HK1009099A1 - Methods and pharmaceutical compositions employing desmethylselegiline - Google Patents

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 entdesmethylselegiline, 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 using desmethylselegiline

Technical Field

The present invention relates to methods and pharmaceutical compositions for using Selegiline (Selegiline) metabolites, desmethylselegiline, and its enantiomers. In particular, the invention provides compositions and methods for the use of these agents in selegiline responsive diseases and disorders.

Background

Two distinct monoamine oxidases are known in the art: monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B). The cdnas encoding these enzymes showed different promoter regions and unique exon portions, indicating that they are independently encoded at different gene positions. Furthermore, analysis of these two proteins has shown differences in their respective amino acid sequences.

The first compound that has been found to selectively inhibit MAO-B is R- (-) -N-methyl-N- (prop-2-ynyl) -2-aminophenylpropane, also known as L- (-) -diprenyl (deprenyl), R- (-) -diprenyl or selegiline. Selegiline has the following structural formula:

the selectivity of selegiline in terms of MAO-B inhibition is important for its safety profile for oral administration. Phencyclamine, an MAO inhibitor introduced more than thirty years ago but subsequently rejected due to its serious hypertensive side effects, is a non-selective MAO inhibitor as opposed to selegiline. Acute toxicity of phencyclylamine results from inhibition of MAO-A, which interferes with tyramine metabolism. Tyramine is normally metabolized by MAO-A in the gastrointestinal tract, but when MAO-A is inhibited, tyramine absorption increases after consumption of tyramine-containing foods such as cheese, beer, herring, etc. This results in the release of catecholamines which can cause the risk of hypertension, producing a "cheese effect". Goodman and Gilman characterized this effect as the most severe toxic effect associated with MAO-A inhibitors.

One of the metabolites of selegiline is its N-nor analogue. Structurally, this metabolite of desmethylselegiline is the R- (-) -enantiomeric form of a secondary amine of the formula:

heretofore, it has not been known that desmethylselegiline is pharmaceutically useful, and that the MAO-related effect, i.e. a strong and selective inhibitory effect on MAO-B, is known. In determining the usefulness of desmethylselegiline for the purposes of the present invention, the MAO-related effects of desmethylselegiline are more fully characterized. This characterization has established that desmethylselegiline has a very weak MAO-B inhibitory effect and has no advantage over selegiline in terms of selectivity towards MAO-B.

For example, this characterization determined the IC of selegiline for MAO-B in human platelets₅₀Value of 5X 10^-9M, IC of desmethylselegiline₅₀Value of 4X 10^-7M, indicating that the latter is about 80-fold less potent as MAO-B inhibitor than the former. Similar characteristics can be seen in the following datA measuring the inhibition of MAO-B and MAO-A in rat cortical-rich mitochondrial fractions:

TABLE 1 inhibition of MAO by selegiline and desmethylselegiline

Concentration of	Percent inhibition
					Selegiline		Desmethylselegiline
	0.003μM0.010μM0.030μM0.100μM0.300μM1.000μM3.000μM10.000μM30.000μM100.000μM	MAO-B	MAO-A	MAO-B					MAO-A
16.7040.2064.7091.8094.5595.6598.10---					----9.7532.5565.5097.75--	3.407.504.606.7026.1554.7386.2795.1597.05-	----0.00.704.1011.75-56.10

As is apparent from the above table, selegiline is about 128-fold more potent as an MAO-B inhibitor than MAO-A, whereas desmethylselegiline is only 97-fold more potent as an MAO-B inhibitor than MAO-A. Thus, desmethylselegiline appears to have roughly equal selectivity for MAO-B (compared to MAO-A) as selegiline, although the potency is greatly reduced.

Similar results were obtained with rat brain tissue. Selegiline shows IC for MAO-B₅₀The value was 0.11X 10^-7M, IC of desmethylselegiline₅₀The value was 7.3X 10^-7M, indicating that desmethylselegiline as MAO-B inhibitor drug effect than selegiline about 70 times less. Both compounds showed low potency in inhibiting MAO-A in rat brain tissue, 0.18X 10 for selegiline^-5Norselegiline is 7.0X 10^-5. Thus, norselegiline is approximately 39-fold less potent than selegiline in inhibiting MAO-A.

Depending on its pharmaceutical profile as described above, desmethylselegiline, which is an MAO-B inhibitor, offers no advantages over selegiline, either in potency or selectivity. In contrast, the ex vivo experimental data above indicated that the amount of desmethylselegiline used as an MAO-B inhibitor required approximately 70 times the amount of selegiline.

Heinonen, E.H. et al reported the efficacy of desmethylselegiline as a live MAO-B inhibitor ("desmethylselegiline, a metabolite of selegiline, is an irreversible inhibitor of MAO-B in human subjects", see academic proceedings "selegiline in Parkinson's disease treatment", report on neurological systems of the university of Turku, Finland Turku, No.33(1995), pp 59-61). Heinonen suggested that the MAO-B inhibitory effect of desmethylselegiline in vivo was only about 1/5 for selegiline, i.e., a dose of 10mg of desmethylselegiline would be required to achieve the same MAO-B effect as 1.8mg of selegiline.

Various diseases and conditions for which selegiline is known to be useful include: depression (us patent 4,861,800); alzheimer's and parkinson's disease, especially by using transdermal dosage forms, including ointments, creams and plasters; macular degeneration (us patent 5,242,950); age-dependent degeneration, including renal function and cognitive function as evidenced by spatial learning ability (U.S. 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 discloses the use of selegiline for the treatment of neuromuscular and neurodegenerative diseases and for the treatment of central nervous system injury due to hypoxia, hypoglycemia, ischemic attacks or trauma.

Although selegiline is known to be effective in treating the above-mentioned conditions, it is not known either the exact number or the nature of the mechanism or mechanism of action. However, there is evidence that selegiline may provide neuroprotection or neuronal rescue by reducing oxidative neuronal damage, increasing the number of superoxide dismutase enzymes, and/or reducing dopamine catabolism. For example, PCT published application WO 92/17169 reports that selegiline works by directly preserving the neural function of an animal, preventing its loss, and/or giving it help.

The biochemical effects of selegiline on neuronal cells have been extensively studied. For example, see Tatton et al, "Selegiline mediates neuronal rescue but not neuronal protection",Movement Disorders8(Supp 1): S20-S30 (1993); tat et al, "rescue of dying neurons",J.Neurosci.Res.30: 662-672 (1991); and Tatton et al "(-) -diprenil prevents mitochondrial depolarization and cell death in rescue auxotrophic cells",11 th Int’l Symp on Parkinson’s Diseaseroman, italy, 26-30 months, 1994.

Selegiline is known to be useful when administered to a subject by a wide variety of routes and dosage forms. For example, U.S. patent 4,812,481(Degussa AG) discloses the use of concomitant selegiline-amantadine in oral, enteral, pulmonary, rectal, nasal, vaginal, lingual, intravenous, intraarterial, intracardiac, intramuscular, intraperitoneal, intradermal, subcutaneous formulations. U.S. patent 5,192,550(Alza corporation) describes a dosage form that includes an outer wall that is impermeable to selegiline but permeable to external fluids. Such a dosage form may be suitable for oral, sublingual or buccal administration of selegiline. Similarly, U.S. patent 5,387,615 discloses a wide variety of selegiline compositions, including tablets, pills, capsules, powders, aerosols, suppositories, dermal patches, parenteral agents, and oral liquids, including oil-water suspensions, solutions and emulsions. Sustained release (long acting) formulations and devices containing selegiline are also disclosed.

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

Thus, despite being a highly potent and selective MAO-B inhibitor, the practical use of selegiline is limited by its dose-dependent specificity for MAO-B and the pharmacology of the selegiline metabolite that is produced after administration.

Summary of The Invention

The present invention relates to the surprising discovery that: i.e., norselegiline ("DMS" or "R (-) DMS") and its enantiomer (endo-norselegiline abbreviated as "Ent-DMS" or "S (+) DMS") are useful in providing subjects with selegiline-like effects, but with greatly reduced MAO-B inhibitory activity and a significant lack of increased selectivity for MAO-B as compared to selegiline. In particular, the present invention relates to the surprising discovery that: i.e. desmethylselegiline, endodesmethylselegiline and isomer mixtures thereof, provide a more advantageous way of achieving a selegiline-responsive disease or condition with a therapeutic effect similar to that of selegiline. Accordingly, the present invention provides novel pharmaceutical compositions employing desmethylselegiline and/or endodesmethylselegiline as active ingredients and novel therapeutic methods involving the administration of desmethylselegiline and endodesmethylselegiline.

Specifically, the present invention provides:

(1) an improved method for obtaining a selegiline-like therapeutic effect in a subject having a selegiline-responsive disease or condition, comprising

Administering to said subject desmethylselegiline, endodesmethylselegiline, or mixtures 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, desmethylselegiline, endodesmethylselegiline, or mixtures thereof, in an amount such that periodic administration of one or more unit doses of said composition is effective to treat one or more selegiline-responsive diseases or conditions in a subject to which said one or more unit doses are administered.

The term "selegiline-responsive disease or condition" as used herein refers to any of a variety of diseases or conditions for which selegiline is known to be useful in mammals, including humans. Specifically, "selegiline responsive diseases or disorders" refers to the various diseases and disorders described above, such as alzheimer's disease, cognitive dysfunction, neuronal rescue, and the like. Similarly, the term "selegiline-like therapeutic effect" refers to one or more health-beneficial effects produced by selegiline in a subject being treated for a selegiline-responsive disease or condition.

Examples of selegiline-responsive diseases or conditions associated with neuronal degeneration or trauma responsive 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, hair loss, senile dementia, hypoxia, cognitive dysfunction, negative symptoms of schizophrenia, amyotrophic lateral sclerosis, Tourette's syndrome, tardive dyskinesia, and toxic neurodegeneration.

The present invention also includes the use of R (-) DMS, S (+) DMS or mixtures thereof to restore and improve immune system function. Such improvement or recovery has been reported to occur when selegiline is administered to an animal. Treatable conditions or diseases include age-dependent immune system dysfunction, AIDS, cancer, and infectious diseases.

Depending on the particular route of administration employed, either desmethylselegiline or endodesmethylselegiline is administered in free base form or as a physiologically acceptable non-toxic acid addition salt. Such salts include those derived from organic and inorganic acids such as, but not limited to, hydrochloric, hydrobromic, phosphoric, sulfuric, methanesulfonic, acetic, tartaric, lactic, succinic, citric, malic, maleic, sorbic, aconitic, salicylic, phthalic, embonic, heptanoic, and the like. The use of salts, especially hydrochloride salts, is particularly desirable when the route of administration employs aqueous solutions as, for example, parenteral administration; the desmethylselegiline or endodesmethylselegiline provided in the free base form is particularly suitable for transdermal administration. Thus, references herein to administration of DMS or Ent-DMS or mixtures thereof include both the free base and acid addition salt forms.

The optimal daily dose of desmethylselegiline and/or endo-desmethylselegiline that can be used 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 disorder being treated, the condition of the subject to which it is administered, the extent of desired therapeutic response, and concomitant therapy of administration to the patient or animal. Generally, however, the attending physician or veterinarian will administer a preliminary dose of at least about 0.0015mg/kg, calculated on the basis of the free secondary amine, followed by escalating doses depending on the response to the treatment. Typically, the daily dose will be about 0.01mg/kg and may extend to about 0.5mg/kg of patient body weight (such doses are also all calculated on the basis of free secondary amine). These guidelines further require the attending physician or veterinarian to carefully prescribe the actual dosage depending upon the age, weight, clinical condition and observed response of the individual patient or animal.

Such daily doses may be administered in a single or divided multiple dosing regimen. Dosage forms and administration regimens may allow for the continuous release of relatively small amounts of an active ingredient from a single dosage unit, such as a transdermal patch, for example, over a course of one or more days. This is particularly desirable for the treatment of chronic disorders such as parkinson's disease, alzheimer's disease and depression. In addition, for conditions such as ischemia or nerve injury, it may also be desirable to administer one or more discrete doses by a more direct systemic route, such as intravenously or by inhalation. In addition, in other cases, such as glaucoma and macular degeneration, topical administration, such as by the intraocular route, may be indicated.

In the case of oral administration, the present invention includes the following unexpected findings: oral administration of desmethylselegiline and/or endodesmethylselegiline is more effective than oral administration of selegiline for a wide variety of conditions and diseases. Thus, in cases where the use of selegiline itself would be contraindicated due to side effects, the subject is administered desmethylselegiline and its enantiomers orally.

Pharmaceutical compositions containing desmethylselegiline and/or endodesmethylselegiline can be prepared according to conventional techniques. For example, a sterile isotonic saline solution can be used for the preparation of desmethylselegiline for parenteral administration by intramuscular, intravenous and intraarterial routes. Sterile isotonic solutions may also be employed for intraocular administration.

Transdermal unit dosage forms of desmethylselegiline and/or endodesmethylselegiline can be prepared using a variety of techniques as previously described (see, e.g., U.S. patents 4,861,800; 4,868,218; 5,128,145; 5,190,763 and 5,242,950; and EP-A404807, EP-A509761 and EP-A593807). For example, a one-piece patch construction may be utilized wherein desmethylselegiline is incorporated directly into the adhesive and the mixture is cast onto a backing sheet.

Furthermore, desmethylselegiline and/or endodesmethylselegiline can be incorporated as an acid addition salt in a multi-layer patch which converts the salt to the free base, for example as described in EP-A593807.

Desmethylselegiline and/or endo-desmethylselegiline can also be administered by a device employing a lyotropic liquid crystal composition, such as a combination of 5-15% desmethylselegiline with a mixture of liquid and solid polyethylene glycols, a polymer, and a nonionic surfactant, optionally with the addition of propylene glycol and an emulsifier. For further details regarding the preparation of such transdermal preparations reference is made to EP-A5509761.

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

The subjects treatable by the present formulations and methods include human subjects and non-human subjects for which a therapeutic effect similar to selegiline is known to be useful. Thus, the above compositions and methods provide particularly useful treatments for mammals, particularly domestic mammals. For example, the present methods and compositions are useful for treating selegiline-responsive diseases or disorders in canine and feline species.

Successful use of the above compositions and methods requires the use of an effective amount of desmethylselegiline, endodesmethylselegiline, or mixtures thereof. Although both desmethylselegiline and endo-desmethylselegiline are much less potent as MAO inhibitors than selegiline, the use of these agents or mixtures of these agents does not require a corresponding increase in dosage to achieve a therapeutic response similar to selegiline. Surprisingly, the dose required to achieve a therapeutic effect similar to that of selegiline is of the same order of magnitude as the known dose of selegiline. Thus, norselegiline and endo-norselegiline offer A much broader safety margin than selegiline with respect to MAO-A related toxicity, since desmethylselegiline and endo-desmethylselegiline show much lower MAO-A inhibition at such doses. Specifically, the deleterious effects of MAO-A inhibition, such as risk of hypertension crisis, are minimized by 40-70 times less potent MAO-A inhibition.

As mentioned above, and despite its demonstrable poor inhibitory properties with respect to MAO-B inhibition, desmethylselegiline and its enantiomers are significantly more effective than selegiline in treating selegiline-responsive disorders such as those caused by neuronal degeneration or neuronal trauma. In this regard, desmethylselegiline and/or its enantiomers, like selegiline itself, are particularly useful when administered by a route that does not rely on upper gastrointestinal or other gastrointestinal absorption. Preferred routes include parenteral, topical, transdermal, intraocular, buccal, sublingual, intranasal, inhalation, vaginal, and rectal routes.

As noted above, the present invention includes another discovery: desmethylselegiline can be employed in either the photoactive or racemic form, i.e., a mixture of enantiomers of desmethylselegiline. Desmethylselegiline, its enantiomers and mixtures thereof are conveniently prepared by methods known in the art, as described in example 1 below.

Brief description of the drawings

FIG. 1: effect of selegiline on neuronal survival. The embryonic day 14 rats were used to prepare midbrain cultures. Cultures were used at approximately 150 million cells per plate and were maintained in either growth medium alone (control culture) or growth medium supplemented with selegiline. On days 1,8 and 15, cells were given immune support to allow the presence of tyrosine hydroxyl enzyme ("TH"). The solid bars represent the results obtained for cultures maintained in the presence of 50 μ M selegiline, and the open bars represent the results for control cultures. In all cases, the results are expressed as the percentage of TH positive cells present in the first day control cultures. The abbreviation "DIV" means "days ex vivo". The asterisk or star above the bar in figure 1 and the figures discussed below indicates that the results differ from the control by a statistically significant amount, i.e., P < 0.05.

FIG. 2: in midbrain cells [ ]³H) -dopamine uptake. The cells cultured as described above for FIG. 1 were subjected to the uptake assay for their marker dopamine, and the results are shown in FIG. 2 as solid bars representing uptake in cells maintained in the presence of 50 μ M selegiline and open bars representing uptake in control cultures.

FIG. 3: effect of selegiline on glutamate receptor dependent neuronal cell death. Rat embryonic mesencephalon cells were cultured as described above. After allowing the culture to stabilize, the medium was changed daily for a period of 4 days to induce glutamate receptor dependent cell death. Depending on the culture, the medium contained 0.5, 5.0 or 50. mu.M selegiline. After the last medium change, cultured cells were immunostained for the presence of tyrosinehydroxylase. Bars on the graph represent results from left to right for control, 0.5, 5.0 and 50 μ M selegiline, respectively.

FIG. 4: effect of selegiline on dopamine uptake in neuronal cultures. The rat midbrain cells were cultured with daily changes of the medium as discussed for fig. 3. Uptake of tritiated dopamine by the cells was measured and the results are shown in the figure. The sequence of the bars from left to right in the figure is the same as in figure 3.

FIG. 5: the effect of R (-) desmethylselegiline on glutamate receptor dependent neuronal cell death. Rat embryo mesencephalon 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 culture was determined. Results are expressed as a percentage of the control group. The bars on the figure show the results from left to right for the control, 0.5, 5 and 50 μ M R (-) DMS.

FIG. 6: effect of R (-) desmethylselegiline on dopamine uptake in neuronal cultures. Cell cultures were prepared as described above in fig. 5, and then tested for tritium-labeled dopamine uptake. The results for the control group and cells maintained in the presence of 0.5. mu.M, 5. mu.M and 50. mu.M desmethylselegiline are shown in the figure from left to right.

FIG. 7: comparison of dopamine uptake by mesencephalon cells cultured in the presence of different monoamine oxidase inhibitors. Rat embryonic mesencephalon cells were prepared as described in FIGS. 3-6 and cultured in the presence of various monoamine oxidase inhibitors. The inhibitor investigated was selegiline; r (-) desmethylselegiline; eugenine (bagelin); and clorgyline (clorgyline) at concentrations of 0.5, 5 and 50 μ M, respectively. In addition, the cells were cultured in the presence of the glutamate receptor blocker MK-801 at a concentration of 10. mu.M. The cultures were tested for tritium-labeled dopamine uptake.

FIG. 8: inhibition of neuronal dopamine neuron reuptake by enantiomers of diprenil and desmethylselegiline. An ex vivo nerve end preparation (synaptosome preparation) was prepared from fresh rat neostriatal tissue. The ability to uptake tritiated dopamine in either buffer alone or in buffers supplemented with different concentrations of selegiline, R (-) desmethylselegiline or S (+) desmethylselegiline was examined for this. The amount of uptake in the presence of each MAO inhibitor was expressed as a percentage of inhibition relative to the amount of uptake in the presence of buffer alone, and the results are shown in figure 8. As indicated in the figure, the ID of each reagent was determined using these curves₅₀. ID of S (+) DMS₅₀Is 20. mu.M; selegiline, 80 μ M; r (-) DMS, about 100. mu.M.

FIG. 9: in vivo MAO-B inhibition of hippocampus of guinea pigs. Guinea pigs were injected with different doses of selegiline, R (-) desmethylselegiline and S (+) desmethylselegiline daily for a5 day period. The animals were then sacrificed and MAO-B activity was measured in hippocampal portions of the brain. Results expressed as percent inhibition of hippocampal MAO-B activity in control animalsThe ratio is also shown in FIG. 9. The ID of each reagent was determined using these curves₅₀And (4) dosage. Selegiline ID₅₀Is about 0.03 mg/kg; both enantiomers of DMS are about 0.3 mg/kg.

FIG. 10: splenocytes from rats injected with selegiline, R (-) DMS or S (+) DMS produce interferon ("IFN") ex vivo. F344 rats were injected intraperitoneally daily with saline, selegiline, R (-) DMS or S (+) DMS for 60 days. All the rats injected are aged rats, namely 18-20 months old. After a 10 day "washout" period without any injection, the rats were sacrificed and their spleens removed. Ex vivo interferon-gamma production by stimulated splenocytes (lymphocytes) was determined and compared to that of uninjected aged rats and young rats (3 months of age). The bars in the figure reflect, from left to right, the results of splenocytes from the following animals: young rats; aged rats, saline injected aged rats; geriatric rats injected with 0.25mg/kg selegiline; geriatric rats injected with 1.0mg/kg selegiline; rats injected with 0.025mg/kgR (-) DMS; rats injected with 0.25mg/kg R (-) DMS; rats injected with 1.0mg/kg R (-) DMS; and rats injected with 1.0mg/kg S (+) DMS. The horizontal line indicates the point where each IFN level is significantly different (P < 0.05) from the level seen with splenocytes from young rats. The results are expressed as units per ml.

FIG. 11: in vitro IFN production by splenocytes from aged rats. The same results shown in fig. 10 are repeated in fig. 11, but excluding the results for splenocytes from young rats. "#" indicates a result that is significantly different (P < 0.05) from that obtained with the spleen of an aged rat injected with saline. "^*"points out the results which are significantly different from the groups except for the aged rats injected with 1.0mg/kg of diprenyl.

FIG. 12: splenocytes from aged rats are produced with isolated Interleukin-2 (Interleukin-2). Rats were injected as described above (see FIG. 10) and the stimulated splenocytes were assayed for production of interleukin-2. The sequence of the bars in the figure from left to right is the same as in figure 10.

FIG. 13: percentage of splenocytes in rats positive for IgM. Rats were injected with saline, selegiline, R (-) DMS or S (+) DMS as described above (see FIG. 10). The spleens of these rats were tested to determine the percentage of IgM positive cells, and the results are shown in the figure. The horizontal line in the graph corresponds to a statistically significant (P < 0.05) reduction in IgM percentage relative to the percentage seen in spleens obtained from young rats. The bars in the figure are in the same order from left to right as in figures 10 and 12.

FIG. 14: percentage of splenocytes in rats positive for CD 5. The experiment of figure 13 was repeated, but instead of determining the percentage of cells positive for IgM, the percentage positive for CD5 was determined.

Detailed description of the invention

The surprising utility of norselegiline and endonorselegiline in the treatment of selegiline-responsive diseases or disorders can be attributed, in part, to their powerful role in preventing dopaminergic neuronal loss by promoting repair and recovery. Thus, reversal of neuronal damage and/or death can be observed at doses as low as 0.01mg/kg, at which little or no MAO-B inhibition is typically observed. Desmethylselegiline and endodesmethylselegiline are valuable for a wide variety of neurodegenerative and neuromuscular diseases because they prevent loss of neuronal cell function and promote recovery thereof. In this regard, desmethylselegiline and endo-desmethylselegiline are much more potent than selegiline, as described more empirically in the examples below. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples of the invention

EXAMPLE 1 preparation of desmethylselegiline and endo-desmethylselegiline

A. Desmethylselegiline

Desmethylselegiline (hereinafter referred to as "R (-) DMS") is prepared by methods known in the artIn (1). Desmethylselegiline, for example, is one of the known chemical intermediates for preparing selegiline described in U.S. patent No.4,925,878. Desmethylselegiline can be prepared as follows: in an inert organic solvent such as toluene, at a slightly elevated temperature (70-90 deg.C) with an equimolar amount of an active propargyl halide such as propargyl bromide Br-CH₂-C.ident.CH treatment of a solution of R (+) -2-aminophenylpropane (L-amphetamine):

optionally, this reaction may be carried out in the presence of an acid acceptor such as potassium carbonate. Then, the reaction mixture is extracted with an aqueous acid solution such as 5% hydrochloric acid, and the extract is made alkaline. The non-aqueous layer formed is separated, for example, by extraction with benzene, dried, and distilled under reduced pressure.

Alternatively, the propargylation may be carried out analogously to the preparation of selegiline described in U.S. Pat. No.4,564,706, using a salt of R (+) -2-aminophenylpropane and a weak acid, such as tartrate, in a biphasic system of a water immiscible solvent and an aqueous base.

B. Endo-desmethylselegiline

Endesmethylselegiline (hereinafter referred to as "S (+) DMS") is derived from the enantiomer S (-) -2-aminophenylpropane (dextroamphetamine), i.e.

Conveniently prepared according to the procedure described above for desmethylselegiline.

C. Mixture of enantiomers

Mixtures of the enantiomeric forms of desmethylselegiline, including racemic desmethylselegiline, are conveniently prepared from mixtures of enantiomers, including racemic mixtures of the aforementioned aminophenylpropane starting materials.

D. Conversion to acid addition salts

N- (prop-2-ynyl) -2-aminophenylpropane, whether in optically active form or in racemic form, may be converted into a physiologically acceptable, non-toxic acid addition salt by conventional techniques, for example by treatment with an inorganic acid. For example, desmethylselegiline hydrochloride is prepared from a solution of hydrogen chloride in isopropanol. Whether as the free base or as a salt, may be further purified by conventional techniques such as recrystallization or chromatography.

Example 2 neuronal survival measured by tyrosine Hydroxygenases

The effect of desmethylselegiline on neuronal survival can be correlated with tyrosine hydroxylase, dopamine biosynthesis rate limiting enzyme. The test was performed by measuring the number of tyrosinehydroxylase positive cells in the brain cells of E-14 embryos cultured for 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 method

Neuronal cultures of brains from embryonic rats at day 14 of gestation were established with chronologically pregnant Sprague-Dawley rats. Separating midbrain without membrane covering layer, and collecting in the presence of Ca⁺⁺And Mg⁺⁺At 4 ℃ in a balanced salt solution. The tissue fragments were gently ground using a small internal diameter pasteur pipette and dissociated in a chemically defined medium. Cell suspension at 1.5X 10⁶Cell/dish density the plates were plated on 35mm Falcon plastic dishes (0.1mg/ml, Sigma) coated with polyornithine. Culture at 10% CO₂A37 ℃ atmosphere of 90% air and 100% relative humidity was maintained and supplemented twice a week with a chemically defined medium consisting of MEM/F12 (1: 1, Gibco), glucose (33mM), HEPES (15mM), NaHCO₃(44.6mM), transferrin (100mg/ml), insulin (25mg/ml), putrescine (60nM), sodium selenite (30nM), progesterone (20nM) and glutamine (2 mM). Control cells were not subjectedTo further additions. The medium for the other cells also includes a test substance such as selegiline at one or more concentrations.

Cultures were fixed with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) for 30 minutes at room temperature, permeabilized with 0.2% Triton X-100 for 30 minutes, and incubated with anti-tyrosine hydroxylase antibody (1: 1000; Eugene Tech) for 48 hours at 4 ℃ in the presence of blocking serum. Then, 3' -diaminobenzidine was stained using a peroxidase-coupled avidin-biotin staining kit (Vectastain ABC kit; Vector Labs) as a chromogen.

The number of dopaminergic neurons in culture was determined by counting cells that had positive immunostaining for the TH antibody. 100 fields (0.5 mm. times.0.5 mm) in two bands orthogonal to the dish diameter were counted at 200 times magnification using a Nikon inverted microscope and accounted for 2.5% of the total area.

B. Results

Using the procedure described above, the following results were obtained:

table 2: effect of selegiline and DMS on survival of TH-Positive cells

Concentration of	Control	SelequiBlue		Desmethylselegiline
						Average	Average	% control	Average	% control
	0.5μM5μM50μM	108.55--	201.70±25.01237.00±12.59292.28±17.41	185.81218.33269.25	246.00±22.76357.95±25.76391.60±34.93						226.62329.76360.76

Example 3 neuronal survival measured by 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. Uptake by cultured brain cells corresponds to axon growth.

A. Test method

Cell cultures created by the methods discussed above and [ ]³H dopamine (0.5 mCi/ml; 37m Ci/mmol; New England Nuclear Co.) in the Presence of ascorbic acid (0.2mg/ml) supplemented with 0.9mM CaCl₂And 0.5mM MgC₂In PBS (pH 7.3), and incubated at 37 ℃ for 15 minutes. After rinsing twice and incubation with fresh buffer for 5 minutes, these cells were allowed to accumulate by incubating the culture with 95% ethanol at 37 ℃ for 30 minutes [ ]³H, releasing dopamine. The formulations were then added to 10ml Ecoscint (National Diagnostics) and counted on a scintillation spectrometer. Non-specific uptake values were obtained by blocking dopaminergic neuronal uptake with 10mM mazindol.

B. Results

Using the above procedure, the results shown in table 3 were obtained.

Table 3: selegiline and DMS pair³Effect of H-dopamine uptake

Concentration of	Control	Selegiline		Desmethylselegiline
						Average	Average	% control	Average	% control
	0.5μM5μM50μM	11982--	14452±21216468±57633018±1317	120.6137.5275.5	24020±80034936±211956826±2656						200.4291.5474.3

C. Conclusion of 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 despite the fact that desmethylselegiline is much less potent than selegiline as a MAO-B inhibitor.

EXAMPLE 4 neuroprotective Effect of desmethylselegiline enantiomers in ex vivo cultured dopamine-containing mesocerebral neurons

In these experiments, the neuroprotective properties of selegiline and R (-) desmethylselegiline were examined with the survival of cultures of dopamine-containing neurons of the midbrain of rat brain tissue. The number of TH positive neurons is proportional to the survival of dopaminergic neurons,³h-dopamine uptake is a measure of axonal growth in these neurons.

A. Effect of selegiline on survival of dopaminergic neurons

Midbrain cultures prepared from embryonic day 14 rats were treated with 0.5, 5 and 50 μ M selegiline for 15 days, from the day of plating. (for a more detailed discussion of cell culture and other methods used in these experiments, see Mytiliou et alJ.Neurochem,61: 1470-1478(1993). ) Survival and growth of dopamine neurons is achieved by immunocytochemistry with tyrosine-Hydroxygenases (TH)³H dopamine uptake, the results are shown in FIGS. 1 and 2.

When selegiline was tested at concentrations of 0.5 and 5 μ M, no effect on neuronal survival was observed. At 50 μ M, selegiline reduced the loss of TH positive neurons at 8 and 15 days post-plating (FIG. 1) and increased dopamine uptake at 15 days (FIG. 2). Pretreatment with 0.5, 5, 50 or 100 μ M selegiline was determined in several independent experiments, in which no inhibition of dopamine uptake was observed under the experimental conditions used.

These results indicate that the uptake values obtained reflect dopamine neuron survival and proud warts, 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 caused neuronal cell death that could be blocked by inhibition of glutamate receptors. In these experiments, cells were plated and allowed to stabilize for several days. The growth medium for these cells is then changed daily to induce cell death, which can be prevented, for example, by blocking glutamate receptors with MK-801. After changing the medium once a day for 4 days, the cultures were subjected to tyrosinehydroxylase staining and tritium-labeled dopamine uptake tests. The results shown in fig. 3 and 4 further support the following conclusions: selegiline can promote the survival of dopaminergic neurons.

C. Effect of desmethylselegiline on dopamine neuron survival

Using the glutamate receptor dependent neuronal death model, an even stronger dopaminergic neuronal protection is provided when selegiline is replaced by desmethylselegiline. Even at the lowest dose tested (0.5 μ M), desmethylselegiline caused a significant decrease in the loss of TH positive neurons (FIG. 5) and a significant increase in dopamine uptake (FIG. 6), both relative to control cultures in which media not supplemented with selegiline or desmethylselegiline was used.

D. Comparison with other MAO inhibitors

The effects of selegiline and desmethylselegiline were compared to two other MAO inhibitors, eugenine and clorgoline, using a glutamate receptor dependent neurotoxicity pattern (figure 7). Consistent with the previous results, the determination of dopamine uptake indicates neuronal protection of 50 μ M diprenil and 5 with 50 μ M desmethylselegiline. The prohibitin apparently did not provide any protection at the concentrations used, whereas clorgyline was protected at 50 μ M. Protection was also achieved by the NMDA receptor blocker MK-801 (10. mu.M), as expected.

E. DMS enantiomer pair³Effect of H-dopamine uptake

The data summarized in Table 4 indicate that both R (-) DMS and S (+) DMS are effective as neuroprotective agents for midbrain dopamine-containing neurons in culture.

TABLE 4 influence of DMS enantiomer on dopamine uptake

³Uptake of H-dopamine

Treatment of (percentage + mean standard error)

Control 100. + -. 14.14%

R(-)DMS(10nM) 140.82±26.20％

S(+)DMS(10nM) 234±38.36％

These results demonstrate 40% and 134% higher axon growth and terminal axon survival after treatment with R (-) DMS and S (+) DMS, respectively, compared to untreated control cells. Thus, S (+) DMS may be an even stronger and/or more therapeutically effective neuroprotective agent than R (-) DMS.

Example 5 desmethylselegiline and endo-desmethylselegiline as dopamine reuptake inhibitors

The biological effects of the brain neurotransmitter dopamine are terminated at the synapse by a high affinity, sodium and energy dependent delivery system (neuronal reuptake) present in the limiting membrane of presynaptic dopamine-containing nerve terminals. Inhibition of this transport mechanism will extend the effect of dopamine on the synapse, thereby enhancing synaptic transmission of dopamine.

A. Test method

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 a valuable indicator of agents in the treatment of diseases where an increase in synaptic dopamine activity is desired. Currently, this will include parkinson's disease, alzheimer's disease, and Attention Deficit Hyperactivity Disorder (ADHD).

The assay system used is essentially that described by Fang et al (Neuropharmacology33: 763-768(1994)). Isolated nerve powderThe apical preparation (synaptosome preparation) was obtained from the brain tissue of a new striatum of fresh rats. Dopamine nerve terminal transport was estimated by measuring tritium-labeled dopamine uptake.

B. Results

As seen in the data set forth in table 5, selegiline, R (-) DMS, and S (+) DMS all inhibited dopamine reuptake by dopamine-containing nerve terminals. Selegiline is approximately equivalent to R (-) DMS. And the drug effect of the S (+) DMS is 4-5 times stronger than that of selegiline or R (-) DMS.

TABLE 5 New striatal brain tissue from rat³H-dopamine uptake

The relative potency can be determined by the concentration (ID) required to inhibit dopamine reuptake by 50%₅₀) To indicate. ID₅₀The values were determined graphically (see fig. 8) and are shown in table 6 below.

TABLE 6 concentrations required to inhibit dopamine uptake by 50%

Medicament	ID
		Selegiline R (-) DMSS (+) DMS	≈80μm≈100μm≈20um

C. Conclusion

These results demonstrate that, at appropriate concentrations, each enantiomer of selegiline and DMS inhibits the transmission of released dopamine at neuronal synapses and increases the relative activity of this neurotransmitter at the synapses. In this respect, S (+) DMS is more potent than selegiline, which in turn is more potent than R (-) DMS. This effect is indicative of a benefit of the agent in the treatment of parkinson's disease, alzheimer's disease and Attention Deficit Hyperactivity Disorder (ADHD). Of the agents tested, S (+) DMS is clearly the most effective in treating ADHD.

Example 6 the R (-) and S (+) enantiomers of Desmethylselegiline (DMS) are useful in humans

Effect of body platelet MAO-B and GuineA pig brain MAO-B and MAO-A Activity

Human platelet MAO exclusively contains the B-isomeric form of the enzyme (isoform). In this study, the inhibition of this enzyme by both enantiomers of DMS was determined and compared to the inhibition caused by selegiline. In addition, the study examined the inhibitory activity of the two enantiomers of DMS on MAO-A and MAO-B in hippocampal tissues of guineA pigs. Ragweed brain tissue is the best animal model to study dopamine metabolism in the brain, the enzyme kinetics of various MAO forms, and the inhibitory properties of novel agents that interact with these enzymes. The various MAO forms in this animal species show the same kinetic properties as the various MAO forms found in human brain tissue. Finally, guinea pigs were injected with the drug to determine the extent to which they could act as in vivo brain MAO inhibitors.

B. Test method

The test system utilizes human platelets and/or guinea pig hippocampal homogenateMAO-A specific substrate: (¹⁴C-serotonin) and MAO-B specific substrate ((M)¹⁴C-phenylethylamine) ex vivo. The conversion rate of each substrate was determined 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. By comparing the 50% Inhibitory Concentration (IC) induced by each agent₅₀Value), the drug effect was evaluated.

In some experiments, R (-) DMS, S (+) DMS or selegiline was administered subcutaneously once daily to A living body over A5 day period, followed by slaughter, preparation of zymophyte homogenate and ex vivo testing for MAO-A and MAO-B activity. These experiments were performed to demonstrate that the DMS enantiomer can enter brain tissue and inhibit MAO activity.

C. Results

Ex vivo MAO-B inhibitory Activity

The results of MAO-B inhibition are shown in tables 7 and 8. IC for MAO-B inhibition₅₀The values and potency compared to selegiline are shown in table 9.

TABLE 7 MAO-B inhibition in human platelets

TABLE 8 MAO-B inhibition in guinea pig hippocampus

TABLE 9 MAO-B inhibited IC₅₀Value of

Treatment of	Human blood platelet	Hippocampus cortex of guinea pig
			Selegiline R (-) DMSS (+) DMS	5nM(1)400nM(80)1400nM(2800)	1nM(1)60nM(60)1200nM(1200)

() The efficacy of the drug compared with selegiline

As observed, R (-) DMS is 20-35 times more potent than S (+) DMS as MAO-B inhibitor, and both enantiomers are less potent than selegiline.

MAO-A inhibitory Activity

The results obtained from experiments investigating MAO-A inhibition in the hippocampus of guineA pigs are summarized in Table 10, the two enantiomers of DMS and the IC of selegiline₅₀The values are shown in table 11.

TABLE 10 MAO-A inhibition in guineA pig hippocampus

TABLE 11 MAO-A inhibited IC₅₀Value of

Treatment of	IC of MAO-A in Hippocampus cortex of GuineA pig
		Selegiline R (-) DMSS (+) DMS	2.5μM(1)50.0μM(20)100.0μM(40)

() The efficacy of the drug is comparable to that of (selegiline)

As MAO-A inhibitor, R (-) DMS has twice the effect of S (+) DMS, and both of them have 20-40 times smaller effect than selegiline. In addition, the efficacy of each of these agents as MAO-A inhibitors in hippocampal brain tissue is 2 to 3 orders of magnitude less than that of MAO-B inhibitors, i.e., 100 to 1000 times. Thus, selegiline and each DMS enantiomer can be classified as a selective MAO-B inhibitor in brain tissue.

Results of in vivo experiments

Each DMS enantiomer was administered to the living body by subcutaneous injection once daily for 5 consecutive days, and inhibition of brain MAO-B activity was then determined. Under these conditions, R (-) DM as MAO-B inhibitorS and S (+) DMS are equivalent, but the potency is 10 times less than selegiline. The results are shown in FIG. 9, IC₅₀The values are summarized in table 12.

TABLE 12 IC of brain MAO-B when injected with drug prior to the experiment₅₀Value of

Treatment of	IC of MAO-B in Hippocampus cortex of Guinea pig
		Selegiline R (-) DMSS (+) DMS	0.03mg/kg0.30mg/kg0.30mg/kg

This experiment demonstrates that each of the DMS enantiomers is able to penetrate the blood-brain barrier and inhibit brain MAO-B following parenteral in vivo administration. It also demonstrates that the difference in potency between each DMS enantiomer observed ex vivo and selegiline as MAO-B inhibitor is reduced in vivo conditions. The dose administered in this experiment was insufficient to inhibit MAO-A activity.

D. Conclusion

Both R (-) DMS and S (+) DMS demonstrated activity as MAO-A inhibitors and MAO-B inhibitors. Each enantiomer is selective for MAO-B. S (+) DMS generally has less potency than R (-) DMS in inhibiting both MAO-A and MAO-B, while both enantiomers of DMS have less potency than selegiline. Both enantiomers demonstrate activity in vivo, indicating that these enantiomers are capable of entering brain tissue following parenteral administration. The ability of these agents to inhibit MAO-A and MAO-B suggests that these agents are valuable as therapeutic agents for parkinson's disease, alzheimer's disease or depression.

EXAMPLE 7 in vivo neuroprotection of the enantiomer of desmethylselegiline

The ability of the DMS enantiomer to prevent neurological deterioration was investigated by administering these agents to wobbler mice, an animal model of motor neuron disease, particularly Amyotrophic Lateral Sclerosis (ALS). The wobbler mice exhibited progressively worsening forelimb weakness, gait disturbances and forelimb muscle flexion contractions.

B. Test method

In a randomized, double-blind study, wobbler mice were administered either R (-) DMS, S (+) DMS, or a control by intraperitoneal injection daily for 30 days. At the end of this time, mice were examined for grip strength, running time, resting motor activity, and rated for semi-quantitative paw posture abnormalities and semi-quantitative walking abnormalities. The researchers who prepared the solution and injected the solution into the animals are different from those who analyzed the behavior change.

The tests and grading are essentially like Mitsumoto et al [ ann. neuron.36: 142, 148(1994) ]. Mouse forepaw grip was determined by having the animal grasp a wire with both paws. The wire was connected to a gram-force meter and the tail of the mouse was pulled until the animal was forced to release the wire. The reading on the force gauge at release was taken as a measure of grip strength.

Running time is defined as the minimum time required to traverse a specified distance, such as 2.5 feet, and the best time to record several trials.

Paw posture abnormalities are graded on a scale based on the degree of contraction, and gait abnormalities are graded on a scale ranging from normal walking to the inability to support the body with the paw.

Locomotor activity was determined by transferring animals to a survey area covered with a square grid. Activity is measured by the number of squares a mouse crosses over at a set time interval, e.g. 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 close to each other at baseline. The weight gain was the same in all three groups, indicating that no major side effects occurred in any animal. Table 13 summarizes the differences observed in the mean grip strength of the test animals:

TABLE 13 treatment with R (-) or S (+) DMS

Average grip strength in wobbler mice

Treatment of	N	Grip strength (gram)
			Control (placebo) R (-) DMSS (+) DMS	1099	9(0-15)20(0-63)14(7-20)

Number of animals analyzed

All animals had a significant decrease in grip strength at the end of the first week. At the end of the study, the grip was the minimum for the control animals, ranging from 0-15 g, with an average of 9 g. The average grip strength of the R (-) group was 20g, and the value ranged from 0 to 63 g. The third group injected with S (+) DMS had an average grip of 14g with individual values ranging from 7 to 20 g. While variability of grip strength in the treated animal group prevented meaningful statistical analysis of this data, the mean grip strength measured in DMS-treated animals was greater than in the control group.

Running time, resting motor activity, semi-quantitative paw posture abnormality grading, and semi-quantitative gait abnormality grading were also tested. However, none of these tests showed data that could indicate that any of these three groups differed from the other.

EXAMPLE 8 immune System recovery of R (-) DMS and S (+) DMS

The age-related decline in immune function that occurs in animals and humans renders older individuals more susceptible to infectious diseases and cancer. Us patents 5,276,057 and 5,387,615 suggest that selegiline is useful in the treatment of immune system dysfunction. The experiments of this example were conducted to determine whether R (-) DMS and S (+) DMS can also be used to treat such dysfunction. It will be appreciated that the ability to support the patient's normal immune defenses would be beneficial in treating a wide variety of acute and chronic diseases, including cancer, AIDS, and bacterial and viral infections.

A. Test procedure

This experiment utilized a rat model to investigate the ability of R (-) DMS and S (+) DMS to restore immune function. The rats were divided into the following experimental groups:

1) young rats (3 months of age, not injected);

2) aged rats (18-20 months old, not injected);

3) aged rats injected with saline;

4) old rats were injected with selegiline. The dosage is 0.25mg/kg body weight;

5) old rats were injected with selegiline. The dosage is 1.0mg/kg body weight;

6) aged rats were injected with R (-) DMS. The dosage is 0.025mg/kg body weight;

7) aged rats were injected with R (-) DMS. The dosage is 0.25mg/kg body weight;

8) aged rats were injected with R (-) DMS. The dosage is 1.0mg/kg body weight;

9) aged rats were injected with S (+) DMS. The dosage is 1.0mg/kg body weight;

rats were injected intraperitoneally once daily over 60 days. They were then maintained for an additional 10 day "flush" period during which no injections were given. At the end of this period, the animals were slaughtered and their spleens removed. The splenocytes are then tested for various factors that are indicative of immune system function. Specifically, the following were determined using standard tests:

1) splenocytes produce interferon-gamma ex vivo;

2) ex vivo production of interleukin-2;

3) percentage of IgM positive splenocytes (IgM is the producer of B lymphocytes);

4) percentage of CD5 positive splenocytes (CD5 was the producer of T lymphocytes).

B. Results

The effect of injection of selegiline, R (-) DMS and S (+) DMS on interferon production by rat splenocytes is shown in FIGS. 10 and 11. As shown in FIG. 10, there was a sharp decrease in the production of interferon cells with age (comparing the production of cells from young animals with the production of cells from older animals or saline-injected older animals). Injection of selegiline, R (-) DMS and S (+) DMS all resulted in partial recovery of the interferon-gamma level, with the greatest magnitude of increase at a dose of 1.0mg/kg body weight.

The same data as shown in fig. 10 are repeated in fig. 11, except for the results omitting the cells of the young rat. This figure shows more clearly the extent to which Dichenille, R (-) DMS and S (+) DMS restore the production of interferon-gamma in splenocytes from aged rats. Interferon-gamma is a multifunctional protein that inhibits viral replication and regulates a wide variety of immune functions. It affects the class of antibodies produced by B-cells, up-regulates MHC class I and class II complex antigens, and increases macrophage-mediated killing efficiency of intracellular parasites.

FIG. 12 shows the effect of each injection on the production of interleukin-2 by splenocytes from rats. It can be seen that both R (-) DMS and S (+) DMS restore the production of interleukin-2 to levels seen in young animal cells.

The effect of each injection on the percentage of IgM positive splenocytes is shown in figure 13. It has been found that both selegiline and R (-) DMS partially restore IgM positive cells to levels closer to those seen in the spleen of young rats. Thus, it is clear that these agents restore B lymphocyte numbers.

FIG. 14 shows that injection of either 0.025mg/kg R (-) DMS or S (+) DMS slightly increased the percentage of CD5 positive cells in the spleen of aged rats. However, it is clear that neither selegiline injection nor 0.25 or 1.0mg/kg R (-) DMS injection has any effect.

C. Conclusion

Taken together, these results support the following conclusions: the DMS enantiomer mimics the effect of selegiline on immune system function. Furthermore, the results obtained with respect to the production of interferon, IL-2 and with respect to the percentage of IgM-positive splenocytes support the following conclusions: the DMS enantiomer can at least partially restore the loss of function of the age-dependent immune system. Thus, it is apparent that R (-) DMS and S (+) DMS will have therapeutically beneficial effects on diseases and conditions resulting from weakened host immunity. This would include cancer, AIDS and all types of infectious diseases.

Example 9 dosage form examples

A. Desselegiline patch

Ingredients by dry weight (mg/cm)²)

Durotak^_87-2194

Adhesive acrylic acid polymer 90 parts by weight

10 parts by weight of desmethylselegiline

The two components were mixed well and cast on a sheet of Scotchpak^_9723 backing sheet of polyester film, drying. Cutting the backing sheet into patches, adding Scotchpak one for each patch^_1022 a fluoropolymer release liner, hermetically sealing the patch in a metal foil envelope. In the treatment of human disorders resulting from neuronal degeneration or neuronal trauma, such as Parkinson's disease, a daily patch is applied to deliver 1-5 mg of desmethylselegiline every 24 hours.

B. Ophthalmic solution

Desmethylselegiline as the hydrochloride salt (0.1g), 1.9g boric acid and 0.004g phenylmercuric nitrate were dissolved in an appropriate amount of sterile water to make a total of 100 ml. Sterilizing the mixture, and sealing. It is useful in ophthalmology for the treatment of conditions resulting from neuronal degeneration or neuronal trauma, such as glaucomatous optic neuropathy and macular degeneration.

Example 3: intravenous solution

A1% solution was prepared by dissolving 1g of desmethylselegiline hydrochloride in 0.9% isotonic saline solution sufficient 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, which can be used to treat conditions resulting from neuronal degeneration or neuronal trauma.

C. Transdermal patch

A self-crosslinking acrylic-based pressure-sensitive adhesive is added to an organic solvent such as a methyl ethyl ketone solution of a copolymer of methacrylic acid and dimethylaminoethyl methacrylate. This was cast onto a first removable metal foil and the solvent was evaporated, the coated metal foil being placed on a polyester backing layer as described in EP-a 593807.

Desmethylselegiline hydrochloride is added to a suitable organic solvent, such as an ethyl acetate solution, for a non-crosslinked acrylic based pressure sensitive adhesive. Additional solvent may be added and heat and agitation may also be employed to facilitate dispersion formation. This was applied to a second removable metal foil and the solvent was evaporated. After the metal foil was removed from the previously prepared polyester backing layer, it was laminated to a coated second removable metal foil. Another portion of the self-crosslinking acrylic-based pressure sensitive adhesive and an organic solvent such as a solution of methacrylic acid and dimethylaminoethyl methacrylate copolymer in methyl ethyl ketone is cast onto a third removable metal foil and the solvent is evaporated. Removing the second and third metal foils, laminating the residues together, cutting the obtained laminate into patch, and packaging. The resulting patch will have a removable release liner, an adhesive layer initially free of desmethylselegiline and a matrix layer containing desmethylselegiline hydrochloride (or other salt). An impermeable backing layer is bonded to the base layer by an intermediate adhesive layer similar to the adhesive layer in contact with the removable release liner. In the treatment of human disorders resulting from neuronal degeneration or neuronal trauma, such as Parkinson's disease, a daily patch is applied to deliver desmethylselegiline in free base form.

D. Oral dosage form

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

1-5 parts of desmethylselegiline

Microcrystalline cellulose 86

Lactose 41.6

0.5-2 parts of citric acid

0.1-2 parts of sodium citrate

Magnesium stearate 0.4

The ratio of citric acid to sodium citrate is about 1: 1.

Claims (21)

1. A compound, namely S (+) desmethylselegiline or a pharmaceutically acceptable acid addition salt thereof, for use in therapy.

2. Use of a compound as defined in claim 1 for the manufacture of a medicament for neuroprotection or neuronal rescue.

3. Use of a compound as defined in claim 1 for the manufacture of a medicament for the restoration or improvement of immune system function.

4. Use of a compound as defined in claim 1 for the manufacture of a medicament for the treatment of a selegiline-responsive disease or condition.

5. Use of a compound as defined in claim 1 for the manufacture of a medicament for the treatment of parkinson's disease, alzheimer's disease or depression.

6. Use of a compound as defined in claim 1 for the manufacture of a medicament for the treatment of attention deficit hyperactivity disorder.

7. Use of a compound as defined in claim 1 for the manufacture of a medicament for the treatment of ischemia or hypoxia.

8. A medicament comprising the compound of claim 1 as an active ingredient.

9. The medicament of claim 8, wherein the medicament is in an injectable dosage form.

10. The medicament of claim 8, wherein the medicament is a transdermal dosage form.

11. The medicament of claim 8, wherein the compound is a hydrochloride salt.

12. A pharmaceutical agent comprising a compound, i.e., R- (-) desmethylselegiline or a pharmaceutically acceptable salt thereof, as an active ingredient, wherein said pharmaceutical agent is in a transdermal dosage form.

13. Use of a compound, i.e. R- (-) desmethylselegiline or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for neuroprotection or neuronal rescue.

14. Use of a compound, i.e. R- (-) desmethylselegiline or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the restoration or improvement of immune system function.

15. Use of a compound, i.e. R- (-) desmethylselegiline or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a selegiline-responsive disease or condition.

16. Use of a compound, i.e. R- (-) desmethylselegiline or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a parkinson's disease, alzheimer's disease or attention deficit hyperactivity disorder.

17. Use of a compound, i.e. R- (-) desmethylselegiline or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of depression.

18. Use of a compound, i.e. R- (-) desmethylselegiline or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of ischemia or hypoxia.

19. Use of a compound, namely R- (-) desmethylselegiline or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of diabetic neuropathy or amyotrophic lateral sclerosis.

20. The use of any one of claims 13-19, wherein the compound is a hydrochloride salt.

21. The use of any one of claims 13-20, wherein the medicament is a non-oral medicament.