CN1178462A - Methods and pharmaceutical compostions employing desmethylselegiline - Google Patents

Abstract

Methods and pharmaceutical compositions for using desmethylselegiline. In particular, the present invention provides novel compositions and methods for using desmethylselegiline for selegiline-responsive diseases and conditions. Diseases and conditions responsive to selegiline include those produced by neuronal degeneration or neuronal trauma and those due to immune system dysfunction. Desmethylselegiline is the R-(-) enantiomer of N-methyl-N-(prop-2-ynyl)-2-aminophenylpropane. Claimed compositions include both the R-(-) isomer and mixtures of the R-(-) and S(+) isomers. Pharmaceutically acceptable acid addition salts may also be used. Effective dosages are a daily dose of at least about 0.015 mg/kg of body weight.

Description

Use the method and the medical composition of S

Invention field

The present invention relates to use selegiline (Selegiline) metabolite is the method and the medical composition of S in S and the enantiomer thereof.Specifically, the invention provides compositions and the method that these medicaments are used for selegiline reactive disorder and disease.

Background of invention

The monoamine oxidase, MAO of two kinds of uniquenesses is technical known: monoamine oxidase A (MAO-A) and monoamine oxidase-B (MAO-B).Show different promoter regions and unique exon part for the cDNA of these enzymes codings, show that they are absolute codings on different gene locations.In addition, the analysis of these two kinds of protein has shown its difference of amino acid sequence separately.

First kind of chemical compound having found energy selectivity inhibition MAO-B is R-(-)-N-methyl-N-(Propargyl)-2-aminophenyl propane, is also referred to as L-(-)-Di Pu Neil (deprenyl), R-(-)-Di Pu Neil or selegiline.Selegiline has following structural formula:

Selegiline is important at the selectivity aspect the MAO-B inhibition for its peroral administration safety image.Tranylcypromine (introduced before three more than ten years but be eliminated because of its serious hypertension side effect subsequently a kind of MAO inhibitor), opposite with selegiline, be a kind of nonselective MAO inhibitor.The acute toxicity of tranylcypromine results from the inhibition of MAO-A, and this has disturbed the metabolism of tyramine.Tyramine is normally metabolic by MAO-A in gastrointestinal tract, but when MAO-A is suppressed, edible contain tyramine food such as cheese, medicated beer, catfish etc. after tyramine absorb and will increase.This causes catecholamine to discharge and can cause the hypertension crisis, produces " cheese effect ".Goodman and Gilman are characterized by this effect the serious toxicity effect that interrelates with the MAO-A inhibitor.

One of metabolite of selegiline is the nor-analog of its N-.Structurally, this S metabolite R-(-)-enantiomeric form that is following formula secondary amine:

Before this, it is useful that people do not know that also S has on the medicine, with the MAO related effect, promptly to the strong and selectivity depression effect of MAO-B.In the definite process of S, S and MAO related effect have been characterized more completely for the serviceability of the object of the invention.This sign determines that S has atomic weak MAO-B depression effect, and compares with selegiline, is not having any advantage aspect the selectivity of MAO-B.

For example, this sign has been determined selegiline IC to MAO-B in human body platelet ₅₀Value is 5 * 10 ^-9M, and the IC of S ₅₀Value is 4 * 10 ^-7M shows that the latter is littler about 80 times than the former as the drug effect of MAO-B inhibitor.MAO-B and the inhibiting of MAO-A can be seen similar characteristics in the column data down in measuring rat cortex pay streak plastochondria fraction:

Table 1 selegiline and S are to the inhibitory action of MAO

Concentration	Suppress percentage rate
					Selegiline		S
	??0.003μM ??0.010μM ??0.030μM ??0.100μM ??0.300μM ??1.000μM ??3.000μM ??10.000μM ??30.000μM ??100.000μM	????MAO-B ????16.70 ????40.20 ????64.70 ????91.80 ????94.55 ????95.65 ????98.10 ????- ????- ????-	????MAO-A ????- ????- ????- ????- ????9.75 ????32.55 ????65.50 ????97.75 ????- ????-	????MAO-B ????3.40 ????7.50 ????4.60 ????6.70 ????26.15 ????54.73 ????86.27 ????95.15 ????97.05 ????-					????MAO-A ????- ????- ????- ????- ????0.0 ????0.70 ????4.10 ????11.75 ????- ????56.10

Apparent from last table, selegiline is as the drug effect of MAO-B inhibitor for MAO-A powerful about 128 times, and S is only strong 97 times for MAO-A as the drug effect of MAO-B inhibitor.Therefore, S obviously have with selegiline about equally, to the selectivity (comparing) of MAO-B, although drug effect reduces greatly with MAO-A.

Obtained similar results with rat cerebral tissue.Selegiline demonstrates the IC to MA0-B ₅₀Value is 0.11 * 10 ^-7M, and the IC of S ₅₀Value is 7.3 * 10 ^-7M shows that S is littler about 70 times than selegiline as the drug effect of MAO-B inhibitor.Portion demonstrates low drug effect aspect two kinds of chemical compound MAO-A in suppressing rat cerebral tissue, and selegiline is 0.18 * 10 ^-5, S is 7.0 * 10 ^-5Therefore, aspect inhibition MAO-A, the drug effect of S is littler about 39 times than selegiline.

According to its aforesaid medicine image,,, compare any advantage that all do not provide with selegiline no matter on the drug effect or on selectivity as the S of MAO-B inhibitor.On the contrary, above-mentioned isolated test data show that S approximately needs 70 times of selegiline quantity as the consumption of MAO-B inhibitor.

Heinonen, E.H. wait the people to report the drug effect (" S; be a kind of metabolite of selegiline; be a kind of irreversible inhibitor of MAO-B in the human experimentation object " of S as live body MAO-B inhibitor, see scientific paper collection " selegiline in the parkinson disease treatment ", Turun Univ's neurological systematic study report, Turku, Finland, No.33 (1995), pp 59-61).Heinonen thinks, the MAO-B depression effect of S has only the about 1/5 of selegiline in the live body, promptly reaches the dosage that the MAO-B effect identical with the 1.8mg selegiline will need the 10mg S.

Known selegiline comprises its useful various diseases and disease: depression (United States Patent (USP) 4,861,800); Alzheimer and parkinson disease especially by using the percutaneous dosage form, comprise ointment, cream and plaster; Degeneration of macula (United States Patent (USP) 5,242,950); The age dependency is degenerated, and comprises renal function and the cognitive function (United States Patent (USP) 5,151,449) that is confirmed by the space learning ability; Hypophysis dependency hypercortisolism (United States Patent (USP) 5,192,808); Immune system malfunction (United States Patent (USP) 5,276,057); And schizophrenia (United States Patent (USP) 5,151,419).The disclosed application of PCT WO 92/17169 discloses selegiline at treatment neuromuscular and neurodegenerative disease with treat purposes aspect the central nervous system injury that causes owing to anoxia, hypoglycemia, ischemia outbreak or wound.

Although known selegiline can be treated above-mentioned disease effectively, or does not know its precise number, or do not know character or mechanism of action that it is machine-processed.Yet, evidence suggests that selegiline may provide neuroprotective or neuron rescue by reducing the infringement of oxidisability neuron, increase the quantity of superoxide dismutase and/or reducing dopamine catabolism.For example, the disclosed application of PCT WO 92/17169 reports, selegiline by direct maintenance animal nerve function, prevent from its loss and/or give its help to work.

Selegiline is extensively studied the biochemical effect of neuronal cell.For example, consult people such as Tatton " neuron rescue of selegiline energy intermediary rather than neuro-protective ", Movement Disorders8 (Supp1): S20-S30 (1993); People such as Tatton " dying neuronic rescue ", J.Neurosci.Res.30:662-672 (1991); With people such as Tatton " (-)-Di Pu Neil prevents the cell death in mitochondrial depolarization and the rescue malnutrition sexual cell ", 11 th Int ' l Symp on Parkinson ' s Disease, Rome, ITA ,-30 days on the 26th March, 1994.

Known selegiline is useful when giving the experimental subject administration by miscellaneous route of administration and dosage form.For example, United States Patent (USP) 4, selegiline-amantadine that 812,481 (Degussa AG) disclose companion's row oral, per os, through intestinal, through lung, per rectum, per nasal, transvaginal, through tongue, through intravenous, through intra-arterial, through intracardiac, through intramuscular, through intraperitoneal, through Intradermal, purposes in subcutaneous prescription.United States Patent (USP) 5,192, a kind of dosage form has been described by 550 (Alza companies), and is impermeable but to the outer wall of outside fluid penetrable to selegiline comprising one.This dosage form may be applicable to per os, the Sublingual of selegiline or contain the agent administration.Similarly, United States Patent (USP) 5,387,615 disclose various selegiline compositionss, comprise tablet, pill, capsule, powder, aerosol, suppository, skin patch, non-, comprise oil-aqueous suspension liquor, solution and emulsion agent through intestinal medicament and per os liquor.Also disclose and contained selegiline sustained release (long-acting) prescription and utensil.

Except that nor-selegiline, selegiline also produces a kind of other main directly metabolite, i.e. mephentermine.S and mephentermine all further are metabolized to amphetamine again.Known back two kinds of metabolite, promptly amphetamine and mephentermine have pair dopamine neuron to produce the potentiality of neurotoxicity effect, are undesirable by-products.Different with selegiline is that S does not produce the mephentermine as metabolite, only produces amphetamine.

Therefore, although be a kind of MAO-B inhibitor that height drug effect and selection arranged, the practical use of selegiline is limited by the pharmacology of its selegiline metabolite that produces after to the dose dependent specificity of MAO-B and administration.

Brief summary of the invention

The present invention relates to surprising discovery: i.e. S (" DMS " or " R (-) DMS ") and enantiomer (interior S thereof, breviary is " Ent-DMS " or " S (+) DMS ") all can be used for providing the effect of similar selegiline to the curee, but compare with selegiline, significantly reduced MAO-B and suppressed the active and obvious increase selectivity that lacks MAO-B.Specifically, the present invention relates to surprising discovery: promptly S, interior S and isomer mixture thereof provide a kind of more favourable approach that makes selegiline reactive disorder or disease obtain similar selegiline therapeutic effect.Therefore, the invention provides and adopt S and/or interior S as the Medicine compositions of effective ingredient with relate to S and the novel method of treatment of interior S administration.

Specifically, the invention provides:

(1) a kind of improved method is used to make the curee who suffers from selegiline reactive disorder or disease to obtain similar selegiline therapeutic effect, and this method comprises

Described curee is used S, interior S or its mixture with the quantity that is enough to produce similar selegiline therapeutic effect; With

(2) a kind of medical composition, wherein, on one or more optional medicines, can accept excipient or the carrier, also contain a certain amount of S, interior S or its mixture, make one or more dosage units of the regular administration of described compositions can treat one or more selegiline reactive disorder or diseases effectively the curee of the described one or more dosage units of its administration.

" selegiline reactive disorder or disease " used herein this term means to comprise that known selegiline is to any in its useful various diseases or the disease in the human mammal.Specifically, " selegiline reactive disorder or disease " means above-mentioned various diseases and disease, for example Alzheimer, understanding malfunction, neuron rescue etc.Similarly, " similar selegiline therapeutic effect " this term means that in the curee who carries out selegiline reactive disorder or treatment for diseases one or more that produced by selegiline are of value to restorative effect.

This method is responded, selegiline reactive disorder or the disease relevant with neuronal degeneration or wound comprise parkinson disease, Alzheimer, depression, glaucoma, degeneration of macula, ischemia, diabetes type neuropathy, the attention-deficient sexual maladjustment, syndrome after the poliomyelitis, multiple sclerosis, sexual impotence, narcolepsy, chronic fatigue syndrome, alopecia, alzheimer disease, anoxia, the understanding malfunction, negative symptoms of schizophrenia, the sclerosis of the amyotrophic lateral sclerosis outside, Tourette syndrome, tardive dyskinesia and toxicity neural degeneration.

The present invention also comprises with R (-) DMS, S (+) DMS or its mixture recovers function of immune system and improvement.Such improvement or recovery take place in existing people's report when animal is used selegiline.Medicable disease or disease comprise age dependent form function of immune system obstacle, AIDS, cancer, and infectious disease.

Different because of the specific administration approach that is adopted, perhaps S or interior S are with free alkali form or as accepting the administration of non-toxic acid addition salts on the physiology.Such salt comprises from organic acid and mineral acid such as but not limited to deutero-salt such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulphuric acid, methanesulfonic acid, acetic acid, tartaric acid, lactic acid, succinic acid, citric acid, malic acid, maleic acid, sorbic acid, equisetic acid, salicylic acid, phthalic acid, seal ripple naphthoic acid (embonic acid), enanthic acid.When route of administration adopts aqueous pharmaceutical as such as non-during through enteral administration, salt, especially the use of hydrochlorate is ideal especially; Use the S or the interior S that are provided to be specially adapted to percutaneous dosing with free alkali form.Therefore, comprise free alkali and these two kinds of forms of acid-addition salts to DMS or Ent-DMS administration or to the lifting manipulation of its mixture here.

Can be used for the S of the object of the invention and/or the best daily dose of interior S determines with technical known method, for example, seriousness according to disease of being treated or disease, to its curee's who treats situation, desirable therapeutic response degree and the companion of patient or animals administer row therapy determined.Yet, in general, the preliminary dosage that the doctor in charge or veterinary are as the criterion administration to calculate with free secondary amine, dosage then because of the different employing of the reaction of this treatment is improved gradually at least about 0.0015mg/kg.Say that typically daily dose will be about 0.01mg/kg and can expand to about 0.5mg/kg weight in patients (such dosage is that benchmark calculates with free secondary amine all also).These guide lines further require the doctor in charge or veterinary because of the different actual dose of carefully deciding of age, body weight, clinical condition and the observed reaction of individual patient or animal.

This daily dose can be by the single or scheme administration of offeing medicine several times.Dosage form and dispensing scheme can allow such as in one day or the many days courses of treatment from the single dose unit such as the percutaneous patch discharge quite a small amount of effective ingredient continuously.This for chronic disease for example the treatment of parkinson disease, Alzheimer and depression be ideal especially.In addition, for diseases such as ischemia or nerve injury, as through intravenous or by the one or more discrete dosage of inhalation, also can be ideal by more direct general approach.In addition, under other situations such as glaucoma and degeneration of macula, can indicate topical, for example by the ophthalmic approach.

Under peroral administration situation, the present invention includes following unexpected the discovery: concerning being used for various diseases and disease, per os uses S and/or interior S to use selegiline more effective than per os.Therefore, can avoid using under the situation of selegiline itself, give curee's oral administration S and enantiomer thereof owing to side effect.

The medical composition that contains S and/or interior S can prepare according to common technology.For example, through intramuscular, can adopt the sterile isotonic common salt aqueous solution through intravenous with through non-S preparations such as intra-arterial approach through the enteral administration approach.Also can adopt sterile isotonic solution to eye drops.

Various technology preparation of describing before the percutaneous unit dosage form of S and/or interior S can utilize (is seen in as United States Patent (USP) 4,861,800; 4,868,218; 5,128,145; 5,190,763 and 5,242,950; With EP-A 404807, EP-A509761 and EP-A 593807).For example, can utilize a kind of monolithic patch structure, wherein, S directly be mixed in the binding agent, and this mixture is cast on the backing light sheet.

In addition, can also mix a kind of can making in the multilamellar patch that this salt changes into free alkali to S and/or interior S as a kind of acid-addition salts, for example, described in EP-A593807.

S and/or interior S also can be by a kind of utensil administrations, this utensil adopts a kind of lyotropic liquid crystal compositions, wherein, randomly add propylene glycol and a kind of emulsifying agent such as the mixture that makes 5～15% Ss and liquid and solid polyethylene glycol, a kind of polymer and the combination of a kind of nonionic surfactant.About the further details of the preparation of this type of percutaneous preparation, can be with reference to EP-A 5509761.

Because " interior S " this term means S (+) isomeric forms of S, thereby, more than the lifting manipulation of the mixture of selegiline and interior S is comprised the racemic mixture and the non-racemic mixture of optical isomer simultaneously.

The medicable object of this preparation and method comprise known class like the selegiline therapeutic effect to its useful human subjects and non-human object.Therefore, above compositions and method are mammal, and especially domestic mammal provides useful especially treatment.For example, this method and compositions can be used for treating selegiline reactive disorder or the disease in Canidae and the feline species.

The successful use of above compositions and method needs the S of empoly effective amount, interior S or its mixture.Although S and interior S are all much smaller than selegiline as the drug effect of MAO inhibitor, adopt the mixture of these medicaments or these medicaments not need corresponding increased dosage amount to obtain the treatment response of similar selegiline.Surprisingly, reach the needed dosage of similar selegiline therapeutic effect and be in the magnitude identical with the selegiline known dose.Therefore, suppress because S and interior S demonstrate much lower MAO-A on such dosage, thus S and interior S provide than selegiline wide Duo, with the xicity related relevant safety scope of MAO-A.Specifically, the ill-effect that MAO-A suppresses such as the risk of hypertension crisis are reduced to bottom line for little 40～70 times because of its MAO-A suppresses drug effect.

As mentioned above, although and its bad inhibition activity of susceptible of proof that suppresses about MAO-B, more more effective significantly than selegiline aspect the disease that S and enantiomer thereof cause in the reactive disease of treatment selegiline such as neuronal degeneration or neuron wound.At this on the one hand, S and/or its enantiomer are the same with selegiline itself, when by a kind of administration that does not rely on upper gastrointestinal or the absorption of other gastrointestinal, are useful especially.Preferably approach comprise parenteral, partial, percutaneous, through ophthalmic, through (the containing agent) of cheek, through the Sublingual, through intranasal, suction, transvaginal and per-rectum approach.

As noted above, the present invention includes another discovery: S can the photolytic activity form can racemic form be the form of mixtures employing of S and interior S also both.S, its enantiomer and composition thereof be with as following example 1 described in, technical known method prepares easily.

Brief Description Of Drawings

Fig. 1: selegiline is to the influence of neuronal survival.Prepare the midbrain culture with 14 days rats of embryo.Culture uses with about 1,500,000 cells of every plate, and or remain in (control cultures) in the independent growth medium, perhaps remain in the growth medium of having added selegiline.In the time of the 1st, 8 and 15 day, pair cell gives immunity and supports to exist to have made it tyrosine hydroxyl enzyme (" TH ").The resulting result of culture that the solid bar representative keeps in the presence of 50 μ M selegilines, hollow bar is represented the result of control cultures.In all cases, the result all is expressed as the percentage rate of the TH positive cell that existed in the control cultures in first day.Abbreviation " DIV " means " isolated test natural law ".The asterisk or the star of rod top point out that this result and matched group differ a statistically evident amount, i.e. P＜0.05 among Fig. 1 and each figure discussed below.

Fig. 2: in the midbrain cell ( ³H)-dopamine uptake.As above cultured cells as described in Fig. 1 is carried out picked-up test of its sign dopamine, the result is illustrated among Fig. 2.Picked-up in the cell that the solid bar representative keeps in the presence of 50 μ M selegilines, hollow bar is represented the picked-up in the control cultures.

Fig. 3: selegiline is to the influence of glutamate receptor dependent form neuronal cell death.The rat embryo midbrain cell is cultivated as previously discussed like that.Make culture reach stable after, change culture medium every day in 4 days by a definite date time, to bring out glutamate receptor dependent form cell death.Different because of culture, culture medium contains 0.5,5.0 or 50 μ M selegilines.After culture medium changes the last time, give immunostaining to cultured cells and exist to have made it tyrosine hydroxyl enzyme.Rod on the figure is from left to right represented the result of matched group, 0.5,5.0 and 50 μ M selegilines respectively.

Fig. 4: selegiline is to the influence of dopamine uptake in the neuron culture.Cultivate the rat midbrain cell, change culture medium every day as Fig. 3 is discussed.Measure the picked-up of cell to the tritiated dopamine, the result represents in the drawings.Each rod order from left to right is identical with Fig. 3 on the figure.

Fig. 5: R (-) S is to the influence of glutamate receptor dependent form neuronal cell death.Rat embryo midbrain culture prepares as the above, and different is to replace selegiline with R (-) DMS.At the 9th day, measure the number of TH positive cell in the culture.Results expression is the percentage rate of matched group.Rod on the figure from left to right shows the result of matched group, 0.5,5 and 50 μ M R (-) DMS.

Fig. 6: R (-) S is to the influence of dopamine uptake in the neuron culture.The above Fig. 5 of cell culture image described such preparation, carry out the tritiated dopamine uptake then and measure examination.The result of matched group and the cell that keeps in the presence of 0.5 μ M, 5 μ M and 50 μ M Ss from left to right is shown among the figure.

Fig. 7: the dopamine uptake of the midbrain cell of cultivating in the presence of different oxidase inhibitor relatively.3～6 described such preparations of rat embryo midbrain cell image pattern, and in the presence of various oxidase inhibitor, cultivate.The inhibitor of being investigated is a selegiline; R (-) S; Pargyline (pargyline); And clorgiline (clorgyline), concentration is 0.5,5 and 50 μ M.In addition, cell is to cultivate in the presence of the glutamate receptor blocker MK-801 of 10 μ M concentration.Culture carries out the tritiated dopamine uptake and measures examination.

Fig. 8: the enantiomer of Di Pu Neil and S is to the inhibition of neuron dopamine neuron reuptake.With a kind of stripped teleneuron preparation of fresh rat neostriatum tissue preparation (synaptosome preparation).This is investigated it in independent buffer agent or added the ability of picked-up tritiated dopamine in the buffer agent of selegiline, R (-) S or S (+) S of variable concentrations.Intake in the presence of each MAO inhibitor is expressed as the inhibition percentage rate with respect to the intake under the buffer agent individualism, and the result is illustrated among Fig. 8.As pointed among the figure, determined the ID of each reagent with these curves ₅₀The ID of S (+) DMS ₅₀Be 20 μ M; Selegiline, 80 μ M; R (-) DMS, about 100 μ M.

Fig. 9: the live body MAO-B of guinea pig hippocampal suppresses.Every day is to selegiline, R (-) S and S (+) S of Cavia porcellus injection various dose in 5 days by a definite date.Slaughter animal then, measure the MAO-B activity in the cerebral hippocampus part.Results expression is with respect to the active inhibition percentage rate of Hippocampus MAO-B in the control animal, and is illustrated among Fig. 9.Determined the ID of every kind of reagent with these curves ₅₀Dosage.The ID of selegiline ₅₀Be about 0.03mg/kg; Two kinds of enantiomer of DMS all are about 0.3mg/kg.

Figure 10: the stripped interferon (" IFN ") of splenocyte of having injected the rat of selegiline, R (-) DMS or S (+) DMS produces.The F344 rat in 60 days by a definite date every day through peritoneal injection saline solution, selegiline, R (-) DMS or S (+) DMS.All rats of injection are senile rat, i.e. 18～20 monthly ages.After the phase, rat being slaughtered, take out its spleen through 10 days " the going out " of not doing any injection.Measure the stripped interferon-output of the splenocyte (lymphocyte) of irriate, and with its with the production ratio of the splenocyte (lymphocyte) of the senile rat of not injection and young rats (3 monthly age).Rod among the figure from left to right reflects the result of following animal splenocyte respectively: young rats; Senile rat has been injected the senile rat of saline solution; Injected the senile rat of 0.25mg/kg selegiline; Injected the senile rat of 1.0mg/kg selegiline; Injected the rat of 0.025mg/kgR (-) DMS; Injected the rat of 0.25mg/kg R (-) DMS; Injected the rat of 1.0mg/kg R (-) DMS; With the rat of having injected 1.0mg/kg S (+) DMS.Horizontal line is pointed out each IFN level and the level point of seeing with the young rats splenocyte that there were significant differences (P＜0.05).Results expression is the units among every ml.

Figure 11: the stripped IFN of senile rat splenocyte produces.Repeat the identical result shown in Figure 10 among Figure 11, but do not comprised the result of young rats splenocyte." # " points out and the result result that there were significant differences (P＜0.05) who obtains with the spleen of senile rat of having injected saline solution." ^*" point out and each the group result that all there were significant differences except that the senile rat of having injected 1.0mg/kg Di Pu Neil (deprenyl).

Figure 12: leukin-2 (Interleukin-2) produced between the senile rat splenocyte exsomatized.Rat is injected (see figure 10) as the above, leukin-2 output between mensuration irriate splenocyte.Rod order from left to right is identical with Figure 10 among the figure.

The percentage rate of the positive rat spleen cells of Figure 13: IgM.Rat is (see figure 10) injection saline solution, selegiline, R (-) DMS or S (+) DMS as the above.Spleen to these rats is tested, and to determine being IgM positive cells percentage rate, the result represents in the drawings.Horizontal line among the figure is corresponding to the significantly IgM percentage rate of (P＜0.05) minimizing on statistics for the percentage rate of seeing the spleen that obtains from young rats.The same in rod among figure order from left to right and Figure 10 and 12.

Figure 14: be the male rat spleen cells percentage rate of CD5.Repeat the experiment of Figure 13, be not IgM positive cells percentage rate, be the male percentage rate of CD5 but measure but do not measure.

Detailed description of the invention

S（+）DMS and interior S（+）DMS can partly repair and recover to prevent the strong effect of dopaminergic neurone loss by promotion owing to them in surprising effectiveness aspect treatment selegiline reactive disorder or the illness. Therefore, with generally observed small or without the low dosage that reaches 0.01mg/kg that MAO-B suppresses, just can observe the reverse of neure damage and/or death. Because S（+）DMS and interior S（+）DMS can prevent the loss of nerve cell function and promote its recovery, thereby they are valuable for miscellaneous neurodegenerative disease and neuromuscular disease. At this on the one hand, the drug effect of S（+）DMS and interior S（+）DMS is more much better than than selegiline, describes as having more empirically in following instance. These examples only are used for the illustrative purpose, are not intended to limit the scope of the invention.

Example

The preparation of example 1 S and interior S

A, S

S (hereinafter referred to as " R (-) DMS ") is to prepare with technical known method.For example, S is a U.S. Patent No. 4,925, a kind of known chemical intermediate of the preparation selegiline described in 878.S can prepare as follows: in a kind of inert organic solvents such as toluene, under high slightly temperature (70～90 ℃), with a kind of active propargylic halide such as the propargyl bromide Br-CH of equimolar amounts ₂-C ≡ CH handles R (+)-2-aminophenyl propane (left-handed amphetamine) solution:

Randomly, this reaction can be carried out in the presence of a kind of acid acceptor such as potassium carbonate.Then, reactant mixture as 5% hcl as extraction agent, makes extract become alkalescence with aqueous acid again.Formed non-water layer is such as using benzene extract and separate, drying, distilling under reduced pressure.

In addition, this propargylization can also be similar to U.S. Patent No. 4,564, the selegiline preparation described in 706, utilize R (+)-2-aminophenyl propane and a kind of faintly acid salt such as tartrate, in the diphasic system of a kind of water immiscible solvent and aqueous alkali, carry out.

B, interior S

Interior S (hereinafter referred to as " S (+) DMS ") is from enantiomer S (-)-2-aminophenyl propane (dextroamphetamine), promptly

According to above the described program of S prepared easily.

C, mixture of enantiomers

The mixture of each enantiomeric form of S comprises the raceme S, is from mixture of enantiomers, comprises the racemic mixture of above-mentioned aminophenyl propane initiation material, prepares easily.

D, change into acid-addition salts

No matter the photolytic activity form still is N-(the Propargyl)-2-aminophenyl propane of racemic form, can change into a kind of physiologically acceptable non-toxic acid addition salts with common technology as using a kind of mineral acid treatment.For example, prepare the S hydrochlorate with isopropanol solution of hydrogen chloride.No matter free alkali or salt can be further purified with common technology such as recrystallization or chromatography again.

The neuronal survival that example 2 usefulness tyrosine hydroxyl enzymes are weighed

S can be that dopamine biosynthesis speed restriction endonuclease is associated with tyrosine hydroxyl enzyme to the influence of neuronal survival.Test is to be undertaken by the number that is determined at tyrosine hydroxyl enzyme positive cell in the E-14 embryo midbrain cell of cultivating in 7～14 days by a definite date.Use various trophic factors, comprise BDNF, GDNF, EGF and β-FGF, seen the protection in this system.

A, test method

With the pregnant Sprague-Dawley rat of timing, set up neuron culture from the 14th day fetal rat brain of gestation.Isolate the midbrain of not being with film coating, be collected in no Ca ⁺⁺And Mg ⁺⁺4 ℃ of balanced salt solutions in.Fragment of tissue grinds gently with little internal diameter pasteur pipet, makes it to dissociate in the culture medium of chemically definition.Cell suspending liquid is with 1.5 * 10 ⁶(0.1mg/ml carries out flat board on Sigma) and cultivates the density of cell/dish being coated with the 35mm Falcon plastics dish of poly ornithine.Culture is at 10%CO ₂Remain in 37 ℃ in the atmosphere of/90% air and 100% relative humidity, add the chemically culture medium of definition weekly twice, its consist of MEM/F12 (1: 1, Gibco), glucose (33mM), HEPES (15mM), NaHCO ₃(44.6mM), transferrins (100mg/ml), insulin (25mg/ml), putrescine (60nM), sodium selenite (30nM), Progesterone (20nM) and glutamine (2mM).Cellular control unit is not subjected to further interpolation.The culture medium that is used for other cell also comprises trier such as selegiline with one or more concentration.

Culture at room temperature uses 4% paraformaldehyde in the 0.1M phosphate buffer (pH7.4) to fix 30 minutes, with 0.2%Triton X-100 infiltration 30 minutes, in the presence of blocking-up serum in 4 ℃ with the antibody of anti-tyrosine hydroxyl enzyme (1: 1000; Eugene Tech) cultivated 48 hours.Then, the avidinbiotin staining test kit of peroxidase (the Vectastain ABC test kit that utilized a kind of coupling; Vector Labs), with 3 ', 3 '-diaminobenzidine is a chromogen, it dyeed.

By counting the cell of TH antibody positive immunostaining is arranged, determine the number of dopaminergic neuron in the culture.With a Nikon inverted microscope with 100 territories (0.5mm * 0.5mm), account for 2.5% of the gross area in 200 times of enlargement ratios counting and orthogonal two cross bands of this dish diameter.

B, result

Utilize the above program, obtain following result:

Table 2: selegiline and DMS are to the influence of TH positive cell survival

Concentration	Contrast	Selegiline		S
						On average	On average	The % contrast	On average	The % contrast
	??0.5μM???????????????108.55?????????????201.70±25.01?????????????185.81???????????????246.00±22.76??????????226.62 ??5μM?????????????????-??????????????????237.00±12.59?????????????218.33???????????????357.95±25.76??????????329.76 ??50μM????????????????-??????????????????292.28±17.41?????????????269.25???????????????391.60±34.93??????????360.76

The neuronal survival that example 3 usefulness dopamine uptake amounts are weighed

In measuring culture, the number (seeing example 2) of TH positive cell, also can determine the protective effect of S by direct mensuration dopamine uptake amount to neuronal cell.The intake of the brain cell of cultivating is corresponding to axon growth.

A, test method

The cell culture of setting up with method discussed above with ( ³H) dopamine (0.5mCi/ml; 37m Ci/mmol; NEN) together, in the presence of ascorbic acid (0.2mg/ml), adding 0.9mM CaCl ₂With 0.5mM MgC ₂PBS (pH7.3) in, cultivated 15 minutes at 37 ℃.After cultivating 5 minutes twice of rinsing with fresh buffer agent, cultivated 30 minutes at 37 ℃ with 95% ethanol by making this culture, make accumulation in these cells ( ³H) dopamine discharges.Then preparation is added among the 10ml Ecoscint (National Diagnostics), count with scintillation spectrometer.By with the picked-up of 10mM mazindol blocking-up dopaminergic neuron, obtain non-single-minded picked-up value.

B, result

Use above program, obtain the result shown in the table 3.

Table 3: selegiline and DMS are right ³The influence of H-dopamine uptake

Concentration	Contrast	Selegiline		S
						On average	On average	The % contrast	On average	The % contrast
	??0.5μM??????????????11982????????????????14452±212?????????????????120.6?????????????24020±800??????????????200.4 ??5μM????????????????-????????????????????16468±576?????????????????137.5?????????????34936±2119?????????????291.5 ??50μM???????????????-????????????????????33018±1317????????????????275.5?????????????56826±2656?????????????474.3

C, example 2 and 3 conclusion

Result described in the example 2 and 3 points out that as a kind of neuroprotective, S is better than selegiline.This is real, although in fact S as a kind of drug effect of MAO-B inhibitor a little less than than selegiline many.

Example 4 S enantiomer containing in the dopamine midbrain neuron in isolated culture

Neuroprotective

In these experiments, the neuroprotective performance of selegiline and R (-) S is investigated in the survival that contains the dopamine neuron culture with the midbrain of rat cerebral tissue.The number of TH positive neuron is proportional to survival of dopaminergic neurons, ³H-dopamine uptake amount is a yardstick of axon growth in these neurons.

The influence that A, selegiline are survived to dopaminergic neuron

Handled 15 days with 0.5,5 and 50 μ M selegilines from the midbrain culture of embryo's rat preparation in the 14th day, cultivate from flat board and count that day.(, see also people such as Mytilineou about the cell culture of use and more going through of other method in these experiments J.Neurochem,61:1470-1478 (1993).) survival of dopamine neuron and growth be with tyrosine hydroxyl enzyme (TH) immunocytochemistry and ( ³H) the dopamine uptake amount is assessed, and the result is illustrated among Fig. 1 and 2.

When selegiline is tested with the concentration of 0.5 and 5 μ M, do not observe neuronal survival is produced any influence.When 50 μ M, selegiline is cultivated the loss (Fig. 1) that reduced the TH positive neuron in back 8 days and 15 days at flat board, has increased dopamine uptake amount (Fig. 2) in the time of 15 days.Determine that in some independent experiments the pretreatment with 0.5,5,50 or 100 μ M selegilines carry out does not suppress the picked-up of dopamine under the experimental condition that uses in these experiments.

These results show that resulting picked-up value reflection dopamine neuron survival and projection wart show that also selegiline has the neuroprotective effect.

B, selegiline are to the influence of glutamate receptor dependent form cell death

Also utilize a kind of experiment example that can cause the neuronal cell death that to be blocked by the inhibition of glutamate receptor, investigated the neuroprotective effect of selegiline.In these experiments, cell carries out flat board to be cultivated, and makes it to stablize some days.All change the growth medium of these cells then every day, to bring out cell death, this death can be such as being prevented with MK-801 blocked glutamic acid receptor.Change culture medium once a day after 4 days, culture carries out tyrosine hydroxyl enzyme to be infected with, and carries out the test of tritiated dopamine uptake.Result displayed is further supported as drawing a conclusion among Fig. 3 and 4: selegiline can promote survival of dopaminergic neurons.

The influence that C, S are survived to dopamine neuron

The neuronal death model that utilizes glutamate receptor to rely on provides a kind of even stronger dopaminergic neuron protection when replacing selegiline with S.Even at the lowest dose level of being tested (0.5 μ M), S has also caused the remarkable minimizing (Fig. 5) of TH positive neuron loss and the remarkable increase (Fig. 6) of dopamine uptake amount, all for control cultures, wherein used the culture medium of not adding selegiline or S.

D, with other MAO inhibitor relatively

The neurotoxicity pattern of utilizing glutamate receptor to rely on, the effect of selegiline and S and two kinds of other MAO inhibitor are that pargyline and clorgiline have been done relatively (Fig. 7).With the unanimity as a result of front, the mensuration of dopamine uptake amount has been pointed out the neuro-protective of 50 μ M Di Pu Neils and 5 and 50 μ M Ss.Pargyline does not obviously provide any protection in employed concentration, and clorgiline has protection at 50 μ M.As expected, protection also obtains by nmda receptor blocker MK-801 (10 μ M).

E, DMS enantiomer are right ³The influence of H-dopamine uptake

The data of summing up in the table 4 show that contain dopamine neuron for the midbrain in the culture, R (-) DMS and S (+) DMS can both be effective as neuroprotective.

Table 4 DMS enantiomer is to the influence of dopamine uptake

³H-dopamine uptake amount

Handle (percentage rate+standard error of mean)

Contrast 100 ± 14.14%

R(-)DMS(10nM)???????????????????????140.82±26.20％

S(+)DMS(10nM)???????????????????????234±38.36％

These results confirm, compare with untreated cellular control unit, after handling with R (-) DMS and S (+) DMS, have respectively to exceed 40% and 134% axon growth and the survival of terminal aixs cylinder.Therefore, S (+) DMS may be a kind of neuroprotective than R (-) DMS even stronger and/or better efficacy.

Example 5 Ss and interior S are as dopamine reuptake inhibitor

The biological action of brain neurotransmitter dopamine is ended in the synapse by a kind of high affinity, sodium and energy dependent form induction system (neuron reuptake) that is present in the restriction film that the presynaptic contains the dopamine teleneuron.The inhibition of this conveyer mechanism can be expanded the effect of dopamine in the synapse, thereby strengthens the dopamine synapse and transmit.

A, test method

To the R (-) of S (DMS) and S (+) enantiomer carry out its aptitude tests that suppress the dopamine reuptake system and with selegiline relatively.Inhibition activity in this test is medicament valuable indication aspect the active disease of treatment needs raising synapse dopamine.At present, this can comprise parkinson disease, Alzheimer and attention-deficient superfunction imbalance (ADHD).

The pilot system of using be basically the described systems of people such as Fang ( Neuropharmacology33:763-768 (1994)).The teleneuron preparation (synaptosome preparation) that exsomatizes obtains from fresh rat neostriatum cerebral tissue.The conveying of dopamine teleneuron is estimated by measuring tritiated dopamine uptake amount.

B, result

As what see in table 5 column data, selegiline, R (-) DMS and S (+) DMS have all suppressed to contain the dopamine reuptake of dopamine teleneuron.Selegiline and R (-) DMS are Approximate Equivalent.And S (+) DMS no matter than selegiline still than R (-) DMS, its drug effect is all strong 4～5 times.

Table 5 rat neostriatum cerebral tissue ³The H-dopamine uptake

Medicament	Concentration	% suppresses x ± SEM
			Dopamine selegiline R (-) DMS S (+) DMS	????1μM ????10μM ????100nM ????1μM ????10μM ????100μM ????100nM ????1μM ????10μM ????100μM ????100nM ????1μM ????10μM ????100μM	????52.0±4.9 ????80.9±0.4 ????7.0±3.6 ????13.9±4.7 ????16.3±3.8 ????59.8±1.0 ????11.5±1.0 ????10.7±2.8 ????20.1±3.1 ????51.3±2.6 ????15.3±7.7 ????24.1±11.7 ????47.0±3.1 ????76.9±1.8

Drug effect can be with making the dopamine reuptake amount suppress 50% needed concentration (ID relatively ₅₀) represent.ID ₅₀Value is the (see figure 8) of determining with diagram method, is presented in the following table 6.

The dopamine uptake amount that makes table 6 suppresses 50% needed concentration

Medicament ID ₅₀

Selegiline ≈ 80 μ m

R(-)DMS??????????????????????≈100μm

S(+)DMS??????????????????????≈20μm

C, conclusion

These results confirm, under debita spissitudo, each enantiomer of selegiline and DMS can both suppress discharge the transmission of dopamine on synapse, and improve the relative activity of this neurotransmitter in this synapse.At this on the one hand, the drug effect of S (+) DMS is stronger than selegiline, and the latter's drug effect is stronger than R (-) DMS again.This effect is that medicament is of value to the treatment parkinson disease, the indication of Alzheimer and attention-deficient superfunction imbalance (ADHD).In the medicament of being tested, S (+) DMS obviously is being the most effective aspect the treatment ADHD.

The R (-) of example 6 Ss (DMS) and S (+) enantiomer are to the people

Body platelet MAO-B and Cavia porcellus brain MAO-B and MAO-A live

The effect of property

Human body platelet MAO exclusively contains the Type B isomeric form (isoform) of this enzyme.In this research, measured of the inhibition of two kinds of enantiomer of DMS, and compared with the caused inhibition of selegiline to this kind of enzyme.In addition, the inhibition activity of two kinds of enantiomer of DMS to MAO-A and MAO-B in the guinea pig hippocampal tissue also investigated in this research.The guinea pig brain tissue be the metabolism of research brain dopamine, multiple MAO form enzyme kinetics and can with the best animal model of the rejection of the pharmaceutical preparations of these enzyme interactings.Multiple MAO form in this animal kind demonstrate with the human body cerebral tissue in the identic kinetic property of multiple MAO found.At last, to the Cavia porcellus injection medicament, can be used as the degree that live body brain MAO inhibitor works to determine them.

B, test method

Pilot system utilized human body platelet and/or guinea pig hippocampal homogenate to the single-minded substrate of MAO-A ( ¹⁴The C-serotonin) and the single-minded substrate of MAO-B ( ¹⁴The C-phenyl ethyl amine) stripped conversion.In the presence of S (+) DMS, R (-) DMS or selegiline, measured the conversion rate of every kind of substrate, and the isozyme activity when not existing with these medicaments is compared.Calculated the inhibition percentage rate from these values.Cause 50% inhibition concentration (IC by more every kind of medicament ₅₀Value), assessed drug effect.

Some the experiment in, R (-) DMS, S (+) DMS or selegiline in 5 days by a definite date once a day through subcutaneous to the live body administration, slaughter, prepare the homogenate of enzyme Hippocampus then and carry out MAO-A and the active isolated test of MAO-B.Carrying out these experiments is to confirm that DMS mapping physical ability enters cerebral tissue and suppresses the MAO activity.

C, result

The MAO-B that exsomatizes suppresses active

The result that MAO-B suppresses is presented in table 7 and 8.The IC that MAO-B suppresses ₅₀Value is presented in the table 9 with the drug effect of comparing with selegiline.

MAO-B in table 7 human body platelet suppresses

Medicament	Concentration	% suppresses x ± SEM
			Selegiline R (-) DMS S (+) DMS	????0.3nM ????5nM ????10nM ????30nM ????100nM ????300nM ????1μM ????100nM ????300nM ????1μM ????3μM ????10μM ????3μM ????300nM ????1μM ????3μM ????10μM ????30μM ????100μM ????1mM	????8.3±3.4 ????50.3±8.7 ????69.0±5.5 ????91.0±1.4 ????96.0±1.6 ????96.0±1.6 ????96.6±1.6 ????14.3±3.6 ????42.1±4.0 ????76.9±1.47 ????94.4±1.4 ????95.8±1.4 ????95.7±2.3 ????6.4±2.8 ????11.1±1.0 ????26.6±1.9 ????42.3±2.3 ????68.2±2.34 ????83.7±0.77 ????94.2±1.36

MAO-B in table 8 guinea pig hippocampal suppresses

Medicament	Concentration	% suppresses x ± SEM
			Selegiline R (-) DMS S (+) DMS	????0.3nM ????5nM ????10nM ????30nM ????100nM ????300nM ????1μM ????100nM ????300nM ????1μM ????3μM ????10μM ????30μM ????300nM ????1μM ????3μM ????10μM ????30μM ????100μM ????1mM	????28.3±8.7 ????81.2±2.6 ????95.6±1.3 ????98.5±0.5 ????98.8±0.5 ????98.8±0.5 ????99.1±0.45 ????59.4±9.6 ????86.2±4.7 ????98.2±0.7 ????98.4±0.95 ????99.1±0.45 ????99.3±0.40 ????18.7±2.1 ????44.4±6.4 ????77.1±6.0 ????94.2±1.9 ????98.3±0.6 ????99.3±0.2 ????99.9±0.1

The IC that table 9 MAO-B suppresses ₅₀Value

Handle Human body platelet The guinea pig hippocampal cortex

Selegiline 5nM (1) 1nM (1)

R(-)DMS???????????????400nM(80)?????????????????????60nM(60)

S(+)DMS??????????????1400nM(2800)?????????????????1200nM(1200)

The drug effect of ()=compare with selegiline

As observed, as the MAO-B inhibitor, the drug effect of R (-) DMS is bigger 20～35 times than (+) DMS, and the drug effect of two kinds of enantiomer is all little than selegiline.

It is active that MAO-A suppresses

The result that the experiment that MAO-A suppresses from investigate guinea pig hippocampal obtains is summarised in the table 10 IC of two kinds of enantiomer of DMS and selegiline ₅₀Value is presented in the table 11.

MAO-A in table 10 guinea pig hippocampal suppresses

Medicament	Concentration	% reduces x ± SEM
			Selegiline R (-) DMS S (+) DMS	????300nM ????1μM ????3μM ????10μM ????100μM ????1mM ????300nM ????1μM ????3μM ????10μM ????100μM ????1mM ????1μM ????3μM ????10μM ????100μM ????1mM ????10mM	????11.95±2.4 ????22.1±1.2 ????53.5±2.7 ????91.2±1.16 ????98.1±1.4 ????99.8±0.2 ????4.8±2.1 ????4.2±1.5 ????10.5±2.0 ????19.0±1.3 ????64.2±1.5 ????96.5±1.2 ????3.3±1.5 ????4.3±1.0 ????10.5±1.47 ????48.4±1.8 ????92.7±2.5 ????99.6±0.35

The IC that table 11 MAO-A suppresses ₅₀Value

In the guinea pig hippocampal cortex

Handle The IC of MAO-A ₅₀

Selegiline 2.5 μ M (1)

R(-)DMS????????????????50.0μM(20)

S(+)DMS???????????????100?0μM(40)

The drug effect of ()=compare with (selegiline)

As the MAO-A inhibitor, the drug effect of R (-) DMS is the twice of S (+) DMS, and both drug effects are all little 20～40 times than selegiline.In addition, each all likens little 2～3 orders of magnitude of drug effect into the MAO-B inhibitor to as the drug effect of MAO-A inhibitor these medicaments in the Hippocampus cerebral tissue, promptly 100～1000 times.Therefore, selegiline and every kind of DMS enantiomer all can be included into selectivity MAO-B inhibitor one class in the cerebral tissue.

The experiment made on the living result

Every kind of DMS enantiomer was injected the live body administration under the percutaneous in continuous 5 days once a day, measured the active inhibition of brain MAO-B then.Under these conditions, as the MAO-B inhibitor, R (-) DMS and S (+) DMS are equivalent, but its drug effect is littler 10 times than selegiline.The result is presented among Fig. 9, IC ₅₀Value is summarised in the table 12.

Table 12 IC of brain MAO-B during injection medicament before test ₅₀Value

In the guinea pig hippocampal cortex

Handle The IC of MAO-B ₅₀

Selegiline 0.03mg/kg

R(-)DMS?????????????????0.30mg/kg

S(+)DMS?????????????????0.30mg/kg

This experiment confirm, each can both penetrate blood-brain barrier and suppress brain MAO-B non-in the DMS enantiomer after the administration of intestinal live body.It confirms that also exsomatizing, the drug effect difference as the MAO-B inhibitor has reduced between observed every kind of DMS enantiomer and the selegiline under condition of living body.The underdosage of administration is to suppress the MAO-A activity in this experiment.

D, conclusion

R (-) DMS and S (+) DMS have confirmed the activity as MAO-A inhibitor and MAO-B inhibitor.Every kind of enantiomer all is optionally to MAO-B.Aspect inhibition MAO-A and MAO-B, the drug effect of S (+) DMS generally is lower than R (-) DMS, and the drug effect of two enantiomer of DMS all is lower than selegiline.Two enantiomer have all confirmed the activity after the live body administration, show that these enantiomer can both enter cerebral tissue non-behind enteral administration.The ability that these medicaments suppress MAO-A and MAO-B shows that these medicaments have the value as the therapeutic agent of parkinson disease, Alzheimer or depression.

The live body neuroprotective of the enantiomer of example 7 Ss

By with these medicaments to the person's of waving mice-motor neuron disease, especially a kind of animal model-administration of amyotrophy outside sclerosis (ALS) has been investigated the DMS enantiomer and has been prevented the ability that the neurological is worsened.The person of waving mice shows forelimb weakness, gait disorder and the forelimb muscle flexing of gradual deterioration and shrinks.

B, test method

In a randomization double-blind study, every day is through peritoneal injection, to the person of waving mice administration R (-) DMS, S (+) DMS or placebo in 30 days by a definite date.When this time finishes, investigate grip, running time, the dormancy locomotor activity of mice, and the unusual and sxemiquantitative abnormal walking classification to its semidefinite measuring jaw attitude.Preparation solution and the research worker of injecting this solution to animal, the research worker that changes with the analysis behavior is different.

Test and classification are to look like Mitsumoto etc. people [Ann.Neurol, 36:142-148 (1994)] is described to carry out like that basically.Mice fore paw grip is by allowing this animal catch a wires to determine with two pawls.This metal wire is connected to a gram level and measures one's own ability on the meter, and mousetail is implemented traction, is forced to unclamp metal wire until this animal.Get reading the measuring on the meter of measuring one's own ability when unclamping as grip.

The running timing definition is for passing certain predetermined distance as 2.5 feet needed shortest times, the golden hour of record several times test.

The pawl attitude is unusually according to a yardstick classification based on shrinkage degree, abnormal walking according to one from normal gait to can not be with the yardstick classification of this scope of pawl body support.

Locomotor activity is by determining in the investigation zone of animal being transferred to the square grid mulched ground of a usefulness plate.Activity be with a mice a setting-up time at interval as the grid number that passes in 9 minutes measure.

C, result

This research when beginning, do not have in each treated animal one group variant on any variable, show that these three groups is approximating on baseline.Weight increase is all identical in all three groups, shows that all not having great side effect in any animal takes place.Table 13 has been summed up observed difference aspect the average grip of experimental animal:

Table 13 is handled with R (-) or S (+) DMS

Average grip in the person of the waving mice

Handle N Grip (gram)

Contrast (placebo) 10 9 (0-15)

R(-)DMS???????????????????????9????????????????????????20(0-63)

S(+)DMS???????????????????????9????????????????????????14(7-20)

The animal number that N=analyzes

Grip all significantly descended when all animals were finished in first week.When this research finished, grip was the control animal minimum, and scope is 0～15g, average 9g.The average grip of R (-) group is 20g, and numerical range is 0～63g.Injected S (+) DMS the 3rd group, its average grip is 14g, and single numerical range is 7～20g.Though the transmutability of grip has hindered the meaningful statistical analysis of this data in the processing animal groups, the average grip of measuring in the DMS processing animal is greater than matched group.

Running time, dormancy locomotor activity, the unusual classification of semidefinite measuring jaw attitude and the classification of sxemiquantitative abnormal walking have also been tested.Yet these are tested none and demonstrate and can show any one group of data that are different from another group in these three groups.

The immune system restitution of example 8 R (-) DMS and S (+) DMS

The decline that the immunologic function that takes place in animal and human's body is relevant with the age makes older individuality be easier to take place infectious disease and cancer.United States Patent (USP) 5,276,057 and 5,387,615 think that selegiline can be used for treating the immune system malfunction.Carry out this example laboratory, is to determine whether R (-) DMS and S (+) DMS also can be used for treating such malfunction.Will be appreciated that the ability that supports the normal immune defence of patient can be of value to the miscellaneous acute and chronic disease of treatment, comprises cancer, AIDS and bacillary and viral infection.

A, test procedure

This experiment has utilized rat model to investigate the ability of R (-) DMS and S (+) DMS recovery immunologic function.Rat is divided into following experimental group:

1) young rats (3 monthly ages, not injection);

2) senile rat (18～20 monthly ages, not injection);

3) senile rat has been injected saline solution;

4) senile rat has been injected selegiline.Dosage is the 0.25mg/kg body weight;

5) senile rat has been injected selegiline.Dosage is the 1.0mg/kg body weight;

6) senile rat has been injected R (-) DMS.Dosage is the 0.025mg/kg body weight;

7) senile rat has been injected R (-) DMS.Dosage is the 0.25mg/kg body weight;

8) senile rat has been injected R (-) DMS.Dosage is the 1.0mg/kg body weight;

9) senile rat has been injected S (+) DMS.Dosage is the 1.0mg/kg body weight;

In 60 days, through intraperitoneal rat is injected once a day.Then, they are kept other one period " flushing " phase of 10 days, do not give any injection in during this period.This section period when finishing slaughters animal, takes out their spleen.Then splenocyte is tested the various factors that those can indicate function of immune system.Specifically, measure following with code test:

1) splenocyte exsomatizes and produces gamma interferon:

2) leukin-2 between stripped the generation;

3) percentage rate (IgM is the maker of bone-marrow-derived lymphocyte) of the positive splenocyte of IgM;

4) percentage rate (CD5 is the lymphocytic maker of T) of the positive splenocyte of CD5.

B, result

Injection selegiline, R (-) DMS and S (+) DMS are illustrated in Figure 10 and 11 influence that rat spleen cells produces interferon.As shown in Figure 10, a kind of cell interferon output that takes place with the age fall sharply (output that contrasts the output and the geriatric animals of young zooblast or injected the geriatric animals cell of saline solution) is arranged.Injection selegiline, R (-) DMS and S (+) DMS all cause the part of gamma interferon level to recover increasing degree maximum when dosage is the 1.0mg/kg body weight.

Repeated among Figure 11 and data identical shown in Figure 10, that different is the result who has omitted the young rats cell.This figure has clearly illustrated that more Di Pu Neil, R (-) DMS and S (+) DMS can make the degree that gamma interferon output is recovered in the senile rat splenocyte.Interferon-is a kind of multifunctional protein, can suppress virus replication and regulate various immunologic functions.It influences that antibody-like that the B-cell is produced, and raises I class and II class MHC complex antigen and improves the mesomeric cytozoon kill efficiency of macrophage.

Figure 12 shows the influence of each injection to leukin-2 between the rat spleen cells generation.Can see that R (-) DMS and S (+) DMS can both make a leukin-2 output return to the level of seeing in young zooblast.

Each injection is illustrated among Figure 13 being the percentile influence of the male splenocyte of IgM.Find that selegiline and R (-) DMS can both make the IgM positive cell partly return to more to approach the sort of level of seeing in the young rats spleen.Therefore, obviously these medicaments recover the bone-marrow-derived lymphocyte number.

Figure 14 shows that injection 0.025mg/kg R (-) DMS or S (+) DMS can improve the percentage rate of CD5 positive cell in the senile rat spleen a little.Yet, inject selegiline still inject 0.25 or 1.0mg/kg R (-) DMS obviously all do not produce any influence.

C, conclusion

Generally speaking, these supports as a result are as drawing a conclusion: the enantiomer of DMS and selegiline are similar to the influence of function of immune system.In addition, about the output of interferon, IL-2 and about the resulting support as a result of percentage rate of the positive splenocyte of IgM as drawing a conclusion: DMS mapping physical ability is recovered age dependent form function of immune system loss at least in part.Therefore, apparent, R (-) DMS and S (+) DMS will have upward useful effect of treatment for weaken the disease and the disease that produce because of host immune.This can comprise cancer, AIDS and all types of infectious disease.

Example 9 dosage form examples

A, S patch

Composition is with dry weight basis (mg/cm ²)

Durotak ^87-2194

Adhesion type acrylate copolymer 90 weight portions

S 10 weight portions

These two kinds of compositions fully mix, and cast in a Scotchpak ^On the 9723 mylar backing light sheets, drying.This backing light sheet is cut into patch, and every subsides add a Scotchpak ^1022 fluoropolymer polymer isolation liner are enclosed this patch in the metal forming big envelope airtightly.In the conditions of human body such as Parkinsonian treatment that are produced by neuronal degeneration or neuron wound, day deposited subsides are to supply with per 24 hours 1～5mg Ss.

B, ophthalmic solution

The S of hydrochloride form (0.1g), 1.9g boric acid and 0.004g nitric acid phenyl mercury are dissolved in an amount of sterilized water, and making total amount is 100ml.Mix sterilization, sealing.It can be used for the treatment of the disease that is produced by neuronal degeneration or neuron wound on ophthalmology, as glaucoma optic neuropathy and degeneration of macula.

Example 3: intravenous solution agent

Being enough to provide final volume is that 0.9% grade of 100ml is oozed dissolving 1g S hydrochlorate in the common salt aqueous solution, prepares a kind of 1% solution.This solution is buffered to pH4, sealing, sterilization with citric acid, and a kind of 1% solution that is suitable for intravenous administration is provided, and can be used for treating the disease that is produced by neuronal degeneration or neuron wound.

C, percutaneous patch

In the organic solvent of the copolymer of methacrylic acid and dimethylaminoethyl methacrylate such as methyl ethyl ketone solution, add a kind of self-crosslinking acrylic based pressure-sensitive adhesive.This thing is cast on removable first metal forming, evaporating solvent, this cated metal forming is placed on the polyester backing layer described in EP-A 593807.

In the suitable organic solvent of non-crosslinked type acrylic contact adhesive such as ethyl acetate solution, add the S hydrochlorate.Can add extra solvent, can also adopt heating and stirring to be beneficial to dispersion liquid and form.This thing is applied on removable second metal forming evaporating solvent.After the polyester backing layer for preparing previously took off metal forming, it is laminated to cated second can remove on the metal forming.Can remove on the metal forming evaporating solvent to the organic solvent of the copolymer of another part self-cross linking type acrylic contact adhesive and methacrylic acid and dimethylaminoethyl methacrylate such as three of methyl ethyl ketone solution-casts to the.Remove second and the 3rd metal forming, these residual layers are forced together, formed laminate is cut into patch, packing.Formed patch will have a removable isolation liner, an adhesive phase and a base layer that contains S hydrochlorate (or other salt) that does not originally have S.An impermeable backing layer is similar to the intermediary adhesive layer that can remove the adhesive phase that isolation liner contacts by one is adhered on this base layer.When conditions of human body that produces by neuronal degeneration or neuron wound in treatment such as parkinson disease, day apply one to paste, to supply with the S of free alkali form.

D, per os dosage form

The tablet and the capsule that contain S from following ingredients (mg/ dosage unit) preparation:

S 1～5

Microcrystalline Cellulose 86

Lactose 41.6

Citric acid 0.5～2

Sodium citrate 0.1～2

Magnesium stearate 0.4

About 1: 1 of the ratio of citric acid and sodium citrate.

Claims (22)

1. medical composition, wherein, except that one or more optional pharmaceutically acceptable excipient or carrier, also contain a certain amount of S, interior S or its mixture, make one or more dosage units regular administration, described compositions can effectively treat one or more selegiline reactive disorder or diseases the curee of the described one or more dosage units of its administration.

2. according to the compositions of claim 1, be used for oral administration.

3. according to the compositions of claim 1, be used for percutaneous dosing.

4. according to the compositions of claim 1, wherein S uses with pure basically stereoisomer form.

5. according to the compositions of claim 1, be applicable to and carry out neuron rescue or neuron guarantor god.

6. according to the compositions of claim 1, be applicable to recovery and improve curee's function of immune system.

7. one kind is used to make the curee who suffers from selegiline reactive disorder or disease to obtain improving one's methods of similar selegiline curative effect, this method comprises: to be enough to produce the quantity of similar selegiline curative effect, described curee is used S, interior S or its mixture.

8. according to the method for claim 7, wherein said curee is the people.

9. according to the method for claim 8, wherein interior S is used for the treatment of ADHD with pure basically stereoisomer form.

10. according to the method for claim 7, wherein similar selegiline curative effect is neuron rescue or neuro-protective.

11. according to the method for claim 7, improvement or recovery that wherein similar selegiline curative effect is a function of immune system.

12. the treatment of conditions method that produces by neuronal degeneration or neuron wound in the mammal, this method comprise with by single dose system or the administration of multiple dose system, every kilogram of weight of mammal is at least about the daily dose of 0.0015mg (is that benchmark calculates with free secondary amine), to this administration S or interior S or its pharmaceutically acceptable acid-addition salts.

13., comprise at least a route of administration that does not rely on gastrointestinal to absorb according to the method for claim 12.

14. according to the method for claim 12, wherein R (-) enantiomer of S is with the free alkali form percutaneous dosing.

15. according to the method for claim 12, wherein said R (-) enantiomer is non-through enteral administration with the form of pharmaceutically acceptable acid-addition salts.

16. the method for claim 15, wherein pharmaceutically acceptable acid-addition salts is a hydrochlorate.

17., wherein press the daily dose of single dose system or multiple dose system administration according to the method for claim 12, be that benchmark calculates with free secondary amine, be every kilogram of about 0.01～about 0.5mg of weight of mammal.

18. according to the method for claim 12, wherein mammal is human.

19. according to the method for claim 12, wherein mammal is a Canis animals.

20. according to the method for claim 12, wherein mammal is a felid.

21. be used for the treatment of the transdermal administration composite of the disease that produces by neuronal degeneration or neuron wound in the mammal, said composition comprises a kind of composite stratified material, at least contain a certain amount of S, interior S or its pharmaceutically acceptable acid-addition salts in one deck at it, be enough to supply with the day percutaneous dosage of every kilogram of weight of mammal at least about the free secondary amine of 0.0015mg.

22. the treatment of conditions method that the immune system malfunction is produced in the mammal, this method comprises with by single dose system or the administration of multiple dose system, every kilogram of weight of mammal is at least about the daily dose of 0.0015mg (is that benchmark calculates with free secondary amine), to this administration S or interior S or its pharmaceutically acceptable acid-addition salts.

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