NO321830B1 - Desmethylselegiline or salt thereof, drug comprising the compound, and uses thereof - Google Patents

Abstract

Methods and pharmaceutical compositions for the use of the selegiline metabolite desmethylselegiline and stereoisomeric forms thereof. The present invention provides in particular new compositions and methods for the use of desmethylselegiline, and/or ent-desmethylselegiline, for selegiline-responsive diseases and conditions. Diseases and conditions responsive to selegiline include those produced by neuronal degeneration or neuronal trauma, and those resulting from immune system dysfunction. Desmethylselegiline, and/or ent-desmethylselegiline, is administered either orally or by a route of administration that does not depend on absorption in the gastrointestinal system. Desmethylselegiline is the R(-)-enantiomer of N-methylN-(prop)-2-ynyl)-2-aminophenylpropane and ent-desmethylselegiline is the S(+)-enantiomer. Claimed mixtures 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 approx. 0.0015mg/kg body weight.

Description

The present invention relates to a compound containing desmethylselegiline or a salt thereof, a drug comprising the compound, as well as uses thereof. The selegiline metabolite desmethylselegiline and its enantiomer, ent-desmethylselegiline, are used in medicines. In particular, the present invention shall provide compounds and drugs for the use of these in selegiline-responsive diseases and conditions.

Two distinct monoamine oxidase enzymes are known in the art. Monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B). The cDNAs that code for these enzymes have different promoter regions and distinct exons, which indicate that they are coded independently in different gene positions. Furthermore, 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 for inhibition of MAO-B is important for its safety profile after oral administration. Tranylcypromine (an MAO inhibitor introduced about 30 years ago but subsequently withdrawn due to its severe hypertensive side effects), unlike selegiline, is a non-selective inhibitor of MAO. The acute toxicity of tranylcypromine comes from inhibition of MAO-A which interferes with the metabolism of tyramine. Tyramine is normally metabolized in the stomach/intestinal tract by MAO-A, but when MAO-A is inhibited, tyramine absorption is increased after the consumption of tyramine-containing foods such as cheese, beer, herring, etc. This leads to the release of catecholamines that can trigger a hypertensive crisis and induce the "cheese effect". This effect is characterized by Goodman and Gilman as the most serious toxic effect in connection with MAO-A inhibitors.

One of the metabolites of selegiline is its N-desmethyl analogue. Structurally, the desmethylselegiline metabolite will be the R(-) enantiomeric form of a secondary amine with the formula:

Previously, desmethylselegiline was not known to have pharmaceutically useful MAO-related effects, potent and selective inhibitory effects on MAO-B. In determining the applicability of desmethylselegiline for the purpose of the present invention, the MAO-related effects of desmethylselegiline were more fully characterized. This characterization has established that desmethylselegiline has very weak MAO-B inhibitory effects, and no advantage in selectivity with respect to MAO-B compared to selegiline.

The present characterization established, for example, that selegiline has an IC50 value against MAO-B in human platelets of 5 x 10"<9> molar, while the IC50 value for desmethylselegiline is 4 x 10~7 molar, which indicates that the latter is approx. . 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 mitochondria-rich fractions:

As can be seen from the table above, selegiline is approx. 128 times more potent as an inhibitor of MAO-B compared to MAO-A, while desmethylselegiline is only 97 times more potent as an inhibitor of MAO-B compared to MAO-A. Accordingly, desmethylselegiline appears to have an approx. equally high selectivity for MAO-B compared to MAO-A as selegiline, albeit with a significantly reduced potency.

Similar results are obtained in rat brain tissue. Selegiline has an IC50 value for MAO-B of 0.11 x 10"<7> molar, while the IC50 value for desmethylselegiline is 7.3 x 10"7 molar, indicating that desmethylselegiline is approx. 70 times less potent as an MAO-B inhibitor than selegiline. Both compounds have low potency for inhibition of MAO-A in rat brain tissue, 0.18 x 10"<5> for selegiline, 7.0 x 10"5 for desmethylselegiline. Desmethylselegiline is thus approx. 39 times less potent than selegiline for inhibiting MAO-A.

Based on its pharmacological profile as set out above, desmethylselegiline as an MAO-B inhibitor will not provide any advantages in either potency or selectivity compared to selegiline. On the contrary, these in vitro data above suggest that the use of desmethylselegiline as an MAO-B inhibitor requires an order of magnitude 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 thesis "Selegiline in the Treatment of Parkinson's Disease" from research reports from the Department of Neurology, University of Turku, Turku, Finland, No. 33 (1995), pages 59-61). According to Heinonen, desmethylselegiline in vivo will have only approx. a fifth of the MAO-B inhibitory effect as selegiline, i.e. that a dose of 10 mg of desmethylselegiline would be necessary 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, (US Patent 4,861,800); Alzheimer's disease and Parkinson's disease, especially when using transdermal dosage forms, including ointments, creams and patches; macular degeneration (US Patent 5,242,950); age-related degeneration, including renal function and cognitive function as manifested by spatial learning ability (US Patent 5,151,449); pituitary-dependent Cushing's disease (US Patent 5,192,808); immune system dysfunction (US Patent 5,276,057); and schizophrenia (US Patent 5,151,419). PCT published application WO 92/17169 describes the use of selegiline in the treatment of neuromuscular and neurodegenerative disease and in the treatment of CNS damage due to hypoxia, hypoglycaemia, ischemic stroke or trauma.

Although selegiline is known to be effective in treating the aforementioned conditions, neither the precise number nor the nature of its mechanism or mechanisms of action is known. However, there is evidence that selegiline provides neuroprotection or neuronal rescue, possibly by reducing oxidative neuronal damage, by increasing the amount of the enzyme superoxide dismutase, and/or reducing dopamine catabolism. PCT published application W092/17169 reports, for example, that selegiline works by directly maintaining, preventing loss of, and/or assisting nerve function in animals.

The biochemical effects of selegiline on neuronal cells have been extensively studied. See for example Tatton et al. "Selegiline Can Mediate Neuronal Rescue Rather than Neuronal Protection." Movement Disorders 8 (Supp 1):S20-S30 (1993); Tatton et al., "Rescue of Dying Neurons," J. Neurosci. Res. 30:666-672 (1991); and Tatton et al. "(-)-Deprenyl Prevents Mitochondrial Depotarization and Reduces Cell Death in Trophically-Deprived Cells," 11th lnt'l Symp. on Parkinson's Disease, Rome, Italy, March 26-30, 1994.

Selegiline is known to be useful when administered to an individual by a number of different routes of administration and dosage forms. US patent 4,812,481 (Degussa AG) for example describes the simultaneous use of selegiline and amantadine in oral, peroral, enteral, pulmonary, rectal, nasal, vaginal, lingual, intravenous, intraarterial, intracardiac, intramuscular, intraperitoneal, intracutaneous and subcutaneous formulations. US 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, US Patent 5,387,615 describes several different selegiline formulations, including tablets, pills, capsules, powders, aerosols, suppositories, skin patches, parenteral drugs and oral liquids, including oil/water suspensions, solutions and emulsions. Also described are selegiline-containing formulations and devices with delayed release that are long-acting.

In addition to desmethylselegiline, selegiline produces another major direct metabolite, methamphetamine. Both desmethylselegiline and methamphetamine are further metabolized to amphetamine. The last two metabolites, amphetamine and methamphetamine, are known to have the potential to produce neurotoxic effects on dopamine neurons, and are unwanted by-products. Unlike selegiline, desmethylselegiline will not produce methamphetamine as a metabolite, only amphetamine.

Consequently, although a highly potent and selective MAO-B inhibitor, the practical use of selegiline will be limited by its dose-dependent specificity for MAO-B, and by the pharmacology of selegiline metabolites formed after administration.

The present invention relates to the surprising discovery that both desmethylselegiline ("DMS" or "R(-)DMS") and its enantiomer (ent-desmethylselegiline, abbreviated as "Ent-DMS" or "S(+)DMS") are useful for to provide selegiline-like effects in subjects, despite dramatically reduced MAO-B inhibitory activity and an apparent lack of increased selectivity for MAO-B compared to selegiline. In particular, it describes the surprising discovery that desmethylselegiline, ent-desmethylselegiline and their isomeric mixtures provide a more advantageous way to achieve selegiline-like therapeutic effects in selegiline-responsive diseases or conditions.

The present invention specifically provides:

In a first aspect, a compound characterized in that it is S(+)-desmethylselegiline or a pharmaceutically acceptable acid addition salt thereof, for therapeutic use. Preferred therapeutic applications are neuroprotection, neuronal rescue, restoration or enhancement of immune system function. The immunodeficiency function could possibly be AIDS, cancer or bacterial or viral infections. The compound according to the present invention can be used in the treatment of a selegiline responsive disease such as Parkinson's disease, Alzheimer's disease, depression, glaucoma, macular degeneration, ischemia, diabetic neuropathy, attention deficit hyperactivity disorder, post-polio syndrome, multiple sclerosis, impotence, narcolepsy, chronic fatigue syndrome, baldness, senile dementia, hypoxia, cognitive dysfunction, negative symptomatology in schizophrenia, amyotrophic lateral sclerosis, Tourette's syndrome, tardive dyskinesia, toxic neurodegeneration, age-related degeneration, Cushing's disease, schizophrenia, CNS damage caused by hypoxia, hypoglycaemia and ischemic stroke or trauma.

In another aspect of the present invention, there is provided a use of the compound of the present invention for the manufacture of a medicament for use in neuroprotection or neuronal rescue, for restoring or improving immune system function, for treating a selegiline-responsive disease such as Parkinson's disease, Alzheimer's disease, depression , glaucoma, macular degeneration, ischaemia, diabetic neuropathy, attention deficit hyperactivity disorder (ADHD), post-polio syndrome, multiple sclerosis, impotence, narcolepsy, chronic fatigue syndrome, baldness, senile dementia, hypoxia, cognitive dysfunction, negative symptomatology in schizophrenia, amyotrophic lateral sclerosis, Tourette's syndrome, tardive dyskinesia, toxic neurodegeneration, age-related degeneration, Cushing's disease, schizophrenia, CNS damage caused by hypoxia, hypoglycaemia and ischemic stroke or trauma. The compound used can preferably be a hydrochloride salt.

In a third aspect, the present invention comprises a drug which comprises as active ingredient a compound according to the invention. In a preferred embodiment, the compound included in the medication is in hydrochloride form. The medication according to the invention is preferably adapted for systemic, parenteral, sublingual, buccal, rectal or transdermal administration. In a further aspect of the present invention, there is provided a use of the compound R(-)-desmethylselegiline or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for use in neuroprotection or neuronal rescue, for restoring or improving immune system function, for treating a selegiline responsive disease such as Parkinson's disease, Alzheimer's disease, depression, glaucoma, macular degeneration, ischemia, diabetic neuropathy, attention deficit hyperactivity disorder, post-polio syndrome, multiple sclerosis, impotence, narcolepsy, chronic fatigue syndrome, baldness, senile dementia, hypoxia, cognitive dysfunction, negative symptomatology in schizophrenia, amyotrophic lateral sclerosis, Tourette's syndrome, tardive dyskinesia, toxic neurodegeneration, age-related degeneration, Cushing's disease, schizophrenia, CNS damage caused by hypoxia, hypoglycaemia and ischemic stroke or trauma.

In preferred embodiments of the use, the compound is a hydrochloride salt and the immunodeficiency function is AIDS, cancer or bacterial or viral infections.

As used herein, the term "selegiline-responsive disease or condition" shall refer 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" shall refer to the various diseases or conditions described above, for example Alzheimer's disease, cognitive dysfunction, neuronal rescue and the like. Similarly, the term "selegiline-like therapeutic effect" shall refer to one or more of the health-promoting effects exerted by selegiline in an individual being treated for a selegiline-responsive disease or condition.

The selegiline-responsive diseases or conditions related to neuronal degeneration or trauma that respond to the present methods include Parkinson's disease, Alzheimer's disease, depression, glaucoma, macular degeneration, ischemia, diabetic neuropathy, attention deficit hyperactivity disorder, post-polio syndrome, multiple sclerosis , impotence, narcolepsy, chronic fatigue syndrome, baldness, senile dementia, hypoxia, cognitive dysfunction, negative symptomatology in schizophrenia, amyotrophic lateral sclerosis, Tourette's syndrome, tardive dyskinesia and toxic neurodegeneration.

The present invention also includes applications for restoring or improving immune system function by R(-)DMS or S(+)DMS. Such improvement or recovery has been reported to occur when selegiline is administered to animals. The conditions or diseases that can be treated include age-related immune system dysfunction, AIDS, cancer and infectious diseases.

Depending on the particular route used, either desmethylselegiline or ent-desmethylselegiline is 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, methanesulfonic acid, acetic acid, tartaric acid, lactic acid, succinic acid, citric acid, malic acid, maleic acid, sorbic acid, acontitic acid, salicylic acid, phthalic acid, embonic acid, enanthic acid and the like. The use of salts, especially hydrochlorides, is particularly desirable when the route of administration uses aqueous solutions, such as parenteral administration; the use of released desmethylselegiline, or ent-desmethylselegiline, in the form of the free base is particularly useful for transdermal administration. Reference herein to the administration of DMS or ent-DMS or mixtures thereof will therefore include both the free base and the 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, for example based on the severity of the disease or condition being treated, the condition of the individual being treated, the desired degree of therapeutic response, and the concomitant therapies administered to the patient or animal. Ordinarily, however, the responsible doctor or veterinarian will administer an initial dose of at least approx. 0.0015 mg/kg, calculated on the basis of the free secondary amine, with the use of progressively higher doses depending on the response to therapy. Typically, the daily dose will be approx. 0.01 mg/kg and can extend to approx. 0.5 mg/kg of the patient's body weight (as all such doses are again calculated on the basis of the free secondary amine). These guidelines further require that the appropriate dose be carefully titrated by the responsible physician or veterinarian depending on the individual patient's or animal's age, weight, clinical condition and observed response.

The daily dose can be administered in a single or multiple dosage regimen. For example, the dosage regimen and regimen may permit the 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 when treating chronic conditions such as Parkinson's disease, Alzheimer's disease and depression. Alternatively, it may be desirable in conditions such as ischemia or neural damage to administer one or more specific doses in a more direct systemic manner such as intravenously or by inhalation. In still other cases such as glaucoma and macular degeneration, localized administration, such as via the intraocular route, may be indicated.

Regarding oral administration, the unexpected discovery is described that the oral use of desmethylselegiline, and/or ent-desmethylselegiline, will be more effective than oral selegiline for use in several different conditions and diseases. Accordingly, desmethylselegiline and its enantiomers are used orally in individuals where one would not use selegiline itself due to side effects.

Pharmaceutical compositions containing desmethylselegiline and/or ent-desmethylselegiline can be prepared according to conventional techniques. For example, preparations for the parenteral route of administration of desmethylselegiline, such as the intramuscular, intravenous, and intraarterial routes, may use sterile isotonic saline solutions. Sterile isotonic solutions can also be used for intraocular administration.

Transdermal dosage unit forms of desmethylselegiline and/or ent-desmethylselegiline can be prepared using several different previously described techniques (see, for example, US 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 plaster structure can be used in which desmethylselegiline is directly incorporated into the adhesive, and this mixture is cast firmly on a backing foil.

Alternatively, desmethylselegiline, and/or ent-desmethylselegiline, can be incorporated as an acid addition salt in a multi-layer plaster which carries out 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 using a lyotropic liquid crystalline mixture 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 addition of propylene glycol and an emulsifier. For further details regarding the preparation of such transdermal preparations reference can be made to EP-A5509761.

Since the term "ent-desmethylselegiline" refers to the S(+) isomeric form of desmethylselegiline, reference above to mixtures of selegiline and ent-desmethylselegiline shall include both racemic and non-racemic mixtures of optical isomers.

Subjects treatable by the present compositions and applications include both human and non-human subjects for which selegiline-like therapeutic effects are known to be applicable. Accordingly, the above compositions and uses will provide particularly useful therapies for mammals, especially domestic mammals. The present applications and mixtures are thus used for the treatment of selegiline-responsive diseases or conditions in canine and feline species.

Successful use of the above compositions and applications requires the use 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, the use of these agents, or a mixture of these agents, will not require a proportionately increased dosage to achieve a selegiline-like therapeutic response. Surprisingly, dosages necessary to achieve a selegiline-like therapeutic effect will be of the same order of magnitude as the known doses of selegiline. Because both desmethylselegiline and ent-desmethylselegiline have a much lower inhibition of MAO-A at such dosages, desmethylselegiline and ent-desmethylselegiline will therefore provide a significantly wider safety margin with respect to MAO-A associated toxicity compared to selegiline. In particular, the risk of the harmful effects of MAO-A inhibition, for example hypertensive crisis, will be minimized due to the 40-70 times reduced potency for MAO-A inhibition.

As described above and despite its demonstrably inferior inhibitory properties with respect to MAO-B inhibition, desmethylselegiline and its enantiomers will be significantly more effective than selegiline for the treatment of selegiline-responsive conditions, such as conditions resulting from neuronal degeneration or neuronal trauma. In this regard, desmethylselegiline and/or its enantiomers, like selegiline itself, will be particularly useful when administered in a manner that does not rely on absorption in the upper gastrointestinal tract or elsewhere in the gastrointestinal tract. Preferred routes of administration include the parenteral, topical, transdermal, intraocular, buccal, sublingual, intranasal, vaginal, rectal and inhalation routes.

Desmethylselegiline can be used both in optically active forms and in racemic form, i.e. as mixtures of desmethylselegiline and ent-desmethylselegiline. Desmethylselegiline, its enantiomers 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-old rats. The cultures were used at approx. 1.5 million cells per plate, and were kept either in growth medium alone (control cultures) or in growth medium supplemented with selegiline. On days 1, 8 and 15, the cells were immunostained for the presence of tyrosine hydroxylase ("TH"). Filled bars represent results obtained for cultures maintained in the presence of 50 (imolar selegiline and open bars represent results for control cultures. In all cases, results are expressed as a percentage of TH-positive cells present in the control cultures on day 1. The abbreviation "DIV" refers to "days in vitro." Asterisks or stars above the bars in both Figure 1 and the figures discussed below indicate a result that differs from the controls by an amount that is statistically significant, that is, 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 the results are shown in Figure 2. Filled columns represent uptake in cells maintained in the presence of 50 smolar 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 for. After the cultures were allowed to stabilize, the culture medium was exchanged daily for a period of 4 days to induce glutamate receptor-dependent cell death. Depending on the culture, the medium contained either 0.5, 5.0 or 50 fxmolar selegiline. After the last medium exchange, cultured cells were immunostained for the presence of tyrosine hydroxylase. From left to right, the bars represent results for controls, 0.5, 5.0 and 50 limolar selegiline. Figure 4: Effect of selegiline on dopamine uptake in neuronal cultures. Rat mesencephalic cells were cultured and the medium was exchanged on a daily basis as discussed for Figure 3. Uptake into the cells of tritiated dopamine was measured and the results are shown in the figure. From left to right, the columns are in the same order as for Figure 3. Figure 5: Effect of R(-)desmethylselegiline on glutamate receptor-dependent neuronal cell death. Rat embryo 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 the cultures was determined. The results are expressed as a percentage of the control. From left to right, the columns show results for controls, 0.5, 5 and 50 umolar 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 the cells maintained in the presence of 0.5 limolar, 5.imolar and 50.imolar 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 embryo mesencephalic cells were prepared as described for Figures 3-6, and incubated in the presence of several different monoamine oxidase inhibitors. The inhibitors investigated were selegiline; R(-)desmethylselegiline; pargyline; and chlorgyline, all at concentrations of 0.5, 5 and 50 µmolar. In addition, the cells were incubated in the presence of the glutamate receptor blocker MK-801 at a concentration of 10 µM. The cultures were tested for uptake of tritiated dopamine. Figure 8: Inhibition of neuronal dopamine neuronal reuptake 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 different concentrations of selegiline, R(-)desmethylselegiline or S(+)desmethylselegiline. Uptake in the presence of each of the MAO inhibitors was expressed as percent inhibition relative to uptake in the presence of buffer alone, and the results are shown in Figure 8. As indicated in the figure, the charts were used to determine the ID50 value for each test agent. The ID 50 value for S(+)DMS was 20 µmolar; for selegiline 80 |amolar; and for R(-)DMS approx. 100 immolars. Figure 9: In vivo MAO-B inhibition of guinea pig hippocampus. Different doses of selegiline, R(-)desmethylselegiline and S(+)desmethylselegiline were injected daily into guinea pigs for a period of 5 days. The animals were then killed, and the MAO-B activity in the hippocampus part of the brain was determined. The results were expressed as percent inhibition in relation to hippocampal MAO-B activity in control animals, and are shown in figure 9. The diagrams were used to determine the ID50 dosage for each mite. The ID50 value for selegiline was approx. 0.03 mg/kg; and for both enantiomers of DMS, approx. 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, ie between 18 and 20 months old. After allowing a 10-day "washout" period during which no injections were made, the rats were euthanized and their spleens removed. In vitro production of interferon-γ from the stimulated spleen cells (lymphocytes) was determined and compared with production from spleen cells (lymphocytes) from non-injected old rats and from young rats (3 months old). From left to right, the bars should 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 selegiline; from aged 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 the IFN level differs significantly (P<0.05) from the level observed using spleen cells from young rats. The results are expressed as units per ml (u/ml). Figure 11: In vitro IFN production in spleen cells from old rats. The same results shown in Figure 10 are repeated in Figure 11, but without including the results for spleen cells from young rats. "#" indicates a result significantly different (P<0.05) from that obtained for spleen cells from old rats injected with saline. "<*>" indicates a result significantly different from all groups except aged rats injected with 1.0 mg/kg deprenyl. Figure 12: In vitro interleukin-2 production from spleen cells from old rats. The rats were injected as described above (see Figure 10) and the production of interleukin-2 from stimulated spleen cells was determined. The order of columns from left to right is the same as for Figure 10. Figure 13: Percentage of rat spleen cells that are IgM-positive. The rats were injected with saline, selegiline, R(-)DMS or S(+)DMS as described above (see Figure 10). The spleens from the rats were analyzed to determine the percentage of cells that were IgM positive, and the results are shown in the figure. The horizontal line in the figure corresponds to the reduction in the IgM percentage which is statistically significant (P<0.05) in relation to the percentage observed in spleens obtained from young rats. The columns 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 in Figure 13 was repeated, but instead of measuring the percentage of cells that are IgM positive, the percentage that is CD5 positive was determined.

Detailed description of the invention

The surprising utility of desmethylselegiline and ent-desmethylselegiline in the treatment of selegiline-responsive diseases or conditions may be attributed in part to their potent action in preventing loss of dopaminergic neurons by promoting repair and healing. Consequently, at doses as low as 0.01 mg/kg, at which little or no MAO-B inhibition is usually observed, a reversal in neuronal damage and/or death can be observed. Because desmethylselegiline and ent-desmethylselegiline prevent loss and facilitate restoration of nerve cell function, they will be of value in a number of different neurodegenerative and neuromuscular diseases. In this respect, desmethylselegiline and ent-desmethylselegiline will be significantly more potent than selegiline, as described more empirically in the examples below.

Examples

Example 1: Preparation of desmethylselegiline and ent-desmethylselegiline

A. Desmethylselegiline

Desmethylselegiline (referred to below as "R(-)DMS") is prepared by methods known in the art. Desmethylselegiline is, for example, a known chemical intermediate for the production of selegiline as described in US patent no.

4,925,878. Desmethylselegiline 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-CH2-C=CH, at a somewhat elevated temperature (70°-90°C). Optionally, the reaction can be carried out 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 made alkaline. The non-aqueous layer that forms is separated, for example by extraction with benzene, dried and distilled under reduced pressure.

Alternatively, the propargylation can be carried out in a two-phase system of a solvent immiscible with water, and aqueous alkali using a salt of R(+)-2-aminophenylpropane with a weak acid such as the tartrate, analogous to the preparation of selegiline as described in US 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.

by 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 to 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 used in the production of desmethylselegiline hydrochloride. Either the free base or the salt can be further purified, again by conventional techniques such as recrystallization or chromatography.

Example 2: Neuronal survival 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 observed with several different trophic factors including BDNF, GDNF, EGF and p-FGF.

A. Test Methods

Timed pregnant Sprague-Dawley rats are used to establish neuronal cultures from embryonic rat brain on the 14th day of pregnancy. Mesencephalon is dissected out without covering the membrane, and collected in Ca" and Mg" free balanced salt solution at 4°C. Tissue fragments are dissociated in chemically defined medium by gentle rubbing with a small-diameter Pasteur pipette. The cell suspension is plated in polyornithine-coated 35 mm Falcon plastic dishes (0.1 mg/ml, Sigma) at a density of 1.5x 10<6> cells/dish. The cultures are maintained at 37°C in an atmosphere of 10% CO2/90°C air and 100% relative humidity, and fed twice a week with chemically defined medium consisting of MEM/F12 (1:1, Gibco), glucose (33 mmolar), HEPES (15 mmolar), NaHCO3 (44.6 mmolar), transferrin (100 mg/ml), insulin (25 mg/ml), putrescine (60 nmolar), sodium selenite (30 nmolar), progesterone (20 nmolar) , and glutamine (2 mmol). The control cells received no further additions. The medium used for other cells also included test substance, for example selegiline, at one or more concentrations.

The cultures are fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min at room temperature, permeabilized with 0.2% Triton X-100 for 30 min 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 stain kit (Vectastain ABC kit; Vector Labs) with 3',3'-diaminobenzidine as chromagen.

The number of dopaminergic neurons in the cultures is determined by counting the cells that are positively immunostained with TH antibodies. 100 areas (0.5 mm x 0.5 mm) in two transverse stripes across the diameter of the dish, which represented 2.5% of the total area, are counted using a Nikon inverted microscope at 200x magnification.

B. Results

By applying the methods described above, the following results were obtained:

Example 3: Neuronal survival 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 can also be determined by directly measuring dopamine uptake. The amount of uptake by the cultured brain cells corresponds to the axonal growth.

A. Test Methods

Cell cultures, established as described above, are incubated with [<3>H]dopamine (0.5 mCi/ml; 37 Ci/mmol; New England Nuclear) for 15 min in the presence of ascorbic acid (0.2 mg/ml) in PBS (pH 7.3), supplemented with 0.9 mmolar CaCl2 and 0.5 mmolar MgCb at 37°C. After two rinses and 5 min incubation with fresh buffer, [<3>H]dopamine accumulated inside the cells is released by incubating the cultures with 95% ethanol for 30 min at 37°C. The preparations are then added to 10 ml of Ecoscint (National Diagnostics) and counted in a scintillation spectrometer. No specific uptake values are obtained by blocking dopaminergic neuronal uptake with 10 mmol mazindole.

B. Results

Using the above method, the results shown in Table 3 were obtained.

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 correct despite the fact that desmethylselegiline is much less potent than selegiline as an inhibitor of

MAO-B.

Example 4: Neuroprotective effect of desmethylselegiline enantiomers i

cultured dopamine-containing mesencephalic neurons in vitro

The survival of mesencephalic dopamine-containing neuronal cultures of rat brain tissue was used in these experiments to investigate the 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 nmolar selegiline for 15 days, beginning on the day of plating.

(For a more detailed description of the cultivation of cells and other procedures used in these experiments see Mytilineou et al., J. Neurochem. 61:1470-1478

(1993). Survival and growth of dopamine neurons were evaluated by tyrosine hydroxylase (TH) immunocytochemistry and [<3>H]dopamine uptake, and the results are shown in Figures 1 and 2 .

No effect was observed on neuronal survival when selegiline was tested at concentrations of 0.5 and 5 nmolar. At 50 nmolar, 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 pretreatment with 0.5, 5, 50 or 100 nmolar selegiline would not inhibit dopamine uptake under the assay conditions used in the experiments.

The results indicate that the uptake values obtained reflect dopamine neuron survival and process outcome, and that selegiline has a neuroprotective effect.

B. Effect of selegiline on glutamate receptor-dependent cell death

The neuroprotective effect of selegiline was also investigated using an experimental paradigm that causes neuronal cell death that can be blocked by inhibition of glutamate receptors. In these experiments the cells were plated out and allowed to stabilize for several days. The growth medium for the cells was then changed on a daily basis to induce cell death which can be prevented by blocking glutamate receptors, for example using MK-801. After 4 days of daily medium changes, the cultures were stained for tyrosine hydroxylase and analyzed for uptake of tritiated dopamine. The results shown in Figures 3 and 4 further support the conclusion that selegiline mediates survival of dopaminergic neurons.

C. Effect of desmethylselegiline on survival of dopamine neurons

Using the glutamate receptor-dependent model of neuron death, an even more powerful protection of dopaminergic neurons was obtained when desmethylselegiline was used instead of selegiline. Even at the lowest tested dose (0.5 nmolar), desmethylselegiline caused a significant reduction in the loss of TH-positive neurons (Figure 5) and a significant increase in dopamine uptake (Figure 6) compared to control cultures in which the medium was used without supplementation with either selegiline or desmethylselegiline.

D. Comparison with other MAO inhibitors

Using the glutamate receptor-dependent neurotoxicity paradigm, the effects of selegiline and desmethylselegiline were compared with two other MOA inhibitors, pargyline and clorgyline (Figure 7). In agreement with previous results, measurement of dopamine uptake could indicate neuron protection at 50 µmolar deprenyl and 5 and 50 nmolar desmethylselegiline. Pargyline did not appear to provide any protection at the concentrations used, while chlorgyline protected at 50 nmolar. As expected, protection was also achieved by the NMDA receptor blocker MK-801 (10 nmolar).

E. Effect of DMS enantiomers on 3H-dopamine uptake

Data summarized in Table 4 suggest that both R(-)DMS and S(+)DMS are effective as neuroprotectants in mesencephalic dopamine-containing neurons in culture.

These results show 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. S(+)DMS may thus be an even more potent and/or effective neuroprotectant than R(-)DMS.

Example 5: Desmethylselegiline and ent-desmethylselegiline as dopamine reuptake inhibitors

The biological actions of the brain neurotransmitter dopamine are terminated at the synapse by a high-affinity, sodium and energy-dependent transport system (neuronal reuptake) present within the limiting membrane of the presynaptic dopamine-containing nerve terminal. Inhibition of this transport mechanism would prolong the effects of dopamine in the synapse, and therefore increase the synaptic transmission of dopamine.

A. Procedure for testing

The R(-) and S(+) enantiomers of desmethylselegiline (DMS) were tested for their ability to inhibit the dopamine reuptake system and compared to selegiline. Inhibitory activity in this assay is indicative of agents of value in the treatment of diseases requiring enhanced synaptic dopamine activity. Currently, this would include Parkinson's disease, Alzheimer's disease and attention deficit hyperactivity disorder (ADHD); Attention Deficit Hyperactivity Disorder.

The analysis system used was essentially as described by Fang et al.

(Neuropharmacology 33:763-768 (1994)). An in vitro nerve terminal preparation (synaptosome preparation) was obtained from healthy rat neostriatal brain tissue. Transport at dopamine nerve endings was estimated by measuring the uptake of tritiated dopamine.

B. Results

As can be seen in the data presented in Table 5, selegiline, R(-)DMS and S(+)DMS all inhibited dopamine reuptake by dopamine-containing nerve endings. Selegiline and R(-)DMS were approximately equally potent. S(+)DMS, on the other hand, was 4-5 times more potent than both selegiline and R(-)DMS.

Relative potency can be expressed in terms of the concentration required to inhibit dopamine reuptake by 50% (ID50). The ID5o values were determined graphically (see Figure 8) and are shown below in Table 6.

C. Conclusions

The results show that at the appropriate concentration, selegiline and each of the enantiomers of DMS will inhibit the transport of released dopamine in the neuronal synapse, and increase the relative activity of this neurotransmitter in the synapse. In this respect, S(+)DMS is more potent than selegiline, which in turn is more potent than R(-)DMS. This effect is an indication of agents that are beneficial in the treatment of Parkinson's disease, Alzheimer's disease and attention deficit hyperactivity disorder ADHD: Attention Deficit Hyperactivity Disorder. Of the agents tested, S(+)DMS seems to be the most preferred with regard to the treatment of ADHD.

Example 6: The effect 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 exclusively covered by the type B isoform of the enzyme. In the present study, the inhibition of this enzyme with the two enantiomers of DMS was determined and compared with the inhibition due to selegiline. In addition, the present study examined the two enantiomers of DMS for inhibitory activity with respect to MAO-A and MAO-B in guinea pig hippocampal tissue. Guinea pig brain tissue is the best animal model for the study of brain dopamine metabolism, the enzyme kinetics of the many forms of MAO and the inhibitory properties of new agents that work together with these enzymes. The many forms of MAO in this animal species have identical kinetic properties to those found in human brain tissue. Finally, agents were injected into guinea pigs to determine the extent to which they may act as inhibitors of brain MAO in vivo.

B. Procedure for Testing:

The test system used in vitro transformation of specific substrates of MAO-A (<14>C-serotonin) and MAO-B (uC-phenylethylamine) by human platelets and/or guinea pig hippocampal homogenates. The rate of conversion of each substrate was measured in the presence of S(+)DMS, R(-)DMS or selegiline, and compared to the isozyme activity in the absence of these agents. Percent inhibition was calculated from these values. Potency was evaluated by comparing the concentration of each agent that caused a 50% inhibition (IC50 value).

In some experiments, R(-)DMS, S(+)DMS or selegiline were administered in vivo subcutaneously (s.c), once a day for 5 days before sacrifice, preparation of enzyme hippocampal homogenates, and the in vitro assay of MAO-A and MAO-B activity. These experiments were carried out to show that the DMS enantiomers were able to enter the brain tissue and inhibit MAO activity.

C. Results

MAO-B inhibitory activity in vitro

Results for MAO-B inhibition are shown in tables 7 and 8. ICso values for MAO-B inhibition and potency compared to selegiline are shown in table 9.

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 investigating inhibition of MAO-A in guinea pig hippocampus are summarized in Table 10, and IC50 values for the two enantiomers of DMS and for selegiline are shown in Table 11.

() 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 was 2-3 orders of magnitude, ie, 100- to 1000-fold, less potent as inhibitors of MAO-A than inhibitors of MAO-B in hippocampal brain tissue. Selegiline and each enantiomer of DMS can therefore 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 determined. Under these conditions, R(-)DMS and S(+)DMS were as potent as MAO-B inhibitors, but ten times less potent than selegiline. Results are shown in figure 9 and IC50 values are summarized in table 12.

This experiment shows 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 MAO-B inhibitor potency differences 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 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 at 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 therapeutic agents 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 investigated by administering the agents to the wobbler mouse, an animal model of motor neuron diseases, particularly amyotrophic lateral sclerosis (ALS). Wobbler mice show progressive worsening of forelimb weakness, gait disturbances, and flexion contractions of the forelimb muscles.

B. Test Procedure

R(-)DMS, S(+)DMS or placebo were administered to wobbler mice by daily intraperitoneal injection for a period of 30 days in a randomized, double-blind study. At the end of this time, the mice were examined for grip strength, running time, resting locomotor activity, and graded for semi-quantitative abnormalities in paw posture and semi-quantitative abnormalities in gait. The scientists who prepared and who injected the solutions into the animals and who analyzed the behavioral changes were different.

Analyzes and grading were performed essentially as described in Mitsumoto et al., Ann. Neurol. 36:142-148 (1994). The grip strength of the forepaws of a mouse was determined by having the animal grasp a wire with both paws. The wire was connected to a gr dynamometer, and a pull was applied to the mouse's tail until the animal was forced to let go of the wire. The reading on the dynamometer at the release point is taken as a measure of grip strength. The run time is defined as the shortest time required to run a specified distance, for example 2.5 feet, and the best time of several attempts is recorded.

Abnormalities in paw position are graded on a scale based on the degree of contraction, and gait abnormalities are graded on a scale ranging from normal walking to inability to support the body using the paws.

Locomotor activity is determined by transferring the animals to an examination area where the floor is covered with square netting. Activity is measured by the number of squares passed by a mouse in a specified time interval, for example 9 minutes.

C. Results

At the beginning of the study, none of the groups differed in any of the variables, indicating that the three groups were comparable at baseline. The weight gain was identical in all three groups, which suggests that no significant side effects occurred in any of the animals. Table 13 summarizes the differences observed in the average grip strength of the test animals:

Grip strength decreased markedly at the end of the first week in all animals. At the end of the study, grip strength was the least in the control animals, in the range from 0-15 gr, with an average of 9 gr. The R(-) group had an average grip strength of 20 gr with values in the range from 0-63 gr. The third group, injected with S(+)DMS had an average grip strength of 14 gr, with individual values from 7 to 20 gr. Although the variability in grip strength in the treated animal groups prevented a meaningful statistical analysis of these data, the average grip strength measured in the DMS-treated animals was higher than that of the controls.

Running time, resting locomotor activity, semi-quantitative abnormality grading of paw position, and semi-quantitative abnormality grading of gait were also tested. However, none of these tests yielded data suggesting that any of these three groups differ from the others.

Example 8: Immune system restoration by R(-)DMS and S(+)DMS

This is an AIDS-related impairment in immunological function that occurs in animals and humans, and which makes older individuals more susceptible to infectious disease and cancer. US 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 applicable in the treatment of such dysfunction. It should be recognized that an ability to enhance the patient's normal immunological defenses would be beneficial in the treatment of a number of different acute and chronic diseases including cancer, AIDS and both bacterial and viral infection.

A. Test Procedure

The present experiments used a rat model to investigate the ability of R(-)DMS and S(+)DMS to restore immunological function. The 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 0.0 mg/kg body weight; 6) aged 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.

The rats were injected ip, daily for 60 days. They were then kept in a further "washout" period of 10 days, during which no injections were given. At the end of this time, the animals were euthanized and their spleens were removed. The spleen cells were then analyzed for several different factors indicative of immune system function. Specifically, standard tests were used to determine the following; 1) in vitro production of γ-interferon in spleen cells; 2) in vitro production of interleukin-2; 3) percentage of IgM-positive spleen cells (IgM is a marker for B-lymphocytes);

4) percentage of CD5-positive spleen cells (CD5 is a marker for 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 decline in cellular interferon production that occurs with age (compare production with cells from young animals with production with 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 increase at doses 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 are omitted. The figure shows more clearly the extent to which deprenyl, R(-)DMS and S(+)DMS are able to restore γ-interferon production in the spleen cells of old rats. Interferon-y is a multifunctional protein that inhibits viral replication and regulates several different immunological functions. It affects the class of antibodies produced by B cells, upregulates class I and class II MHC complex antigens and increases the efficiency of macrophage-mediated killing of intracellular parasites.

Figure 12 shows the effects of injections on the production of interleukin-2 in rat spleen cells. It can be observed that both R(-)DMS and S(+)DMS are able to restore production to a level observed in cells from young animals.

The effect of injections on the percentage of spleen cells that are IgM-positive is shown in Figure 13. Both selegiline and R(-)DMS were found to partially restore IgM-positive cells to a level closer to that observed in the spleen of young rats . It thus appears that these agents will restore B-lymphocyte cell numbers.

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 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

The results generally support the conclusion that the enantiomers of DMS mimic the effects of selegiline and immune system function. The results obtained with regard to the production of interferon IL-2 and on the percentage of IgM-positive spleen cells further support the conclusion that the DMS enantiomers are able to at least partially restore the age-dependent loss of immune system function. It thus appears that R(-)DMS and S(+)DMS will have a therapeutically beneficial effect for diseases and conditions caused by reduced host immunity. This would include cancer, AIDS and infectious diseases of all kinds.

Example 9: Examples of dosage forms

A. Desmethylselegiline patch

The two components are thoroughly mixed and cast onto a Scotchpak<®> 9723 polyester film backing sheet and dried. The backing foil is then cut into patches, a Scotchpak<®> 1022 fluoropolymer release liner is applied, and the patches are hermetically sealed in metal foil envelopes. A patch is applied daily to deliver 1-5 mg of desmethylselegiline per 24 hours when treating conditions in a person caused by neuronal degeneration or neuronal trauma such as Parkinson's disease.

B. Eye solution

Desmethylselegiline (0.1 g) as the hydrochloride, 1.9 g of boric acid and 0.004 g of phenylmercury II nitrate are dissolved in sterile water qs 100 ml. The mixture is sterilized and sealed. It can be used ophthalmologically in the treatment of conditions caused by neuronal degeneration or neuronal trauma, such as, for example, optic neuropathy in glaucoma and macular degeneration.

Example 3: Intravenous solution

A 1% solution is prepared by dissolving 1 g of desmethylselegiline as the hydrochloride in sufficient 0.9% isotonic saline to give 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 metal foil, the solvents are evaporated, and the coated metal foil is placed on a polyester backing layer, as disclosed in EP-A 593807.

Desmethylselegiline hydrochloride is added to a solution of non-crosslinked acrylic-based pressure-sensitive adhesive in a suitable organic solvent, such as ethyl acetate. Additional solvents may be added, and heat and agitation may be used to facilitate dispersion formation. This is coated on a second metal foil which can be removed, and the solvent evaporated. After removing the metal foil from the previously produced polyester coated layer, it is laminated to the coated second metal foil which can be removed. 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 is cast onto a third removable metal foil and the solvent evaporated. The second and third metal foils are removed, the remaining layers laminated, and the resulting laminate cut into patches and packaged. The resulting patches will have a release liner that can be removed, an adhesive layer that is initially free of desmethylselegiline, and a matrix layer containing desmethylselegiline as the hydrochloride or other salt. An impermeable backing layer is adhered to the matrix layer through an intermediate adhesive layer in line with the adhesive layer closest to the release liner which can be removed. A patch is applied daily to deliver desmethylselegiline as the free base in the treatment of conditions in a human caused by neuronal degeneration or neuronal trauma, such as Parkinson's disease.

D. Oral dosage form

Tablets and capsules containing desmethylselegiline are prepared from the following ingredients (mg/unit dose);

with a ratio of approx. 1:1 of citric acid and sodium citrate.

Claims (19)

1. Compound, characterized in that it is S(+)-desmethylselegiline or a pharmaceutically acceptable acid addition salt thereof, for therapeutic use. 2. Compound according to claim 1, for use in neuroprotection or neuronal rescue. 3. Compound according to claim 1, for use in restoring or improving immune system function. 4. A compound according to claim 3, wherein the immunodeficiency function is AIDS, cancer or bacterial or viral infections. 5. Compound according to claim 1, for use in the treatment of a selegiline-responsive disease such as Parkinson's disease, Alzheimer's disease, depression, glaucoma, macular degeneration, ischemia, diabetic neuropathy, attention deficit hyperactivity disorder, post-polio syndrome, multiple sclerosis, impotence, narcolepsy, chronic fatigue syndrome, baldness, senile dementia, hypoxia, cognitive dysfunction, negative symptomatology in schizophrenia, amyotrophic lateral sclerosis, Tourette syndrome, tardive dyskinesia, toxic neurodegeneration, age-related degeneration, Cushing's disease, schizophrenia, CNS damage caused by hypoxia, hypoglycaemia and ischemic stroke or trauma. 6. Compound according to claim 5, for use in the treatment of Parkinson's disease, Alzheimer's disease or depression. 7. Compound according to claim 5, for use in the treatment of ischemia or hypoxia. 8. Compound according to claim 5, for use in the treatment of attention deficit hyperactivity disorder (ADHD). A compound according to any one of the preceding claims, which is a hydrochloride salt. 10. Use of a compound as defined in claim 1, for the preparation of a drug for use in neuroprotection or neuronal rescue, for the restoration or improvement of the immune system function, for the treatment of a selegiline-responsive disease such as Parkinson's disease, Alzheimer's disease, depression, glaucoma, macular degeneration, ischemia, diabetic neuropathy, attention deficit hyperactivity disorder (ADHD), post-polio syndrome, multiple sclerosis, impotence, narcolepsy, chronic fatigue syndrome, baldness, senile dementia, hypoxia, cognitive dysfunction, negative symptomatology in schizophrenia, amyotrophic lateral sclerosis, Tourette syndrome, tardive dyskinesia, toxic neurodegeneration, age-related degeneration, Cushing's disease, schizophrenia, CNS damage caused by hypoxia, hypoglycaemia and ischemic stroke or trauma. 11. Use according to claim 10, where the compound is a hydrochloride salt. 12. Medicine, characterized in that it comprises as an active ingredient a compound as defined in claim 1. 13. Medicine according to claim 12, adapted for systemic administration. 14. Medicine according to claim 13, adapted for parenteral, sublingual, buccal or rectal administration. 15. Medicine according to claim 12, adapted for transdermal administration. 16. Medicine according to claims 12-15, where the compound is the hydrochloride salt. 17. Use of the compound R(-)-desmethylselegiline or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for use in neuroprotection or neuronal rescue, for the restoration or improvement of immune system function, for the treatment of a selegiline responsive disease such as Parkinson's disease, Alzheimer's disease, depression, glaucoma, macular degeneration, ischemia, diabetic neuropathy, attention deficit hyperactivity disorder, post-polio syndrome, multiple sclerosis, impotence, narcolepsy, chronic fatigue syndrome, baldness, senile dementia, hypoxia, cognitive dysfunction, negative symptomatology in schizophrenia, amyotrophic lateral sclerosis, Tourette's syndrome, tardive dyskinesia, toxic neurodegeneration, age-related degeneration, Cushing's disease, schizophrenia, CNS damage caused by hypoxia, hypoglycaemia and ischemic stroke or trauma. 18. Use according to claim 17, wherein the compound is a hydrochloride salt. 19. Use according to claim 17, where the immunodeficiency function is AIDS, cancer or bacterial or viral infections.