U.S. patent application number 09/885365 was filed with the patent office on 2001-12-20 for s(+) desmethylselegiline and drug withdrawal.
Invention is credited to Disanto, Anthony R..
Application Number | 20010053798 09/885365 |
Document ID | / |
Family ID | 23226301 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053798 |
Kind Code |
A1 |
Disanto, Anthony R. |
December 20, 2001 |
S(+) desmethylselegiline and drug withdrawal
Abstract
The present invention provides novel compositions and methods
for using the S-(+) enantiomer of desmethylselegiline
(N-methyl-N-(prop-2-ynyl)-2-a- minophenylpropane), for the
treatment of 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. Effective dosages are a daily dose of
at least about 0.0015 mg/kg of body weight.
Inventors: |
Disanto, Anthony R.;
(Gobles, MI) |
Correspondence
Address: |
Margaret J. Sampson
VINSON & ELKINS LLP
2300 First City Tower
1001 Fannin
Austin
TX
77002-6760
US
|
Family ID: |
23226301 |
Appl. No.: |
09/885365 |
Filed: |
June 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09885365 |
Jun 20, 2001 |
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09315840 |
Nov 3, 2000 |
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09315840 |
Nov 3, 2000 |
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08679328 |
Jul 12, 1996 |
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6033682 |
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08679328 |
Jul 12, 1996 |
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PCT/US96/01561 |
Jan 11, 1996 |
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08679328 |
Jul 12, 1996 |
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08372139 |
Jan 13, 1995 |
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60001979 |
Jul 31, 1995 |
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Current U.S.
Class: |
514/649 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 31/00 20130101; A61K 31/135 20130101; A61K 31/135 20130101;
A61K 31/137 20130101; A61K 45/06 20130101; A61K 31/135
20130101 |
Class at
Publication: |
514/649 |
International
Class: |
A61K 031/137 |
Claims
What is claimed is:
1. In a method for obtaining a selegiline-like therapeutic effect
in a patient undergoing drug withdrawal, the improvement which
comprises administering to said patient the S-(+) enantiomer of
desmethylselegiline employing a dosage regimen effective to to
treat one or more withdrawal symptoms.
2. The improvement of claim 1, wherein said S-(+) enantiomer of
desmethylselegiline is administered to treat one or more withdrawal
symptoms resulting from an addictive psychostimulant.
3. The improvement of claim 2, wherein said addictive
psychostimulant is cocaine.
4. The improvement of claim 2, wherein said addictive
psychostimulant is selected from the group consisting of:
amphetamines; methamphetamines; dextroamphetamines;
chlorphentermine, methylphenidate, pipradol, p-hydroxymorphedrine,
fenfluramine, 1-(2,5-dimethoxy-4-methylphenyl)-2-am- inopropane,
bupropion and pemoline.
5. The improvement of claim 1, wherein said S-(+) enantiomer of
desmethylselegiline is administered to treat one or more withdrawal
symptoms resulting from an addictive opiate.
6. The improvement of claim 5, wherein said addictive opiate is
selected from the group consisting of: opium; morphine; and
heroin.
7. The improvement of claim 1, wherein said S-(+) enantiomer of
desmethylselegiline is administered to treat one or more withdrawal
symptoms resulting from an addictive narcotic.
8. The improvement of claim 7, wherein said addictive narcotic is
selected from the group consisting of: alphaprodine, anileridine,
bezitramide, codeine, dihydrocodeine, diphenoxylate, fentanyl,
hydrocodone, hydromorphone, isomethadone, levomethorphan,
levorphanol, metazocine, methadone, metopon, oxycodone,
oxymorphone, pethidine, phenazocine, piminodine, racemethorphan,
racemorphon and thebaine.
9. The improvement of claim 1, wherein said S-(+) enantiomer of
desmethylselegiline is administered to treat one or more withdrawal
symptoms resulting from an addictive barbiturate.
10. The improvement of claim 9, wherein said addictive barbiturate
is selected from the group consisting of: allobarbital,
amylbarbital, butabarbital, hexabarbital, mephobarbital,
methohexital, pentobarbital, phenobarbital, phenethylbarbital,
secobarbital, talbutal and thiopental.
11. The improvement of claim 1, wherein said S-(+) enantiomer of
desmethylselegiline is administered to treat one or more withdrawal
symptoms resulting from alcohol.
12. The improvement of claim 1, wherein said S-(+) enantiomer of
desmethylselegiline is administered to treat one or more withdrawal
symptoms resulting from nicotine.
13. The improvement of claim 1, wherein said withdrawal symptoms
are selected from the group consisting of: drug craving;
depression, irritability; anergia; amotivation; appetite changes;
nausea; shaking; psychomotoric retardation; and irregular sleep
patterns.
14. The method of any one of claims 1-13, wherein said S-(+)
enantiomer of desmethylselegiline is administered orally.
15. The method of any one of claims 1-13, wherein said S-(+)
enantiomer of desmethylselegiline is administered non-orally.
16. A pharmaceutical composition, comprising: a) S-(+) enantiomer
of desmethylselegiline; and b) a second drug useful in the
treatment of the symptoms associated with drug withdrawal; wherein
said S-(+) enantiomer of desmethylselegiline and said second drug
are present in an amount such that one or more unit doses of said
composition are effective in the symptomatic treatment of drug
withdrawal.
17. The composition of claim 16, wherein said second drug is
selected from the group consisting of naloxone, naltrexone or
nalorphine
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
application Ser. No. 08/679,328 which is a continuation-in-part of
PCT/US96/01561, with an international filing date of Jan. 11, 1996,
a continuation-in-part of U.S. Provisional Application No.
60/001,979, filed Jul. 31, 1995 and a continuation-in-part of U.S.
application Ser. No. 08/372,139, filed Jan. 13, 1995.
FIELD OF THE INVENTION
[0002] The present invention provides a novel S-(+) isomer of
desmethylselegiline, i.e., a compound of the formula: 1
[0003] and further provides novel pharmaceutical and other
compositions containing this isomer. In addition, the present
invention provides novel methods for using this enantiomer in the
treatment of selegiline-responsive diseases and conditions in
animals, including humans.
BACKGROUND OF THE INVENTION
[0004] Two distinct monoamine oxidase enzymes are known in the art:
monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B). The
cDNAs encoding these enzymes show different promoter regions and
distinct exon portions, indicating they are encoded independently
at different gene positions. In addition, analysis of the two
proteins has shown differences in their respective amino acid
sequences.
[0005] The first compound found to selectively inhibit MAO-B was
R-(-)-N-methyl-N-(prop-2-ynyl)-2-aminophenylpropane, also known as
L-(-)-deprenyl, R-(-)-deprenyl, or selegiline. Selegiline has the
following structural formula: 2
[0006] The selectivity of selegiline in the inhibition of MAO-B is
important to its safety profile following oral administration.
Inhibition of MAO-A may cause toxic side effects by interfering
with the metabolism of tyramine. Tyramine is normally metabolized
in the gastrointestinal tract by MAO-A but when MAO-A is inhibited,
tyramine absorption is increased following consumption of
tyramine-containing foods such as cheese, beer, herring, etc. This
results in the release of catecholamines which can precipitate a
hypertensive crisis, producing the "cheese effect." This effect is
characterized by Goodman and Gilman as the most serious toxic
effect associated with MAO-A inhibitors.
[0007] One of the metabolites of selegiline is its N-desmethyl
analog. Structurally, the desmethylselegiline metabolite is the R
(-) enantiomeric form of a secondary amine of the formula: 3
[0008] Heretofore, desmethylselegiline was not known to have
pharmaceutically useful MAO-related effects, i.e., potent and
selective inhibitory effects on MAO-B. In the course of determining
the usefulness of desmethylselegiline for the purposes of the
present invention, the MAO-related effects of desmethylselegiline
were more completely characterized. This characterization has
established that desmethylselegiline has exceedingly weak MAO-B
inhibitory effects and no advantages in selectivity with respect to
MAO-B compared to selegiline.
[0009] For example, the present characterization established that
selegiline has an IC.sub.50 value against MAO-B in human platelets
of 5.times.10.sup.-9 M whereas desmethylselegiline's IC.sub.50
value is 4 .times.10.sup.-7 M, indicating the latter is
approximately 80 times less potent as an MAO-B inhibitor than the
former. Similar characteristics can be seen in the following data
measuring inhibition of MAO-B and MAO-A in rat cortex
mitochondrial-rich fractions:
1TABLE 1 Inhibition of MAO by Selegiline and Desmethylselegiline
Percent Inhibition selegiline desmethylselegiline Conc. MAO-B MAO-A
MAO-B MAO-A 0.003 .mu.M 16.70 -- 3.40 -- 0.010 .mu.M 40.20 -- 7.50
-- 0.030 .mu.M 64.70 -- 4.60 -- 0.100 .mu.M 91.80 -- 6.70 -- 0.300
.mu.M 94.55 9.75 26.15 0.0 1.000 .mu.M 95.65 32.55 54.73 0.70 3.000
.mu.M 98.10 65.50 86.27 4.10 10.000 .mu.M -- 97.75 95.15 11.75
30.000 .mu.M -- -- 97.05 -- 100.000 .mu.M -- -- -- 56.10
[0010] As is apparent from the above table, selegiline is
approximately 128 times more potent as an inhibitor of MAO-B
relative to MAO-A, whereas desmethylselegiline is about 97 times
more potent as an inhibitor of MAO-B relative to MAO-A.
Accordingly, desmethylselegiline appears to have an approximately
equal selectivity for MAO-B compared to MAO-A as selegiline, albeit
with a substantially reduced potency.
[0011] Analogous results are obtained in rat brain tissue.
Selegiline exhibits an IC.sub.50 for MAO-B of 0.11.times.10.sup.-7
M whereas desmethylselegiline's IC.sub.50 value is
7.3.times.10.sup.-7 M, indicating desmethylselegiline is
approximately 70 times less potent as an MAO-B inhibitor than
selegiline. Both compounds exhibit low potency in inhibiting MAO-A
in rat brain tissue, 0.18.times.10.sup.-5 for selegiline,
7.0.times.10.sup.-5 for desmethylselegiline. Thus, in vitro,
desmethyl-selegiline is approximately 39 times less potent than
selegiline in inhibiting MAO-A.
[0012] Based on its pharmacological profile as set forth above,
R(-)desmethylselegiline as an MAO-B inhibitor provides no
advantages in either potency or selectivity compared to selegiline.
To the contrary, the above in vitro data suggest that use of
desmethylselegiline as an MAO-B inhibitor requires on the order of
70 times the amount of selegiline.
[0013] The potency of desmethylselegiline as an MAO-B inhibitor in
vivo has been reported by Heinonen, E. H., et al.
("Desmethylselegiline, a metabolite of selegiline, is an
irreversible inhibitor of MAO-B in human subjects," referenced in
Academic Dissertation "Selegiline in the Treatment of Parkinson's
Disease," from Research Reports from the Department of Neurology,
University of Turku, Turku, Finland, No. 33 (1995), pp. 59-61).
According to Heinonen, desmethylselegiline in vivo has only about
one-fifth the MAO-B inhibitory effect as selegiline, i.e., a dose
of 10 mg of desmethylselegiline would be required for the same
MAO-B effect as 1.8 mg of selegiline. In rats, Borbe reported
R(-)desmethylselegiline to be an irreversible inhibitor of MAO-B,
with a potency about 60 fold lower than selegiline in vitro and
about 3 fold lower ex vivo (Borbe, H. O., J. Neural Trans. (Suppl.)
32:131 (1990)).
[0014] The various diseases and conditions for which selegiline is
disclosed as being to be useful include: depression (U.S. Pat. No.
4,861,800); Alzheimer's disease and Parkinson's disease,
particularly through the use of transdermal dosage forms, including
ointments, creams and patches, macular degeneration (U.S. Pat. No.
5,242,950); age-dependent degeneracies, including renal function
and cognitive function as evidenced by spatial learning ability
(U.S. Pat. No. 5,151,449); pituitary-dependent Cushing's disease in
humans and nonhumans (U.S. Pat. No. 5,192,808); immune system
dysfunction in both humans (U.S. Pat. No. 5,387,615) and animals
(U.S. Pat. No. 5,276,057); age-dependent weight loss in mammals
(U.S. Pat. No. 5,225,446); and schizophrenia (U.S. Pat. No.
5,151,419). PCT Published Application WO 92/17169 discloses the use
of selegiline in the treatment of neuromuscular and
neurodegenerative disease and in the treatment of CNS injury due to
hypoxia, hypoglycemia, ischemic stroke or trauma. In addition, the
biochemical effects of selegiline on neuronal cells have been
extensively studied. For example, see Tatton et al., "Selegiline
Can Mediate Neuronal Rescue Rather than Neuronal Protection,"
Movement Disorders 8 (Supp. 1):S20-S30 (1993); Tatton, et al.,
"Rescue of Dying Neurons," J. Neurosci. Res. 30:666-672 (1991); and
Tatton, et al., "(-)-Deprenyl Prevents Mitochondrial Depolarization
and Reduces Cell Death in Trophically-Deprived Cells," 11th Int'l
Symp. on Parkinson's Disease, Rome, Italy, Mar. 26-30, 1994.
[0015] Although selegiline is reported as being effective in
treating the foregoing conditions, neither the precise number or
nature of its mechanism or mechanisms of action are known. However,
there is evidence that selegiline provides neuroprotection or
neuronal rescue, possibly by reducing oxidative neuronal damage,
increasing the amount of the enzyme superoxide dismutase, and/or
reducing dopamine catabolism. For example, PCT Published
Application WO 92/17169 reports that selegiline acts by directly
maintaining, preventing loss of, and/or assisting in, the nerve
function of animals.
[0016] Selegiline is disclosed as being useful when administered to
a subject through a wide variety of routes of administration and
dosage forms. For example U.S. Pat. No. 4,812,481 (Degussa AG)
discloses the use of concomitant selegiline-amantadine in oral,
peroral, enteral, pulmonary, rectal, nasal, vaginal, lingual,
intravenous, intraarterial, intracardial, intramuscular,
intraperitoneal, intracutaneous, and subcutaneous formulations.
U.S. Pat. No. 5,192,550 (Alza Corporation) describes a dosage form
comprising an outer wall impermeable to selegiline but permeable to
external fluids. This dosage form may have applicability for the
oral, sublingual or buccal administration of selegiline. Similarly,
U.S. Pat. No. 5,387,615 discloses a variety of selegiline
compositions, including tablets, pills, capsules, powders,
aerosols, suppositories, skin patches, parenterals, and oral
liquids, including oil-aqueous suspensions, solutions, and
emulsions. Also disclosed are selegiline-containing sustained
release (long acting) formulations and devices.
[0017] The present invention is directed to the novel, S(+)
enantiomeric form of desmethylselegiline. This isomer has striking
and wholly unexpected pharmacological effects and, accordingly, is
surprisingly and unexpectedly useful in treating
selegiline-responsive diseases and conditions. Assay results
suggest that, in at least some respects, the S(+) enantiomer is
considerably more effective than either selegiline or the R(-)
enantiomer of desmethylselegiline. For example, results suggest
that the S(+) enantiomer may be 4-5 times more effective than these
other agents at inhibiting dopamine re-uptake by neurons and, in
certain cell culture models, it has a greater neuroprotective
effect than either R(-)desmethylselegiline or selegiline. Thus, the
S(+) isomer is the form of choice in the treatment of conditions
which require enhanced synaptic dopamine activity or neuronal
protection/rescue. Such conditions include Parkinson's disease;
Alzheimer's disease and attention deficit hyperactivity disorder
(ADHD); dementia; depression; schizophrenia; and dysautonomia.
SUMMARY OF THE INVENTION
[0018] The present invention provides in part:
[0019] (a) the S-(+) enantiomer of desmethylselegiline, a compound
of the formula: 4
[0020] including the S-(+) enantiomer in substantially isomerically
pure form;
[0021] (b) a pharmaceutical composition comprising the S-(+)
enantiomer of desmethylselegiline, wherein one or more unit doses
of said composition, administered on a periodic basis, are
effective to treat a selegiline-responsive disease or condition in
a animal to whom said unit dose or unit doses are administered;
[0022] (c) in a method for obtaining a selegiline-like therapeutic
effect in a subject suffering from a selegiline-responsive disease
or condition, the improvement which comprises:
[0023] administering to said subject the S-(+) enantiomer of
desmethylselegiline in a dosage regimen sufficient to produce said
selegiline-like therapeutic effect.
[0024] (d) a method of treating a condition in a mammal produced by
neuronal degeneration or neuronal trauma which comprises
administering to said mammal the S-(+) enantiomer of
desmethylselegiline or a pharmaceutically acceptable acid addition
salt thereof, at a daily dose, administered in a single or multiple
dosage regimen, of at least about 0.0015 mg, calculated on the
basis of the free secondary amine, per kg of the mammal's body
weight;
[0025] (e) a transdermal delivery composition for use in treating a
condition in a mammal produced by neuronal degeneration or neuronal
trauma which comprises a layered composite containing in at least
one layer an amount of the S-(+) enantiomer of desmethylselegiline,
or a pharmaceutically acceptable acid addition salt thereof,
sufficient to supply a daily transdermal dose of at least about
0.0015 mg of the free secondary amine, per kg of the mammal's body
weight; and
[0026] (f) a method of treating a condition in a mammal produced by
immune system dysfunction, which comprises administering to the
mammal the S-(+) enantiomer of desmethylselegiline, or a
pharmaceutically acceptable acid addition salt thereof, at a daily
dose, administered in a single or multiple dosage regimen, of at
least about 0.0015 mg, calculated on the basis of the free
secondary amine, per kg of the mammal's body weight.
[0027] The present invention is based upon the discovery that both
desmethylselegiline ("DMS" or "R(-)DMS") and its enantiomer
(ent-desmethylselegiline, abbreviated as "ent-DMS" or "S-(+) DMS")
are useful in providing selegiline-like effects in subjects,
notwithstanding dramatically reduced MAO-B inhibitory activity and
an apparent lack of enhanced selectivity for MAO-B compared to
selegiline. It has been discovered that desmethylselegiline,
ent-desmethylselegiline and their isomeric mixtures provide a more
advantageous way of obtaining selegiline-like therapeutic effects
in selegiline-responsive diseases or conditions. This is
particularly true for diseases and conditions characterized by
neuronal degeneration, neuronal trauma or which are
hypodopaminergic in nature, i.e. diseases or conditions
characterized by reduced dopamine release and formation.
[0028] Thus, the present invention is directed to compositions
comprising the S(+) enantiomer of desmethylselegiline either alone
or together with the R(-) enanatiomer. In preferred embodiments,
the S(+) enantiomer is present at a greater concentration than the
concentration of the R(-) enantiomer or, alternatively, in
substantially isomerically pure form. S(+) and R(-) forms of
desmethylselegiline can be conveniently prepared by methods known
in the art, as described below in Example 1. The characteristics of
a preparation of purified S(+) enantiomer have been determined and
are described in Example 3. Its chemical structure is as follows:
5
[0029] The S(+) enantiomer, S(+)DMS, is used as an active
ingredient in novel pharmaceutical compositions. In this regard, a
quantity of the isomer is employed such that one or more unit doses
of the composition is effective in the treatment of one or more
selegiline-responsive diseases or conditions in a subject
administered the composition on a periodic basis. If desired, the
R(-) enantiomer may be included. Compositions may be designed so
that they are suitable for oral, topical, transdermal, sublingual,
buccal, parenteral or other conventional routes of administration.
So formulated and administered, S(+)DMS compositions are useful for
effecting an increase in dopaminergic activity, neuronal
rescue/protection and can be administered in a manner so as to
improve immune system function in a subject or to treat
hypodopaminergic related diseases.
[0030] In addition, the present invention provides for an improved
method for obtaining a selegiline-like therapeutic effect in a
subject suffering from a selegiline-responsive disease or
condition, which comprises administering to the subject the S(+)
enantiomer of desmethylselegiline in an amount sufficient to
produce the selegiline-like therapeutic effect. The S(+)DMS may be
present in substantially isomerically pure form, a form preferred
for the treatment of ADHD, or it may be present together with
R(-)DMS.
[0031] As used herein the term "selegiline-responsive disease or
condition" refers to any of the various diseases or conditions in
mammals, including humans, for which selegiline is disclosed in the
prior art as being useful. In particular, a "selegiline-responsive
disease or condition" refers to the various diseases and conditions
described above, e.g., Alzheimer's disease, cognitive dysfunction,
neuronal rescue or protection, and the like. The term also refers
to the use of selegiline as an appetite suppressant. Similarly, the
term "selegiline-like therapeutic effect" refers to one or more of
the salutary effects reported as being exerted by selegiline in
subjects being treated for a selegiline-responsive disease or
condition.
[0032] The selegiline-responsive diseases or conditions related to
neuronal degeneration or trauma which respond to the present
methods include Parkinson's disease, Alzheimer's disease,
depression, glaucoma, macular degeneration, ischemia, diabetic
neuropathy, attention deficit disorder, post polio syndrome,
multiple sclerosis, impotence, narcolepsy, chronic fatigue
syndrome, alopecia, senile dementia, hypoxia, cognitive
dysfunction, negative symptomatology of schizophrenia, amyotrophic
lateral sclerosis, Tourette's syndrome, tardive dyskinesia, and
toxic neurodegeneration.
[0033] In preferred embodiments, the invention is directed to a
method of treating a mammal for a hypodopaminergic condition or
neuronal trauma/neuronal degeneration by administering the S(+)
enantiomer of desmethylselegiline, or a pharmaceutically acceptable
acid addition salt of this enantiomer, at a daily dose of at least
about 0.015 mg per kg body weight. The dosage is based upon the
weight of the free secondary amine and may be administered in
either a single or multiple dosage regimen.
[0034] In addition, the invention includes the control of
selegiline-responsive withdrawal symptoms (e.g., drug craving,
depression, irritability, anergia, amotivation, appetite changes,
nausea, shaking, psychomotoric retardation and irregular sleep
patterns) associated with a variety of drugs including: addictive
psychostimulants (e.g. cocaine, amphetamines, methamphetamines,
dextroamphetamines, chlorphentermine, methylphenidate, pipradol,
p-hydroxymorphedrine, fenfluramine,
1-(2,5-dimethoxy-4-methylphenyl) -2-aminopropane, bupropion and
pemoline), addictive opiates (e.g. opium, morphine and heroin),
addictive narcotics (e.g. alphaprodine, anileridine, bezitramide,
codeine, dihydrocodeine, diphenoxylate, fentanyl, hydrocodone,
hydromorphone, isomethadone, levomethorphan, levorphanol,
metazocine, methadone, metopon, oxycodone, oxymorphone, pethidine,
phenazocine, piminodine, racemethorphan, racemorphon and thebaine),
addictive barbiturates (e.g. allobarbital, amylbarbital,
butabarbital, hexabarbital, mephobarbital, methohexital,
pentobarbital, phenobarbital, phenethylbarbital, secobarbital,
talbutal and thiopental), alcohol and nicotine. The
desmethylselegilne may be administered either orally or non-orally
and may be used in conjunction with other drugs. Specifically
included within the invention are pharmaceutical compositions
containing desmethylselegiline together with a second drug useful
in the treatment of drug addiction and in which the
desmethylselegiline and second drug are present in an amount such
that one or more unit doses of the composition are effective in the
symptomatic treatment of drug withdrawal. Specific second drugs
that can be used include naloxone, naltrexone or nalorphine.
[0035] The present invention also encompasses the restoration or
improvement of immune system function by S(+)DMS. Again, the S(+)
enantiomer may be administered either as a substantially pure
isomer or in combination with R(-)DMS. The conditions or diseases
treatable include age-dependent immune system dysfunction, AIDS,
immunological loss due to cancer chemotherapy and infectious
diseases. In a preferred embodiment, the invention is directed to a
method of treating a condition in a mammal produced by immune
system dysfunction, by administering the S(+) enantiomer of
desmethyl-selegiline, or a pharmaceutically acceptable acid
addition salt thereof, at a daily dose of at least about 0.015 mg,
calculated on the basis of the free secondary amine, per kg of the
mammal's body weight.
[0036] Depending upon the particular route employed,
R-(+)desmethylselegiline is administered in the form of a free base
or as a physiologically acceptable non-toxic acid addition salt.
Acid addition salts include those derived from organic and
inorganic acids such as, without limitation, hydrochloric acid,
hydrobromic acid, phosphoric acid, sulfuric acid, methanesulphonic
acid, acetic acid, tartaric acid, lactic acid, succinic acid,
citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,
salicylic acid, phthalic acid, embonic acid, enanthic acid, and the
like. The use of salts, especially the hydrochloride, is
particularly desirable when the route of administration employs
aqueous solutions, as for example parenteral administration. Use of
delivered S(+)-desmethylselegiline, in the form of the free base is
especially useful for transdermal administration. Reference herein
to the administration of DMS or ent-DMS or to mixtures thereof
encompasses both the free base and acid addition salt forms.
[0037] The optimal daily dose of S(+)-desmethylselegiline useful
for the purposes of the present invention is determined by methods
known in the art, e.g., based on the severity of the disease or
condition being treated, the condition of the subject to whom
treatment is being given, the desired degree of therapeutic
response, and the concomitant therapies being administered to the
patient or animal. Ordinarily, however, the attending physician or
veterinarian will administer an initial dose of at least about
0.015 mg/kg, calculated on the basis of the free secondary amine,
with progressively higher doses being employed depending upon the
route of administration and the subsequent response to the therapy.
Typically the daily dose will be about 0.10 mg/kg and may extend to
about 1.0 mg/kg of the patient's body weight (all such doses again
being calculated on the basis of the free secondary amine). These
guidelines further require that the actual dose be carefully
titrated by the attending physician or veterinarian depending on
the age, weight, clinical condition, and observed response of the
individual patient or animal.
[0038] The daily dose can be administered in a single or multiple
dosage regimen. Oral dosage forms will most typically be used and
are preferred but other dosage forms may also be employed and may
permit, for example, a continuous release of relatively small
amounts of the active ingredient from a single dosage unit, such as
a transdermal patch, over the course of one or more days. This is
particularly desirable in the treatment of chronic conditions such,
as Parkinson's disease, Alzheimer's disease, and depression.
Alternatively, it may be desirable in conditions such as ischemia
or neural damage to administer one or more discrete doses by a more
direct systemic route such as intravenously or by inhalation. In
still other instances such as glaucoma and macular degeneration,
localized administration, such as via the intraocular route, can be
indicated.
[0039] Pharmaceutical compositions containing
S(+)-desmethylselegiline can be prepared according to conventional
techniques. For example, preparations for parenteral routes of
administration of S(+)-desmethylselegiline, e.g., intramuscular,
intravenous and intraarterial routes, can employ sterile isotonic
saline solutions. Sterile isotonic buffered solutions can also be
employed for intraocular administration.
[0040] Transdermal dosage unit forms of S(+)-desmethylselegiline
can be prepared utilizing a variety of previously described
techniques (see e.g., U.S. Pat. Nos. 4,861,800; 4,868,218;
5,128,145; 5,190,763; and 5,242,950; and EP-A 404807, EP-A 509761,
and EP-A 593807). For example, a monolithic patch structure can be
utilized in which S(+)-desmethylselegiline is directly incorporated
into the adhesive and this mixture is cast onto a backing sheet.
Alternatively S(+)-desmethylselegiline, can be incorporated as an
acid addition salt into a multilayer patch which effects a
conversion of the salt to the free base, as described for example
in EP-A 593807. In a preferred embodiment, the present invention is
directed to a transdermal delivery composition for use in treating
a condition in a mammal produced by neuronal degeneration or
neuronal trauma and for treating hypodopaminergic diseases.
[0041] Subjects treatable by the present preparations and methods
include both human and non-human subjects for which selegiline-like
therapeutic effects are known to be useful. Accordingly, the
compositions and methods above provide especially useful therapies
for mammals, especially domesticated mammals. Thus, the present
methods and compositions are used in treating selegiline-responsive
diseases or conditions in canine and feline species.
[0042] Successful use of the compositions and methods above
requires employment of an effective amount of
S(+)-desmethylselegiline. Although both R(-) desmethylselegiline
and S(+)-desmethylselegiline are dramatically less potent than
selegiline as inhibitors of MAO, employment of these agents, or a
mixture of these agents, does not require a commensurately
increased dosage to obtain a selegiline-like therapeutic response.
Surprisingly, dosages necessary to attain a selegiline-like
therapeutic effect appear to be on the same order as the known
doses of selegiline. Accordingly, because both desmethylselegiline
and ent-desmethylselegiline exhibit a much lower inhibition of
MAO-A at such dosages, desmethylselegiline and
ent-des-methylselegiline provide a substantially wider margin of
safety with respect to MAO-A associated toxicity compared to
selegiline. In particular, the risk of the adverse effects of MAO-A
inhibition, e.g., hypertensive crisis, are minimized due to the
reduced potency for MAO-A inhibition.
[0043] As described above and notwithstanding its demonstrably
inferior inhibitory properties with respect to MAO-B inhibition,
S(+)-desmethylselegiline appears to be at least as effective as
selegiline in treating certain selegiline-responsive conditions,
e.g., conditions resulting from reduced dopamine release and
formation, neuronal degeneration or neuronal trauma. Although the
oral route of administration will generally be most convenient,
drug may be administered by the parenteral, topical, transdermal,
intraocular, buccal, sublingual, intranasal, inhalation, vaginal,
rectal or other routes as well.
BRIEF DESCRIPTION OF THE FIGURES
[0044] FIG. 1: HPLC Chromatogram of Purified R(-)DMS (Microsorb MV
Cyano Column). The purity of a preparation of R(-)DMS was
determined by HPLC on a Microsorb MV Cyano column and results are
shown in FIG. 1. The column had dimensions of 4.6 mm.times.15 cm
and was developed at a flow rate of 1.0 ml/min using a mobile phase
containing 90% 0.01M H.sub.3PO.sub.4 (pH 3.5) and 10% acetonitrile.
The column was run at a temperature of 40.degree. C. and effluent
was monitored at a wavelength of 215 nm. The chromatogram shows one
major peak appearing at a time of 6.08 minutes and having 99.5% of
the total light-absorbing material eluted from the column. No other
peak had greater than 0.24%.
[0045] FIG. 2: BPLC Elution Profile of R(-)DMS (Zorbax Mac-Mod C18
Column). The same preparation that was analyzed in the experiments
discussed in FIG. 1 was also analyzed for purity by HPLC on a
Zorbax Mac-Mod SB-C18 column (4.6 mm.times.75 mm). Effluent was
monitored at 215 nm and results can be seen in FIG. 2. Greater than
99.6% of the light-absorbing material appeared in the single large
peak eluting at a time of between 2 and 3 minutes.
[0046] FIG. 3: Mass Spectrum of R(-)DMS. A mass spectrum was
obtained for purified R(-)DMS and results are shown in FIG. 3. The
spectrum is consistent with a molecule having a molecular weight of
209.72 and a molecular formula of C.sub.12H.sub.15N.HCl.
[0047] FIG. 4: Infrared Spectrum (KBr) of Purified R(-)DMS.
Infrared spectroscopy was performed on a preparation of R(-)DMS and
results are shown in FIG. 4. The solvent used was CDCl.sub.3.
[0048] FIG. 5: NMR Spectrum of Purified R(-)DMS. A preparation of
purified R(-)DMS was dissolved in CDCl.sub.3 and .sup.1H NMR
spectroscopy was performed at 300 MHZ. Results are shown in FIG.
5.
[0049] FIG. 6: HPLC Chromatogram of S(+)DMS. The purity of a
preparation of S(+)DMS was examined by reverse phase HPLC on a 4.6
mm.times.75 mm Zorbax Mac-Mod SB-C18 column. The elution profile,
monitored at 215 nm, is shown in FIG. 6. One major peak appears in
the profile at a time of about 3 minutes and contains greater than
99% of the total light-absorbing material that eluted from the
column.
[0050] FIG. 7: Mass Spectrum of Purified S(+)DMS. Mass spectroscopy
was performed on the same preparation examined in FIG. 6. The
spectrum is shown in FIG. 7 and is consistent with the structure of
S(+)DMS.
[0051] FIG. 8: Infrared Spectrum (KBr) of Purified S(+)DMS. The
preparation of S(+)DMS discussed in connection with FIGS. 6 and 7
was examined by infrared spectroscopy and results are shown in FIG.
8.
[0052] FIG. 9: Effect of Selegiline on Neuron Survival.
Mesencephalic cultures were prepared from embryonic 14 day rats.
Cultures were used at about 1.5 million cells per plate and were
maintained either in growth medium alone (control cultures) or in
growth medium supplemented with selegiline. On day 1, 8 and 15,
cells were immunostained for the presence of tyrosine hydroxylase
("TH"). Striped bars represent results obtained for cultures
maintained in the presence of 50 .mu.M selegiline and open bars
represent results for control cultures. In all cases, results are
expressed as a percentage of TH positive cells present in control
cultures on day 1. The abbreviation "DIV" refers to "days in
vitro." Asterisks or stars above bars both in FIG. 9 and the
figures discussed below indicate a result that differs from
controls in an amount that is statistically significant, i.e.
P<0.05.
[0053] FIG. 10: [.sup.3H]-Dopamine Uptake in Mesencephalic Cells.
Cells, cultured as described above for FIG. 9, were tested for
their uptake of labeled dopamine and results are shown in FIG. 10.
Striped bars represent uptake in cells maintained in the presence
of 50 .mu.M selegiline and open bars represent uptake in control
cultures.
[0054] FIG. 11: Effect of Selegiline on Glutamate Receptor
Dependent Neuronal Cell Death. Rat embryonic mesencephalic cells
were cultured as described above. After allowing cultures to
stabilize, the culture medium was changed daily for a period of 4
days to induce glutamate receptor-dependent cell death. Depending
on the culture, medium contained either 0.5, 5.0 or 50 .mu.M
selegiline. After the final medium change, cultured cells were
immunostained for the presence of tyrosine hydroxylase. From left
to right, bars represent results for controls, 0.5, 5.0 and 50
.mu.M selegiline.
[0055] FIG. 12: Effect of Selegiline on Dopamine Uptake in Neuronal
Cultures. Rat mesencephatic cells were cultured and medium was
changed on a daily basis as discussed for FIG. 11. Uptake of
tritiated dopamine by cells was measured and results are shown in
the figure. From left to right, bars are in the same order as for
FIG. 11.
[0056] FIG. 13: Effect of R(-)Desmethylselegiline on Glutamate
Receptor Dependent Neuronal Cell Death. Rat embryonic mesencephalic
cultures were prepared as described above except that R(-)DMS was
used instead of selegiline. On day 9, the number of TH positive
cells in cultures was determined. Results are expressed as a
percentage of control. From left to right, bars show results for
controls, 0.5, 5 and 50 .mu.M R(-)DMS.
[0057] FIG. 14: Effect of R(-)Desmethylselegiline on Dopamine
Uptake in Neuronal Cultures. Cell cultures were prepared as
described above for FIG. 13 and then tested for uptake of tritiated
dopamine. Results for controls and for cells maintained in the
presence of 0.5 .mu.M, 5 .mu.M and 50 .mu.M desmethylselegiline are
shown from left to right in the figure.
[0058] FIG. 15: Comparison of Dopamine Uptake in Mesencephalic
Cells. Incubated in the Presence of Different Monoamine Oxidase
Inhibitors. Rat embryonic mesencephalic cells were prepared as
described for FIGS. 11-14 and incubated in the presence of a
variety of monoamine oxidase inhibitors. The inhibitors examined
were selegiline; R(-) desmethylselegiline; pargyline; and
clorgyline, all at concentrations of 0.5, 5 and 50 .mu.M. In
addition, cells were incubated in the presence of the glutamate
receptor blocker MK-801 at a concentration of 10 .mu.M. Cultures
were tested for uptake of tritiated dopamine.
[0059] FIG. 16: Relative Effectiveness of R(-) and S(+)DMS in
Maintaining [.sup.3H]-Dopamine Uptake by Cultured Mesencephalic
Cells (NMDA Model). Preparations of R(-) and S(+)DMS were assayed
for their effect on [.sup.3H]-dopamine uptake by cultured rat
mesencephalic cells exposed to the toxin N-methyl-D-aspartate
(NMDA). Results were expressed as a percentage of the uptake seen
in control cultures not exposed to NMDA and are shown in FIG. 16.
From the left, the bars represent: cells incubated with medium
alone; medium +5 .mu.M deprenyl; medium +0.5 .mu.M R(-)DMS; medium
+5 .mu.M R(-)DMS; medium +50 .mu.M R(-)DMS; medium +0.5 .mu.M
S(+)DMS; medium +5 .mu.M S(+)DMS; and medium +50 .mu.M S(+)DMS. All
of the cell cultures shown in the figure were exposed to 100 .mu.M
NMDA. Statistical significance was determined by ANOVA followed by
Dunnett's test. One star above a bar indicates a percentage uptake
that differs significantly from control uptake at the 0.05
confidence level. Two stars indicate a result that differs at the
0.01 confidence level.
[0060] FIG. 17: Relative Effectiveness of R(-) and S(+)DMS on
Survival of Cultured Mesencephalic Cells (NMDA Model). Rat
mesencephalic cell cultures were exposed to 100 .mu.M NMDA and
incubated as described above in connection with FIG. 16. The effect
of DMS enantiomers on the survival TH positive cells is shown in
FIG. 17. The bars are in the same order as for FIG. 16 and results
are expressed as a percentage of control. One star indicates
p<0.05 and two stars indicates p<0.01 when results are
compared to those obtained for cells exposed to NMDA and then
incubated in unsupplemented medium.
[0061] FIG. 18: Inhibition of Neuronal Dopamine Re-Uptake by
Deprenyl and the Two Enantiomers of Desmethylselegiline. An in
vitro nerve terminal preparation (synaptosome preparation) was
prepared using fresh rat neostriatal tissue. This was examined for
its ability to take up tritiated dopamine in buffer alone or in
buffer supplemented with various concentrations of selegiline,
R(-)desmethylselegiline or S(+)desmethylselegiline. Uptake in the
presence of each MAO inhibitor, expressed as a percent inhibition
vs. log concentration of inhibitor is shown in FIG. 18. As
indicated, the plot was used to determine the IC.sub.50 for each
test agent.
[0062] FIG. 19: Determination of IC.sub.50 Values for Inhibition of
Dopamine Re-Uptake. The experiment of FIG. 18 was repeated in a
concentration range designed to more accurately provide an
IC.sub.50 value and results are shown in FIG. 19. Using the log C
vs. probit graphs, as shown in the figure, the IC.sub.50 for
S(+)DMS was determined to be about 11 .mu.M; for selegiline, about
46 .mu.M; and for R(-)DMS about 54 .mu.M.
[0063] FIG. 20: In Vivo MAO-B Inhibition in Guinea Pig Hippocampus.
Various doses of selegiline, R(-)desmethylselegiline, and
S(+)desmethylselegiline were administered daily to guinea pigs for
a period of 5 days. Animals were then sacrificed and the MAO-B
activity in the hippocampus portion of the brain was determined.
Results were expressed as a percent inhibition relative to
hippocampus MAO-B activity in control animals and are shown in FIG.
20. The plots were used to estimate the ID.sub.50 dosage for each
agent. The ID.sub.50 for selegiline was about 0.008 mg/kg; for
R(-)DMS, it was about 0.2 mg/kg; and for S(+)DMS, it was about 0.5
mg/kg.
DETAILED DESCRIPTION OF THE INVENTION
[0064] The surprising utility of S(+)desmethylselegiline and
desmethylselegiline in treating certain selegiline-responsive
diseases or conditions is attributable in part to their powerful
action in preventing loss of dopaminergic neurons by promoting
repair and recovery. Hence, at doses at which little or no MAO-B
inhibition is generally observed, a reversal in neuronal damage
and/or death can be observed. Because S(+)-desmethylselegiline is a
more potent inhibitor of dopamine uptake and also can prevent loss
and facilitate recovery of nerve cell function, it is of value in a
wide variety of hypodopaminergic, neurodegenerative and
neuromuscular diseases. In this regard, S(+)DMS is substantially
more potent than R(-)desmethylselegiline, selegiline in certain
pharmacological effects, as described more empirically in the
examples below.
EXAMPLES
Example 1
Preparation of Desmethylselegiline and Ent-desmethylselegiline
[0065] A. Desmethylselegiline
[0066] Desmethylselegiline (designated below as "R(-)DMS") is
prepared by methods known in the art. For example,
desmethylselegiline is a known chemical intermediate for the
preparation of selegiline as described in U.S. Pat. No. 4,925,878.
Desmethylselegiline can be prepared by treating a solution of
R(-)-2-aminophenylpropane (levoamphetamine): 6
[0067] in an inert organic solvent such as toluene with an
equimolar amount of a reactive propargyl halide such as propargyl
bromide, Br--CH.sub.2--C.ident.CH, at slightly elevated
temperatures (70.degree.-90.degree. C.). Optionally the reaction
can be conducted in the presence of an acid acceptor such as
potassium carbonate. The reaction mixture is then extracted with
aqueous acid, for example 5% hydrochloric acid, and the extracts
are rendered alkaline. The nonaqueous layer which forms is
separated, for example by extraction with benzene, dried, and
distilled under reduced pressure.
[0068] Alternatively the propargylation can be conducted in a
two-phase system of a water-immiscible solvent and aqueous alkali,
utilizing a salt of R(+)-2-aminophenylpropane with a weak acid such
as the tartrate, analogously to the preparation of selegiline as
described in U.S. Pat. No. 4,564,706.
[0069] B. Ent-Desmethylselegiline
[0070] Ent-desmethylselegiline (designated below as "S(+)DMS") is
conveniently prepared from the enantiomeric
S(+)-2-aminophenylpropane (dextroamphetamine), i.e., 7
[0071] C. Mixtures of Enantiomers
[0072] Mixtures of enantiomeric forms of desmethylselegiline,
including racemic desmethylselegiline, are conveniently prepared
from enantiomeric mixtures, including racemic mixtures of the above
amninophenylpropane starting material.
[0073] D. Conversion Into Acid Addition Salts
[0074] N-(prop-2-ynyl)-2-aminophenylpropane in either optically
active or racemic form can be converted to a physiologically
acceptable non-toxic acid addition salt by conventional techniques
such as treatment with a mineral acid. For example, hydrogen
chloride in isopropanol is employed in the preparation of
desmethylselegiline hydrochloride. Either the free base or salt can
be further purified, again by conventional techniques such as
recrystallization or chromatography.
Example 2
Characteristics of Substantially Pure R(-)DMS
[0075] A preparation of substantially pure R(-)DMS has the
appearance of a white crystalline solid with a melting point of
162-163.degree. C. and an optical rotation of
[.alpha.].sub.D.sup.23.degree. C.=-15.2+/-2.0 when measured at a
concentration of 1.0M using water as solvent. R(-)DMS appeared to
be 99.5% pure when analyzed by HPLC on a Microsorb MV Cyano column
(see chromatogram in FIG. 1) and 99.6% pure when analyzed by HPLC
on a Zorbax Mac-Mod SB-C18 column, (see chromatogram in FIG. 2). No
single impurity is present at a concentration greater than or equal
to 0.5%. Heavy metals are present at a concentration of less than
10 ppm and amphetamine hydrochloride at a concentration of less
than 0.03%. The last solvents used for dissolving the preparation,
ethyl acetate and ethanol are both present at a concentration of
less than 0.1%. A mass spectrum performed on the preparation (see
FIG. 3) is consistent with a compound having a molecular weight of
209.72 and a formula of C.sub.12H.sub.15N.HCl. Infrared and NMR
spectra are shown in FIGS. 4 and 5 respectively. These are also
consistent with the known structure of R-(-)-DMS.
Example 3
Characteristics of Substantially Pure S(+)DMS
[0076] A preparation of substantially pure S(+)DMS has the
appearance of a white powder with a melting point of approximately
160.04.degree. C. and a specific rotation of +15.1 degrees when
measured at 22.degree. C. in water, at a concentration of 1.0M.
When examined by reverse phase HPLC on a Zorbax Mac-Mod SB-C18
column the preparation appears to be about 99.9% pure (FIG. 6).
Amphetamine hydrochloride is present at a concentration of less
than 0.13% (w/w). A mass spectrum is performed on the preparation
and is consistent with a compound having a molecular weight of
209.72 and a molecular formula of C.sub.12H.sub.15N.HCl (see FIG.
7). Infrared spectroscopy is performed and also provides results
consistent with the structure of S(+)DMS (see FIG. 8).
Example 4
Neuronal Survival as Measured Using Tyrosine Hydroxylase
[0077] The effect of desmethylselegiline on neuron survival can be
correlated to tyrosine hydroxylase, the rate limiting enzyme in
dopamine biosynthesis. Assays are performed by determining the
number of tyrosine hydroxylase positive cells in cultured E-14
embryonic mesencephalic cells over a period of 7 to 14 days.
Protection in this system has been seen with a variety of trophic
factors including BDNF, GDNF, EGF, and .beta.-FGF.
[0078] A. Test Methods
[0079] Timed pregnant Sprague-Dawley rats are used to establish
neuronal cultures from embryonic rat brain on the 14th day of
gestation. Mesencephalon is dissected out without the membrane
coverings and collected in Ca.sup.++ and Mg.sup.++ free balanced
salt solution at 4.degree. C. Tissue fragments are dissociated in
chemically defined medium by mild trituration with a small bore
pasteur pipette. Cell suspension is plated in polyornithine-coated
35 mm Falcon plastic dishes (0.1 mg/ml, Sigma) at a density of
1.5.times.10.sup.6 cells/dish. Cultures are maintained at
37.degree. C. in an atmosphere of 10% CO.sub.2:90% air and 100%
relative humidity, and fed twice weekly with chemically defined
medium consisting of MEM/F12 (1:1, Gibco), glucose (33 mM), HEPES
(15 mM), NaHCO.sub.3 (44.6 mM), transferrin (100 mg/ml), insulin
(25 mg/ml), putrescine (60 nM), sodium selenite (30 nM),
progesterone (20 nM), and glutamine (2 mM). Control cells receive
no further additions. The medium used for other cells also includes
test substance, e.g. selegiline, at one or more concentrations.
[0080] Cultures are fixed in 4% paraformaldehyde in 0.1M phosphate
buffer (pH 7.4) for 30 minutes at room temperature, permeabilized
with 0.2% Triton X-100 for 30 minutes and incubated with an
antibody against tyrosine hydroxylase (1:1000; Eugene Tech) for 48
hours at 4.degree. C. in the presence of a blocking serum. They are
then stained using a peroxidase-coupled avidin-biotin staining kit
(Vectastain ABC kit; Vector Labs) with 3',3'-diaminobenzidine as a
chromagen.
[0081] The number of dopaminergic neurons in cultures is determined
by counting the cells positively immunostained with TH antibodies.
100 fields (0.5 mm.times.0.5 mm) in two transverse strips across
the diameter of the dish, representing 2.5% of the total area, are
counted using a Nikon inverted microscope at 200.times.
magnification.
[0082] B. Results
[0083] Using the procedures described above, the following results
were obtained:
2TABLE 2 Effect of Selegiline and DMS on the Survival of TH
Positive Cells Control Selegiline Desmethylselegiline Mean Mean
Mean Conc. cells/cm.sup.2 cells/cm.sup.2 % cont. cells/cm.sup.2 %
cont. 0.5 .mu.M 108.55 201.70 .+-. 25.01 185.81 246.00 .+-. 22.76
226.62 5 .mu.M -- 237.00 .+-. 12.59 218.33 357.95 .+-. 25.76 329.76
50 .mu.M -- 292.28 .+-. 17.41 269.25 391.60 .+-. 34.93 360.76
Example 5
Neuronal Survival as Measured Using Dopamine Uptake
[0084] In addition to determining the number of TH positive cells
in culture (see Example 4), the protective effect of
desmethylselegiline on neuronal cells also can be determined by
directly measuring dopamine uptake. The amount of uptake by the
cultured brain cells corresponds to axonal growth.
[0085] A. Test Methods
[0086] Cell cultures, established in the manner discussed above,
are incubated with [.sup.3H]dopamine (0.5 mCi/ml; 37 Ci/mmol; New
England Nuclear) for 15 minutes in the presence of ascorbic acid
(0.2 mg/ml) in PBS (pH 7.3), supplemented with 0.9 mM CaCl.sub.2
and 0.5 mM MgCl.sub.2 at 37.degree. C. After two rinses and a 5
minute incubation with fresh buffer, [.sup.3H]dopamine accumulated
within the cells is released by incubating the cultures with 95%
ethanol for 30 minutes at 37.degree. C. Preparations are then added
to 10 ml Ecoscint (National Diagnostics) and counted in a
scintillation spectrometer. Nonspecific uptake values are obtained
by blocking dopaminergic neuronal uptake with 10 mM mazindol.
[0087] B. Results
[0088] Using the above procedure, the results shown in Table 3 were
obtained.
3TABLE 3 Effect of Selegiline and DMS on .sup.3H-Dopamine Uptake
Cont. Selegiline Desmethylselegiline Conc. Mean Mean % Cont Mean %
Cont 0.5 .mu.M 11982 14452 .+-. 212 120.6 24020 .+-. 800 200.4 5
.mu.M -- 16468 .+-. 576 137.5 34936 .+-. 2119 291.5 50 .mu.M --
33018 .+-. 1317 275.5 56826 .+-. 2656 474.3
[0089] C. Conclusions from Examples 4 and 5
[0090] The results described in Examples 4 and 5 indicate that
desmethylselegiline is substantially more potent superior to
selegiline as a neuroprotective agent. This is true notwithstanding
the fact that desmethylselegiline in much less potent than
selegiline as an inhibitor of MAO-B.
Example 6
Neuroprotective Action of Desmethylselegiline Enantiomers in
Cultured Dopamine-Containing Mesencephalic Neurons In Vitro
[0091] The survival of mesencephalic, dopamine-containing neuronal
cultures of rat brain tissue was used in these experiments to
examine neuroprotective properties of selegiline and
R(-)desmethylselegiline. The number of TH positive neurons is
directly proportional to the survival of dopaminergic neurons and
.sup.3H-dopamine uptake is a measure of axonal growth in these
neurons.
[0092] A. Effect of Selegiline on the Survival of Dopaminergic
Neurons
[0093] Mesencephalic cultures prepared from embryonic day 14 rats
were treated with 0.5, 5 or 50 .mu.M selegiline for 15 days,
beginning on the day of plating. (For a more detailed discussion of
the culturing of cells and other methods used in these experiments
see Mytilineou et al., J. Neurochem. 61:1470-1478 (1993)). Survival
and growth of dopamine neurons was evaluated by tyrosine
hydroxylase (TH) immunocytochemistry and [.sup.3H]dopamine uptake
and results are shown in FIGS. 9 and 10.
[0094] B. Effect of Selegiline on Glutamate Receptor Dependent Cell
Death
[0095] The neuroprotective effect of selegiline was also examined
using an experimental paradigm that causes neuronal cell death that
can be blocked by inhibition of glutamate receptors. In these
experiments, cells were plated and allowed to stabilize for several
days. The growth medium of the cells was then changed on a daily
basis to induce cell death that can be prevented by blocking
glutamate receptors, e.g. using MK-801. After 4 days of daily
medium changes, cultures were stained for tyrosine hydroxylase and
assayed for uptake of tritiated dopamine. The results shown in
FIGS. 11 and 12 further support the conclusion that selegiline
promotes the survival of dopaminergic neurons.
[0096] C. Effect of Desmethylselegiline on the Survival of Dopamine
Neurons
[0097] Using the glutamate receptor dependent model of neuron
death, an even more potent protection of dopaminergic neurons was
provided when desmethylselegiline was used in place of selegiline.
Even at the lowest dose tested (0.5 EM), desmethylselegiline caused
a significant reduction in the loss of TH positive neurons (FIG.
13) and a significant increase in dopamine uptake (FIG. 14)
relative to control cultures in which medium was used without
supplementation with either selegiline or desmethylselegiline.
[0098] D. Comparison With Other MAO Inhibitors
[0099] Using the glutamate receptor dependent model of
neurotoxicity, the effects of selegiline and desmethylselegiline
were compared with two other MAO inhibitors, pargyline and
clorgyline (FIG. 15). In agreement with previous results,
measurement of dopamine uptake indicated neuron protection by 50
.mu.M deprenyl and 5 and 50 .mu.M desmethylselegiline. Pargyline
did not appear to offer any protection at the concentrations used,
while clorgyline protected at 50 .mu.M. As expected, protection was
also obtained by the NMDA receptor blocker MK-801 (10 .mu.M).
[0100] E. Effect of DMS Enantiomers on .sup.3H-Dopamine Uptake
[0101] The data summarized in Table 4 suggests that both (R-)DMS
and S(+)DMS are effective as neuroprotectants in mesencephalic
dopamine-containing neurons in culture.
[0102] Table 4: Effect of DMS Enantiomers on Dopamine Uptake
4TABLE 4 Effect of DMS Enantiomers on Dopamine Uptake
.sup.3H-Dopamine uptake Treatment as a percentage + SEM Control 100
.+-. 14.4% R(-)DMS (10 .mu.M) 140.82 .+-. 26.20% S(+)DMS (10 .mu.M)
234 .+-. 38.36%
[0103] These results were obtained using the medium change model of
cell death. Compared to untreated control cells, there was 40% and
134% more axonal growth and terminal axonal survival after
treatment with R(-)DMS and S(+)DMS, respectively. In this study,
S(+)DMS showed greater potency as a neuroprotectant than
R(-)DMS.
Example 7
Comparison of the Neuroprotective Effect of R(-)DMS and S(+)DMS
[0104] The neuroprotective effect of R(-)DMS and S(+)DMS on
cultured rat mesencephalic cells was examined using two models of
neuronal cell death. In the first model, cells were exposed to 100
.mu.M N-methyl-D-aspartate (NMDA), an agent which causes cell death
by binding to glutamate receptors. Cells exposed to NMDA were
incubated in the presence of either medium alone; medium
supplemented with 50 .mu.M deprenyl; medium with 0.5, 5, or 50
.mu.M R(-)DMS; or medium containing 0.5, 5 or 50 .mu.M S(+)DMS. The
effect of these treatments on [.sup.3H]-dopamine uptake and the
survival of TH positive cells was determined and results are shown
in Tables 5-8 and FIGS. 16 and 17. It can be seen that both forms
of DMS had a neuroprotective effect, with S(+)DMS being the most
effective treatment to a statistically significant degree as
determined by tritiated dopamine uptake. Experiments examining the
neuroprotective effect of DMS enantiomers were also performed using
the medium change model of cell death described previously (see
Example 6). As can be seen in Tables 9-12, both the R(-) and S(+)
enantiomers significantly enhanced [.sup.3H]-dopamine uptake and
the survival of TH positive cells. In this model, the relative
potency of both enantiomers appears to be equal to treatment with
50 .mu.M selegiline.
5TABLE 5 R(-)DMS: Dopamine Uptake After 100 .mu.M NMDA Exposure
Control R(-)DMS (0.5 .mu.M) R(-)DMS (5.0 .mu.M) R(-)DMS (50 .mu.M)
Deprenyl (50 .mu.M) counts/min counts/min % control counts/min %
control counts/min % control counts/min % control 6013 9385 138.9
13509 199.9 23090 341.8 18479 273.5 6558 8976 132.9 11471 169.8
21530 318.7 16958 251.0 7462 9028 133.6 13786 204.0 17520 259.3
17550 259.8 6432 8133 120.4 10229 151.4 22963 339.9 18572 274.9
7317 11304 167.3 11014 163.0 17708 262.1 15410 228.1 Mean 6756.4
9365.2 138.6 12001.8 177.6 20562.2 304.3 17393.8 257.4 St. Dev.
614.3 1177.2 17.4 1569.7 23.2 2761.0 40.9 1295.7 19.2
[0105]
6TABLE 6 S(+)DMS: Dopamine Uptake After 100 .mu.M NMDA Exposure
Control R(-)DMS (0.5 .mu.M) R(-)DMS (5.0 .mu.M) R(-)DMS (50 .mu.M)
Deprenyl (50 .mu.M) counts/min counts/min % control counts/min %
control counts/min % control counts/min % control 6013 12092 179.0
20313 300.6 25944 384.0 18479 273.5 6558 12269 181.6 16579 245.4
28545 422.5 16958 251.0 7462 16399 242.7 15929 235.8 39042 577.9
17550 259.8 6432 11435 169.2 15052 222.8 33024 488.8 18572 274.9
7317 11096 164.2 15535 229.9 25101 371.5 15410 228.1 Mean 6756.4
12658.2 187.4 16681.6 246.9 30331.2 448.9 17393.8 257.4 St. Dev.
614.3 2144.9 31.7 2105.6 31.2 5764.6 85.3 1295.7 19.2
[0106]
7TABLE 7 R(-)DMS: TH Immunochemistry After 100 .mu.M NMDA Exposure
Control R(-)DMS (0.5 .mu.M) R(-)DMS (5.0 .mu.M) R(-)DMS (50 .mu.M)
Deprenyl (50 .mu.M) cells/cm.sup.2 cells/cm.sup.2 % control
cells/cm.sup.2 % control cells/cm.sup.2 % control cells/cm.sup.2 %
control 95.0 95.0 100.9 142.5 151.3 237.5 252.2 230.0 244.2 90.0
75.0 79.6 122.5 130.1 170.0 180.5 287.5 305.3 97.5 105.0 111.5
130.0 138.1 102.5 108.8 187.5 199.1 117.5 124.8 115.0 122.1 177.5
188.5 Mean 94.17 91.67 97.3 128.13 136.1 156.25 165.9 220.63 234.3
St. Dev. 3.8 15.3 16.2 10.9 11.5 61.6 65.4 50.1 53.2
[0107]
8TABLE 8 S(+)DMS: TH Immunochemistry After 100 .mu.M NMDA Exposure
Control S(+)DMS (0.5 .mu.M) S(+)DMS (5.0 .mu.M) S(+)DMS (50 .mu.M)
Deprenyl (50 .mu.M) cells/cm.sup.2 cells/cm.sup.2 % control
cells/cm.sup.2 % control cells/cm.sup.2 % control cells/cm.sup.2 %
control 95.0 127.5 135.4 192.5 204.4 297.5 315.9 230 244.2 90.0 210
223.0 187.5 199.1 202.5 215.0 287.5 305.3 97.5 177.5 188.5 192.5
204.4 317.5 337.2 187.5 199.1 172.5 183.2 222.5 236.3 177.5 188.5
Mean 94.17 171.67 182.3 186.25 197.8 260 276.1 220.63 234.3 St.
Dev. 41.6 44.1 9.5 10.1 56.1 59.5 50.1 53.2
[0108]
9TABLE 9 R(-)DMS: Dopamine Uptake, Medium Change Model Control
R(-)DMS (0.5 .mu.M) R(-)DMS (5.0 .mu.M) R(-)DMS (50 .mu.M) Deprenyl
(50 .mu.M) counts/min counts/min % control counts/min % control
counts/min % control counts/min % control 17880 29885 142.3 32577
155.2 37440 178.3 38053 181.2 21500 32002 152.4 29831 142.1 39200
186.7 34130 162.6 23471 29934 142.6 36370 173.2 39126 186.3 36810
175.3 21134 27382 130.4 30342 144.5 40013 190.6 33863 161.3 Mean
20996.25 29800.75 141.9 32280 153.7 38944.75 185.5 35714 170.1 St.
Dev. 2317.2 1890.4 9.0 2976.0 14.2 1080.7 5.1 2050.0 9.8
[0109]
10TABLE 10 S(+)DMS: Dopamine Uptake, Medium Change Model Control
S(+)DMS (0.5 .mu.M) S(+)DMS (5.0 .mu.M) S(+)DMS (50 .mu.M) Deprenyl
(50 .mu.M) counts/min counts/min % control counts/min % control
counts/min % control counts/min % control 17880 35830 170.6 35976
171.3 26002 123.8 38053 181.2 21500 32074 152.8 36476 173.7 37320
177.7 34130 162.6 23471 33042 157.4 38143 181.7 30725 146.3 36810
175.3 21134 39516 188.2 40964 195.1 38020 181.1 33863 161.3 Mean
20996.25 35115.5 167.2 37889.75 180.5 33016.75 157.3 35714 170.1
St. Dev. 2317.2 3337.9 15.9 2249.2 10.7 5715.7 27.2 2050.0 9.8
[0110]
11TABLE 11 R(-)DMS: TH Immunochemistry, Medium Change Model Control
R(-)DMS (0.5 .mu.M) R(-)DMS (5.0 .mu.M) R(-)DMS (50 .mu.M) Deprenyl
(50 .mu.M) cells/cm.sup.2 cells/cm.sup.2 % control cells/cm.sup.2 %
control cells/cm.sup.2 % control cells/cm.sup.2 % control 270.0
340.0 129.0 322.5 122.3 310.0 117.6 385.0 146.0 237.0 310.0 117.6
342.5 129.9 442.5 167.9 327.5 124.2 280.0 330.0 125.2 362.5 137.5
380.0 144.1 320.0 121.4 267.5 362.5 365.0 138.5 395.0 149.8 Mean
263.63 335.63 123.9 348.13 132.1 381.88 144.9 344.17 130.6 St. Dev.
18.6 21.8 5.8 19.8 7.5 54.8 20.8 35.6 13.5
[0111]
12TABLE 12 S(+)DMS: TH Immunochemistry, Medium Change Model Control
S(+)DMS (0.5 .mu.M) S(+)DMS (5.0 .mu.M) S(+)DMS (50 .mu.M) Deprenyl
(50 .mu.M) cells/cm.sup.2 cells/cm.sup.2 % control cells/cm.sup.2 %
control cells/cm.sup.2 % control cells/cm.sup.2 % control 270.0
402.5 152.7 342.5 129.9 307.5 116.6 385.0 146.0 237.0 330.0 125.2
357.5 135.6 250.0 94.8 327.5 124.2 280.0 402.5 152.7 325.0 123.3
312.5 118.5 320.0 121.4 267.5 477.5 352.5 133.7 287.5 109.1 Mean
263.6 403.1 143.5 344.4 130.6 289.4 109.8 344.2 130.6 St. Dev. 18.6
60.2 15.9 14.3 5.4 28.4 10.8 35.6 13.5
Example 8
Desmethylselegiline and Ent-Desmethylselegiline as Inhibitors of
Dopamine Re-Uptake
[0112] The biological actions of the brain neurotransmitter
dopamine are terminated at the synapse by a high-affinity, sodium
and energy-dependent transport system (neuronal re-uptake) present
within the limiting membrane of the presynaptic dopamine-containing
nerve terminal. Inhibition of this transport mechanism would extend
the actions of dopamine at the synapse and therefore enhance
dopamine synaptic transmission.
[0113] A. Method of Testing
[0114] The R(-) and S(+) enantiomers of desmethylselegiline (DMS)
were tested for their ability to inhibit the dopamine re-uptake
system and compared to selegiline. Inhibitory activity in this
assay is indicative of agents of value in the treatment of diseases
which require enhanced synaptic dopamine activity. Presently this
would include Parkinson's disease, Alzheimer's disease and
attention deficit hyperactivity disorder (ADHD).
[0115] The assay system used was essentially that described by Fang
et al. (Neuropharmacology 33:763-768 (1994)). An in vitro
nerve-terminal preparation (symptosome-preparation) was obtained
from fresh rat neostriatal brain tissue. Transport by dopamine
nerve-terminals was estimated by measuring the uptake of tritiated
dopamine.
[0116] B. Results
[0117] As seen in the data presented in Table 13, selegiline,
R(-)DMS and S(+)DMS all inhibited dopamine re-uptake by
dopamine-containing nerve terminals. Selegiline and R(-) DMS were
approximately equipotent. In contrast, S(+)DMS was 4-5 times more
potent than either selegiline or R(-)DMS.
13TABLE 13 .sup.3H-Dopamine Uptake By Rat Neostriatal Brain Tissue
% Reduction Agent Concentration {overscore (x)} .+-. SEM Dopamine 1
.mu.M 52.0 .+-. 4.9 10 .mu.M 80.9 .+-. 0.4 Selegiline 100 nM 7.0
.+-. 3.6 1 .mu.M 13.9 .+-. 4.7 10 .mu.M 16.3 .+-. 3.8 100 .mu.M
59.8 .+-. 1.0 R(-)DMS 100 nM 11.5 .+-. 1.0 1 .mu.M 10.7 .+-. 2.8 10
.mu.M 20.1 .+-. 3.1 100 .mu.M 51.3 .+-. 2.6 S(+)DMS 100 nM 15.3
.+-. 7.7 1 .mu.M 24.1 .+-. 11.7 10 .mu.M 47.0 .+-. 3.1 100 .mu.M
76.9 .+-. 1.8
[0118] Relative potency can be expressed in terms of the
concentration required to inhibit dopamine re-uptake by 50%
(IC.sub.50). The IC.sub.50 values were determined graphically (see
FIG. 18) and are shown below in Table 14.
14TABLE 14 Concentrations Needed to Inhibit Dopamine Uptake by 50%
Agent IC50 Relative Potency Selegiline .apprxeq.80 .mu.M 1 R(-)DMS
.apprxeq.100 .mu.M 0.8 S(+)DMS .apprxeq.20 .mu.M 4
[0119] The experiment described above was repeated in a
concentration range designed to more accurately describe IC.sub.50
values and results are shown in FIG. 19. ID.sub.50 values
determined based upon the graph are shown in Table 15.
15TABLE 15 Concentrations Needed to Inhibit Dopamine Uptake by 50%
Potency Relative to Compound ID.sub.50 Selegiline S(+)DMS 11 .mu.M
4.2 selegiline 46 .mu.M 1 R(-)DMS 54 .mu.M 1.2
[0120] C. Conclusions
[0121] The results demonstrate that, at the appropriate
concentration, selegiline and each of the enantiomers of DMS
inhibit transport of released dopamine at the neuronal synapse and
enhance the relative activity of this neurotransmitter at the
synapse. In this regard, S(+)DMS is more potent than selegiline
which, in turn, is more potent than R(-)DMS. Of the agents tested,
S(+)DMS is the most preferred with regard to the treatment of
hypodopaminergic conditions such as ADHD.
Example 9
Actions of the R(-) and S(+) enantiomers of Desmethylselegiline
(DMS) on Human Platelet MAO-B and Guinea Pig Brain MAO-B and MAO-A
Activity
[0122] Human platelet MAO is comprised exclusively of the type-B
isoform of the enzyme. In the present study, the in vitro and in
vivo inhibition of this enzyme by the two enantiomers of DMS was
determined and compared with inhibition due to selegiline. The
present study also examined the two enantiomers of DMS for
inhibitory activity with respect to the MAO-A and MAO-B in guinea
pig hippocampal tissue. Guinea pig brain tissue is an excellent
animal model for studying brain dopamine metabolism, the enzyme
kinetics of the multiple forms of MAO and the inhibitory properties
of novel agents that interact with these enzymes. The multiple
forms of MAO in this animal species show similar kinetic properties
to those found in human brain tissue. Finally, the test agents were
administered to guinea pigs and the extent to which they might act
as inhibitors of brain MAO in vivo was assessed.
[0123] A. Method of Testing
[0124] In vitro: The test system utilized the in vitro conversion
of specific substrates of MAO-A (.sup.14C-serotonin) and MAO-B
(.sup.14C-phenylethylamine) by human platelets and/or guinea pig
hippocampal homogenates. The rate of conversion of each substrate
was measured in the presence of S(+)DMS, R(-)DMS or selegiline and
compared to the isozyme activity in the absence of these agents. A
percent inhibition was calculated from these values. Potency was
evaluated by comparing the concentration of each agent which caused
a 50% inhibition (IC.sub.50 value).
[0125] In vivo: R(-)DMS, S(+)DMS or selegiline was administered in
vivo subcutaneously (sc), once a day for 5 days prior to sacrifice,
preparation of enzyme hippocampal homogenates, and the in vitro
assay of MAO-A and MAO-B activity. These experiments were performed
to demonstrate that the DMS enantiomers were capable of entering
brain tissue and inhibiting MAO activity.
[0126] B. Results
[0127] MAO-B Inhibitory Activity In Vitro
[0128] Results for MAO-B inhibition are shown in Tables 16 and 17.
IC.sub.50 values for MAO-B inhibition and potency as compared to
selegiline is shown in Table 18.
16TABLE 16 MAO-B Inhibition in Human Platelets Concentration %
Inhibition Agent Concentration {overscore (x)} .+-. SEM Selegiline
0.3 nM 8.3 .+-. 3.4 5 nM 50.3 .+-. 8.7 10 nM 69.0 .+-. 5.5 30 nM
91.0 .+-. 1.4 100 nM 96.0 .+-. 1.6 300 nM 96.0 .+-. 41.6 1 .mu.M
96.6 .+-. 1.6 R(-)DMS 100 nM 14.3 .+-. 3.6 300 nM 42.1 .+-. 4.0 1
.mu.M 76.9 .+-. 1.47 3 .mu.M 94.4 .+-. 1.4 10 .mu.M 95.8 .+-. 1.4 3
.mu.M 95.7 .+-. 2.3 S(+)DMS 300 nM 6.4 .+-. 2.8 1 .mu.M 11.1 .+-.
1.0 3 .mu.M 26.6 .+-. 1.9 10 .mu.M 42.3 .+-. 2.3 30 .mu.M 68.2 .+-.
2.34 100 .mu.M 83.7 .+-. 0.77 1 mM 94.2 .+-. 1.36
[0129]
17TABLE 17 MAO-B Inhibition in Guinea Pig Hippocampus % Inhibition
Agent Concentration {overscore (x)} .+-. SEM Selegiline 0.3 nM 28.3
.+-. 8.7 5 nM 81.2 .+-. 2.6 10 nM 95.6 .+-. 1.3 30 nM 98.5 .+-. 0.5
100 nM 98.8 .+-. 0.5 300 nM 98.8 .+-. 0.5 1 .mu.M 99.1 .+-. 0.45
R(-)DMS 100 nM 59.4 .+-. 9.6 300 nM 86.2 .+-. 4.7 1 .mu.M 98.2 .+-.
0.7 3 .mu.M 98.4 .+-. 0.95 10 .mu.M 99.1 .+-. 0.45 30 .mu.M 99.3
.+-. 0.40 S(+)DMS 300 nM 18.7 .+-. 2.1 1 .mu.M 44.4 .+-. 6.4 3
.mu.M 77.1 .+-. 6.0 10 .mu.M 94.2 .+-. 1.9 30 .mu.M 98.3 .+-. 0.6
100 .mu.M 99.3 .+-. 0.2 1 mM 99.9 .+-. 0.1
[0130]
18TABLE 18 IC.sub.50 Values for the Inhibition of MAO-B Guinea Pig
Treatment Human Platelets Hippocampal Cortex Selegiline 5 nM (l) 1
nM (1) R(-)DMS 400 nM (80) 60 nM (60) S(+)DMS 1400 nM (2800) 1200
nM (1200) ()= reduction in potency compared to selegiline
[0131] As observed, R(-)DMS was 20-35 times more potent than
S(+)DMS as an MAO-B inhibitor and both enantiomers were less potent
than selegiline.
[0132] MAO-A Inhibitory Activity In Vitro
[0133] Results obtained from experiments examining the inhibition
of MAO-A in guinea pig hippocampus are summarized in Table 19. The
IC.sub.50 values for the two enantiomers of DMS and for selegiline
are shown in Table 20.
19TABLE 19 MAO-A Inhibition in Guinea Pig Hippocampus % Reduction
Agent Concentration x .+-. SEM Selegiline 300 nM 11.95 .+-. 2.4 1
.mu.M 22.1 .+-. 1.2 3 .mu.M 53.5 .+-. 2.7 10 .mu.M 91.2 .+-. 1.16
100 .mu.M 98.1 .+-. 1.4 1 mM 99.8 .+-. 0.2 R(-)DMS 300 nM 4.8 .+-.
2.1 1 .mu.M 4.2 .+-. 1.5 3 .mu.M 10.5 .+-. 2.0 10 .mu.M 19.0 .+-.
1.3 100 .mu.M 64.2 .+-. 1.5 1 mM 96.5 .+-. 1.2 S(+)DMS 1 .mu.M 3.3
.+-. 1.5 3 .mu.M 4.3 .+-. 1.0 10 .mu.M 10.5 .+-. 1.47 100 .mu.M
48.4 .+-. 1.8 1 mM 92.7 .+-. 2.5 10 mM 99.6 .+-. 0.35
[0134]
20TABLE 20 IC.sub.50 Values for the Inhibition of MAO-A IC.sub.50
for MAO-A in Guinea Treatment Pig Hippocampal Cortex Selegiline 2.5
.mu.M (1) R(-)DMS 50.0 .mu.M (20) S(+)DMS 100.0 .mu.M (40) () =
reduction in potency compared to selegiline
[0135] R(-)DMS was twice as potent as S(+)DMS as an MAO-A inhibitor
and both were 20-40 times less potent than selegiline. Moreover,
each of these agents were 2-3 orders of magnitude, i.e., 100 to
1000 times, less potent as inhibitors of MAO-A than inhibitors of
MAO-B in hippocampal brain tissue. Therefore, selegiline and each
enantiomer of DMS can be classified as selective MAO-B inhibitors
in brain tissue.
[0136] Results of In Vivo Experiments
[0137] Each enantiomer of DMS was administered in vivo by
subcutaneous injection once a day for five consecutive days, and
inhibition of brain MAO-B activity was then determined. In
preliminary studies, selegiline was found to have an ID.sub.50 of
0.03 mg/kg and both R(-)DMS and S(+)DMS were determined to be about
10 times less potent. More recent studies, performed on a larger
group of animals, indicates that R(-)DMS is actually about 25 times
less potent than selegiline as an inhibitor of MAO-B and that
S(+)DMS is about 50 times less potent. Results are shown in FIG. 20
and ID.sub.50 values are summarized in Table 21.
21TABLE 21 ID.sub.50 Values for Brain MAO-B Following 5 Days of
Administration ID.sub.50 for MAO-B in Guinea Treatment Pig
Hippocampal Cortex Selegiline 0.008 mg/kg R(-)DMS 0.20 mg/kg
S(+)DMS 0.50 mg/kg
[0138] This experiment demonstrates that the enantiomers of DMS
penetrate the blood brain-barrier and inhibit brain MAO-B after in
vivo administration. It also demonstrates that the potency
differences as an MAO-B inhibitor observed in vitro between each of
the DMS enantiomers and selegiline are substantially reduced under
in vivo conditions.
[0139] In experiments examining the effect of 5 s.c. treatments on
MAO-A activity in guinea pig cortex (hippocampus), it was found
that selegiline administration at a dose of 1.0 mg/kg resulted in a
36.1% inhibition of activity. R(-)DMS resulted in an inhibition of
29.8% when administered at a dose of 3.0 mg/kg. S(+)DMS
administration did not cause any observable inhibition at the
highest dose tested (10 mg/kg) indicating that it has significantly
less cross reactivity potential.
[0140] C. Conclusions
[0141] In vitro, R(-)DMS and S(+)DMS both exhibit activity as MAO-B
and MAO-A inhibitors. Each enantiomer was selective for MAO-B.
S(+)DMS was less potent than R(-)DMS and both enantiomers of DMS
were less potent than selegiline in inhibiting both MAO-A and
MAO-B.
[0142] In vivo, both enantiomers demonstrated activity in
inhibiting MAO-B, indicating that these enantiomers are able to
pass through the blood-brain barrier. The ability of these agents
to inhibit MAO-B suggests that these agents may be of value as
therapeutics for hypodopaminergic diseases such as ADHD and
dementia.
Example 10
In Vivo Neuroprotection by the Enantiomers of
Desmethylselegiline
[0143] The ability of the enantiomers of DMS to prevent
neurological deterioration was examined by administering the agents
to the wobbler mouse, an animal model of motor neuron diseases,
particularly amyotrophic lateral sclerosis (ALS). Wobbler mice
exhibit progressively worsening forelimb weakness, gait
disturbances, and flexion contractions of the forelimb muscles.
[0144] A. Test Method
[0145] A 0.1 mg/kg dose of R(-)DMS, S(+)DMS or placebo was
administered to wobbler mice by daily intra-peritoneal injection
for a period of 30 days in a randomized, doubleblind study. At the
end of this time mice were examined for grip strength, running
time, resting locomotive activity and graded for semi-quantitative
paw posture abnormalities, and semi-quantitative walking
abnormalities. The investigators who prepared and administered the
test drugs to the animals were different than those who analyzed
behavioral changes.
[0146] Assays and grading were performed essentially as described
in Mitsumoto et al., Ann. Neurol. 36:142-148 (1994). Grip strength
of the front paws of a mouse was determined by allowing the animal
to grasp a wire with both paws. The wire is connected to a gram
dynamometer and traction is applied to the tail of the mouse until
the animal is forced to release the wire. The reading on the
dynamometer at the point of release is taken as a measure of grip
strength.
[0147] Running time is defined as the shortest time necessary to
traverse a specified distance, e.g. 2.5 feet and the best time of
several trials is recorded.
[0148] Paw posture abnormalities are graded on a scale based upon
the degree of contraction and walking abnormalities are graded on a
scale ranging from normal walking to an inability to support the
body using the paws.
[0149] Locomotive activity is determined by transferring animals to
an examination area in which the floor is covered with a square
grid. Activity is measured by the number of squares traversed by a
mouse in a set time interval, e.g., 9 minutes.
[0150] B. Results
[0151] At the beginning of the study, none of the groups were
different in any variables, indicating that the three groups were
comparative at the baseline. Weight gain was identical in all three
groups, suggesting that no major side effects occurred in any
animals. Table 22 summarizes differences that were observed in the
mean grip strength of the test animals:
22TABLE 22 Mean Grip Strength in Wobbler Mice Treated with R(-) or
S(+)DMS Treatment N Grip Strength (gm) Control (placebo) 10 9(0-15)
R(-)DMS 9 20(0-63) S(+)DMS 9 14(7-20) N = number of animals
analyzed
[0152] Grip strength dropped markedly at the end of the first week
in all animals. At the end of the study, grip strength was the
least in control animals. The variability in grip strength in the
treated animal groups prevented a meaningful statistical analysis
of this data, however, at a dose of 0.1 mg/kg, the mean grip
strength measured in the DMS-treated animals was greater than for
the controls. These results suggest that the dose may have been too
low, and that a higher dose study should be performed.
[0153] Running time, resting locomotive activity, semiquantitative
paw posture abnormality grading, and semi-quantitative walking
abnormality grading were also tested. None of these tests, however,
showed any difference among the three groups tested.
Example 11
Immune System Restoration by R(-)DMS and S(+)DMS
[0154] There is an age-related decline in immunological function
that occurs in animals and humans which makes older individuals
more susceptible to infectious disease and cancer. U.S. Pat. Nos.
5,276,057 and 5,387,615 suggest that selegiline is useful in the
treatment of immune system dysfunction. The present experiments
were undertaken to determine whether R(-)DMS and S(+) are also
useful in the treatment of such dysfunction. It should be
recognized that an ability to bolster a patient's normal
immunological defenses would be beneficial in the treatment of a
wide variety of acute and chronic diseases including cancer, AIDS,
and both bacterial and viral infections.
[0155] A. Test Procedure
[0156] The present experiments utilized a rat model to examine the
ability of R(-)DMS and S(+)DMS to restore immunological function.
Rats were divided into the following experimental groups:
[0157] 1) young rats (3 months old, no treatment);
[0158] 2) old rats (18-20 months old, no treatment);
[0159] 3) old rats injected with saline;
[0160] 4) old rats treated with selegiline at a dosage of 0.25
mg/kg body weight;
[0161] 5) old rats treated with selegiline at a dosage of 1.0 mg/kg
body weight;
[0162] 6) old rats treated with R(-)DMS at a dosage of 0.025 mg/kg
body weight;
[0163] 7) old rats treated with R(-)DMS at a dosage of 0.25 mg/kg
body weight;
[0164] 8) old rats treated with R(-)DMS at a dosage of 1.0 mg/kg
body weight;
[0165] 9) old rats treated with S(+)DMS at a dosage of 1.0 mg/kg
body weight.
[0166] Rats were administered saline or test agent ip, daily for 60
days. They were then maintained for an additional "wash out" period
of 10 days during which time no treatment was given. At the end of
this time, animals were sacrificed and their spleens were removed.
The spleen cells were then assayed for a variety of factors which
are indicative of immune system function. Specifically, standard
tests were employed to determine the following:
[0167] 1) in vitro production of .gamma.-interferon by concanavalin
A-stimulated spleen cells;
[0168] 2) in vitro concanavalin A-induced production of
interleukin-2;
[0169] 3) percentage of IgM positive spleen cells (IgM is a marker
of B lymphocytes);
[0170] 4) percentage of CD5 positive spleen cells (CD5 is a marker
of T lymphocytes).
[0171] B. Results
[0172] The effect of administration of selegiline, R(-)DMS and
S(+)DMS on concanavalin A-induced interferon production by rat
spleen cells is shown in Tables 23 and 24. Table 23 shows that
there is a sharp decline in cellular interferon production that
occurs with age. Administration of selegiline, R(-)DMS and S(+)DMS
all led to a restoration of .gamma.-interferon levels with the most
dramatic increases occurring at dosages of 1.0 mg/kg body
weight.
23TABLE 23 Effect of Age on T Cell Function* IL-2 IFN-.gamma.
Groups U/ml std. error U/ml std. error young 59.4 18.27 12297 6447
old 19.6 7.52 338 135 *T cell activities were assessed after
stimulation of rat spleen cells with concanavalin A. TH, cytokines,
IL-2 and IFN-.gamma. were measured. Young vs. old, p = 0.0004
[0173]
24TABLE 24 Mean and % control IL-2 and IFN g IL-2 U/ml IFN-.gamma.
U/ml Groups mean % control mean % control control* 19.64 100 351
100 control 41.22 210 339 96 R(-)DMS 55.17 281 573 163 R(-)DMS
64.54 329 516 147 R(-)DMS 43.7 223 2728 777 S(+)DMS 57.12 291 918
261 Sel 0.25 109.6 558 795 226 Sel. 1.0 73.78 376 1934 550 *Old
rats (22 months old) with no treatment.
[0174] Table 24 shows the extent to which R(-)DMS, S(+)DMS and
selegiline are capable of restoring .gamma.-interferon production
in the spleen cells of old rats. Interferon-.gamma. is a cytokine
associated with T cells that inhibit viral replication and regulate
a variety of immunological functions. It influences the class of
antibodies produced by B-cells, upregulates class I and class II
MHC complex antigens and increases the efficiency of
macrophage-mediated killing of intracellular parasites.
[0175] Histological immunofluorescence studies show a dramatic loss
of innervation in rat spleens with age. When rats are treated with
R(-)DMS, there is a significant increase in innervation in the
spleens of animals and this increase occurs in a dose-response
manner. S(+)DMS did not show any effect on histological
examination, despite a modest increase in interferon-.gamma.
production. IL-2 production was not enhanced by treatment with
R(-)DMS or S(+)DMS, suggesting that the effects of these agents may
be limited to IFN-.gamma. production.
[0176] C. Conclusions
[0177] The results obtained with respect to histological
examination, the production of interferon, and the percentage of
IgM positive spleen cells support the conclusion that the DMS
enantiomers are capable of at least partially restoring the
age-dependent loss of immune system function. The results observed
with respect to IFN-.gamma. are particularly important. In both
humans and animals, IFN-.gamma. production is associated with the
ability to successfully recover from infection with viruses and
other pathogens. In addition, it appears that R(-)DMS and S(+)DMS
will have a therapeutically beneficial effect for diseases and
conditions mediated by weakened host immunity. This would include
AIDS, response to vaccines, infectious diseases and adverse
immunological effects caused by cancer chemotherapy.
Example 12
Examples of Dosage Forms
[0178] A. Desmethylselegiline Patch.
25 Dry Weight Basis Component (mg/cm.sup.2) Durotak .RTM. 87-2194
90 parts by weight adhesive acrylic polymer Desmethylselegiline 10
parts by weight
[0179] The two ingredients are thoroughly mixed, cast on a film
backing sheet (e.g., Scotchpak.RTM. 9723 polyester) and dried. The
backing sheet is cut into patches a fluoropolymer release liner
(e.g., Scotchpak.RTM. 1022) is applied, and the patch is
hermetically sealed in a foil pouch. One patch is applied daily to
supply 1-10 mg of desmethylselegiline per 24 hours in the treatment
of conditions in a human produced by neuronal degeneration or
neuronal trauma.
[0180] B. Ophthalmic Solution
[0181] Desmethylselegiline (0.1 g) as the hydrochloride, 1.9 g of
boric acid, and 0.004 g of phenyl mercuric nitrate are dissolved in
sterile water qs 100 ml. The mixture is sterilized and sealed. It
can be used ophthalnologically in the treatment of conditions
produced by neuronal degeneration or neuronal trauma, as for
example glaucomatous optic neuropathy and macular degeneration.
[0182] C. Intravenous Solution
[0183] A 1% solution is prepared by dissolving 1 g of
desmethylselegiline as the HCl in sufficient 0.9% isotonic saline
solution to provide a final volume of 100 ml. The solution is
buffered to pH 4 with citric acid, sealed, and sterilized to
provide a 1% solution suitable for intravenous administration in
the treatment of conditions produced by neuronal degeneration or
neuronal trauma.
[0184] D. Oral Dosage Form
[0185] Tablets and capsules containing desmethylselegiline are
prepared from the following ingredients (mg/unit dose):
26 desmethylselegiline 1-5 microcrystalline cellulose 86 lactose
41.6 citric acid 0.5-2 sodium citrate 0.1-2 magnesium stearate
0.4
[0186] with an approximately 1:1 ratio of citric acid and sodium
citrate.
* * * * *