EP0578687A1 - Utilisation du deprenyle pour maintenir ou recuperer la fonction de cellules nerveuses ou pour empecher la perte de cette fonction - Google Patents
Utilisation du deprenyle pour maintenir ou recuperer la fonction de cellules nerveuses ou pour empecher la perte de cette fonctionInfo
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- EP0578687A1 EP0578687A1 EP92907543A EP92907543A EP0578687A1 EP 0578687 A1 EP0578687 A1 EP 0578687A1 EP 92907543 A EP92907543 A EP 92907543A EP 92907543 A EP92907543 A EP 92907543A EP 0578687 A1 EP0578687 A1 EP 0578687A1
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- deprenyl
- mptp
- somata
- loss
- neurons
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- A61P25/00—Drugs for disorders of the nervous system
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/08—Antiepileptics; Anticonvulsants
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- G—PHYSICS
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Definitions
- the present invention relates to the use of deprenyl or derivatives or analogues of deprenyl to maintain, prevent loss and/or recover nerve cell function in an animal; to pharmaceutical compositions containing deprenyl adapted for such use; and, to methods for the treatment of disorders of the nervous system by maintaining, preventing loss or recovering nerve cell function.
- the invention also relates to methods for testing drugs for their activity in maintaining, preventing loss and/or recovering nerve cell function in an animal.
- Deprenyl was first used as an adjunct to conventional drug therapy (L-dihydroxyphenylalanine (L-DOPA) plus a peripheral decarboxylase inhibitor) of Parkinson's disease (PD) in Europe over a decade ago on the basis that as a selective monoamine oxidase-B (MAO-B) inhibitor, it would elevate brain dopamine levels and potentiate the pharmacologic action of dopamine formed from L-DOPA, and yet prevent the tyramine-pressor effect observed with non-selective MAO inhibitors.
- L-DOPA L-dihydroxyphenylalanine
- MAO-B selective monoamine oxidase-B
- deprenyl an MAO-B inhibitor
- the present invention relates to the use of deprenyl or derivatives or analogues of deprenyl to maintain, prevent loss of and/or recover nerve cell function in an animal.
- the invention also relates to a pharmaceutical composition for use in the treatment of disorders of the nervous system by maintaining, preventing loss or recovering nerve cell function comprising deprenyl or derivatives or analogues of deprenyl as an active agent.
- the invention further relates to a method for the treatment of disorders of the nervous system by maintaining, preventing loss or recovering nerve cell function comprising administering to a patient an effective amount of deprenyl or derivatives or analogues of deprenyl.
- the invention also relates to methods for testing a drug for activity in maintaining, preventing loss and/or recovering nerve cell function in a patient.
- a method for testing a drug for activity in maintaining, preventing loss and/or recovering nerve cell function in a patient comprising administering an agent having neurotoxic activity to a test animal; administering the drug to the test animal; sacrificing the test animal within a period of 20 days from completion of administration of the agent; taking serial sections of the brain through the substantia nigra compacta; determining the number of tyrosine hydroxlyase positive somata in alternate serial sections of the brain through the substantia nigra compacta and determining the number of Nissl stained positive substantia nigra compacta in intervening serial sections; and comparing the number of tyrosine hydroxlyase positive somata and Nissl stained somata determined with the number of tyrosine hydroxlyase positive somata and Nisss
- a method for testing a drug for activity in maintaining, preventing loss and/or recovering nerve cell function in a patient comprising carrying out an axotomy on a test animal; administering the drug to the test animal; determining the number of choline acetyl transferase positive somata for histological sections from the site of the axotomy; and comparing the number of choline acetyl transferase positive somata determined with a number of choline acetyl transferase positive somata in serial sections from the site of the axotomy in a control animal which has not been administered the drug.
- Figure 1 is a graph showing the numbers of tyrosine hydroxylase immunopositive (TH+) neurons in the substantia nigra compacta (SNc) following the administration of MPTP.;
- Figure 2 are joint plots of the counts of TH+ and Nissl stained SNc somata from corresponding areas of immediately adjacent sections for Saline Only treated (A1,A2,A3), MPTP-Saline treated (B1,B2,B3) and MPTP- Deprenyl treated animals (C1,C2,C3) with the data pooled from 3 animals in each group at 20 days following the MPTP treatment;
- Figure 3 is a graph showing the cumulative counts of TH+ SNC neurons versus section number for individual representative SNc nuclei taken from alternate 10 micron serial sections throughout the entire nucleus;
- Figure 4 is a graph showing the mean and SEM values for the MPTP, MPTP-Saline and MPTP-deprenyl treated mice;
- Figure 5 shows a spectral analysis of locomotory activity for mice injected with MPTP;
- Figure 6 shows high resolution power spectra for LD and DD preinjection control period from a saline injected mouse
- Figure 7 shows a high resolution power spectra for control and MPTP mice
- Figure 8 is a graph showing the normalized sum % peak power versus median day;
- Figure 9A and 9B show SNc sections for glued brains from animals treated with MPTP or saline;
- Figure 10A, B, C, and D are graphs showing the counts of TH+ SNc and VTA neuronal somata following MPTP treatment taken through whole nuclei expressed as a percentage of the mean counts for the corresponding saline-injected animals (A); the concentration of striatal
- DOPAC/DA ratio (D) for saline and MPTP injected mice DOPAC/DA ratio (D) for saline and MPTP injected mice
- Figure 11 is a graph showing the mean OD/mean O.D. for saline background versus days after MPTP injections;
- Figure 12 shows photomicrographs of adjacent ChAT im unoreacted (Al and BI) and Nissl stained (A2 and B2) sections through the facial nucleus ipsilateral to transection of the facial nerve;
- Figure 13 is a bar graph for the counts of ChAT+ somata for the facial nuclei for the different lesion and treatment groups (bars-means, error bars - standard deviations);
- Figure 14 are graphs showing joint Nissl/ChAT+ counts of adjacent sections for the no lesion groups
- Figure 14B is a bar graph showing MAO-A and MAO-B measurements at 24 hours (d4) after the first administration of deprenyl (0.25 mg/kg or 0.01 mg/kg) and 18 days later (d22).
- the present inventors have studied the time course of neuronal death induced by the neurotoxin l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine (MPTP) .
- MPTP is oxidized, under the action of monoamine oxidase-B (MAO-B), via a dihydropyridium intermediate (MPDP+) to its toxic metabolite l-methyl-4-phenyl-pyridinium ion (MPP+) . It is believed that MPTP is converted to MPP+ in nondopaminergic cells, released and then taken up into dopaminergic neurons where it exerts its neurotoxic effects (see Vincent S.R. Neuroscience, 1989, 28 p. 189- 199, Pintari, J.E.
- MPTP is rapidly metabolized and cleared in the mouse (Johannessen, J.N. et al. Life Sci. 1985, 36: p. 219-224, Markey, S.P. et al. Nature, 1984, 311 p. 465- 467, Lau, Y.S. et al. Life Sci. 1988, 43(18): p. 1459- 1464).
- MPTP (30 mg/kg/d) was administered i.p.
- mice for five consecutive days total cumulative dose 150 mg/kg to produce a loss of approximately 50% of TH-immunopositive (TH+) neurons in the substantia nigra compacta (SNc) and ventral tegmental area (VTA)
- TH+ TH-immunopositive
- SNc substantia nigra compacta
- VTA ventral tegmental area
- TH+ somata 20-30% were lost by the five days after the completion of the administration of MPTP; loss of TH+ neurons continued over the next ten to fifteen days with no detectable loss thereafter. This continual loss of TH+ neurons could not be accounted for by the presence of MPP+, based on the excretion data referred to above. Joint plots of counts of TH+ and Nissl stained SNc somata also confirmed that the loss of TH+ somata represented the death of SNc neurons rather than a loss of TH immunoreactivity. In tandem with the loss of TH+ SNc somata the present inventors have also found changes in immunodensity of TH protein in SNc and the ventral teg ental area (VTA) .
- VTA ventral teg ental area
- Cytoplasmic TH immunodensity was 40% lower in the somata of the remaining TH+ DNS neurons for MPTP-treated animals at day 5 in comparison to saline treated controls. Average somal TH-immunodensity increased over time and had reached control levels by 20 days following MPTP. Alterations in striatal DA concentrations and dopamine- dependent behaviours such as locomotion were found to parallel the changes in TH-immunochemistry. Further, the present inventors found that an increase in striatal DA content and DA synthesis as estimated by DOPAC/DA ratios also appeared to parallel behaviourial recovery and indicated increased DA content and synthesis in the VTA and SNc neurons surviving MPTP exposure.
- the present inventors have significantly found that following MPTP-induced neuronal damage, there is a critical 20 day period in which TH+ SNc neurons either undergo effective repair and recovery or else they die.
- deprenyl Most studies with deprenyl have been designed to demonstrate that inhibition of MAO-B activity in vivo blocks the conversion of MPTP to MPP+ and the neurotoxicity of MPTP. As a consequence, deprenyl was usually given either several hours or for several days prior to and then throughout MPTP administration to ensure that MAO-B activity was inhibited during the time of MPTP exposure (for example, see Cohen, G., et al., Eur. J. Pharmacol., 1984. 106: p. 209-210, Heikkila, R.E., et al., Eur. J. Pharmacol, 1985. 116(3): p. 313-318, Heikkila, R.E., et al.. Nature, 1984.
- MPTP- treated mice (cumulative dose of 150 mg/kg) received deprenyl (0.01, 0.25, 10 mg/kg i.p.; 3 times per week) from day 3 to day 20 following MPTP administration. Deprenyl administration was withheld until day 3 to ensure that all mice were exposed to comparable levels of MPP+ and that all MPTP and its metabolytes had been eliminated from the central nervous system. Clorgiline, an MAO-B inhibitor, was also administered to the MPTP-treated mice.
- the present inventors found that in saline treated mice, about 38% of dopaminergic substantia nigracompacta (DSN) neurons died progressively over the twenty days.
- the number of DSN neurons was found to be statistically the same in the MPTP-Saline and MPTP- Clorgiline treated mice.
- deprenyl increased the number of DSN neurons surviving MPTP-induced damage (16% loss - 0.01 mg/kg, 16% loss - 0.25 mg/kg, and 14% loss - lOmg/kg), with all doses being equipotent.
- deprenyl could rescue dying neurons and increase their probability of undergoing effective repair and re-establishing their synthesis of enzymes, such as tyrosine hydroxylase, necessary for dopamine synthesis. This is believed to be the first report of a peripherally or orally administered treatment which reverses the sequence of damage to death in neurons which would have otherwise died.
- the present inventors also measured MAO-A and MAO-B 24 hours after deprenyl (0.25 mg/kg or 0.01 mg/kg) administration and 18 days later (i.e. corresponding to day 21 which is just after the animals were sacrified for immunochemistry at day 20) to provide measurement of MAO-A and MAO-B activity at the beginning and end of the treatment period. It was surprisingly found that the 0.01 mg/kg dose did not produce any significant MAO-A or MAO-B inhibition at the two time periods. Thus, the marked rescue of DSN neurons with 0.01 mg/kg deprenyl appears not to be attributable to MAO-B inhibition.
- deprenyl could help prevent the death of all neurons in the brain that respond to glial trophic factors, rather than just influencing dopaminergic neurons alone.
- it would also be effective in other neurodegenerative and neuromuscular diseases and in brain damage due to hypoxia, ischemia, stroke or trauma and may even slow the progressive loss of neurons associated with brain aging (Coleman, P.D. & Flood D.G., Neurobiol. Aging 8, 521-845 (1987); McGeer, P.L. et al. in Parkinsonism and Aging (eds. D.B. Calne, D,C, - G. Comi and R. Horowski) 25-34 (Plenum, New York, 1989). It may also be useful in stimulating muscle reinnervation in traumatic and non- traumatic peripheral nerve damage.
- the present invention relates to the use of deprenyl or derivatives or analogues of deprenyl to maintain, prevent loss of and/or recover nerve cell function in an animal; to pharmaceutical compositions containing deprenyl adapted for such use; and, to methods for the treatment of disorders of the nervous system by maintaining, preventing loss of or recovering nerve cell function.
- Deprenyl preferably L-deprenyl (see The Merck Index, 11th ed. 2893), derivatives of deprenyl, preferably pharmaceutically acceptable salts and esters of deprenyl; or, analogues of deprenyl, preferably structural analogues of deprenyl or functional analogues of deprenyl such as pargyline, AGN-1133, AGN-1135 and MD 240928, or other agents which may or may not inhibit MAO-B such as imipramine, chlorpromazine, amitriptyline, (-)2,3- dichloro- ⁇ -methylbenzylamine, N-cyclopropyl-substituted arylalkylamines, may be used in the present invention.
- L-deprenyl is used in the pharmaceutical compositions, therapeutic uses and methods of the present invention.
- deprenyl or derivatives or analogues of deprenyl may maintain, prevent loss, and/or recover nerve cell function in an animal and thus may be used for the treatment of neurodegenerative and neuromuscular diseases and in brain damage due to hypoxia, hypoglycemia, ischemic stroke or trauma and may be used to slow the progressive loss of neurons associated with brain aging. More specifically, deprenyl may be used to treat Parkinson's disease, ALS, head trauma or spinal cord damage, patients immediately following an ischemic stroke, hypoxia due to ventilatory deficiency, drowning, prolonged convulsion, cardiac arrest, carbon monoxide exposure, exposure to toxins, or viral infections.
- Deprenyl or derivatives or analogues of deprenyl may also be used to stimulate muscle reinnervation in traumatic and non- traumatic peripheral nerve damage.
- the pharmaceutical compositions of the invention contain deprenyl or derivatives or analogues of deprenyl, either alone or together with other active substances.
- Such pharmaceutical compositions can be for oral, topical, rectal, parenteral, local, inhalant or intracerebral use. They are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets.
- those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection can be used, and can therefore be prepared as solutions of the active compounds or as powders of the active compounds to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity which is compatible with the physiological fluids.
- those preparations in the form of creams or ointments for topical use or in the form of sprays should be considered; for inhalant uses, preparations in the form of sprays, for example nose sprays, should be considered.
- the preparations of the invention can be intended for administration to humans or animals.
- Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration, but daily dosages to humans by subcutaneous, intramuscular or intracerebral injection generally vary between .1 and 10 mg of active substance per day, preferably between 1 to 10 mg per day, most preferably between 5 and 10 mg per day.
- compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
- Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
- compositions include, albeit not exclusively, solutions of the deprenyl or derivatives or analogues of deprenyl in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
- the invention also relates to methods for testing a drug for activity in maintaining, preventing loss and/or recovering nerve cell function in a patient.
- a method for testing a drug for activity in maintaining, preventing loss and/or recovering nerve cell function in a patient comprising administering an agent having neurotoxic activity to a test animal; administering the drug to the test animal; sacrificing the test animal within a period of 20 days from completion of administration of the agent; taking serial sections of the brain through the substantia nigra compacta; determining the number of tyrosine hydroxlyase positive somata in alternate serial sections of the brain through the substantia nigra compacta and determining the number of Nissl stained positive substantia nigra compacta in intervening serial sections; and comparing the number of tyrosine hydroxlyase positive somata and Nissl stained somata determined with the number of tyrosine hydroxlyase positive somata and Nissl stained somata in serial sections of the brain through the substantia nigra compacta of a control animal which has not been
- the agent having neurotoxic activity is MPTP, most preferably in an amount of 30 mg/kg/d, which ⁇ is administered i.p. to 8 week old mice, preferably C57BL mice, for five consecutive days (days -5 to 0; total preferred cumulative dose of 150 mg/kg) .
- days 0 Three days following cessation of MPTP adminstration (day 0), treatment with saline (control animal) or the appropriate doses of the drug (test animals) is commenced.
- the administration of the drug is withheld until day 3 to ensure that all mice are exposed to comparable levels of MPP+ and that all MPTP and its metabolites have been eliminated from the central nervous system.
- mice are killed by anaesthetic overdose (pentobarbital) followed by paraformaldehyde perfusion 20 days following their last MPTP injection.
- Brains are bisected longitudinally along the midline and the half brains are glued together using Tissue-Tek so that surface landmarks are in longitudinal register.
- the glued brains are frozen in -70°C methylbutane and 10 ⁇ m serial sections are cut through the entire longitudinal length of each SNc.
- the number of tyrosine hydroxlyase positive somata in serial sections of the brain through the substantia nigra compacta may preferably be determined using a polycional TH antibody as the primary antibody and a standard avidin-biotin reaction with diaminobenzidine (DAB) as the chromogen for visualization as generally described in Seniuk, N.A. et al. Brain Res. 527, 7-20 (1990) and Tatton, W.G. et al. Brain Res. 527, 21-32 (1990) which are incorporated herein by reference, and modified as follows. Slide-mounted sections are incubated with unlabelled primary TH antisera in 0.2% Triton/0.1 M phosphate buffer at 4°C overnight.
- DAB diaminobenzidine
- Tissues are washed with phosphate buffer then incubated for 1 hour with biotinylated goat anti-rabbit IgG secondary antibody followed by avidin-HRP incubation.
- a 0.05% solution of DAB in 0.01% hydrogen peroxide is used to visualize the immunoreactive somata.
- Intervening sections may be Nissl stained to define nuclear outlines following the procedure set forth in Seniuk et al., Brain Res. 527: 7, 1990 and Tatton et al. Brain Res. 527:21, 1990 which are incorporated herein by reference.
- a method for testing a drug for activity in maintaining, preventing loss and/or recovering nerve cell function in a patient comprising carrying out an axotomy on a test animal; administering the drug to the test animal; determining the number of choline acetyl transferase positive somata for histological sections from the site of the axotomy; and comparing the number of choline acetyl transferase positive somata determined with a number of choline acetyl transferase positive somata in serial sections from the site of the axotomy in a control animal which has not been administered the drug.
- a unilateral facial nerve transection is carried out on the test animal, preferably a rat, and paired lesion and no lesion groups are treated with saline (control animal) or appropriate doses of the drug (test animal) .
- the rats are sacrificed at 21 days after axotomy and serial coronal histological sections of the brainstem at the level of the facial nuclei are processed for choline acetyl transferase (ChAT) immunocytochemistry using the procedure of Tatton W.G.et al, Brain Res. 527:21, 1990, which is incorporated herein by reference.
- ChAT choline acetyl transferase
- Example 1 This example demonstrates the loss of tyrosine hydroxylase immunopositive (TH+) neurons from the substantia nigra compacta (SNc) following the administration of MPTP and their rescue by deprenyl.
- Dissected brains were immersed in 4% paraformaldehyde in 0.1 m phosphate buffer overnight and placed in 20% sucrose.
- Deprenyl adminstration was withheld until day 3 to ensure that all mice were exposed to comparable levels of MPP+ and that all MPTP and its metabolites had been eliminated from the central nervous system.
- Doses of deprenyl were chosen to reflect those used in studies demonstrating that deprenyl can prolong the lifespan of the rat and inhibit MAO-B activity by approximately 75% but have no effect on MAO-B activity (0.25 mg/kg) or cause inhibition of both MAO-B and MAO-A (10 mg/kg) (Knoll, J. Mt. Israel J. Med. 55, 67-74 (1988) and Knoll, J. Mech. Ageing Dev. 46, 237- 262 (1988), Demarest, K.T. and Azzarg A.J.
- brains were bisected longitudinally along the midline and the half brains were glued together using Tissue-Tek so that surface landmarks were in longitudinal register.
- the glued brains were frozen in -70°C methylbutane and then 10 ⁇ m serial sections were cut through the entire longitudinal length of each SNc.
- Tissues were washed with phosphate buffer then incubated for 1 hour with biotinylated goat anti-rabbit IgG secondary antibody followed by avidin-HRP incubation. A 0.05% solution of DAB in 0.01% hydrogen peroxide was used to visualize the immunoreactive somata.
- sections from control and MPTP-treated brains were mounted on the same slide to reduce the effect of slide to slide variability in the assay procedure and were processed for immunocytochemistry.
- the number of TH+ SNc neurons was obtained by counts of number coded alternate serial sections through each entire nucleus. Sections were recounted by multiple blind observers to check any observer bias. The values were corrected for section thickness (Konigsmark, B.W. In: Nauta, W.H., Ebesson S.O.E. ed Contemporary Research Methods in Neuroanatomy, New York, Springer Verlag, p. 315-380, 1970). , The mean value plus or minus the standard error of the mean was computed for the saline injected control mice. Subsequent data was then expressed as a percentage of this mean number as shown in Figure 1.
- Nissl somata containing a nucleolus within the outline were counted according to three size groups (small - 140 to 280 ⁇ m 2 , medium - 300 to 540 ⁇ m 2 and large - 540 to 840 ⁇ m 2 ) , excluding glial profiles (40 to 100 ⁇ m 2 ), using criteria similar to those of the rat SNc (Poirier et al. 1983 Brain Res. Bull. 11:371). Numbers of TH+ somata were plotted against numbers of Nissl somata for the corresponding areas of 20 immediately adjacent sections.
- the joint Nissl/TH+ counts provide a means for determining whether reductions in the numbers of TH+ SNc somata are due to neuronal destruction or a loss of TH immunoreactivity by surviving neurons (see Seniuk et al. 1990, supra, for details as to rationale for the procedure) .
- Figure 1 shows a loss of TH+ somata from the SNc rom days 0 to 20 post MPTP, with no decline thereafter. 20 to 30% of TH+ somata were lost by five days after completion of the injection schedule (day 5); loss of TH+ neurons continued over the next ten to fifteen days with no further disappearance thereafter.
- Figure 2B2 and 2B3 show that even though the counts of Nissl and TH+ somata are reduced from 21.6+/-15.5 and 20.6+/-15.5 per section to 12.4+/-8.0 and 11.4+/-7.2 for the medium- sized and large-sized somata respectfully (values are means +/- 1.0 standard deviation), the almost equal value relationships between the counts were maintained. If the SNc neurons were losing TH immunoreactivity but not dying, the scatter of the joint plots would be expected to shift to loci above the equal value diagonal (Seniuk et al. Brain Res. 527:7, 1990).
- Figure 2B1 shows that the numbers of small-sized Nissl stained somata decreased slightly (26.2+/-18.3 to 22.4+/-12.5 per section) in accord with the reduction (4.1+/-2.8 to 2.3+/- 1.6 per section) in the TH+ component of the small-sized SNc somata. If some of the losses of medium and large sized SNc somata were due to atrophy so that their cross- sectional areas no longer fell within the medium and large size ranges in response to the MPTP treatment, one would expect an increase in the numbers of small sized Nissl stained somata.
- Figure 3 presents the raw counts of TH+ SNc somata for individual SNc nuclei taken from alternate 10 micron serial sections throughout the entire rostro-caudal length of each nucleus and expressed as a cumulative frequency distribution.
- Four representative trials for each treatment are presented in Figure 3.
- Values for neuronal counts from mice treated with saline alone, MPTP (150 mg/kg) and saline and MPTP plus deprenyl (0.25 mg/kg, 3 times per week) are shared with those presented in histogram fashion in Figure 4.
- the cumulative frequency distribution curves for all SNc nuclei have a similar pattern indicating that the loss of TH+ somata following MPTP and their rescue by deprenyl occurred in all parts of the nuclei although it appears to be greatest in the rostral portion of the nuclei (sections 10-40) that contains neurons which are relatively more resistant to the toxin.
- Figure 4 also illustrates that there is no overlap in individual frequency distribution curves between the three groups of animals.
- raw counts of TH+ somata were converted to neuronal numbers using a correction factor of 2.15 as described by Konigsmark, B.W., in Contemporary Research Methods in Neuroanatomy (eds. Nauta, W.H. and Ebesson SOE) 315-380 (Springer Verlag, New York, 1970).
- Figure 4 shows an increased number of TH+ SNc somata in the deprenyl treated mice relative to animals receiving MPTP alone, suggesting that deprenyl prevented a portion of the neuronal loss associated with MPTP-induced toxicity. Both low and high doses of deprenyl were equipotent in preventing the TH+ SNc neuronal loss.
- Figure 4 shows that the mean corrected numbers of TH+ somata found for animals treated with saline only of 3014+/-304 (mean +/- SEM) were significantly reduced (Mann-Whitney Test, p ⁇ 0.001) in the animals treated with MPTP only (1756+/-161) and the MPTP- Saline groups (1872+/-187, 1904+/-308 and 1805 ⁇ 185). Therefore MPTP caused average losses of 36, 38 and 42% of TH+ somata in those three MPTP pretreated groups (black bars in Figure 4) . All the MPTP saline control groups are statistically the same (p>0.05).
- Figure 4 also shows that Clorgiline an MAO-A inhibitor does not rescue the neurons since the MPTP-Saline (1706 +/- 155) and MPTP-Clorgiline (1725 +/- 213.6) values are statistically the same.
- MAO-A and MAO-B measurements were obtained in accordance with the method set out below 24 hours after the first 0.25 mg/kg or 0.01 mg/kg deprenyl administration and 18 days later (corresponding to day 21 which would be just after the animals were sacrificed for the immunochemistry at day 20).
- MAO activity was assayed in fresh tissue homogenates by the method of Wurtman, R. J. and Axelrod, J., (Biochem Pharmacol 1963;12:1439-1444), with a modification of substrates in order to distinguish between MAO-A- and MAO-B.
- This method relies on the extraction of acidic metabolites of either (14-C)-serotonin (for MAO-A) or (14-C) phenylethylamine (for MAO-B) in toluene/ethyl acetate.
- Tissue homogenates were incubated in potassium phosphate buffer containing either radiolabelled serotonin (100 micromolar) or phenylethylamine (12.5 micromolar) for 30 minutes at 37°C
- the reaction was stopped by the addition of HCl and acid metabolites extracted into toluene/ethyl acetate. Radioactivity in the toluene/ethyl acetate layer is determined by liquid scintillation spectrometry.
- Blanks are obtained from either boiled tissue homogenates or form reaction mixtures containing enzyme (Crane, S. Bs and Greenwood, C.E. Dietary Fat Source Influences Mitochondrial Monoamine Oxidase Activity and Macronutrient Selection in Rats. Pharmacol Biochem Behav 1987;27:1-6) .
- Figure 15 presents the MAO-A and MAO-B measurements for 24 hours after the first 0.25 mg/kg or 0.01 mg/kg and 18 days later (corresponding to day 21 which would be just after the animals were sacrificed for the immunocytochemistry at day 20).
- the KS probability shown in the brackets above each pair represents the results of the Kolmogorov-Smirnov two sample non- parametric statistical testing (Siegel, S. Non Parametric Statistics for the Behavioral Sciences, McGraw-Hill Book Company, New York, 1956, pp. 127-136) to determine if the deprenyl-saline pairs are drawn from the same population.
- the probability value indicates the probability that the data comes from the same population. A value of p ⁇ 0.5 is required to detect any significant differences and p ⁇ .01 is preferable.
- the 0.01 mg/kg dose did not produce any significant MAO-A or MAO-B inhibition at d4 and d22.
- the marked rescue with 0.01 mg/kg is equipotent to that with 0.25 mg/kg but cannot be due to MAO-B inhibition. Therefore, deprenyl may activate a receptor through a 3D structure which may not be related to the structure which blocks MAO-B.
- mice Male, C57BL/6J mice obtained at five weeks of age from Jackson Labs (Bar Harbour, Maine) were housed in individual cages and allowed food and water ad libitum. Mice were given an initial two week acclimatization period to a 12:12 hour light:dark (LD) cycle in an isolated room kept at a constant temperature of 21°C Subjective 'day' began at 8:00 hours while subjective 'night' began at 20:00 hours. Light levels were maintained at 200 lux during the subjective day. Locomotory movements were selectively quantified with a Stoelting Electronic Activity Monitor, individual sensor boxes being placed under each cage. Higher frequency signal interruption such as feeding or grooming events were not recorded.
- LD light:dark
- Locomotory movements for individual mice were continuously monitored under continual darkness (DD) or under LD conditions for 90 to 120 days. After approximately 20 days the mice were treated with twice daily injections for 5 days (pre injection days -5 to 0) of saline or MPTP (to achieve cumulative doses of 37.5, 75, 150 and 300 mg/kg). Injections were always given during the subjective day, the first injection occurring 4 hours after 'lights on' and the second, 4 hours before 'lights off.
- DD continual darkness
- MPTP to achieve cumulative doses of 37.5, 75, 150 and 300 mg/kg
- the activity values were treated with a split-cosine-bell taper to reduce leakage from strong components into other components. These values were then padded with zeros to 512 samples. The mean was then removed from these values and the Fourier transform was calculated for 100 lags to encompass hours/cycle values of 5.12 to 512. The magnitudes were squared to determine the power of each component and the power for each hour/cycle value was expressed as a percentage of the total power.
- Neurochemical assays were performed at 5, 10, 15 and 20 days following the last of the MPTP injections. The mice were sacrificed by cervical dislocation and the brain removed. Striatal tissue was dissected so as to include the nucleus accumbens and the caudate.
- the tissue was frozen in 2-methylbutane (Kodak) at -70°C until their catecholamine concentrations were measured by reverse- phase ion-pair high performance liquid chromatography (HPLC) with electrochemical detection.
- Tissue samples were weighed, then homogenized in 0.2 N perchloric acid containing dihydroxybenzylamine as internal standard and extracted onto alumina (Mefford, I.N.J. Neurosci. Meth. 1981, 3, 207-224).
- the catecholamines were desorbed into 0.1 N phosphoric acid, filtered and injected onto an Ultrasphere 0DS 5 urn column.
- the mobile phase contained 7.1g/l Na2HP04, 50 mg/1 EDTA, 100 mg/1 sodium octyl sulphate and 10% methanol.
- the detector potential was +0.72 versus a Ag-AgCl reference electrode. Interrun variability was approximately 5%.
- Figure 5 shows 92 days of typical recording and the black bar indicates the interval of MPTP injection (150 mg/kg in total, 30 mg/kg daily for five days). Each vertical bar on the activity trace represents the sum of activity for one hour. Note that there is a slower rhythm with a period between 100-200 hours superimposed on a faster (about 24 hour) circadian rhythm which introduces a cyclic variation into the amplitude of the activity peaks. The regularity of these patterns, as well as the amplitude of activity, was significantly affected during the MPTP injection period (675h-842h), but seemed to "recover" by 1200 hours, viz. between days 15-20 post- injection.
- panel A shows that interruption of the animals' endogenous activity by saline injections was sufficient to reduce the percentage power of the P22-26 relative to pre-injection and post-injection days.
- activity changes like those in Panel B could not be reliably interpreted for the MPTP injection period.
- Saline injections did not produce any changes in the P22- 26 in the post-injection period (Panel C for an example) .
- the 150 and 300 mg/kg doses resulted in marked depression of the P22-26 which recovered by days 12 to 20 (Panels B and D) .
- Figure 8 shows that saline and 37.5 or 75 mg/kg
- the DOPAC/DA ratio shows a marked increase and rapid decline over Days 5-10 for the MPTP injected animals and then maintains a constant Level at about 2 times that of the saline injected animals.
- a computer optical density (OD) system was used to measure somal cytoplasmic TH immunoreactivity and the background immunoreactivity in the immediately adjacent tissue for randomly chosen SNc and VTA somata (Tatton, W.G. et al. Brain Res. 1990, 527, 21-32) for the glued brain sections. Background OD per unit area was subtracted from somal OD per unit area for each cell to obtain an estimate of cytoplasmic TH immunodensity per unit area.
- the mean background OD for the saline injected half of each glued section was used to normalize the values for the MPTP background OD and the saline and MPTP cytoplasmic ODs.
- Figure 10 presents distributions for the normalized background and cytoplasmic measurements for TH+ SNc somata at Days 5-20 after saline or 150 mg/kg MPTP injections. In this and other studies using the glued brains, background values did not differ significantly (p ⁇ .05) for the saline injected and MPTP injected halves thereby allowing valid comparisons of the cytoplasmic values.
- the control distributions for the saline injected animals often revealed a bimodal distribution of TH immunodensity for the SNc somata ranging from 0.5 to 6 times mean background levels with modes at about 2 and 4 times mean background level.
- the inventors have adapted spectral analysis techniques with fast Fourier transforms to the analysis of long term locomotory activity in mice treated with MPTP. This provides both highly sensitive and reproducible data that is not dependent on subjective assessment of animals that have been aroused by recent handling or the presence of observers.
- MPTP did not produce motor deficits in rodents due to the view that rat and mouse SNc neurons were resistant to the toxin. This was based largely on neurochemical data that reported only transient changes in striatal dopamine following MPTP (Ricuarte, G.A. et al. Brain Res. 1986, 376, 117-124, and Walters, A., et al. Biogenic Amines 1984, 1, 297-302).
- Locomotory activity as measured by the power under the P22-26 peak, striatal DA concentration and TH immunodensity in SNc and VTA somata are correlated in their recovery toward normal after MPTP treatment.
- the numbers of SNc and VTA somata with detectable TH immunoreactivity decay to a steady state level over the first 20 days after MPTP treatment.
- dopamine content in the striatum is increasing while the number of SNc and VTA neurons with detectable TH content is decreasing.
- the rapid rise and fall of the DOPAC/DA ratio likely is related to the death of DA terminals in the striatum with loss of DA into the extracellular space. Yet the ratio is maintained at an increased level after Day 15 in support of the earlier findings suggesting that DA synthesis is increased in SNc neurons surviving MPTP exposure.
- the affinity constants for the inventors' polycional antibodies and those for the immunoreaction between the primary and secondary antibodies may not provide for a linear relationship between the concentration of the epitope and the concentration of avidin molecules. Yet, the results probably do indicate a recovery in TH concentrations in the somata of VTA and SNc surviving MPTP exposure.
- the recovery of TH immunodensity parallels the increases in striatal DA content which suggests that a recovery of TH synthesis is a factor in the recovery of DA content and possibly increased DA synthesis by individual surviving neurons.
- Neostriatal dopaminergic and other ctecholaminergic systems in rodents have been related to the generation of locomotory activity (Tabar J. , et al. Pharmacol Biochem Behav 1989, 33, 139-146, Oberlander, C, et al. Neurosci. Lett. 1986, 67, 113-118, Melnick, M.E. et al. 17th Annual Meeting Of The Society For Neuroscience, New Orleans, Louisiana, USA, November 1987, 13, Marek, G.J., et al. Brain Res 1990, 517, 1-7, Kostowski, W., et al. Acta Physiol. Pol.
- Example 4 An experiment was carried out to determine whether deprenyl can reduce the death of other axonally- damaged neuronal phenotypes e.g. rat motoneurons.
- the proportion of rat motoneurons which die after axotomy is maximal during the first 4 days of life (80-90% loss) and then diminishes to adult levels (20-30% loss) over the next 3 to 4 weeks (Sendtner et al. Nature, 345, 440-441, 1990, Snider W.D. and Thanedar, S.J. Compl. Neuro 1, 270,489, 1989).
- a lesioned and an unlesioned group were begun on deprenyl 10 mg/kg intraperitoneally every ⁇ econd day until ⁇ acrifice.
- the other lesioned and unlesioned groups were given identical injections with saline.
- the rats were killed by anaesthetic overdose followed by perfusion with isotonic saline and 4% paraformaldehyde in phosphate buffer.
- Brains from the unlesioned groups were bisected longitudinally along the midline and the half brains from saline treated and deprenyl treated animals were glued together using Tissue-Tek so that the surface landmarks coincided.
- the glued brains for the unlesioned animals and the intact brains for the lesioned animals were frozen in -70°C methylbutane and 10 ⁇ m serial sections were cut through the portion of the medulla containing the facial nuclei.
- Figure 12 shows photomicrographs of adjacent ChAT immunoreacted (Al and BI) and Nissl stained (A2 and B2) sections through the facial nucleus ipsilateral to transection of the facial nerve.
- Al and A2 are for saline treated animals and BI and B2 are for deprenyl treated animals.
- Figure 13 is a bar graph for the counts of ChAT+ somata for the facial nuclei for the different lesion and treatment groups (bars-mean ⁇ , error bar ⁇ - ⁇ tandard deviations) .
- ChAT immunoreactive somata containing nuclear profiles were counted from every third section taken serially through entire facial nuclei. The value at the top of each bar is the mean.
- the Ipsi.Lesion and Contra.Lesion indicate the nuclei located ipsilaterally and contralaterally to the facial nerve transection respectively.
- the counts were not adjusted to estimate the total numbers of ChAT+ somata in the facial nuclei, so the numbers for unlesioned groups are approximately one third of values reported for counts of Nissl stained somata. The values were compared statistically in a pairwise fashion using the Mann Whitney U test.
- Figure 14 show ⁇ the joint Ni ⁇ l/ChAT+ counts of adjacent sections.
- One of each pair of intervening sections between those that were immunoreacted for ChAT was Nis ⁇ l stained.
- With the aid of a camera lucida the number of ChAT+ somata and Nissl-stained nucleolus- containing somata (Oppenheim,R.W. J.Comp. Neurol. 246:281, 1986 for criteria) were counted in matching areas of adjacent sections on 20 randomly-chosen sections through the length of each nucleus for each animal.
- Nissl counts were then plotted against ChAT+ counts for the adjacent sections (values from three animals in each lesion- treatment group were pooled) .
- Nissl and ChAT+ somal counts were done to determine whether decrease ⁇ in the number of immunopositive somata reflected the death of the motoneurons or los ⁇ of immunoreactivity.
- deprenyl can prevent the death of motoneurons and is consistent with the work indicating that deprenyl can reduce the death of axonally-da aged neurons.
- the death of axotomized motoneurons in immature rats is believed to reflect a dependency of the motoneurons for trophic support from the muscles they innervate (Crews, L. and Wigston, D.J.; J. Neurosci 10, 1643, 1990; Snider, W>D. and Thanedar, S. supra) .
- Recent studies have shown that some neuronotrophic factors can reduce the loss of the motoneurons supporting that concept (Sendtner, M. et al, Nature 345,440, 1990). This study suggests that deprenyl has the capacity to activate some mechanism which compensates for the loss of target derived trophic agents. Part of the action of deprenyl in neurodegenerative diseases may reflect a similar compensation for reduced trophic support.
- Axotomy initiates transient changes in protein ⁇ ynthesi ⁇ in facial motoneurons (Tetzlaff, W. et al. Neuro Sci. 8, 3191 (1988)) which include a decrease in choline acetyl transferase (Hoeover, D. R. & Hancock, J.C Neuroscience 15, 481, 1985).
- deprenyl (10 mg/kg) was sufficient to block the majority of MAO-B activity and some MAO-A activity as well (Demarest, K.T., Aazzaro, A. J. in Monoamine Oxidase: Structure, Function and Altered Functions (eds. Singer, T.P., Korff, R.W. and Murphy, D.L.) 423-340, Academic Press, New York, 1979) hence the reduction in motoneuron death may be due to MAO-B or MAO-A inhibition or may be independent of both enzymes. However, it is expected that a 0.01 mg/kg deprenyl dose will produce a reduction in motoneuron death similar to that obtained with the 10 mg/kg dose.
- the 0.01 mg/kg dose does not produce any ⁇ ignficant MAO-A or MAO-B inhibition indicating that the rescue with 0.01 mg/dg deprenyl is not due to MAO-A or MAO-B inhibition. (See example 2) Thus, it is more likely that the reduction in motoneuron death will be independent of MAO-B or MAO-A.
- MAO-inhibitors may be more effective then deprenyl in reducing the necrosis of dorsal striatal neurons after a transient interruption of the arterial blood supply to that region (Matsui, Y. and Kamagae, Y. , Neurossci lett 126, 175-178, 1991). Yet deprenyl dose ⁇ (0.25 mg/kg) too low to produce inhibition of MAO-A but ⁇ ufficient to product 20-75% inhibition of MAO-B in mice are as effective as a 10 mg/kg dose in preventing the death of SNc neurons.
- MAO-B is largely concentrated in glial cells although present in some serotonergic and histaminergic neurons (Vincent, S.R.
- glial cells may be involved in deprenyl- induced prevention of neuronal death.
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EP03029904A Withdrawn EP1413299A3 (fr) | 1991-04-04 | 1992-03-31 | Utilisation de l'analogues du deprenyle pour maintenir ou recuperer la fonction de cellules nerveuses ou pour empecher la perte de cette fonction |
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US5844003A (en) * | 1991-04-04 | 1998-12-01 | Innovations Foundation | Use of deprenyl compounds to maintain, prevent loss, or recover nerve cell function |
FR2704756B1 (fr) * | 1993-05-04 | 1995-07-28 | Sanofi Elf | Utilisation de la sélégiline en médecine vétérinaire. |
ATE194911T1 (de) * | 1993-05-04 | 2000-08-15 | Ceva Sante Animale | Anwendung von selegilin in tiermedizin |
DE4428444A1 (de) * | 1994-08-11 | 1996-02-15 | Dresden Arzneimittel | Verwendung von Selegilin zur Behandlung von epileptischen Erkrankungen |
US6319954B1 (en) | 1995-01-13 | 2001-11-20 | Somerset Pharmaceuticals, Inc. | S-(+)-desmethylselegiline and its use in the therapeutic methods and pharmaceutical compositions |
US6033682A (en) * | 1995-01-13 | 2000-03-07 | Somerset Pharmaceuticals, Inc. | S(+) desmethylselegiline and its use in therapeutic methods and pharmaceutical compositions |
US6299901B1 (en) | 1995-01-13 | 2001-10-09 | Somerset Pharmaceuticals, Inc. | Methods and pharmaceutical compositions employing desmethylselegiline |
US6348208B1 (en) | 1995-01-13 | 2002-02-19 | Somerset Pharmaceuticals, Inc. | Methods and pharmaceutical compositions employing desmethylselegiline |
AU723047B2 (en) | 1995-02-10 | 2000-08-17 | University Of Toronto Innovations Foundation, The | Deprenyl compounds for treatment of glaucoma |
CA2244253C (fr) | 1996-03-15 | 2002-11-12 | Somerset Pharmaceuticals, Inc. | Methode de prevention et de traitement de la neuropathie peripherique par administration de selegiline |
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US6417160B1 (en) * | 1997-10-14 | 2002-07-09 | Nadine A. Tatton | Methods for increasing schwann cell survival |
AU2472499A (en) * | 1998-01-27 | 1999-08-09 | Thomas N. Thomas | Methods of treatment using mao-a and mao-b inhibitors such as l-deprenyl |
CA2547053C (fr) | 2003-11-25 | 2014-05-27 | Technion Research & Development Foundation Ltd. | Compositions et procedes pour traiter des troubles et des maladies cardiovasculaires |
US8263655B2 (en) | 2005-10-06 | 2012-09-11 | Technion Research And Development Foundation Ltd | Methods for treatment of renal failure |
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