Note: Descriptions are shown in the official language in which they were submitted.
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WO 96/22068 PCT/US96/01561
Methods and Pharmaceutical Compositions
Employing Desmethylselegiline
Field of the Invention
The present invention pertains to methods and pharmaceutical compositions for
using the selegiline metabolite desmethylselegiline and its enantiomer, ent-
desmethyl-
selegiline. In particular, the present invention provides compositions and
methods for using
these agents for selegiline-responsive diseases and conditions.
Bacl~ground of the Invention
Two distinct monoamine oxidise enzymes are known in the art: monoamine
oxidise A (MAO-A) and monoamine oxidise 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.
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:
CH3
CHI-C N CH2-C-CH
N CH
3
The selectivity of selegiline in the inhibition of MAO-B is important to its
safety
profile following oral administration. Tranylcypromine (an MAO inhibitor
introduced
some thirty years ago but subsequently withdrawn due to its severe
hypertensive side
effects), in contrast to selegiline, is a non-selective inhibitor of MAO. The
acute toxicity of
tranylcypromine arises from inhibition of MAO-A, which interferes 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
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WO 96/22068 PCT/US96/01561
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. °
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:
CH3
CH - C NH - CH - C- CH
2 2
N
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.
For example, the present characterization established that selegiline has an
IC50 -
value against MAO-B in human platelets of 5 x 10-9 M whereas
desmethylselegiline's ICso
value is 4 x 10-' 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:
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w0 96122068 PCT/US96/01561
Table 1: Inhibition of MAO by Selegiline and Desmethylselegiline
Percent
Inhibition
Conc.
a selegiline desmethylselegiline
MAO-B MAO-A MAO-B MAO-A
0.003,uM 16.70 - 3.40 -
O.OlO~cM 40.20 - 7.50 -
0.030,uM 64.70 - 4.60 -
0.100~cM 91.80 - 6.70 -
0.300,uM 94.55 9.75 26.15 0.0
1.OOO,uM 95.65 32.55 54.73 0.70
3.OOO~cM 98.10 65.50 86.27 4.10
10.000~cM - 97.75 95.15 11.75
30.OOO,uM - - 97.05 -
100.000,uM - - - 56.10
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 only
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.
Analogous results are obtained in rat brain tissue. Selegiline exhibits an
ICso for
MAO-B of 0.11 x 10-7 M whereas desmethylselegiline's ICSO value is 7.3 x 10-7
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 x 10-5 for selegiline, 7.0 x 10-5 for desmethylselegiline. Thus,
desmethyl-
selegiline is approximately 39 times less potent than selegiline in inhibiting
MAO-A.
Based on its pharmacological profile as set forth above, 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.
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
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WO 96122068 PCTILTS96J01561
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.
The various diseases and conditions for which selegiline is known to be useful
include: depression (U.S. patent 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. patent 5,242,950); age-dependent
degeneracies,
including renal function and cognitive function as evidenced by spatial
learning ability
(U.S. patent 5,151,449); pituitary-dependent Cushing's disease (U.S. patent
5,192,808);
immune system dysfunction (U.S. patent 5,276,057); and schizophrenia (U.S.
patent
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.
Although selegiline is known to be 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.
The biochemical effects of selegiline on neuronal cells has 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 Mitochondria! Depolarization and Reduces Cell Death in
Trophically-
.. Deprived Cells," 11th Int'l Symp. on Parkinson's Disease, Rome, Italy,
March 26-30, -
1994.
Selegiline is known to be useful when administered to a subject through a wide
n
variety of routes of administration and dosage fozms. For example U.S. patent
4,812,481
(Degussa AG) discloses the use of concomitant selegiline-amantadine in oral,
peroral, -
>> -
.x
CA 02209892 2000-11-14
enteral, pulmonary, rectal, nasal, vaginal, lingual, intravenous,
intraarterial, intracardial,
intramuscular, intraperitoneal, intracutaneous, and subcutaneous formulations.
U.S. patent
5,192,550 (Alza Corporation) describes a dosage form comprising an outer wall
impermeable to selegiline but permeable to external fluids. This dosage form
may have
applicability for the oral, sublingual or buccal administration of selegiline.
Similarly, U.S.
patent 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.
In addition to desmethylselegiline, selegiline produces one other principal
direct
metabolite, methamphetamine. Both desmethylselegiline and methamphetamine are
further
metabolized to amphetamine. The last two metabolites, amphetamine and
methamphetamine, are known to have the potential to exert neurotoxic effects
on dopamine
neurons, and are undesirable by-products. Unlike selegiline,
desmethylselegiline does not
produce methamphetamine as a metabolite, only amphetamine.
Accordingly, although a highly potent and selective MAO-B inhibitor,
selegiline's
practical use is circumscribed by its dose-dependent specificity for MAO-B,
and the
pharmacology of selegiline metabolites generated after administration.
Summary of the Invention
In one aspect, this invention relates to S(+) -desmethylselegiline or a
pharmaceutically-acceptable acid addition salt thereof, for use in obtaining
selegiline-like
therapeutic effect, or neuroprotection or neuronal rescue or restoring or
improving
immune system function or the treatment of a selegiline-responsive disease or
condition, or
the treatment of Parkinson's disease, Alzheimer's disease or ADHD.
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In another aspect, this invention relates to use of S(+) -desmethylselegiline
or a
pharmaceutically-acceptable acid addition salt thereof for the manufacture of
a medicament
for use in neuroprotection or neuronal rescue, or restoring or improving
immune system
function or the treatment of a selegiline-responsive disease or condition, or
the treatment of
Parkinson's disease, Alzheimer's disease or ADHD.
Yet in another aspect, this invention relates to a non-oral medicament
comprising, as
an active ingredient, a compound which is S(+) or R(-) desmethylselegine or a
pharmaceutically-acceptable acid addition salt thereof or a mixture thereof.
This invention further relates to the R(-) enantiomer of desmethylselegine,
for use in
obtaining selegiline-like therapeutic effect, or neuroprotection or neuronal
rescue or
restoring or improving immune system function or the treatment of a selegiline-
responsive
disease or condition, or the treatment of Parkinson's disease, Alzheimer's
disease or
ADHD.
Yet in another aspect, this invention relates to use of R(-) enantiomer of
desmethyselegiline, for the manufacture of a medicament for use in
neuroprotection or
neuronal rescue, or restoring or improving immune system function, or the
treatment of a
selegiline-responsive disease or condition, or the treatment of Parkinson's
disease,
Alzheimer's disease or ADHD.
The present invention relates to the surprising discovery that both
desmethylselegiline ("DMS" or "R(-)DMS") and its enantiomer (ent-
desmethylselegiline,
abbreviated as "Ent-DMS" or "S(+)DMS") are useful 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. In particular,
the present
invention relates to the surprising discovery 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.
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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 known to
be useful, including Alzheimer's disease, cognitive
dysfunction, neuronal rescue, and the like. Similarly, the
term "selegiline-like therapeutic effect" refers to one or more
of the salutary effects exerted by selegiline in a sub~ect
being treated for a selegiline-responsive disease or condition.
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 or schizophrenia,
amyotrophic lateral sclerosis, Tourette's syndrome, tardive
dyskinesia, and toxic neurodegeneration.
The present invention also encompasses the restoration or
improvement of immune system function by R(-)DMS, S(+)DMS or
mixtures thereof. Such improvement or restoration has been
reported to occur when selegiline is administered to animals.
The conditions or diseases treatable may include age-dependent
immune system dysfunction, AIDS, cancer and infectious
diseases.
Depending upon the particular route employed, either
desmethyselegiline or ent-desmethylselegiline may be
administered in the form of a free base or as a physiologically
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CA 02209892 1997-07-03
acceptable non-toxic acid addition salt. Such salts include those derived from
organic and
inorganic acids such as, without limitation, hydrochloric acid, hydrobromic
acid,
phosphoric acid, sulfuric acid, methanesulphonic acid, acetic acid, tartaric
acid, lactic acid,
succini~ acid, citric acid, malic acid, malefic 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
desmethylselegiline, or
ent-desmethylselegiline, in the form of the free base is especially useful for
transdermal
administration. Accordingly, reference herein to the administration of DMS or
ent-DMS or
to mixtures thereof encompasses both the free base and acid addition salt
forms.
The optimal daily dose of desmethylselegiline and/or ent-desmethylselegiline
useful
for the purposes of the present invention is determined IZy 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 0.0015
mg/kg, calculated on the basis of the free secondary amine, with progressively
higher doses
being employed depending upon the response to the therapy. Typically the daily
dose will
be about 0.01 mg/kg and may extend to about 0.5 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 ox animal.
The daily dose can be administered in a single or multiple dosage regimen. The
dosage form and regimen 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 can be desirable in conditions such as ischemia or neural
damage to
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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.
In the case of oral administration, the oral use of
desmethylselegiline, and/or ent-desmethylselegiline, is more
effective than oral selegiline for use in a variety of
conditions and diseases. Accordingly, desmethylselegiline and
its enantiomer may be employed orally in subjects where the use
of selegiline itself would be contraindicated due to side effects.
Pharmaceutical compositions containing desmethylselegiline and/or ent-
desmethylselegiline can be prepared according to conventional techniques. For
example,
preparations for parenteral routes of administration for desmethylselegiline,
e.g.,
intramuscular, intravenous and intraarterial routes, can employ sterile
isotonic saline
solutions. Sterile isotonic solutions can also be employed for intraocular
administration.
Transdermal dosage unit forms of desmethylselegiline and/or ent-
desmethylselegiline can be prepared utilizing a variety of previously
described techniques
(see e.g., U.S. Patent Nos. 4,861,800; 4,868,218; 5,128,145; 5,190,763; and
5,242,950;
and EP-A 404807, EP-A 509761, and EP-A 593807). For example, a monolithic
patch
structure can be utilized in which desmethylselegiline is directly
incorporated into the
adhesive and this mixture is cast onto a backing sheet.
Alternatively desmethylselegiline, and/or ent- 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.
Desmethylselegiline and/or ent-desmethylselegiline can also be administered by
a
device employing a lyotropic liquid crystalline composition in which, for
example, 5 to
15 % of desmethylselegiline is combined with a mixture of liquid and solid
polyethylene
glycols, a polymer, and a nonionic surfactant, optionally with the addition of
propylene
glycol and an emulsifying agent. For further details on the preparation of
such transdermal
preparations, reference can be made to EP-A 5509761.
Since the term "ent-desmethylselegiline" refers to the S(+)-isomeric form of
desmethylselegiline, reference above to mixtures of selegiline and ent-
desmethylselegiline
includes both racemic and non-racemic mixtures of optical isomers.
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CA 02209892 1997-07-03
Preferably, sub.~ects t reatable by the present invent ion
include both human and non-human subjects for which
selegiline-like therapeutic effects are known to be useful.
Accordingly, the present invention may provide especially
useful therapies for mammals, especially domesticated
mammals. Thus, the present invention may be used in
treating selegiline-responsive diseases or conditions in
canine and feline species.
Successful use of the present invention requires
employment of an. effective amount of desmethylselegiline,
ent-desmethylselegiline or a mixture thereof.
Although both desmethylselegiline and ent-desmethylselegiline are dramatically
less potent
than selegiline as inhibitors of MAO, 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 are 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-desmethylselegiline 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,
~e fed due to the 40-70 fold reduced potency for MAO-A inhibition.
As described above and notwithstanding its demonstrably inferior inhibitory
proper-
ties with respect to MAO-B inhibition, desmethylselegiline and its enantiomer
are signifi-
cantly more effective than selegiline in treating selegiline-responsive
conditions, e.g.,
conditions resulting from neuronal degeneration or neuronal trauma. In this
regard
desmethylselegiline and/or its enantiomer, like selegiline itself, may
be particularly useful when administered by a route which does not rely
upon upper GI tract or other gastrointestinal absorption. Preferred
routes include the parenteral, topical, transdermal, intraocular,
buccal, sublingual, intranasal, inhalation, vaginal, and rectal routes.
As noted above, the present invention encompasses the additional discovery
that
desmethylselegiline can be employed in both optically active forms and in
racemic form,
i.e., as mixtures of desmethylselegiline and ent-desmethylselegiline.
Desmethylselegiline,
its enantiomer and mixtures thereof are conveniently prepared by methods known
in the
art, as described below in Example 1.
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Brief Description of the Figures
Figure 1: 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"). Solid bars represent results
obtained for -
cultures maintained in the presence of 50 ,uM 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 Figure 1 and the figures discussed below
indicates a
result that differs from controls in an amount that is statistically
significant, i.e. P < 0.05
Figure 2: [3H]-Dopamine Uptake in Mesencephalic Cells. Cells, cultured as
described above for Figure 1, were tested for their uptake of labeled dopamine
and results
are shown in Figure 2. Solid bars represent uptake in cells maintained in the
presence of
50 ,uM selegiline and open bars represent uptake in control cultures
Figure 3: Effect of Selegiline on Glutamate Receptor Dependent Neuronal Cell -
Death. Rat embryonic mesencephalic cells were cultured as described above.
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 ,uM 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 SO ,uM selegiline
Figure 4: Effect of Selegiline on Dopamine Uptake in Neuronal Cultures. Rat
mesencephalic cells were cultured and medium was changed on a daily basis as
discussed
for Figure 3. 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 Figure 3.
Figure 5: 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 ~M R(-)DMS.
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Figure 6: Effect of R(-)Desmethylselegiline on Dopamine Uptake in Neuronal
Cultures. Cell cultures were prepared as described above for Figure 5 and then
tested for
uptake of tritiated dopamine. Results for controls and for cells maintained in
the presence
of 0.5 ,uM, 5 ,uM and 50 ,uM desmethylselegiline are shown from left to right
in the figure.
S Figure 7: 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 Figures 3-6 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 ,uM.
In addition, cells were incubated in the presence of the glutamate receptor
blocker MK-801
at a concentration of 10 ,uM. Cultures were tested for uptake of tritiated
dopamine.
Figure 8: Inhibition of Neuronal Dopamine Neuronal Re-Uptake by Deprenyl and
the Enantiomers of Desmethylselegiline. An in vitro nerve terminal preparation
(synaptosome preparation) was prepared using fresh rat neostriatal tissue.
This was
examined for its ability to take up tritiated dopamine in buffer alone or in
buffer
supplemented with various concentrations of selegiline, R(-
)desmethylselegiline or
- S(+)desmethylselegiline. Uptake in the presence of each of the MAO
inhibitors was
expressed as a percent inhibition relative to uptake in the presence of buffer
alone and
results are shown in Figure 8. As indicated in the figure, the plots were used
to determine
the IDSO for each test agent. The IDso for S(+)DMS was 20 ~cM; for selegiline,
80 ~.M;
and for R(-)DMS about 100 ,uM.
Figure 9: In Vivo MAO-B Inhibition in Guinea Pig Hippocampus. Various doses of
selegiline, R(-)desmethylselegiline, and S(+)desmethylselegiline were injected
daily into
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
Figure 9. The plots were used to determine the IDso dosage for each agent. The
ID50 for
selegiline was about 0.03 mg/kg; and for both enantiomers of DMS, about 0.3
mg/kg.
Figure 10: In Vitro Interferon ("IFN") Production in Spleen Cells from Rats
Injected with Selegiline, R(-)DMS or S(+)DMS. F34.4 rats were injected ip,
daily for 60
days with saline, selegiline, R(-)DMS or S(+)DMS. All injected rats were old,
i.e.,
.:
:$
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between 18 and 20 months of age. After allowing a 10 day "wash out" period in
which no
injections were made, rats were killed and their spleens removed. The in vitro
production -
of interferon-y by the stimulated spleen cells (lymphocytes) was determined
and compared
with production by spleen cells (lymphocytes) from non-injected old rats and
from young > -
rats (3 months old). From left to right, the bars reflect results for spleen
cells from young
rats; from old rats, from old rats injected with saline; from old rats
injected with 0.25 ~ -
mg/kg of selegiline; from old rats injected with 1.0 mg/kg of selegiline; from
rats injected
with 0.025 mg/kg of R(-)DMS; from rats injected with 0.25 mg/kg of R(-)DMS;
from rats
injected with 1.0 mg/kg of R(-)DMS; and from rats injected with 1.0 mg/kg of
S(+)DMS.
The horizontal line indicates the point at which IFN levels differ
significantly (P < .OS)
from the levels seen using young rat spleen cells. Results are expressed as
units oer ml.
Figure 11: In Vitro IFN Production in Spleen Cells From Old Rats: The same
results shown in Figure 10 are repeated 11 but without the inclusion of the
results for
young rat spleen cells. "#" indicates a result significantly different (P <
0.05) from that
obtained for spleens from old rats injects with saline. "*" indicates a result
significantly -
diferent from all groups except old rats injected with 1.0 mg/kg of deprenyl.
Figure 12: In Vitro Interleukin-2 Production by Spleen Cells from Old Rats.
Rats
were injected as described above (see Figure 10) and the production of
interleukin-2 by -
stimulated spleen cells was determined. The order of bars from left to right
is the same as
for Figure 10.
Figure 13: Percentage of Rat Spleen Cells that are IgM Positive. Rats were
injected -
with saline, selegiline, R(-)DMS or S(+)DMS as described above (see Figure
10). The
spleens from the rats were assayed to determine the percentage of cells that
were IgM
positive and results are shown in the figure. The horizontal line in the
figure corresponds
to the decrease in IgM percentage that is statistically significant (P < 0.05)
relative to the
percentage seen in spleens obtained from young rats. The bars are in the same
order from
left to right as in Figures 10 and 12.
Figure 14: Percentage of Rat Spleen Cells that are CDS Positive. The
experiment
of Figure 13 was repeated but instead of measuring the percentage of cells
that are IgM
positive, the percentage that are CDS positive was determined.
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Detailed Description of the Invention
The surprising utility of desmethylselegiline and ent-desmethylselegiline in
treating
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 as low as O.OI mg/kg, at which little or no MAO-B inhibition is
generally observed,
a reversal in neuronal damage and/or death can be observed. Because
desmethylselegiline
and ent-desmethylselegiline prevent loss and facilitate recovery of nerve cell
function, they
are of value in a wide variety of neurodegenerative and neuromuscular
diseases. In this
regard, desmethylselegiline and ent-desmethylselegiline are substantially more
potent than
selegiline, as described more empirically in the examples below. The Examples
are for
illustrative purposes only and are not intended to limit the scope of the
invention.
Examples
Example l: Preparation of Desmethylselegiline and Ent-desmethylselegiline
A. Desmethylselegiline
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. Patent No. 4,925,878.
Desmethyl-
selegiline can be prepared by treating a solution of R(+)-2-aminophenylpropane
(levoamphetamine):
CH3
CH -C NH
2 2
H
in an inert organic solvent such as toluene with an equimolar amount of a
reactive
propargyl halide such as propargyl bromide, Br-CHZ-C---CH, at slightly
elevated tem-
peratures (70°-90°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
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nonaqueous layer which forms is separated, for example by extraction with
benzene, dried,
and distilled under reduced pressure.
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. Patent No. 4,564,706.
B. Ent-Desmethylselegiline
Ent-desmethylselegiline (designated below as "S(+)DMS") is conveniently
prepared
from the enantiomeric S(-)-2-aminophenylpropane (dextroamphetamine), i.e.,
CH3
CH2-C NH2
H
following the procedures set forth above for desmethylselegiline.
C. Mixtures of Enantiomers
Mixtures of enantiomeric forms of desmethylselegiline, including racemic -
desmethylselegiline, are conveniently prepared from enantiomeric mixtures,
including
racemic mixtures of the above aminophenylpropane starting material.
D. Conversion Into Acid Addition Salts
N-(prop-2-ynyl)-2-aminophenylpropane in either optically active or racemic
form
can be converted to a physiologically acceptable non-toxic acid addition salt
by
conventional techniques such as treatment with a mineral acid. For example,
hydrogen
chloride in isopropanol is 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: Neuronal Survival as Measured Using Tyrosine Hydroxylase
The effect of desmethylselegiline on neuron survival can be correlated to
tyrosine
hydroxylase, the rate limiting enzyme in dopamine biosynthesis. Assays are
performed by
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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 (3-FGF.
A. Test Methods
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++ and Mg++ free balanced salt
solution at
4°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 1.5X106 cells/dish.
Cultures are
maintained at 37 °C in an atmosphere of 10 % C02/90 % air and 100 %
relative humidity,
and fed twice weekly with chemically defined medium consisting of MEM/Fl2
(l:l,
Gibco), glucose (33 mM), HEPES (15 mM), NaHC03 (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 included test substance, e.g. selegiline, at one or
more
concentrations.
Cultures are fixed in 4 % paraformaldehyde in 0.1 M 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°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.
The number of dopaminergic neurons in cultures is determined by counting the
cells
positively immunostained with TII antibodies. 100 fields (0.5 mm X 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 200X magnification.
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B. Results
Using the procedures described above, the following results were obtained:
Table 2: Effect of Selegiline and DMS on the Survival of TH Positive Cells '
Control Selegiline Desmethylselegiline . -
Mean Mean % Cont Mean % Cont
Conc.
O.S~M 108.55 201.70 f 25.01 185.81 246.00 t 22.76 226.62
5 ,uM - 237.00 t 12.59 218.33 357.95 t 25.76 329.76
50 ~cM - 292.28 t 17.41 269.25 391.60 t 34.93 360.76 -
Example 3: Neuronal Survival as Measured Using Dopamine Uptake
In addition to determining the number of TH positive cells in culture (see
Example
2) the protective effect of desmethylselegiline on neuronal cells also can be
determined by
directly measuring dopamine uptake. The amount of uptake by the cultured brain
cells
corresponds to axonal growth. -
A. Test Methods
Cell cultures, established in the manner discussed above, are incubated with
[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 CaCl2
and 0.5 mM MgCl2 at 37°C. After two rinses and a 5 minute incubation
with fresh -
buffer, [3H]dopamine accumulated within the cells is released by incubating
the cultures
with 95% ethanol for 30 minutes at 37°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.
B. Results
Using the above procedure, the results shown in Table 3 were obtained.
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Table 3: Effect of Selegiline and DMS on 3H-Dopamine Uptake
Cont. I Selegiline I Desmethylselegiline
Mean Mean ~ % Cont Mean ~ % Cont
Conc. ~
O.S,uM 11982 14452 120.6 24020 ~ 800 200.4
t 212
,uM - 16468 137.5 34936 t 2119 291.5
~ 576
5 50 ,uM - 33018 275.5 56826 t 2656 474.3
t 1317
C. Conclusions from Examples 2 and 3
The results described in Examples 2 and 3 indicate that desmethylselegiline is
superior to selegiline as a neuroprotective agent. This is true
notwithstanding the fact that
desmethylselegiline in much less potent than selegiline as an inhibitor of MAO-
B.
Example 4: Neuroprotective Action of Desmethylselegiline Enantiomers in
Cultured
Dopamine-Containing Mesencephalic Neurons In Vitro
The survival of mesencephalic, dopamine-containing neuronal cultures of rat
brain
tissue was used in these experiments to examine neuroprotective properties of
selegiline
and R(-) desmethylselegiline. The number of TH positive neurons is directly
proportional
to the survival of dopaminergic neurons and 3H-dopamine uptake is a measure of
axonal
growth in these neurons
A. Effect of Selegiline on the Survival of Dopaminergic Neurons.
Mesencephalic cultures prepared from embryonic day 14 rats were treated with
0.5,
5 or 50 ~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
[3H]dopamine uptake and results are shown in figures 1 and 2.
No effect was observed on neuron survival of when selegiline was tested at
- 25 concentrations of 0.5 and 5 ,uM. At 50 ,uM, selegiline reduced the loss
of TH-positive
neurons at 8 and 15 days after plating (Figure 1) and increased dopamine
uptake at 15 days
(Figure 2). In separate experiments it was determined that pre-treatment with
0.5, 5, 50 or
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100 ,uM selegiline did not inhibit the uptake of dopamine under the assay
conditions used
in the experiments.
The results indicate that the uptake values obtained reflect dopamine neuron
survival and process outgrowth and that selegiline has a neuroprotective
effect.
B. Effect of Selegiline on Glutamate Receptor Dependent Cell Death.
The neuroprotective effect of selegiline was also examined using an
experimental
paradigm that 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 Figures 3 and 4 further
support the
conclusion that selegiline promotes the survival of dopaminergic neurons.
C. Effect of Desmethylselegiline on the Survival of Dopamine Neurons.
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 ~cM),
desmethylselegiline
caused a significant reduction in the loss of TH positive neurons (Figure 5)
and a
significant increase in dopamine uptake (Figure 6) relative to control
cultures in which
medium was sued without supplementation with either selegiline or
desmethylselegiline.
D. Comparison With Other MAO Inhibitors.
Using the glutamate receptor dependent paradigm of neurotoxicity, the effects
of
selegiline and desmethylselegiline were compared with two other MOA
inhibitors,
pargyline and clorgyline (Figure 7). In agreement with previous results,
measurement of
dopamine uptake indicated neuron protection by SO ,uM deprenyl and 5 and SO ~M
desmethylselegiline. Pargyline did not appear to offer any protection at the
concentrations
used, while clorgyline protected at 50 ~M. As expected protection was also
obtained by the
NMDA receptor blocker MK-801 (10 ,uM).
E. Effect of DMS Enantiomers on 3H-Dopamine Uptake -
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.
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- Table 4: Effect of DMS Enantiomers on Dopamine Uptake
3H-Dopamine uptake
~~ as a ercen age + SEM
' Control 100 t 14.14
R(-) DMS ( l OnM) 140. 82 t 26. 20
S(+) DMS (lOnM) 234 t 38.36%
These results demonstrate that, compared to untreated control cells, there was
40
and 134 % more axonal growth and terminal axonal survival after treatment with
R(-) DMS
and S(+) DMS, respectively, Thus, S(+) DMS may be an even more potent and/or
efficacious neuroprotectant than R(-) DMS.
Example 5: Desmethylselegiline and Ent-Desmethylselegiline as Inhibitors of
Dopamine Re-Uptake
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 Qf
dopamine at the synapse and therefore enhance dopamine synaptic transmission.
A. Method of Testing
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 dopamime activity. Presently this would include
Parkinson's
Disease, Alzheimer's Disease and Attention Deficit Hyperactivity Disorder
(ADHD).
The assay system used was essentially that described by Fang et al. (Neuro-
pharmacology 33:763-768 (1994)). An in vitro nerve-terminal preparation
(synaptosome
preparation) was obtained form fresh rat neostriatal brain tissue. Transport
by dopamine
nerve-terminals was estimated by measuring the uptake of tritiated dopamine.
B. Results
As seen in the data presented in Table 5, selegiline, R(-) DMS and S(+) DMS
all
inhibited dopamine re-uptake by dopamine-containing nerve terminals.
Selegiline and R(-)
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DMS were approximately equipotent.
In contrast, S(+) DMS was
4-5 times more potent
than either selegiline or -
R(-) DMS.
Table 5: 3H-Dopamine Uptake
By Rat Neostriatal Brain
Tissue ' -
Agent Concentration %
Reduction
X~SEM
Dopamine l,uM 52.0 ~ 4.9
lO,uM 80.9 ~ 0.4
Selegiline 100nM 7.0 ~ 3.6
l,uM 13.9 ~ 4.7
lO,uM 16.33.8
100,uM 59.8 ~ 1.0
R(-) DMS 100nM 11.5 ~ 1.0
l,uM 10.7 ~ 2.8
lO,uM 20.1 ~ 3.1
100~M 51.3 2.6
S(+) DMS 100nM 15.3 ~ 7.7
1 ~cM 24.1 ~ 11. 7
lO~cM 47.0 ~ 3.1
100~cM 76.9
~
1.8
Relative potency can be
expressed in terms of the
concentration required
to
inhibit dopamine re-uptake
by SO% (IDSO). The IDSO
values were determined
graphically
(see Figure 8) and are shown
below in Table 6.
Table 6: Concentrations
Needed to Inhibit Dopamine
Uptake by 50
Ag~ul II2so
Selegiline ~
80
,um
R(-) DMS ~
100
,um
S(+) DMS ~
20
~cm
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C. Conclusions
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. This
effect is indicative of agents beneficial in the treatment of Parkinson's
Disease, Alzheimer's
Disease and Attention Deficit Hyperactivity Disorder (ADHD). Of the agents
tested, S(+)
DMS appears to be the most preferred with regard to the treatment of ADHD.
Example 6: 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
Human platelet MAO is comprised exclusively of the type -B isoform of the
enzyme. In the present study, the inhibition of this enzyme by the two
enantiomers of
DMS was determined and compared with inhibition due to selegiline. In
addition, the
present study 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 the
best 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 identical
kinetic
properties to those found in human brain tissue. Finally, agents were injected
into guinea
pigs to determine the extent to which they might act as inhibitors of brain
MAO in vivo.
B. Method of Testing:
The test system utilized the in vitro conversion of specific substrates of MAO-
A
(14C-serotonin) and MAO-B (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 (ICso value).
In some experiments, R(-) DMS, S(+) DMS or selegiline was administered in vivo
subcutaneously (s.c.), once a day for 5 days prior to sacrifice, preparation
of enzyme
g:
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hippocampal homogenates,
and the in vitro assay
of MAO-A and MAO-B activity.
These
experiments were performed were capable of
to demonstrate that the
DMS enantiomers
entering brain tissue and
inhibiting MAO activity.
,
C. Results '
MAO-B Inhibitory Activity
In Vitro
Results for MAO-B inhibition
are shown in Tables 7
and 8. ICSO values for
MAO-B
inhibition and potency
as compared to selegiline
is shown in Table 9.
Table 7: MAO-B Inhibition
in Human Platelets
Agent Concentration % Inhibition
x t SEM
Selegiline 0.3nM 8.3 ~ 3.4
SnM 50.3 ,~ 8.7
lOnM 69.0 ~ 5.5 -
30nM 91.0 ~ 1.4
100nM 96.0 ~ 1.6 -
300nM 96.0 1.6
l~M 96.6 ~ 1.6
R(-) DMS 100nM 14.3 ,f 3.6
300nM 42.1 ~ 4.0
l~cM 76.9 ~ 1.47
3~cM 94.4 ~ 1.4 -
lO~cM 95.8 ~ 1.4
3,uM 95.7 ~ 2.3
S(+) DMS 300nM 6.4 ~ 2.8
l,uM 11.1 ~ 1.0
3,uM 26.6 ~ 1.9
lO~cM 42.3 ~ 2.3
30,uM 68.2 .~ 2.34 _
100~cM 83.7 ~ 0.77
1mM 94.2 1.36
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Table 8: MAO-B Inhibition in Guinea Pig Hippocampus
Agent Concentration % Inhibition
x tSEM
Selegiline 0. 3 nM 28 . 3,18 .
7
5nM 81.2+ 2.6
lOnM 95.6 + 1.3
30nM 98.5 + 0.5
100nM 98.8 + 0.5
300nM 98.8 ~ 0.5
l,uM 99.1 ~ 0.45
R(-) DMS 100nM 59.4 ~ 9.6
300nM 86.2 ~ 4.7
l,uM 98.2 X0.7
3~M 98.4 X0.95
lO,uM 99.1 ~ 0.45
30~cM 99.3 X0.40
S(+) DMS 300nM 18.7 ~. 2.1
l~cM 44.4 ~ 6.4
3~cM 77.1 ~ 6.0
lO,uM 94.2 ~ 1.9
30,uM 98.3 0.6
100,uM 99.3 ~ 0.2
1 mM 99. 9 ~ 0.1
Table 9: ICSO Values for the Inhibition of MAO-B
Guinea Pig
Treatment Human Platelets Hi~pocampal Cortex
Selegiline 5 nM (1) 1 nM (1)
R(-) DMS 400 nM (80) 60 nM (60)
S(+) DMS 1400 nM (2800) 1200 nM (1200)
( ) = potency compared to selegiline
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As observed, R(-) DMS was 20-35 times more potent than S(+) DMS as an MAO-
B inhibitor and both enantiomers were less potent than selegiline.
MAO A Inhibitory Activity
Results obtained from experiments examining the inhibition of MAO-A in guinea -
pig hippocampus are summarized in Table 10 and ICso values for the two
enantiomers of
Y -
DMS and for selegiline are shown in Table 11. -
Table 10: MAO-A Inhibition in
Guinea Pig Hippocampus
Agent Concentration % Reduction
XtSEM
Selegiline 300nM 11.95 ~ 2.4
l,uM 22.1 ~ 1.2
3,uM 53.5 ~ 2.7
lOuM 91.2 ~ 1.16
100,uM 98.1 ~ 1.4
1 mM 99. 8 ~ 0.2 -
R(-) DMS 300nM 4.8 + 2.1
l,uM 4.2 ~ 1.5 -
3,uM 10.5 ~ 2.0
lO,uM 19.0 .~ 1.3
100,uM 64.2 ,~ 1.5
1mM 96.5 ~ 1.2 -
S(+) DMS l,uM 3.3 ~ 1.5 -
3,uM 4.3 ~ 1.0
lO,uM 10.5 ~ 1.47
100 ~cM 48.4 ~ 1.8
1 rnM 92 . 7 ,~ 2. 5 _
lOmM 99.6,10.35
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Table 11: ICso Values for the Inhibition of MAO-A
I~sa for MAO-A in Guinea Pig
Treatment ~~pocampal Cortex
Selegiline 2.5 ,uM ( 1 )
R(-) DMS 50.0 lcM (20)
S(+) DMS 100.0 ,uM (40)
( ) = potency compared to selegiline
R(-) DMS was twice as potent as S(+) DMS as an MAO-A inhibitor and both were
20-4.0 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.
Results of In Vivo Experiments
Each enantiomer of DMS was administered in vivo by subcutaneous injection once
a
day for five consecutive days, and inhibition of brain MAO-B activity was then
determined. Under these conditions, R(-) DMS and S(+) DMS were equipotent as
MAO-
B inhibitors but ten times less potent than selegiline. Results are shown in
Figure 9 and
ICso values are summarized in Table 12.
Table 12: ICSO Values for Brain MAO-B When Agents are Injected Prior to Assay
. ~5~ for MAO-B in Guinea Pig
Treatment H~ppocamnal Cortex
Selegiline 0.03 mglkg
R(-) DMS 0.30 mg/kg
S(+) DMS 0.30 mg/kg
This experiment demonstrates that each of the enantiomers of DMS will
penetrate
the blood brain-barrier and inhibit brain MAO-B after parenteral in vivo
administration. It
also demonstrates that the potency differences as an MAO-B inhibitor observed
in vitro
between each of the DMS enantiomers and selegiline, are reduced under in vivo
conditions.
i
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The dosages administered in this experiment, were not sufficient to inhibit
MAO-A
activity.
D. Conclusions:
R(-) DMS and S(+) DMS both demonstrate activity as an MAO-B and MAO-A
inhibitors. Each enantiomer was selective for MAO-B. S(+) DMS was generally
less
potent than R(-) DMS and both enantiomers of DMS were less potent than
selegiline in
inhibiting both MAO-A and MAO-B. Both enantiomers demonstrated activity after
in vivo
administration, indicating that these enantiomers are able to enter brain
tissue after -
parenteral administration. The ability of these agents to inhibit MAO-A or MAO-
B
suggests that these agents are of value as therapeutics for Parkinson's
disease, Alzheimer's
disease or depression.
Example 7: In Vivo Neuroprotection by the Enantiomers of Desmethylselegiline
The ability of the enantiomers of DMS to prevent neurological deterioration
was
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.
B. Test Method
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, double-blind
study. At the end
of this time mice were examined for grip strength, running time, resting
locomotive
activity and graded for semiquantitative paw posture abnormalities, and
semiquantitative
walking abnormalities. The investigators who prepared and injected the
solutions to the
animals and who analyzed behavioral changes were different.
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 was
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.
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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.
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
S to an inability to support the body using the paws.
Locomotive activity is determined by transferring animals to an examination
area in
which the floor is covered with a a square grid.. Activity is measured by the
number of
squares traversed by a mouse in a set time interval, e.g. 9 minutes.
C. Results
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 13 summarizes differences that were observed in the mean grip strength
of the test
animals
Table 13: Mean Grip Strength in Wobbler Mice Treated with R(-) or S(+) DMS
Tr atm n LV .C'~rig r ngth (gm)
Control (placebo) 10 9(0-15)
R(-)DMS 9 20(0 - 63)
S(+)DMS 9 14(7 - 20)
N = number of animals analyzed
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, ranging from
0 to 15 g,
with a mean of 9 g. The R(-) group had a mean grip strength of 20 g with
values ranging
from 0 to 63 g. The third group, injected with S(+) DMS, had a mean grip
strength of 14
' 25 g, with individual values ranging from 7 to 20 g. While the variability
in grip strength in
the treated animal groups prevented a meaningful statistical analysis of this
data, the mean
grip strength measured in the DMS-treated animals was greater than for the
controls.
Running time, resting locomotive activity, semiquantitative paw posture
abnormality grading, and semiquantitative walking abnormality grading were
also tested.
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None of these tests, however, showed data suggesting that any one of these
three groups -
differ from the other.
Example 8: Immune System Restoration by R(-)DMS and S(+)DMS
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. patents 5,276,057 and 5,387,615 suggest that selegiline is useful in the
treatment of
immune system dysfunction. The present experiments were undertaken to
determine
whether R(-)DMS and S(+) are also 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.
A. Test Procedure
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:
1) young rats (3 months old, not injected);
2) old rats (18-20 months old, not injected);
3) old rats injected with saline;
4) old rats injected with selegiline at a dosage of 0.25 mg/kg body weight;
5) old rats injected with selegiline at a dosage of 1.0 mg/kg body weight;
6) old rats injected with R(-)DMS at a dosage of 0.025 mg/kg body weight;
7) old rats injected with R(-)DMS at a dosage of 0.25 mg/kg body weight;
8) old rats injected with R(-)DMS at a dosage of 1.0 mg/kg body weight;
9) old rats injected with S(+)DMS at a dosage of 1.0 mg/kg body weight.
Rats were injected ip, daily for 60 days. They were then maintained for an
additional "wash out" period of 10 days during which time no injections were
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:
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1) in vitro production of y-interferon by spleen cells;
- 2) in vitro production of interleukin-2;
- 3) percentage of IgM positive spleen cells (IgM is a marker of B
lymphocytes);
4) percentage of CDS positive spleen cells (CDS is a marker of T lymphocytes).
- 5 B. Results
The effect of injections of selegiline, R(-)DMS and S(+)DMS on interferon
production by rat spleen cells is shown in Figures 10 and 11. As shown in
Figure 10,
there is a sharp decrease in cellular interferon production that occurs with
age (compare
production by cells from young animals with production by cells from old
animals or from
= 10 old animals injected with saline. Injections of selegiline, R(-)DMS and
S(+)DMS all led to
a partial restoration of y-interferon levels with the most dramatic increases
occurring at
- dosages of 1.0 mg/kg body weight.
The same data as shown in Figure 10 is repeated in Figure 11 except that
results for
cells from young rats is omitted. The figure more clearly shows the extent to
which
15 deprenyl, R(-)DMS and S(+)DMS are capable of restoring 'y-interferon
production in the
spleen cells of old rats. Interferon-y is a multifunctional protein that
inhibits viral
replication and regulates a variety of immunological functions. It influences
the class of
antibodies produced by B-cells, up-regulates class I and class II MHC complex
antigens
and increases the efficiency of macrophage-mediated killing of intracellular
parasites.
20 Figure 12 shows the effect of injections on the production of interleukin-2
by the rat
spleen cells. It can be seen that both R(-)DMS and S(+)DMS are capable of
restoring
production to levels seen in cells from young animals.
The effect of injections on the percentage of spleen cells that are IgM
positive is
shown in Figure 13. It was found that both selegiline and R(-)DMS partially
restored IgM
25 positive cells to a level closer to that seen in the spleens of young rats.
Thus, it appears that
these agents are restoring B lymphocyte cell number.
Figure 14 suggests that injection of either 0.025 mg/kg of R(-)DMS or S(+)DMS
may have slightly increased the percentage of CDS positive cells in the
spleens obtained
from old rats. However, neither injections with selegiline nor with 0.25 or
1.0 mg/kg of
30 R(-)DMS appeared to have any effect.
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C. Conclusions
Overall, the results support the conclusion that the enantiomers of DMS mimic
the
effects of selegiline on immune system function. Moreover, the results
obtained with
respect to the production of interferon, IL-2 and on the percentage of IgM
positive spleen
S cells support the conclusion that the DMS enantiomers are capable of at
least partially -
restoring the age-dependent loss of immune system function. Thus, it appears
that R(-)
DMS and S(+)DMS will have a therapeutically beneficial effect for diseases and
conditions
facilitated by weakened host immunity. This would include cancer, AIDS, and
infectious
diseases of all types.
Example 9: Examples of Dosage Forrns
A. Desmethylselegiline Patch.
Dry Weight Basis
Component (mg/cm2) -
Durotak~ 87-2194
adhesive acrylic polymer 90 parts by weight
Desmethylselegiline 10 parts by weight
The two ingredients are thoroughly mixed and cast on a Scotchpak~ 9723
polyester
film backing sheet, and dried. The backing sheet is cut into patches, a
Scotchpak~ 1022
fluoropolymer release liner is applied, and the patch is hermetically sealed
in foil
envelopes. One patch is applied daily to supply 1-5 mg of desmethylselegiline
per 24
hours in the treatment of conditions in a human produced by neuronal
degeneration or
neuronal trauma, as for example Parkinson's disease.
B. Ophthalmic Solution
Desmethylselegiline (0.1 g) as the hydrochloride, 1.9 g of boric acid, and
.004 g of
phenyl mercuric nitrate are dissolved in sterile water qs 100m1. The mixture
is sterilized
and sealed. It can be used ophthalmologically in the treatment of conditions
produced by
neuronal degeneration or neuronal trauma, as for example glaucomatous optic
neuropathy
and macular degeneration.
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Example 3: Intravenous Solution.
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
- 5 for intravenous administration in the treatment of conditions produced by
neuronal
degeneration or neuronal trauma.
- C. Transdermal Patch.
- A self crosslinking acrylic based pressure sensitive adhesive is added to a
solution
of a copolymer of methacrylic acid and dimethylaminoethyl methacrylate in an
organic
solvent such as methyl ethyl ketone. This is cast onto a first removable foil,
the solvents
- are evaporated, and the coated foil placed on a polyester backing layer, as
set forth in EP-
_ A 593807.
Desmethylselegiline hydrochloride is added to a solution of a non-crosslinking
acrylic based pressure sensitive adhesive in a suitable organic solvent, as
for example ethyl
- 15 acetate. Additional solvent can be added and heat and stirring can be
applied to facilitate
formation of the dispersion. This is coated onto a second removable foil and
the solvent
evaporated. After removing the foil from the previously prepared polyester
backed layer,
- it is laminated to the coated second removable foil. Additional self
crosslinking acrylic
based pressure sensitive adhesive and copolymer of methacrylic acid and
dimethylaminoethyl methacrylate in an organic solvent such as methyl ethyl
ketone are cast
onto a third removable foil and the solvent evaporated. The second and third
foils are
removed, the residual layers laminated, and the resulting laminate cut into
patches and
packaged. The resulting patches will have a removable release liner, an
adhesive layer
initially free of desmethylselegiline, and a matrix layer containing
desmethylselegiline as
the hydrochloride (or other salt). An impenetrable backing layer is adhered to
the matrix
layer through an intermediate adhesive layer similar to the adhesive layer
contiguous to the
removable release liner. One patch is applied daily to supply
desmethylselegiline as the
free base in the treatment of conditions in a human produced by neuronal
degeneration or
neuronal trauma, as for example Parkinson's disease. ,
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D. Oral Dosage Form
Tablets and capsules containing desmethylselegiline are prepared from
the
following ingredients
(mg/unit dose): -
desmethylselegiline 1-5
microcrystalline cellulose86
lactose 41.6
citric acid 0.5-2
sodium citrate 0.1-2
magnesium stearate 0.4
with an approximately 1:1 ratio of citric acid and sodium citrate.
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