaken to determine whether R(-)DMS and S(+) are also useful in the treatment of such dysfunction. It should be recognized that an ability to bolster a patient's normal immunological defenses would be beneficial in the treatment of a wide variety of acute and chronic diseases including cancer, AIDS, and both bacterial and viral infections. A. Test Procedure The present experiments utilized a rat model to examine the ability of R(-)DMS and S(+)DMS to restore immunological function. Rats were divided into the following experimental groups: 1) young rats (3 months old, not injected); 2) old rats (18-20 months old, not injected); 3) old rats injected with saline; 4) old rats injected with selegiline at a dosage of 0.25 mg/kg body weight; 5) old rats injected with selegiline at a dosage of 1.0 mg/kg body weight; 6) old rats injected with R(-)DMS at a dosage of 0.025 mg/kg body weight; 7) old rats injected with R(-)DMS at a dosage of 0.25 mg/kg body weight; 8) old rats injected with R(-)DMS at a dosage of 1.0 mg/kg body weight; 9) old rats injected with S(+)DMS at a dosage of 1.0 mg/kg body weight. Rats were injected ip, daily for 60 days. They were then maintained for an additional "wash out" period of 10 days during which time no injections were given. At the end of this time, animals were sacrificed and their spleens were removed. The spleen cells were then assayed for a variety of factors which are indicative of immune system function. Specifically, standard tests were employed to determine the following: 1) in vitro production of γ-interferon by spleen cells; 2) in vitro production of interleukin-2; 3) percentage of IgM positive spleen cells (IgM is a marker of B lymphocytes); 4) percentage of CD5 positive spleen cells (CD5 is a marker of T lymphocytes). B. Results The effect of injections of selegiline, R(-)DMS and S(+)DMS on interferon production by rat spleen cells is shown in Figures 10 and 11. As shown in Figure 10, there is a sharp decrease in cellular interferon production that occurs with age (compare production by cells from young animals with production by cells from old animals or from old animals injected with saline. Injections of selegiline, R(-)DMS and S(+)DMS all led to a partial restoration of γ-interferon levels with the most dramatic increases occurring at dosages of 1.0 mg/kg body weight. The same data as shown in Figure 10 is repeated in Figure 11 except that results for cells from young rats is omitted. The figure more clearly shows the extent to which deprenyl, R(-)DMS and S(+)DMS are capable of restoring γ-interferon production in the spleen cells of old rats. Interferon-γ is a multifunctional protein that inhibits viral replication and regulates a variety of immunological functions. It influences the class of antibodies produced by B-cells, up-regulates class I and class II MHC complex antigens and increases the efficiency of macrophage-mediated killing of intracellular parasites. Figure 12 shows the effect of injections on the production of interleukin-2 by the rat spleen cells. It can be seen that both R(-)DMS and S(+)DMS are capable of restoring production to levels seen in cells from young animals. The effect of injections on the percentage of spleen cells that are IgM positive is shown in Figure 13. It was found that both selegiline and R(-)DMS partially restored IgM positive cells to a level closer to that seen in the spleens of young rats. Thus, it appears that these agents are restoring B lymphocyte cell number. Figure 14 suggests that injection of either 0.025 mg/kg of R(-)DMS or S(+)DMS may have slightly increased the percentage of CD5 positive cells in the spleens obtained from old rats. However, neither injections with selegiline nor with 0.25 or 1.0 mg/kg of R(-)DMS appeared to have any effect. C. Conclusions Overall, the results support the conclusion that the enantiomers of DMS mimic the effects of selegiline on immune system function. Moreover, the results obtained with respect to the production of interferon, IL-2 and on the percentage of IgM positive spleen cells support the conclusion that the DMS enantiomers are capable of at least partially restoring the age-dependent loss of immune system function. Thus, it appears that R(-) DMS and S(+ )DMS will have a therapeutically beneficial effect for diseases and conditions facilitated by weakened host immunity. This would include cancer, AIDS, and infectious diseases of all types. Example 9: Examples of Dosage Forms A. Desmethylselegiline Patch. Dry Weight Basis Component (mg/cm^) Durotak ® 87-2194 adhesive acrylic polymer 90 parts by weight Desmethylselegiline 10 parts by weight The two ingredients are thoroughly mixed and cast on a Scotchpak ® 9723 polyester film backing sheet, and dried. The backing sheet is cut into patches, a Scotchpak ® 1022 fluoropolymer release liner is applied, and the patch is hermetically sealed in foil envelopes. One patch is applied daily to supply 1-5 mg of desmethylselegiline per 24 hours in the treatment of conditions in a human produced by neuronal degeneration or neuronal trauma, as for example Parkinson's disease. B. Ophthalmic Solution Desmethylselegiline (0.1 g) as the hydrochloride, 1.9 g of boric acid, and .004 g of phenyl mercuric nitrate are dissolved in sterile water qs 100ml. The mixture is sterilized and sealed. It can be used ophthalmologically in the treatment of conditions produced by neuronal degeneration or neuronal trauma, as for example glaucomatous optic neuropathy and macular degeneration. Example 3: Intravenous Solution. A 1 % solution is prepared by dissolving 1 g of desmethylselegiline as the HC1 in sufficient 0.9% isotonic saline solution to provide a final volume of 100 ml. The solution is buffered to pH 4 with citric acid, sealed, and sterilized to provide a 1 % solution suitable for intravenous administration in the treatment of conditions produced by neuronal degeneration or neuronal trauma. C. Transdermal Patch. A self-crosslinking acrylic based pressure sensitive adhesive is added to a solution of a copolymer of methacrylic acid and dimethylaminoethyl methacrylate in an organic solvent such as methyl ethyl ketone. This is cast onto a first removable foil, the solvents are evaporated, and the coated foil placed on a polyester backing layer, as set forth in EP- A 593807. Desmethylselegiline hydrochloride is added to a solution of a non-crosslinking acrylic based pressure sensitive adhesive in a suitable organic solvent, as for example ethyl acetate. Additional solvent can be added and heat and stirring can be applied to facilitate formation of the dispersion. This is coated onto a second removable foil and the solvent evaporated. After removing the foil from the previously prepared polyester backed layer, it is laminated to the coated second removable foil. Additional self-crosslinking acrylic based pressure sensitive adhesive and copolymer of methacrylic acid and dimethylaminoethyl methacrylate in an organic solvent such as methyl ethyl ketone are cast onto a third removable foil and the solvent evaporated. The second and third foils are removed, the residual layers laminated, and the resulting laminate cut into patches and packaged. The resulting patches will have a removable release liner, an adhesive layer initially free of desmethylselegiline, and a matrix layer containing desmethylselegiline as the hydrochloride (or other salt). An impenetrable backing layer is adhered to the matrix layer through an intermediate adhesive layer similar to the adhesive layer contiguous to the removable release liner. One patch is applied daily to supply desmethylselegiline as the free base in the treatment of conditions in a human produced by neuronal degeneration or neuronal trauma, as for example Parkinson's disease. D. Oral Dosage Form Tablets and capsules containing desmethylselegiline are prepared from the following ingredients (mg/unit dose): desmethylselegiline 1-5 microcrystalline cellulose 86 lactose 41.6 citric acid 0.5-2 sodium citrate 0.1-2 magnesium stearate 0.4 with an approximately 1 : 1 ratio of citric acid and sodium citrate.

Claims: What is claimed is: 1. A pharmaceutical composition comprising, in addition to one or more optional pharmaceutically acceptable excipients or carriers, an amount of desmethylselegiline, ent-desmethylselegiline, or mixtures thereof, such that one or more unit doses of said composition, administered on a periodic basis, is effective to treat one or more selegiline-responsive diseases or conditions in a subject to whom said unit dose or unit doses are administered. 2. A composition according to claim 1, for oral administration. 3. A composition according to claim 1, for transdermal administration. 4. A composition according to claim 1, wherein desmethylselegiline is employed as a substantially pure stereoisomer. 5. A composition according to claim 1, adapted for effecting neuronal rescue or neuronal protection. 6. A composition according to claim 1, adapted for restoring or improving immune system function in a subject. 7. An improved method for obtaining selegiline-like therapeutic effects in a subject suffering from a selegiline-responsive disease or condition, which comprises: administering to said subject desmethylselegiline, ent-desmethylselegiline, or a mixture thereof, in an amount sufficient to produce a selegiline-like therapeutic effect. 8. A method according to claim 7, wherein said subject is human. 9. A method according to claim 8, wherein ent-desmethylselegiline is employed as a substantially pure stereoisomer in the treatment of ADHD. 10. A method according to claim 7, wherein the selegiline-like therapeutic effect is neuronal rescue or neuronal protection. 11. A method according to claim 7, wherein the selegiline-like therapeutic effect is an improvement or restoration of immune system function. 12. A method of treating a condition in a mammal produced by neuronal degeneration or neuronal trauma which comprises administering to the mammal desmethylselegiline or ent-desmethylselegiline, or a pharmaceutically acceptable acid addition salt thereof, at a daily dose, administered in a single or multiple dosage regimen, of at least about 0.0015 mg, calculated on the basis of the free secondary amine, per kg of the mammal's body weight. 13. A method according to claim 12, comprising at least one route of administration which does not rely upon gastrointestinal absorption. 14. The method of claim 12, wherein the R-(-) enantiomer of desmethylselegiline is administered transdermally as the free base. 15. The method of claim 12, wherein said R-(-) enantiomer is administered parenterally as a pharmaceutically acceptable acid addition salt. 16. The method of claim 15, wherein the pharmaceutically acceptable acid addition salt is the hydrochloride salt. 17. The method according to claim 12, wherein the daily dose, administered in a single or multiple dosage regimen, is from about 0.01 to about 0.5 mg, calculated on the basis of the free secondary amine, per Kg of the mammal's body weight. 18. The method according to claim 12, wherein the mammal is a human. 19. The method according to claim 12, wherein the mammal is a canine. 20. The method according to claim 12, wherein the mammal is a feline. 21. A transdermal delivery composition for use in treating a condition in a mammal produced by neuronal degeneration or neuronal trauma which comprises a layered composite containing in at least one layer an amount of desmethylselegiline, ent- desmethylselegiline or a pharmaceutically acceptable acid addition salt thereof, sufficient to supply a daily transdermal dose of at least about 0.0015 mg of the free secondary amine, per Kg of the mammal 's body weight. 22. A method of treating a condition in a mammal produced by immune system dysfunction which comprises administering to the mammal desmethylselegiline or ent-desmethylselegiline, or a pharmaceutically acceptable acid addition salt thereof, at a daily dose, administered in a single or multiple dosage regimen, of at least about 0.0015 mg, calculated on the basis of the free secondary amine, per kg of the mammal's body weight.

Inventors: BLUME CHERYL D, DISANTO ANTHONY R, BLUME, CHERYL, D., DISANTO, ANTHONY, R.
Applicants: BLUME CHERYL D, DISANTO ANTHONY R, SOMERSET PHARMACEUTICALS INC, BLUME, CHERYL, D., DISANTO, ANTHONY, R., SOMERSET PHARMACEUTICALS, INC.
Application year: 1996
Main classification: A61K31
IPC codes: A61K31, A61P25, A61P37, A61P43