U.S. patent application number 11/595726 was filed with the patent office on 2007-05-03 for use of r-enantiomer of n-propargyl-1-aminoindan, salts, compositions and uses thereof.
This patent application is currently assigned to Teva Pharmaceutical Industries, Ltd.. Invention is credited to Tirtsah Berger-Paskin, John P.M. Finberg, David Lerner, Ruth Levy, Jeffrey Sterling, Alexander Veinberg, Izila Yeilin, Haim Yellin, Moussa B.H. Youdim.
Application Number | 20070100001 11/595726 |
Document ID | / |
Family ID | 23772591 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070100001 |
Kind Code |
A1 |
Youdim; Moussa B.H. ; et
al. |
May 3, 2007 |
Use of R-enantiomer of N-propargyl-1-aminoindan, salts,
compositions and uses thereof
Abstract
The subject invention provides methods of treating a subject
afflicted with Parkinson's disease, memory disorder, depression,
hyperactive syndrome, Attention Deficit Disorder, dementia, brain
ischemia, stroke, head trauma injury, spinal trauma injury,
neurotrauma, neurodegenerative disease, neurotoxic injury, multiple
sclerosis, nerve damage, affective illness, schizophrenia or
symptoms of withdrawal from an addictive substance, using the
mesylate salt of R(+)-N-propargyl-1-aminoindan.
Inventors: |
Youdim; Moussa B.H.; (Haifa,
IL) ; Finberg; John P.M.; (Iivon, IL) ; Levy;
Ruth; (Tel-Aviv, IL) ; Sterling; Jeffrey;
(Jersusalem, IL) ; Lerner; David; (Jerusalem,
IL) ; Berger-Paskin; Tirtsah; (Raanana, IL) ;
Yellin; Haim; (Ramat-Gan, IL) ; Yeilin; Izila;
(Ramat-Gan, IL) ; Veinberg; Alexander; (Rehovot,
IL) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Assignee: |
Teva Pharmaceutical Industries,
Ltd.
Technion Research
Development Foundation Ltd.
|
Family ID: |
23772591 |
Appl. No.: |
11/595726 |
Filed: |
November 10, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11155864 |
Jun 17, 2005 |
|
|
|
11595726 |
Nov 10, 2006 |
|
|
|
10305478 |
Nov 27, 2002 |
6956060 |
|
|
11155864 |
Jun 17, 2005 |
|
|
|
10016268 |
Oct 26, 2001 |
6630514 |
|
|
10305478 |
Nov 27, 2002 |
|
|
|
08952705 |
Nov 16, 1998 |
6316504 |
|
|
PCT/US96/07465 |
May 22, 1996 |
|
|
|
10016268 |
Oct 26, 2001 |
|
|
|
08446439 |
May 22, 1995 |
5744500 |
|
|
08952705 |
Nov 16, 1998 |
|
|
|
08411398 |
Mar 28, 1995 |
5532415 |
|
|
08446439 |
May 22, 1995 |
|
|
|
08139517 |
Oct 18, 1993 |
|
|
|
08411398 |
Mar 28, 1995 |
|
|
|
08063455 |
May 18, 1993 |
5387612 |
|
|
08139517 |
Oct 18, 1993 |
|
|
|
07632184 |
Dec 21, 1990 |
|
|
|
08063455 |
May 18, 1993 |
|
|
|
Current U.S.
Class: |
514/657 |
Current CPC
Class: |
C07C 211/42 20130101;
C07C 2602/08 20170501; A61K 31/205 20130101; C07C 211/30 20130101;
A61P 9/00 20180101; A61P 25/20 20180101; A61P 25/18 20180101; A61P
25/00 20180101; A61P 9/10 20180101; A61P 25/08 20180101; A61K 45/06
20130101; A61K 31/215 20130101; A61P 25/28 20180101; A61K 31/135
20130101; A61P 25/24 20180101; A61K 31/195 20130101; A61P 25/16
20180101; A61K 31/195 20130101; A61K 31/135 20130101; A61K 31/215
20130101; A61K 31/135 20130101; A61K 31/195 20130101; A61K 2300/00
20130101; A61K 31/215 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/657 |
International
Class: |
A61K 31/135 20060101
A61K031/135 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 3, 1990 |
IL |
92,952 |
Claims
1-41. (canceled)
42. A method of treating a subject afflicted with dementia which
comprises administering to the subject a pharmaceutical composition
consisting of an amount of the mesylate salt of
R(+)-N-propargyl-1-aminoindan effective to treat the subject and at
least one pharmaceutically acceptable carrier.
43. The method of claim 42, wherein the dementia is of the
Alzheimer type.
44. The method of claim 42, wherein the subject is human and the
effective amount is from about 0.1 mg to about 100 mg per day.
45. The method of claim 44, wherein the effective amount is from
about 1 mg to about 10 mg per day.
46. The method of claim 45, wherein the effective amount is 1 mg
per day.
47. The method of claim 42, wherein the pharmaceutical composition
is administered orally, rectally, intravenously, transdermally, or
parenterally.
48. The method of claim 47, wherein the pharmaceutical composition
is administered orally.
49. The method of claim 46, wherein pharmaceutical composition is
administered orally, rectally, intravenously, transdermally, or
parenterally.
50. The method of claim 49, wherein the pharmaceutical composition
is administered orally.
51. A method of treating a subject afflicted with dementia which
comprises administering to the subject a pharmaceutical composition
consisting essentially of an amount of the mesylate salt of
R(+)-N-propargyl-1-aminoindan effective to treat the subject and at
least one pharmaceutically acceptable carrier.
52. The method of claims 51, wherein the dementia is of the
Alzheimer type.
53. The method of claim 51, wherein the subject is human and the
effective amount is from about 0.1 mg to about 100 mg per day.
54. The method of claim 53, wherein the effective amount is from
about 1 mg to about 10 mg per day.
55. The method of claim 54, wherein the effective amount is 1 mg
per day.
56. The method of claim 51, wherein the pharmaceutical composition
is administered orally, rectally, intravenously, transdermally, or
parenterally.
57. The method of claim 56, wherein the pharmaceutical composition
is administered orally.
58. The method of claim 55, wherein the pharmaceutical composition
is administered orally, rectally, intravenously, transdermally, or
parenterally.
59. The method of claim 58, wherein the pharmaceutical composition
is administered orally.
60. A method of treating a subject afflicted with dementia which
comprises administering to the subject a pharmaceutical composition
comprising an amount of the mesylate salt of
R(+)-N-propargyl-1-aminoindan effective to treat the subject and at
least one pharmaceutically acceptable carrier.
61. The method of claims 60, wherein the dementia is of the
Alzheimer type.
62. The method of claim 60, wherein the subject is human and the
effective amount is from about 0.1 mg to about 100 mg per day.
63. The method of claim 62, wherein the effective amount is from
about 1 mg to about 10 mg per day.
64. The method of claim 63, wherein the effective amount is 1 mg
per day.
65. The method of claim 60, wherein the pharmaceutical composition
is administered orally, rectally, intravenously, transdermally, or
parenterally.
66. The method of claim 65, wherein the pharmaceutical composition
is administered orally.
67. The method of claim 64, wherein the pharmaceutical composition
is administered orally, rectally, intravenously, transdermally, or
parenterally.
68. The method of claim 67, wherein the pharmaceutical composition
is administered orally.
Description
BACKGROUND OF THE INVENTION
[0001] I. The subject invention is in the field of selective
irreversible inhibitors of the enzyme monoamine oxidase
(hereinafter MAO) and provides the R(+) enantiomer of
N-propargyl-1-aminoindan (also referred to herein as PAI) which is
a selective irreversible inhibitor of the B-form of monoamine
oxidase enzyme (hereinafter MAO-B). The subject invention also
provides pharmaceutical compositions containing R(+)PAI which are
particularly useful for the treatment of Parkinson's disease, a
memory disorder, dementia, depression, hyperactive syndrome, an
affective illness, a neurodegenerative disease, a neurotoxic
injury, stroke, brain ischemia, a head trauma injury, a spinal
trauma injury, neurotrauma, schizophrenia, an attention deficit
disorder, multiple sclerosis, and withdrawal symptoms.
[0002] II. Parkinson's disease is widely considered to be the
result of degradation of the pre-synaptic dopaminergic neurons in
the brain, with a subsequent decrease in the amount of the
neurotransmitter dopamine being released. Inadequate dopamine
release, therefore, leads to the onset of disturbances of voluntary
muscle control, which disturbances are symptomatic of Parkinson's
disease.
[0003] Various methods of treating Parkinson's disease have been
established and are currently in widespread use, including, for
example, the administration of L-DOPA together with a decarboxylase
inhibitor such as L-carbidopa or benserazide. The decarboxylase
inhibitor protects the L-DOPA molecule from peripheral
decarboxylation and thus ensures L-DOPA uptake by the remaining
dopaminergic neurons in the striatum of the brain. Here, the L-DOPA
is converted into dopamine resulting in increased levels of
dopamine in these neurons. In response to physiological impulses,
these neurons are therefore capable of releasing larger amounts of
dopamine at levels which approximate the normal required levels.
L-DOPA treatment thus alleviates the symptoms of the disease and
contributes to the well-being of the patient.
[0004] However, L-DOPA treatment has its drawbacks, the main one
being that its effectiveness is optimal only during the first few
years of treatment. After this period, the clinical response
diminishes and is accompanied by adverse side effects which include
dyskinesia, fluctuation in efficacy throughout the day ("on-off
effect") and psychiatric symptoms such as confusional states,
paranoia, and hallucinations. This decrease in the effect of L-DOPA
treatment is attributed to a number of factors, including the
natural progression of the disease, alteration in dopamine
receptors as a consequence of increased dopamine production or
increased levels of dopamine metabolites, and pharmacokinetic
problems of L-DOPA absorption (reviewed by Youdim, et al., Progress
in Medicinal Chemistry, 21, 138-167 (1984)).
[0005] In order to overcome the drawbacks of L-DOPA treatment,
various treatments have been devised in which L-DOPA is combined
with MAO inhibitors with the aim of reducing the metabolic
breakdown of newly formed dopamine (see for example, Chiese, P.,
U.S. Pat. No. 4,826,875, issued May 2, 1989).
[0006] MAO exists in two forms known as MAO-A and MAO-B which are
selective for different substrates and inhibitors. For example,
MAO-B more efficiently metabolizes substrates such as
2-phenylethylamine, and is selectively and irreversibly inhibited
by (-)-deprenyl as described below.
[0007] It should be noted, however, that treatments combining
L-DOPA with an inhibitor of both MAO-A and MAO-B are undesirable,
as they lead to adverse side effects related to an increased level
of catecholamines throughout the neuraxis. Furthermore, complete
inhibition of MAO is also undesirable as it potentates the action
of sympathomimetic amines such as tyramine, leading to the
so-called "cheese effect" (reviewed by Youdim et al., Handbook of
Experimental Pharmacology, ed. by Trendelenburg and Weiner,
Springer-Verlag, 90, ch. 3 (1988)). As MAO-B was shown to be the
predominant form of MAO in the brain, selective inhibitors for this
form are thus considered to be a possible tool for achieving a
decrease in dopamine breakdown on the one hand, together with a
minimization of the systemic effects of total MAO inhibition on the
other.
[0008] Many inhibitors of MAO are chiral molecules. Although one
enantiomer often shows some stereoselectivity in relative potency
towards MAO-A and -B, a given enantiomeric configuration is not
always more selective than its mirror image isomer in
discriminating between MAO-A and MAO-B.
[0009] Table I lists the IC.sub.50 (mmol/L) of enantiomeric pairs
of propargyl amines in a rat brain preparation of MAO. These
results show small differences in potency in MAO-B inhibition
between the R and S enantiomers. (B. Hazelhoff, et al.,
Naunyn-Schmeideberg's Arch. Pharmacol., 330, 50 (1985)). Both
enantiomers are selective for MAO-B. In 1967, Magyar, et al.
reported that R-(-)-deprenyl is 500 times more potent than the
S-(+) enantiomer in inhibiting the oxidative deamination of
tyramine by rat brain homogenate. (K. Magyar, et al., Act. Physiol.
Acad. Sci., Hung., 32, 377 (1967)).
[0010] In rat liver homogenate, R-deprenyl is only 15 times as
potent as the S enantiomer. In other pharmacological activity
assays, such as for the inhibition of tyramine uptake, deprenyl
shows different stereoselectivities. The S form is in certain cases
the more potent epimer. (J. Knoll and K. Magyar, Advances in
Biochemical Psychopharmacology, 5, 393 (1972)).
[0011] N-Methyl-N-propargyl-1-aminotetralin (2-MPAT) is a close
structural analogue of deprenyl. The absolute stereo-chemistry of
2-MPAT has not been assigned. However, the (+) isomer is selective
for MAO-B and the (-) isomer is selective for MAO-A. The difference
in potency between the 2-MPAT enantiomers is less than 5-fold. (B.
Hazelhoff, et al., id.). The enantiomers of
N-propargyl-1-aminotetralin (1-PAT) are also similar in activity.
The lack of data in Table I showing clear structure-activity
relationships between isolated (+) or (-)-2-MPAT makes it
impossible to predict the absolute stereochemistry thereof.
[0012] After extensive computer modeling, Polymeropoulos recently
predicted that (R)-N-methyl-N-propargyl-1-aminoindan (R-1-MPAI)
would be more potent than (S) as a MAO-B inhibitor. (E.
Polymeropoulos, Inhibitors of Monoamine Oxidase B, I. Szelenyi,
ed., Birkhauser Verlag, p. 110 (1993)). However, experiments
described show that R-1-MPAI is a slightly more potent inhibitor of
MAO-B than S-1-MPAI, but is an even more potent inhibitor of MAO-A.
Both the selectivity between MAO-A and -B and the relative potency
of the R and S epimers are low. Thus, contrary to expectations in
the art, 1-MPAI is useless as a pharmaceutical agent.
[0013] The data presented below demonstrate that high selectivity
for MAO of one enantiomer versus the other cannot be predicted. The
structure of the MAO active site is not well enough understood to
permit the prediction of the relative potency or selectivity of any
given compound or pair of enantiomers thereof.
[0014] III. Brain stroke is the third leading cause of death in the
developed countries. Survivors often suffer from neurological and
motor disabilities. The majority of CNS strokes are regarded as
localized tissue anemia following obstruction of arterial blood
flow which causes oxygen and glucose deprivation. Occlusion of the
middle cerebral artery in the rat (MCAO) is a common experimental
procedure that is assumed to represent stroke in humans. It has
been proposed that the neurological lesion caused by proximal
occlusion of this artery in the rat corresponds to a large focal
cerebral infarct in humans (Yamori et al., 1976). This
correspondence has been based on similarities between cranial
circulation in the two species. Other animal models of stroke have
been described by Stefanovich (1983).
[0015] The histological changes described by Tamura et al. (1981)
who were the first to introduce the MCAO procedure, were commonly
seen in the cortex of the frontal (100%), sensimotor (75%) and
auditory (75%) areas and to a lesser extent in the occipital lobe
cortex (25%). In addition, damage was observed in the lateral
segment of the caudate nucleus (100%), and only to a variable
extent in its medial portion (38%). Concomitantly, the following
disorders were reported in MCAO animals: neurological deficits
(Menzies et al., 1992), cognitive disturbances (Yamamoto et al.,
1988), brain edema (Young et al., 1993; Matsui et al., 1993; Saur;
et al., 1993), decreased cerebral blood flow (Teasdale et al.,
1983), catecholamine fluctuations. (Cechetto et al., 1989). Any of
these disorders might be indicative of the severity and extent of
brain damage that follow MCAO in the rat. Conversely, a drug with a
potential to limit or abort a given disorder may be considered as a
candidate for the treatment of stroke in humans. TABLE-US-00001
TABLE IA IC.sub.50 (mmol/L) Data for Rat Brain MAO Inhibition by
Propargylamines RELATIVE INHIBITION POTENCY COMPOUND REF EPIMER A B
A/B +/- 2-MPAI a + 140 16 8.8 A B - 46 88 0.5 3 0.2 R/S DEPRENYL a
S 3600 16 120 80 2.6 R 450 6 75 1-MPAI b S 70 50 1.4 23 5 R 3 10
0.3 1-PAT c S 3800 50 76 4 0.5 R 900 90 10 a. B. Hazelhoff, et al.,
Naunyn-Schmeideberg's Arch. Pharmacol., 330, 50 (1985). b. European
Patent Application 436,492 A2, published Jul. 10, 1991. c. Present
inventors.
[0016] One selective MAO-B inhibitor, (-)-deprenyl, has been
extensively studied and used as a MAO-B inhibitor to augment L-DOPA
treatment. This treatment with (-)-deprenyl is generally favorable
and does not cause the "cheese effect" at doses causing nearly
complete inhibition of MAO-B (Elsworth, et al., Psychopharmacology,
57, 33 (1978)). Furthermore, the addition of (-)-deprenyl to a
combination of L-DOPA and a decarboxylase inhibitor administered to
Parkinsons's patients leads to improvements in akinesia and overall
functional capacity, as well as the elimination of "on-off" type
fluctuations (reviewed by Birkmayer & Riederer in "Parkinson's
Disease," Springer-Verlag, pp. 138-149 (1983)). Thus, (-)-deprenyl
(a) enhances and prolongs the effect of L-DOPA, and (b) does not
increase the adverse effects of L-DOPA treatment.
[0017] However, (-)-deprenyl is not without its own adverse sides
effects, which include activation of pre-existing gastric ulcers
and occasional hypertensive episodes. Furthermore, (-)-deprenyl is
an amphetamine derivative and is metabolized to amphetamine and
methamphetamines, which substances may lead to undesirable side
effects such as increased heart rate (Simpson, Biochemical
Pharmacology, 27, 1951 (1978); Finberg, et al., in "Monoamine
Oxidase Inhibitors--The State of the Art," Youdim and Paykel, eds.,
Wiley, pp. 31-43 (1981)).
[0018] Other compounds have been described that are selective
irreversible inhibitors of MAO-B but which are free of the
undesirable effects associated with (-)-deprenyl. One such
compound, namely N-propargyl-1-aminoindan.HCl (racemic PAI.HCl),
was described in GB 1,003,666 and GB 1,037,014 and U.S. Pat. No.
3,513,244, issued May 19, 1970. Racemic PAI.HCl is a potent,
selective, irreversible inhibitor of MAO-B, is not metabolized to
amphetamines, and does not give rise to unwanted sympathomimetic
effects.
[0019] In comparative animal tests, racemic PAI was shown to have
considerable advantages over (-)-deprenyl. For example, racemic PAI
produces no significant tachycardia, does not increase blood
pressure (effects produced by doses of 5 mg/kg of (-)-deprenyl),
and does not lead to contraction of nictitating membrane or to an
increase in heart rate at doses of up to 5 mg/kg (effects caused by
(-)-deprenyl at doses over 0.5 mg/kg). Furthermore, racemic PAI.HCl
does not potentiate the cardiovascular effects of tyramine
(Finberg, et al., in "Enzymes and Neurotransmitters in Mental
Disease," pp. 205-219 (1980), Usdin, et al., Eds., Wiley, N.Y.;
Finberg, et al. (1981), in "Monoamine Oxidase Inhibitors--The State
of the Art," ibid.; Finberg and Youdim, British Journal Pharmacol.,
85, 451 (1985)).
[0020] One underlying object of this invention was to separate the
racemic PAI compounds and to obtain an enantiomer with MAO-B
inhibition activity which would be free of any undesirable side
effects associated with the other enantiomer.
[0021] Since deprenyl has a similar structure to PAI and it is
known that the (-)-enantiomer of deprenyl, i.e. (-)-deprenyl, is
considerably more pharmaceutically active than the (+)-enantiomer,
the (-) enantiomer of PAI would be expected to be the more active
MAO-B inhibitor.
[0022] However, contrary to such expectations, upon resolution of
the enantiomers, it was found that the (+)-PAI enantiomer is in
fact the active MAO-B inhibitor while the (-)-enantiomer shows
extremely low MAO-B inhibitory activity. Furthermore, the (+)-PAI
enantiomer also has a degree of selectivity for MAO-B inhibition
surprisingly higher than that of the corresponding racemic form,
and should thus have fewer undesirable side effects in the
treatment of the indicated diseases than would the racemic mixture.
These findings are based on both in vitro and in vivo experiments
as discussed in greater detail infra.
[0023] It was subsequently shown that (+)-PAI has the R absolut
configuration. This finding was also surprising based o the
expected structural similarity of (+)-PAI analogy wit deprenyl and
the amphetamines.
[0024] The high degree of stereoselectivity of pharmacologica
activity between R(+)-PAI and the S(-) enantiomer a discussed
hereinbelow is also remarkable. The compoun R(+)-PAI is nearly four
orders of magnitude more active tha the S(-) enantiomer in MAO-B
inhibition. This ratio i significantly higher than that observed
between the tw deprenyl enantiomers (Knoll and Magyar, Adv. Biochem
Psychopharmacol., 5, 393 (1972); Magyar, et al., Act Physiol. Acad.
Sci. Hung., 32, 377 (1967)). Furthermore, i some physiological
tests, (+)-deprenyl was reported to hav activity equal to or even
higher than that of the (- enantiomer (Tekes, et al., Pol. J.
Pharmacol. Pharm., 40 653 (1988)).
[0025] MPAI is a more potent inhibitor of MAO activity, but wit
lower selectivity for MAO-B over A (Tipton, et al., Biochen
Pharmacol., 31, 1250 (1982)). As only a small degree c difference
in the relative activities of the two resolve enantiomers was
surprisingly observed with MPAI, t remarkable behavior of R(+)PAI
is further emphasized (S Table 1B).
[0026] The subject invention also provides methods of using t
pharmaceutically active PAI-enantiomer alone (without DOPA) for
treatment of Parkinson's disease, a memo disorder, dementia,
depression, hyperactive syndrome, affective illness, a
neurodegenerative disease, a neurotox injury, brain ischemia, a
head trauma injury, a spin trauma injury, schizophrenia, an
attention deficit disorde multiple sclerosis, or withdrawal
symptoms (see review Youdim, et al., in Handbook of Experimental
Pharmacology, Trendelenberg and Wiener, eds., 90/I, ch. 3
(1988)).
[0027] The subject invention further provides a method of using the
pharmaceutically active PAI-enantiomer alone for pre-treatment of
Parkinson's disease. The subject invention also provides
pharmaceutical compositions comprising R(+)PAI and synergistic
agents such as levodopa. The use of such agents has been studied
with respect to (-)-deprenyl which was shown to be effective when
administered alone to early Parkinson's patients, and may also have
a synergistic effect in these patients when administered together
with .alpha.-tocopherol, a vitamin E derivative (The Parkinson's
Study Group, New England J. Med., 321(20), 1364-1371 (1989)).
[0028] In addition to its usefulness in treating Parkinson's
disease, (-)-deprenyl has also been shown to be useful in the
treatment of patients with dementia of the Alzheimer type (DAT)
(Tariot, et al., Psychopharmacology, 91, 489-495 (1987)), and in
the treatment of depression (Mendelewicz and Youdim, Brit. J.
Psychiat. 142, 508-511 (1983)). The R(+)PAI compound of this
invention, and particularly the mesylate salt thereof, has been
shown to restore memory. R(+)PAI thus has potential for the
treatment of memory disorders, dementia, especially of the
Alzheimer's type, and hyperactive syndrome in children.
[0029] Finally, the subject invention provides highly stable salts
of R(+)PAI with superior pharmaceutical properties. The mesylate
salt is especially stable, shows unexpectedly greater selectivity,
and shows significantly fewer side effects than do the
corresponding racemic salts.
SUMMARY OF THE INVENTION
[0030] The subject invention provides R(+)-N-propargyl-1-aminoindan
having the structure: ##STR1##
[0031] The subject invention further provides a pharmaceutically
acceptable salt of R(+)-N-propargyl-1-aminoindan.
[0032] The subject invention further provides a pharmaceutical
composition which comprises a therapeutically effective amount of
R(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable salt
thereof and a pharmaceutically acceptable carrier.
[0033] The subject invention further provides a method of treating
a subject afflicted with Parkinson's disease which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat
Parkinson's disease in the subject.
[0034] The subject invention further provides a method of treating
a subject afflicted with a memory disorder which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the memory
disorder in the subject.
[0035] The subject invention further provides a method of treating
a subject afflicted with dementia which comprises administering to
the subject an amount of R(+)-N-propargyl-1-aminoindan or the
pharmaceutically acceptable salt thereof of the subject invention
effective to treat dementia in the subject. In one embodiment, the
dementia is of the Alzheimer type (DAT).
[0036] The subject invention further provides a method of treating
a subject afflicted with depression which comprises administering
to the subject an amount of R(+)-N-propargyl-1-aminoindan or the
pharmaceutically acceptable salt thereof of the subject invention
effective to treat depression in the subject.
[0037] The subject invention further provides a method of treating
a subject afflicted with hyperactive syndrome which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat
hyperactive syndrome in the subject.
[0038] The subject invention further provides a method of treating
a subject afflicted with an affective illness which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
affective illness in the subject.
[0039] The subject invention further provides a method of treating
a subject afflicted with a neurodegenerative disease which
comprises administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
neurodegenerative disease in the subject.
[0040] The subject invention further provides a method of treating
a subject afflicted with a neurotoxic injury which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
neurotoxic injury in the subject.
[0041] The subject invention further provides a method of treating
a subject afflicted with brain ischemia which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat brain
ischemia in the subject.
[0042] The subject invention further provides a method of treating
a subject afflicted with a head trauma injury which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the head
trauma injury in the subject.
[0043] The subject invention further provides a method of treating
a subject afflicted with a spinal trauma injury which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the spinal
trauma injury in the subject.
[0044] The subject invention further provides a method of treating
a subject afflicted with schizophrenia which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat
schizophrenia in the subject.
[0045] The subject invention further provides a method of treating
a subject afflicted with an attention deficit disorder which
comprises administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
attention deficit disorder in the subject.
[0046] The subject invention further provides a method of treating
a subject afflicted with multiple sclerosis which comprises
administering to the subject an amount of
R(+)-N-propargyl-3-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat multiple
sclerosis in the subject.
[0047] The subject invention further provides a method of
preventing nerve damage in a subject which comprises administering
to the subject an amount of R(+)-N-propargyl-1-aminoindan or the
pharmaceutically acceptable salt thereof of the subject invention
effective to prevent nerve damage in the subject.
[0048] The subject invention further provides a method of treating
a subject suffering from symptoms of withdrawal from an addictive
substance which comprises administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
symptoms of withdrawal in the subject.
[0049] The subject invention further provides a method for
preparing R(+)-N-propargyl-1-aminoindan which comprises contacting,
in the presence of an organic or inorganic base, R(-)-aminoindan
with either propargyl bromide or propargyl chloride so as to form
R(+)-N-propargyl-1-aminoindan, and isolating the
R(+)-N-propargyl-1-aminoindan formed thereby.
[0050] The subject invention further provides a method for
preparing racemic N-propargyl-1-aminoindan which comprises
contacting, in the presence of an organic or inorganic base,
racemic 1-aminoindan with propargyl bromide or propargyl chloride
so as to form racemic N-propargyl-1-aminoindan, and isolating the
racemic N-propargyl-1-aminoindan formed thereby.
[0051] Finally, the subject invention provides a method of
preparing an R(+)-N-propargyl-1-aminoindan salt which comprises
contacting racemic N-propargyl-1-aminoindan with an optically
active acid so as to form two diastereomeric
N-propargyl-1-aminoindan salts, and isolating
R(+)-N-propargyl-1-aminoindan salt from the diastereomeric
N-propargyl-1-aminoindan salts so formed.
BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1 is a graphic representation of the results according
to Example 22 showing in vitro MAO-A inhibitory activity.
[0053] FIG. 2 is a graphic representation of the results according
to Example 22 showing in vitro MAO-B inhibitory activity.
[0054] FIG. 3 is a graphic representation of the results according
to Example 22 showing MAO activity in human cortical tissue.
[0055] FIG. 4 is a graphic representation of the results according
to Example 23 showing acute inhibition (i.p.) of MAO-A in
brain.
[0056] FIG. 5 is a graphic representation of the results according
to Example 23 showing acute inhibition (i.p.) of MAO-B in
brain.
[0057] FIG. 6 is a graphic representation of the results according
to Example 23 showing acute inhibition (i.p.) of MAO-A in
liver.
[0058] FIG. 7 is a graphic representation of the results according
to Example 23 showing acute inhibition (i.p.) of MAO-B in
liver.
[0059] FIG. 8 is a graphic representation of the results according
to Example 23 showing acute inhibition (per os) of MAO-A in
brain.
[0060] FIG. 9 is a graphic representation of the results according
to Example 23 showing acute inhibition (per os) of MAO-B in
brain.
[0061] FIG. 10 is a graphic representation of the results according
to Example 23 showing acute inhibition (per os) of MAO-A in
liver.
[0062] FIG. 11 is a graphic representation of the results according
to Example 23 showing acute inhibition (per os) of MAO-B in
liver.
[0063] FIG. 12 is a graphic representation of the results according
to Example 24 showing chronic inhibition of MAO-A in brain.
[0064] FIG. 13 is a graphic representation of the results according
to Example 24 showing chronic inhibition of MAO-B in brain.
[0065] FIG. 14 is a graphic representation of the results according
to Example 24 showing chronic inhibition of MAO-A in liver.
[0066] FIG. 15 is a graphic representation of the results according
to Example 24 showing chronic inhibition of MAO-B in liver.
[0067] FIG. 16 is a graphic representation of the results according
to Example 25 showing MAO-B activity in rat brain as a function of
time following i.p. administration of R(+)PAI.
[0068] FIG. 17 is a graphic representation of the results according
to Example 32 showing restoration of normokinesia in mice that had
received haloperidol 6 mg/kg s.c. Mice received each of the test
drugs i.p. at the indicated dose. 2 hours later they received
haloperidol. Kinetic scores were taken 3 hours after haloperidol.
These scores consisted of the ability to move horizontally along a
rod, the ability to descend a vertical rod, and the shortening of
catalepsia. In the absence of haloperidol, the maximum score is 12,
with haloperidol alone, 6.6.+-.0.03. Statistical significance was
calculated by the Student's "t" test: * p.ltoreq.0.05; **
p.ltoreq.0.01; * * *p.ltoreq.0.001 with respect to haloperidol
alone. The scores of (R)-PAI are significantly different from those
of racemic-PAI at 5 mg/kg (p.ltoreq.0.05), at 10 mg/kg
(p.ltoreq.0.01), and at 15 mg/kg (p.ltoreq.0.05), (n=5.6). The
dosage shown is for the free base of PAI (and not the mesylate
salt).
[0069] FIG. 18 is a graphic representation of the results according
to Example 32 showing restoration of motor activity in rats treated
with .alpha.-methyl-p-tyrosine at 100 mg/kg i.p. Rats received the
test drug i.p. at the indicated doses. After two hours they
received .alpha.-Mpt and were immediately placed in activity cages.
Total motor activity was recorded for the duration of 10 hours.
Control rats, treated with saline, only scored 15, 862+1424. With
.alpha.-Mpt alone, they scored 8, 108.+-.810. Statistical
significance by the Student's "t" test: *p.ltoreq.0.05;
**p.ltoreq.0.01; ***p.ltoreq.0.001 with respect to .alpha.-MpT
alone. The scores of (R)-PAI are significantly different from
racemic-PAI at 2 mg/kg (p.ltoreq.0.01), (n=6). Dosage shown is for
the free base of PAI and not the mesylate salt.
[0070] FIG. 19 is a graph showing the NADH response to 2 minutes of
anoxia measured 30 minutes after injury and at half-hour intervals
thereafter.
[0071] FIG. 20 Ischemic brain lesion evaluation with MRI T2-scan 48
hours after MCA-O and [R] (+)PAI Mesylate Treatment in rats: The
middle cerebral artery was surgically occluded as described in
Example 38. [R](+)PAI Mesylate was administered as follows: 1.0
mg/kg ip immediately after surgery; 0.5 mg/kg ip, 2 hrs after
surgery; 1.0 mg/kg ip, 24 hrs after surgery. Infarct volume
(mm.sup.3) was determined by MRI 48 hours following-surgery.
[0072] FIG. 21: neurological evaluation of Wistar rats subjected to
MCA-O and [R](+)PAI Mesylate Treatment: The middle cerebral artery
was surgically occluded and [R](+)PAI Mesylate administered as in
FIG. 20. At 24 hours post surgery a neurological score was taken as
described in Example 38.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The subject invention provides R(+)-N-propargyl-1-aminoindan
having the structure: ##STR2##
[0074] As demonstrated in the Experimental Examples hereinbelow,
R(+)PAI is nearly 7,000 times more active as an inhibitor of MAO-B
than is S(-)PAI. In view of known MAO-B inhibitors in the art which
possess low selectivity between MAO-A and MAO-B, and which do not
show predictable trends in potency as a function of R or S
configuration, the selectivity of R(+)PAI is unexpected.
[0075] R(+)PAI may be obtained by optical resolution of racemic
mixtures of R- and S-enantiomers of PAI. Such a resolution can be
accomplished by any conventional resolution method well known to a
person skilled in the art, such as those described in J. Jacques,
A. Collet and S. Wilen, "Enantiomers, Racemates and Resolutions,"
Wiley, N.Y. (1981). For example, the resolution may be carried out
by preparative chromatography on a chiral column. Another example
of a suitable resolution method is the formation of diastereomeric
salts with a chiral acid such as tartaric, malic, mandelic acid or
N-acetyl derivatives of amino acids, such as N-acetyl leucine,
followed by recrystallisation to isolate the diastereomeric salt of
the desired R enantiomer.
[0076] The racemic mixture of R and S enantiomers of PAI may be
prepared, for example, as described in GB 1,003,676 and GB
1,037,014. The racemic mixture of PAI can also be prepared by
reacting 1-chloroindan with propargylamine. Alternatively, this
racemate may be prepared by reacting propargylamine with 1-indanone
to form the corresponding imine, followed by reduction of the
carbon-nitrogen double bond of the imine with a suitable agent,
such as sodium borohydride.
[0077] In accordance with this invention, the R enantiomer of PAI
can also be prepared directly from the optically active
R-enantiomer of 1-aminoindan by reaction with propargyl bromide or
propargyl chloride in the presence of an organic or inorganic base,
and optionally in the presence of a suitable solvent.
[0078] Suitable organic or inorganic bases for use in the above
reaction include, by way of example, triethylamine, pyridine,
alkali metal carbonates, and bicarbonates. If the reaction is
conducted in the presence of a solvent, the solvent may be chosen
from, e.g., toluene, methylene chloride, and acetonitrile. One
method of preparing R(+)PAI is to react R-1-aminoindan with
propargyl chloride using potassium bicarbonate as a base and
acetonitrile as solvent.
[0079] The above-described reaction of 1-aminoindan generally
results in a mixture of unreacted primary amine, the desired
secondary amine and the tertiary amine N,N-bispropargylamino
product. The desired secondary amine, i.e.,
N-propargyl-1-aminoindan, can be separated from this mixture by a
conventional separation method including, by way of example,
chromatography, distillation and selective extraction.
[0080] The R-1-aminoindan starting material can be prepared by
methods known in the art which include, by way of example, the
method of Lawson and Rao, Biochemistry, 19, 2133 (1980), methods in
references cited therein, and the method of European Patent No.
235,590.
[0081] R-1-aminoindan can also be prepared by resolution of a
racemic mixture of the R and S enantiomers, which involves, for
example, the formation of diastereomeric salts with chiral acids,
or any other known method such as those reported in J. Jacques, et
al., ibid. Alternatively, R-1-aminoindan may be prepared by
reacting 1-indanone with an optically active amine, followed by
reduction of the carbon nitrogen double bond of the resulting imine
by hydrogenation over a suitable catalyst, such as palladium on
carbon, platinum oxide or Raney nickel. Suitable optically active
amines include, for example, one of the antipodes of phenethylamine
or an ester of an amino acid, such as valine or phenylalanine. The
benzylic N--C bond may be cleaved subsequently by hydrogenation
under non-vigorous conditions.
[0082] An additional method for preparing R-1-aminoindan is the
hydrogenation of indan-1-one oxime ethers as described above,
wherein the alkyl portion of the ether contains an optically pure
chiral center. Alternatively, a non-chiral derivative of
indan-1-one containing a carbon-nitrogen double bond, such as an
imine or oxime, can be reduced with a chiral reducing agent, e.g.,
a complex of lithium aluminum-hydride and ephedrine.
[0083] The subject invention further provides a pharmaceutically
acceptable salt of R(+)-N-propargyl-1-aminoindan.
[0084] In the practice of this invention, pharmaceutically
acceptable salts include, but are not limited to, the mesylate,
maleate, fumarate, tartrate, hydrochloride, hydrobromide, esylate,
p-toluenesulfonate, benzoate, acetate, phosphate and sulfate
salts.
[0085] In one embodiment, the salt is selected from the group
consisting of the mesylate salt of R(+)-N-propargyl-1-aminoindan,
the esylate salt of R(+)-N-propargyl-1-aminoindan, and the sulfate
salt of R(+)-N-propargyl-1-aminoindan.
[0086] As demonstrated in the Experimental Examples hereinbelow,
the mesylate salt is highly stable to thermal degradation, and
shows unexpectedly superior selectivity for MAO-B over the racemic
salt.
[0087] For the preparation of pharmaceutically acceptable acid
addition salts of the compound of R(+)PAI, the free base can be
reacted with the desired acids in the presence of a suitable
solvent by conventional methods. Similarly, an acid addition salt
may be converted to the free base form in a known manner.
[0088] A preferred mode of preparing the mesylate salt of (R)-PAI
comprises (a) adding an aqueous solution of 15% sodium hydroxide to
a solution of propargyl benzenesulfonate (or tosylate or mesylate)
in toluene; (b) stirring for 5 hours; (c) adding additional toluene
and water; (d) separating and washing the organic phase with 10%
sodium hydroxide, and then diluting with water; (e) adjusting the
pH of the mixture to 3.2 by adding 10% aqueous sulfuric acid; (f)
separating the aqueous phase and adjusting the pH to 7.3 with 10%
sodium hydroxide; (g) extracting three times with toluene while
maintaining constant pH; (h) concentrating combined organic layers
in vacuo to give a yellow oil; (i) dissolving the oil and
L-tartaric acid in isopropanol; (j) heating to reflux for 1 hour;
(k) cooling to room temperature and collecting the precipitate by
filtration; (l) recrystallizing the crude di-propargylaminoindan
tartrate from methanol/isopropanol (1:1) to give
di(R(+)-N-propargyl-1-aminoindan) tartrate; (m) dissolving the
tartrate salt and methanesulfonic acid in isopropanol, and heating
to reflux for 30 minutes; and (n) cooling to room temperature, and
collecting the precipitated R(+)-N-propargyl-1-aminoindan.
[0089] The subject invention further provides a pharmaceutical
composition which comprises a therapeutically effective amount of
R(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable salt
thereof and a pharmaceutically acceptable carrier. The
"therapeutically effective amount" of the
R(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable salt
thereof may be determined according to methods well known to those
skilled in the art.
[0090] Possible salts useful for such compositions include
hydrochloride, phosphate, maleate, fumarate, tartrate, mesylate,
esylate, and sulfate salts.
[0091] These compositions may be prepared as medicaments to be
administered orally, parenterally, rectally, or transdermally.
[0092] In one embodiment, the pharmaceutically acceptable carrier
is a solid and the pharmaceutical composition is a tablet. The
therapeutically effective amount may be an amount from about 0.1 mg
to about 100 mg. The therapeutically effective amount may also be
an amount from about 1 mg to about 10 mg.
[0093] Suitable forms for oral administration include tablets,
compressed or coated pills, dragees, sachets, hard or soft gelatin
capsules, sublingual tablets, syrups and suspensions.
[0094] In an alternative embodiment, the pharmaceutically
acceptable carrier is a liquid and the pharmaceutical composition
is an injectable solution. The therapeutically effective amount may
be an amount from about 0.1 mg/ml to about 100 mg/ml. The
therapeutically effective amount may also be an amount from about 1
mg/ml to about 10 mg/ml. In one embodiment, the dose administered
is an amount between 0.5 ml and 1.0 ml.
[0095] In a further alternative embodiment, the carrier is a gel
and the pharmaceutical composition is a suppository.
[0096] For parenteral administration the invention provides
ampoules or vials that include an aqueous or non-aqueous solution
or emulsion. For rectal administration there are provided
suppositories with hydrophilic or hydrophobic vehicles. For topical
application as ointments and transdermal delivery there are
provided suitable delivery systems as known in the art.
[0097] In the preferred embodiment, the pharmaceutically acceptable
salt is a mesylate salt.
[0098] These compositions may be used alone to treat the
above-listed disorders, or alternatively, as in the case of
Parkinson's disease, for example, they may be used as an adjunct to
the conventional L-DOPA treatments.
[0099] The preferred dosages of the active ingredient, i.e., R-PAI,
in the above compositions are within the following ranges. For oral
or suppository formulations, 0.1-100 mg per dosage unit may be
taken daily, and preferably 1-10 mg per dosage unit is taken daily.
For injectable formulations, 0.1-100 mg/ml per dosage unit may be
taken daily, and preferably 1-10 mg/ml per dosage unit is taken
daily.
[0100] In one embodiment, the pharmaceutical composition further
comprises a therapeutically effective amount of Levodopa. In
another embodiment, the pharmaceutical composition still further
comprises an effective amount of a decarboxylase inhibitor.
[0101] The amount of decarboxylase inhibitor administered in
combination with (R)-PAI or a pharmaceutically acceptable salt
thereof is an amount effective to ensure L-DOPA uptake in the
subject.
[0102] The decarboxylase inhibitor may be L-Carbidopa. In one
embodiment, the therapeutically effective amount of
R(+)-N-propargyl-1-aminoindan is about 0.1 mg to about 100 mg, the
therapeutically effective amount of Levodopa is about 50 mg to
about 250 mg, and the effective amount of L-Carbidopa is about 10
mg to about 25 mg.
[0103] The decarboxylase inhibitor may also be benserazide. In one
embodiment, the therapeutically effective amount of
R(+)-N-propargyl-1-aminoindan is about 0.1 mg to about 100 mg, the
therapeutically effective amount of Levodopa is about 50 mg to
about 200 mg, and the effective amount of benserazide is about 12.5
mg to about 50 mg.
[0104] The subject invention further provides a method of treating
a subject afflicted with Parkinson's disease which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat
Parkinson's disease in the subject.
[0105] Methods of treatment of Parkinson's disease which combine
the use of (R)-PAI with other drugs, such as dopamine agonists,
bromocryptine, pergolide, lisuride, as well as catecholamine
oxidase methyl transferase inhibitors are within the scope of the
subject invention.
[0106] In the preferred embodiment, the pharmaceutically acceptable
salt is a mesylate salt.
[0107] The administering may comprise orally administering,
rectally administering, transdermally administering, or
parenterally administering.
[0108] In one embodiment, the method of the subject invention
further comprises administering to the subject a therapeutically
effective amount of Levodopa. In another embodiment, the method of
the subject invention still further comprises administering to the
subject an effective amount of a decarboxylase inhibitor.
[0109] The decarboxylase inhibitor may be L-Carbidopa.
Alternatively, the decarboxylase inhibitor may be benserazide.
[0110] The subject invention further provides a method of treating
a subject afflicted with a memory disorder which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the memory
disorder in the subject.
[0111] The subject invention further provides a method of treating
a subject afflicted with dementia which comprises administering to
the subject an amount of R(+)-N-propargyl-1-aminoindan or the
pharmaceutically acceptable salt thereof of the subject invention
effective to treat dementia in the subject. In one embodiment, the
dementia is of the Alzheimer type (DAT).
[0112] The subject invention further provides a method of treating
a subject afflicted with depression which comprises administering
to the subject an amount of R(+)-N-propargyl-1-aminoindan or the
pharmaceutically acceptable salt thereof of the subject invention
effective to treat depression in the subject.
[0113] The subject invention further provides a method of treating
a subject afflicted with hyperactive syndrome which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat
hyperactive syndrome in the subject.
[0114] The administering may comprise orally administering,
rectally administering, or parenterally administering.
[0115] The subject invention further provides a method of treating
a subject afflicted with an affective illness which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
affective illness in the subject.
[0116] The subject invention further provides a method of treating
a subject afflicted with a neurodegenerative disease which
comprises administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
neurodegenerative disease in the subject.
[0117] The subject invention further provides a method of treating
a subject afflicted with a neurotoxic injury which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
neurotoxic injury in the subject.
[0118] The subject invention further provides a method of treating
a subject afflicted with brain ischemia which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat brain
ischemia in the subject.
[0119] This invention provides a method of treating brain ischemia
or stroke in a subject which comprises administering to the subject
an amount of R(+)-N-propargyl-3-aminoindan or a pharmaceutically
acceptable salt thereof effective to treat brain ischemia or stroke
in the subject.
[0120] In an embodiment of the method for treatment of brain
ischemia or stroke, the pharmaceutically acceptable salt of
R(+)-N-propargyl-1-aminoindan is selected from the group consisting
of: the mesylate salt; the ethylsulfonate salt; the sulfate salt;
and the hydrochloride salt. Preferably, the pharmaceutically
acceptable salt is the mesylate salt of
R(+)-N-propargyl-1-aminoindan.
[0121] The effective amount can be determined using techniques
known to those of skill in the art, such as titration. In an
embodiment of this invention, the effective amount is from about
0.5 milligrams per kilogram body weight of the subject to about 2.5
milligrams per kilogram body weight of the subject. The
R(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable salt
thereof is administered using techniques known to those of skill in
the art. For example, it may be administered intravenously, orally,
rectally, transdermally, or parenterally.
[0122] The subject is preferably a mammal, such as a dog, cat,
mouse, rat, rabbit, pig, horse, goat, sheep, cow, ape or monkey. In
a particular embodiment the subject is human.
[0123] In an embodiment of this invention, the effective amount is
from about 0.01 mg to 50.0 mg per day. In a more specific
embodiment, the effective amount is from 0.1 to 10.0 mg per
day.
[0124] In one embodiment of the above-described method, the area of
the brain ischemia is reduced by about thirty-five percent.
[0125] The subject invention further provides a method of treating
a subject afflicted with a head trauma injury which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the head
trauma injury in the subject.
[0126] The subject invention further provides a method of treating
a subject afflicted with a spinal trauma injury which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the spinal
trauma injury in the subject.
[0127] This invention further provides a method of treating
neurotrauma in a subject which comprises administering to the
subject an amount of R(+)-N-propargyl-1-aminoindan or a
pharmaceutically acceptable salt thereof effective to treat
neurotrauma in the subject.
[0128] In the treatment of head trauma injury, spinal trauma injury
or neurotrauma, the pharmaceutically acceptable salt of
R(+)-N-propargyl-1-aminoindan is selected from the group consisting
of: the mesylate salt; the ethylsulfonate salt; the sulfate salt;
and the hydrochloride salt. Preferably, the pharmaceutically
acceptable salt is the mesylate salt of
R(+)-N-propargyl-1-aminoindan.
[0129] The effective amount can be determined using techniques
known to those of skill in the art, such as titration. In an
embodiment of this invention, the effective amount is from about
0.5 milligrams per kilogram body weight of the subject to about 2.5
milligrams per kilogram body weight of the subject. The
R(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable salt
thereof is administered using techniques known to those of skill in
the art. For example, it may be administered intravenously, orally,
rectally, transdermally, or parenterally.
[0130] The subject is preferably a mammal, such as a dog, cat,
mouse, rat, rabbit, pig, horse, goat, sheep, cow, ape or monkey. In
a particular embodiment the subject is human.
[0131] In an embodiment of this invention, the effective amount is
from about 0.01 mg to 50.0 mg per day. In a more specific
embodiment, the effective amount is from 0.1 to 10.0 mg per
day.
[0132] The subject invention further provides a method of treating
a subject afflicted with schizophrenia which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat
schizophrenia in the subject.
[0133] The subject invention further provides a method of treating
a subject afflicted with an attention deficit disorder which
comprises administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
attention deficit disorder in the subject.
[0134] The subject invention further provides a method of treating
a subject afflicted with multiple sclerosis which comprises
administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat multiple
sclerosis in the subject.
[0135] The subject invention further provides a method of
preventing nerve damage in a subject which comprises administering
to the subject an amount of R(+)-N-propargyl-1-aminoindan or the
pharmaceutically acceptable salt thereof of the subject invention
effective to prevent nerve damage in the subject.
[0136] In one embodiment, the nerve damage is structural nerve
damage. In another embodiment, the structural nerve damage is optic
nerve damage.
[0137] The subject invention further provides a method of treating
a subject suffering from symptoms of withdrawal from an addictive
substance which comprises administering to the subject an amount of
R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable
salt thereof of the subject invention effective to treat the
symptoms of withdrawal in the subject.
[0138] As used herein, the term "symptoms of withdrawal" refers to
physical and/or psychological symptoms, including drug craving,
depression, irritability, anergia, amotivation, appetite change,
nausea, shaking and sleep irregularity.
[0139] As used herein, the term "addictive substance" includes, by
way of example, (a) addictive opiates such as opium, heroin and
morphine, (b) psychostimulants such as cocaine, amphetamines and
methamphetamines, (c) alcohol, (d) nicotine, (e) barbiturates and
(f) narcotics such as fentanyl, codeine, diphenoxylate and
thebaine.
[0140] In one embodiment, the addictive substance is cocaine. In
another embodiment, the addictive substance is alcohol.
[0141] The subject invention further provides a method for
preparing R(+)-N-propargyl-1-aminoindan which comprises contacting,
in the presence of an organic or inorganic base, R(-)-aminoindan
with either propargyl bromide or propargyl chloride so as to form
R(+)-N-propargyl-1-aminoindan, and isolating the
R(+)-N-propargyl-1-aminoindan formed thereby.
[0142] The subject invention further provides a method for
preparing racemic N-propargyl-1-aminoindan which comprises
contacting, in the presence of an organic or inorganic base,
racemic 1-aminoindan with propargyl bromide or propargyl chloride
so as to form racemic N-propargyl-1-aminoindan, and isolating the
racemic N-propargyl-1-aminoindan formed thereby.
[0143] Finally, the subject invention provides a method of
preparing an R(+)-N-propargyl-1-aminoindan salt which comprises
contacting racemic N-propargyl-1-aminoindan with an optically
active acid so as to form two diastereomeric
N-propargyl-1-aminoindan salts, and isolating
R(+)-N-propargyl-1-aminoindan salt from the diastereomeric
N-propargyl-1-aminoindan salts so formed.
[0144] In one embodiment, the isolating comprises isolating by
fractional crystallization.
[0145] The following Experimental Details are set forth to aid in
an understanding of the invention, and are not intended, and should
not be construed, to limit in any way the invention set forth in
the claims which follow thereafter.
[0146] Experimental Details
EXAMPLE 1
Racemic N-propargyl-1-aminoindan hydrochloride
[0147] 10.0 g of racemic 1-aminoindan and 10.49 of potassium
carbonate were added to 75 ml of acetonitrile. The resulting
suspension was heated to 60.degree. C. and 4.5 g of propargyl
chloride was added dropwise.
[0148] The mixture was stirred at 60.degree. C. for 16 hours,
whereafter most of the volatiles were removed by distillation in
vacuo. The residue was partitioned between 10% aqueous sodium
hydroxide and methylene chloride.
[0149] The organic phase was dried and the solvent removed by
distillation. The residue was flash chromatographed on silica gel,
eluting with 40%; ethyl acetate/60% hexane. The fractions
containing the title compound as a free base were combined and the
eluant replaced by ether. The ethereal solution was treated with
gaseous HCl, the precipitate formed was isolated by suction
filtration and recrystallized from isopropanol to yield 7.3 g of
the title compound, m.p. 182-4.degree. C.
[0150] Chromatographic and spectroscopic data were in accordance
with U.S. Pat. No. 3,513,244, issued May 19, 1970, and an authentic
sample and were as follows: NMR .delta. (CDCl.sub.3): 2.45 (2H, m),
2.60 (1H, t), 2.90 (1H, m), 3.45 (1H, m), 3.70 (2H, d), 4.95 (1H,
t), 7.5 (4H, m) ppm.
EXAMPLE 2
S-(-)-N-Propargyl-1-aminoindan hydrochloride
[0151] The title compound in free base form was isolated by
resolving the racemic mixture of the free base of Example 1 on a
Chiracel OJ (cellulose tris [p-methylbenzoate]) preparative HPLC
column eluting with 10% isopropanol/90% hexane and collecting the
first eluted major peak. The resulting oil was converted to the
title compound (hydrochloride) by treatment of a 10% diethyl ether
solution of the oil with gaseous HCl, and the resulting precipitate
was collected by suction filtration. [a].sub.D-29.2.degree. (1%,
ethanol), m.p. 182-184.degree. C. Other chromatographic and
spectroscopic properties were identical with the hydrochloride salt
of Example 1.
EXAMPLE 3
R-(')-N-Propargyl-1-aminoindan hydrochloride
[0152] The title compound was prepared as in Example 2 above,
except that the second eluted peak from the preparative HPLC was
collected: [a].sub.D+29.1.degree. (0.8%, ethanol), m.p.
179-181.degree. C. Other chromatographic and spectroscopic
properties were identical with the hydrochloride salt of Example
1.
EXAMPLE 4
R-(+)-N-Propargyl-1-aminoindan hydrochloride
[0153] 12.4 g of R-(-)-1-Aminoindan and 12.9 g of potassium
carbonate were added to 95 ml of acetonitrile. The resulting
suspension was heated to 60.degree. C. and 5.6 g of propargyl
chloride was added dropwise. The mixture was stirred at 60.degree.
C. for 16 hours, whereafter most of the volatiles were removed by
distillation in vacuo. The residue was partitioned between 10%
aqueous sodium hydroxide and methylene chloride.
[0154] The organic phase was dried and the solvent removed in
vacuo. The residue was flash chromatographed on silica get eluting
with 40% ethyl acetate/60% hexane. Fractions containing the free
base of the title compound were combined and the solvent replaced
by ether. The ethereal solution was treated with gaseous HCl and
the resulting precipitate was isolated by suction filtration and
recrystallized from isopropanol to yield 6.8 g of the title
compound, m.p. 163-185.degree. C., [a].sub.D+30.90 (2% ethanol).
Spectral properties were identical to those reported for the
compound of Example 1.
EXAMPLE 5
S-(-)-N-Propargyl-1-aminoindan hydrochloride
[0155] The title compound was prepared by the method of Example 4,
except that S-(+)-1-aminoindan was used as starting material. The
product exhibited [a].sub.D-30.3 (2% ethanol), m.p. 183-5.degree.
C. Spectral properties were identical to those reported for the
compound of Example 1.
EXAMPLE 6A
Di(R-(+)-N-propargyl-1-aminoindan) L-tartrate
[0156] To a solution of tartaric acid (4.4 g) in 48 ml of boiling
methanol was added a solution of R-(+)-N-propargyl-1-aminoindan
free base (5.0 g) in methanol (48 ml). The solution was heated to
reflux and 284 ml of t-butylmethyl ether was added over 20 minutes.
The mixture was heated for an additional 30 minutes, cooled, and
the resulting precipitate was isolated by suction filtration to
yield 6.7 g of the title compound: m.p. 175-177.degree. C.;
[.alpha.].sub.D (1.5, H.sub.2O)=+34.3; Anal. calcd. for
C.sub.28H.sub.32O.sub.6N.sub.2; C, 68.26, H, 6.56, N, 5.69. Found:
C, 68.76; H, 6.57; N, 5.61.
EXAMPLE 6B
R-(+)-N-propargyl-1-aminoindan mesylate
[0157] a) To a solution of propargyl benzenesulfonate (78.4 g) and
racemic aminoindan (63.2 g) in toluene (240 mL) at 20.degree. C.
was added dropwise an aqueous solution of 15% sodium hydroxide (108
mL). After 5 hours of stirring, additional toluene (80 mL) and
water (200 mL) were added with stirring. The organic phase was
separated and washed with 10% aqueous sodium hydroxide and then
diluted with water. The pH of the mixture was adjusted to 3.2 by
the addition of 10% aqueous sulfuric acid. The aqueous phase was
separated and its pH was adjusted to 7.3 with 10% sodium hydroxide
and extracted three times with toluene while maintaining constant
pH. The combined organic layers were concentrated in vacuo to 40.7
g of a yellow oil.
[0158] b) The above crude racemic propargylaminoindan and
L-tartaric acid (10 g) were dissolved in isopropanol (1 L) and
heated to reflux for 1 hour. The reaction was then allowed to cool
to room temperature with stirring and the precipitate collected by
filtration. The crude di-propargylaminoindan tartrate was
recrystallized from 1 L of 1:1 methanol/isopropanol to give
di(R-(+)-N-propargyl-1-aminoindan)-L-tartrate with physical and
spectral properties identical to that of the compound of Example
6A.
[0159] c) A solution of di-(R-(+)-N-propargyl-1-aminoindan)
tartrate (15 g) and methanesulfonic acid (6 g) in isopropanol (150
mL) was heated to reflux for 30 minutes The reaction was allowed to
cool to room temperature and the resulting precipitate isolated by
suction filtration to give the title compound (11.1 g) with m.p.
157.degree. C. and [.alpha.].sub.D=22.degree..
EXAMPLE 7
R-(+)-N-Methyl-N-propargyl-1-aminoindan hydrochloride
[0160] The free base form of R-(+)-N-propargyl-1-aminoindan from
Example 4 (1.2 grams), potassium carbonate (0.97 grams) and methyl
iodide (1 gram) were added to 15 ml of acetone and the resulting
suspension heated to reflux under a nitrogen atmosphere for 8
hours. Thereafter the volatiles were removed under reduced pressure
and the residue partitioned between 10% aqueous sodium hydroxide
(30 ml) and methylene chloride (30 ml). The organic phase was dried
and the solvent removed in vacuo. The residue was flash
chromatographed on silica gel eluting with 40% ethyl acetate/60%
hexane. Fractions containing the title compound as a free base were
combined and the solvent replaced by diethyl ether. The etheral
solution was treated with gaseous HCl. The volatiles were removed
in vacuo, and the residue recrystallized from isopropanol to yield
400 mg of the title compound as a white crystalline solid, m.p.
134-136.degree. C., [.alpha.].sub.D+31.40 (ethanol). NMR .delta.
(CDCl.sub.3): 2.55 (2H, m); 2.7 (1H, br.s); 2.8 (3H, s); 3.0 (1H,
m); 3.4 (1H, m); 3.9 (2H, br.s); 5.05 (1H, m); 7.7 (4H, m) ppm.
EXAMPLE 8
S-(-)-N-Methyl-N-propargyl-1-aminoindan hydrochloride
[0161] The title compound was prepared as in Example 7 above,
except that S-(-)-N-propargyl-1-aminoindan (free base) from Example
5 was used as the starting material. All of the physical and
spectral properties of the title compound were identical to those
in Example 7 except for the [.alpha.].sub.D-34.9.degree. C.
(ethanol).
EXAMPLE 9
[0162] TABLE-US-00002 Tablet Composition
N-Propargyl-1(R)-aminoindan Hydrochloride 7.81 mg* Pregelatinized
starch NF 47.0 mg Lactose NF hydrous 66.0 mg Microcrystalline
cellulose NF 20.0 mg Sodium starch glycolate NF 2.99 mg Talc USP
1.5 mg Magnesium stearate NF 0.7 mg *Equivalent to 5.0 mg of
N-propargyl aminoindan base.
EXAMPLE 10
[0163] TABLE-US-00003 Tablet Composition
N-Propargyl-1(R)-aminoindan Hydrochloride 1.56 mg* Lactose hydrous
50.0 mg Pregelatinized starch 36.0 mg Microcrystalline cellulose
14.0 mg Sodium starch glycolate 2.14 mg Talc USP 1.0 mg Magnesium
stearate NF 0.5 mg *Equivalent to 1.0 mg of N-propargyl aminoindan
base.
EXAMPLE 11
[0164] TABLE-US-00004 Capsule Composition
N-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mg Pregelatinized
starch 10.0 mg Starch 44.0 mg Microcrystalline cellulose 25.0 mg
Ethylcellulose 1.0 mg Talc 1.5 mg Purified water added as required
for granulation.
EXAMPLE 12
[0165] TABLE-US-00005 Injection Composition
N-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mg Dextrose anhydrous
44.0 mg HCl added to pH 5 Purified water added as required for 1
ml
EXAMPLE 13
[0166] TABLE-US-00006 Injection Composition
N-Propargyl-1(R)-aminoindan Hydrochloride 1.0 mg Sodium chloride
8.9 mg HCl added to pH 5 Purified water added as required for 1
ml
EXAMPLE 14
[0167] TABLE-US-00007 Injection Composition
N-Propargyl-1(R)-aminoindan Hydrochloride 2.0 mg Sodium chloride
8.9 mg HCl added to pH 5 Purified water added as required for 1
ml
EXAMPLE 15
[0168] TABLE-US-00008 Syrup Composition N-Propargyl-1(R)-aminoindan
Hydrochloride 5.0 mg Sucrose 2250.0 mg Saccarin sodium 5.0 mg
Methylparaben 6.0 mg Propylparaben 1.0 mg Flavor 20.0 mg Glycerin
USP 500 mg Alcohol 95% USP 200 mg Purified water as required to 5.0
ml
EXAMPLE 16
[0169] TABLE-US-00009 Sublingual Tablets
N-Propargyl-1(R)-aminoindan Hydrochloride 2.5 mg Microcrystalline
cellulose 20.0 mg Lactose hydrous 5.0 mg Pregelatinized starch 3.0
mg Povidone 0.3 mg Coloring agent q.s. Flavor q.s. Sweetener q.s.
Talc 0.3 mg
[0170] Blend the excipients and the active and granulate with an
ethanol solution of Providone. After drying and weighing, it is
blended with the talc and compressed.
EXAMPLE 17
[0171] TABLE-US-00010 PAI Sublingual Tablets
N-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mg Microcrystalline
cellulose 15.0 mg Pregelatinized starch 12.0 mg Ethyl cellulose 0.3
mg Talc 0.3 mg Purified water added as required for
granulation.
EXAMPLE 19
[0172] TABLE-US-00011 Tablet Composition
N-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mg Levodopa 100.0 mg
Carbidopa 25.0 mg Pregelatinized starch 24.0 mg Starch 40.0 mg
Microcrystalline cellulose 49.5 mg Col. D & C Yellow No. 10 0.5
mg Col. D & C Yellow No. 6 0.02 mg Alcohol USP added as
required for granulation.
EXAMPLE 19
[0173] TABLE-US-00012 Tablet Composition
N-Propargyl-1(R)-aminoindan Mesylate 7.81 mg* Pregelatinized starch
NF 47.0 mg Lactose NF hydrous 66.0 mg Microcrystalline cellulose NF
20.0 mg Sodium starch glycolate NF 2.99 mg Talc USP 1.5 mg
Magnesium stearate NF 0.7 mg *Equivalent to 5.0 mg of N-propargyl
aminoindan base.
EXAMPLE 20
[0174] TABLE-US-00013 Tablet Composition
N-Propargyl-1(R)-aminoindan Mesylate 1.56 mg* Lactose hydrous 50.0
mg Pregelatinized starch 36.0 mg Microcrystalline cellulose 14.0 mg
Sodium starch glycolate 2.14 mg Talc USP 1.0 mg Magnesium stearate
NF 0.5 mg *Equivalent to 1.0 mg of N-propargyl aminoindan base.
EXAMPLE 21
[0175] TABLE-US-00014 Capsule Composition
N-Propargyl-1(R)-aminoindan Mesylate 5.0 mg Pregelatinized starch
10.0 mg Starch 44.0 mg Microcrystalline cellulose 25.0 mg
Ethylcellulose 1.0 mg Talc 1.5 mg Purified water added as required
for granulation.
[0176] The following Examples and the accompanying Tables and
Figures relate to biological experiments carried out in accordance
with this invention.
EXAMPLE 22
Inhibition of MAO Activity in vitro
[0177] Experimental protocol
[0178] The MAO enzyme source was a homogenate of rat brain in 0.3M
sucrose, which was centrifuged at 600 g for 15 minutes. The
supernatant was diluted appropriately in 0.05M phosphate buffer,
and pre-incubated with serial dilutions of compounds: R(+)-PAI,
S(-)-PAI and racemic PA: for 20 minutes at 37.degree. C.
.sup.14C-Labelled substrates (2-phenylethylamine, hereinafter PEA;
5-hydroxytryptamine, hereinafter 5-HT) were then added, and the
incubation continued for a further 20 minutes (PEA), or 30-45
minutes (5-HT). Substrate concentrations used were 50 .mu.M (PEA)
and 1 mM (5-HT). In the case of PEA, enzyme concentration was
chosen so that not more than 10% of the substrate was metabolized
during the course of the reaction. The reaction was then stopped by
addition of tranylcypromine (to a final concentration of 1 mM), and
the incubate filtered over a small column of Amberlite CG-50
buffered to pH 6.3. The column was washed with 1.5 ml water, the
eluates pooled and the radioactive content determined by liquid
scintillation spectrometry. Since the amine substrates are totally
retained on the column, radioactivity in the eluate indicates the
production of neutral and acidic metabolites formed as a result of
MAO activity. Activity of MAO in the sample was expressed as a
percentage of control activity in the absence of inhibitors after
subtraction of appropriate blank values. The activity determined
using PEA as substrate is referred to as MAO-B, and that determined
using 5-HT as MAO-A.
[0179] Results
[0180] Inhibitory activity of R(+)-PAI, S(-)-PAI and racemic-PAI
were examined separately in vitro, and the results of typical
experimental runs are shown in FIGS. 1 and 2. The entire experiment
was repeated three times. Concentrations of inhibitor producing 50%
inhibition of substrate metabolism (IC-50) were calculated from the
inhibition curves, and are shown in Table 1B. From this data it can
be seen that: [0181] (a) the R(+)-PAI is twice as active as the
racemate for inhibition of MAO-B; [0182] (b) the R(+)-PAI is 29
times more active for inhibition of MAO-B than MAO-A;
[0183] (c) the S(-)-PAI is only 1/6, 800 as active as the R(+)PAI
for inhibition of MAO-B, and shows little or no selectivity between
MAO-B and MAO-A. TABLE-US-00015 TABLE 1A IC-50 (nM) VALUES FOR
INHIBITION OF MAO-A AND MAO-B BY RACEMIC-PAI AND THE R(+) AND S(-)
ENANTIOMERS THEREOF IN RAT BRAIN HOMOGENATE IN VITRO IC-50 (nM)
MAO-A MAO-B S(-)PAI R(+)PAI Rac S(-)PAI R(+)PAI Rac 26000 73 140
17000 2.5 5
[0184] The results of the same experiments using R(+) and S(-) MPAI
(N-methyl-N-propargyl-1-aminoindan) are reported in Table 1B. Each
of the enantiomers of MPAI is less selective in MAO-A and MAO-B
inhibition than R(+) PAI. Furthermore, R(+)-MPAI is only five times
as active as S(-)-MPAI in MAO-B inhibition, in contrast to R(+)-PAI
which is about 7000 times as active as S(-)-PAI in this assay.
TABLE-US-00016 TABLE 1B IC-50 (nM) VALUES FOR INHIBITION OF MAO-A
AND MAO-B BY T R(+) AND S(-) ENANTIOMERS OF MPAI IN RAT BRAIN
HOMOGENAT IN VITRO IC-50 (nM) MAO-A MAO-B S(-)MPAI R(+)MPAI
S(-)MPAI R(+)MPAI Compound: 70 3 50 10
[0185] Some experiments were also carried out with human cerebr
cortical tissues obtained 6 hours post-mortem, and treat as
described above. The results of such an experiment a shown in FIG.
3, where R(+)-PAI, S(-)-PAI, and racemic. P are as defined
herein.
EXAMPLE 23
Inhibition of MAO Activity in vivo: Acute Treatment
[0186] Experimental protocol
[0187] Rats (male Sprague-Dawley-derived) weighing 250.+-.20 g we
treated with one of the enantiomers or the racemic form PAI by
intraperitoneal injection (ip) or oral gavage (p and decapitated 1
h or 2 h later respectively. Groups three rats were used for each
dose level of inhibitor, a MAO activity determined in brain and
liver using the gene technique described above. The amount of
protein in ea incubation was determined using the Folin-Lowry
method, enzyme activity calculated as nmol of substrate metaboliz
per hour of incubation for each mg of protein. Activity MAO in
tissues from animals treated with inhibitors expressed as a
percentage of the enzyme activity in a gr of control animals
administered vehicle (water for o administration, 0.9% saline for
ip injection) and killed as above.
[0188] Results
[0189] None of the dose levels used with the inhibitor drugs
produced any obvious behavioral alteration. The results are
depicted in FIGS. 4 to 11. Following i.p. administration, compound
R(+)PAI produced 90% inhibition of brain MAO-B activity at a dose
of 0.5 mg/kg. The same dose produced only 20% inhibition of MAO-A
activity. By oral administration, the same dose of R(+)PAI produced
80% inhibition of MAO-B with no detectable inhibition of MAO-A.
Essentially similar results were seen for inhibition of hepatic
MAO, as for brain MAO. The doses producing 50% inhibition of MAO-A
and MAO-B (IC-50) were calculated from the inhibition curves, and
are shown in Table 2. These data show: (a) that MAO inhibitory
activity of R(+)PAI is maintained in vivo in the rat; (b) that
selectivity for inhibition of MAO-B, as opposed to MAO-A, by
R(+)PAI is maintained in vivo; (c) that the much greater activity
of the (+)-enantiomer as opposed to the (-)-enantiomer, is
maintained in vivo; (d) that the compounds are effectively absorbed
after oral administration; and (e) that the compounds effectively
pass the blood-brain barrier, and effectively inhibit brain MAO.
The fact that R(+)-PAI was about twice as active as the racemic
compound for inhibition of MAO-B is a reflection of the extremely
low activity of S(-)-PAI for inhibition of MAO-B. TABLE-US-00017
TABLE 2 IC-50 VALUES (mg/kg) FOR INHIBITION OF MAO-A AND MAO-B BY
R(+)-PAI, S(-)-PAI OR RACEMIC-PAI, IN THE RAT FOLLOWING
INTRAPERITONEAL (I.P.) INJECTION OR ORAL ADMINISTRATION (P.O.)
IC-50 (mg/kg) MAO-A MAO-B Compound: S(-)PAI R(+)PAI Rac S(-)PAI
R(+)PAI Rac I.P. BRAIN >10 1.2 2.5 >10 0.07 0.22 I.P. LIVER
>10 5 5 >10 0.06 0.11 P.O. BRAIN >10 >5 >5 >10
0.17 0.29 P.O. LIVER >10 >5 >5 >10 0.05 0.09 (Rac =
Racemic PAI)
EXAMPLE 24
Inhibition of MAO Activity in vivo: Chronic Treatment
[0190] Experimental protocol
[0191] Rats (specifications as in Example 23, 4 animals for each
dose level) were treated with R(+)PAI or the racemic mixture at
three dose levels (0.05, 0.1 and 0.5 mg/kg) by oral administration,
one dose daily for 21 days, and decapitated 2 hours after the last
dose. The activities of MAO types A and B were determined in brain
and liver as described in Example 23.
[0192] Results
[0193] A daily dose of 0.1 mg/kg of compound R(+)PAI produced a
good degree of selective inhibition, with more than 80% inhibition
of brain MAO-B and 20% or less inhibition of brain MAO-A. At the
higher dose of 0.5 mg/kg daily, MAO-A was still inhibited by less
than 50% (FIGS. 12 and 13). Hepatic MAO showed a similar degree of
selective inhibition (FIGS. 14 and 15). Compound R(+)PAI was again
more potent than the racemic mixture by a factor of about twofold.
In the case of brain MAO, R(+)PAI had a better degree of
selectivity for inhibition of MAO-B than did the racemic
mixture.
[0194] These results show that selectivity of MAO-B inhibition can
be maintained following chronic treatment with the compounds. As
with other irreversible inhibitors, the degree of enzyme inhibition
is greater with chronic treatments than that following a single
dose of the drug. Compound R(+)PAI shows a better degree of
selectivity for inhibition of brain MAO-B than the racemic
mixture.
EXAMPLE 25
Irreversible Nature of MAO Inhibition
[0195] Experimental protocol
[0196] A single dose of compound R(+)PAI (1 mg/kg) was administered
by i.p. injection to groups of 4 rats, and the animals killed 2, 6,
18, 24, 48 and 72 hours later. Activity of MAO-B was determined in
whole brain tissues as described hereinabove.
[0197] Results
[0198] The results are shown in FIG. 16. Maximal inhibition of
MAO-B was attained at 6 hours after injection. MAO activity had
only returned to 30% of control activity at 72 hours after
injection. This experiment demonstrates the irreversible nature of
the MAO inhibition by R(+)PAI.
EXAMPLE 26
Potentiation of Tyramine Pressor Effect in Conscious Rats
[0199] Experimental protocol
[0200] Rats were anesthetized with a mixture of pentobarbital (30
mg/kg) and chloral hydrate (120 mg/kg) by intraperitoneal
injection. The left carotid artery and jugular vein were cannulated
with fine polytene tubing (artery) or fine silicone rubber tubing
connected to polyethylene tubing (vein), the distal end of which
was brought under the skin to an anchor point behind the neck. The
tubing was filled with heparinized saline solution, and plugged
with a fine steel rod. The animals were treated with 20 mg
chloramphenicol by intramuscular injection and allowed to recover
from the operation overnight. The following day, the rats were
placed in a high-walled container permitting free movement. The
arterial catheter was connected to a pressure transducer via a 100
cm length of saline-filled, fine-bore polyethylene tubing, and the
venous catheter connected to a 1 ml syringe via a similar length of
tubing, which, together with the syringe, contained a solution of
tyramine hydrochloride in saline (1 mg/ml). Following an
equilibration period of 30 to 40 minutes, tyramine injections (50
or 100 .mu.g) were given, and blood pressure responses recorded. An
interval of at least 15 minutes was maintained between injections
after return of blood pressure to control values. Control pressor
responses were established, then one of the drugs was injected
intraperitoneally, and tyramine responses were repeated over the
next 4 hours. The area under the blood pressure response curve was
estimated, and the ratio of this area after treatment to before
treatment and to 1 to 3 hours after injection of the compounds, was
determined using the average of 3 to 4 values obtained in the
control period.
[0201] Results
[0202] The results are shown in Table 3. Compound R(+)PAI at a dose
of 1 mg/kg (which causes complete inhibition of MAO-B in brain and
liver, and 40 to 50% inhibition of MAO-A in these tissues) caused
no significant potentiation of tyramine pressor response. At the
higher R(+)PAI dose of 5 mg/kg (which causes more extensive
inhibition of MAO-A in brain and periphery), there was a
significant potentiation of the tyramine pressor response, which
was similar in extent to that produced by the same dose of
deprenyl, and less than that produced by clorgyline (at a dose
which inhibits hepatic MAO-A activity by over 85%). TABLE-US-00018
TABLE 3 POTENTIATION OF TYRAMINE PRESSOR EFFECT IN CONSCIOUS RATS
BY MAO INHIBITORS Ratio Area Under Dose No. of rats Pressor
Response Inhibitor (mg/kg) (n) Curve; After/Before SEM* Saline 12
1.25 0.28 Clorgyline 2 6 10.39 2.13 (-)Deprenyl 1 2 1.15
(+)Deprenyl 5 3 2.36 0.16 R(+)PAI 1 3 1.38 0.7 R(+)PAI 5 3 3.49
0.98 *SEM = standard error of the mean
[0203] From this experiment it can be concluded that compound
R(+)PAI causes no potentiation of the tyramine pressor effect at a
dose which effectively inhibits MAO-B.
EXAMPLE 27
Suppression of MPTP-Induced Dopaminergic Toxicity by R(+)PAI
[0204] 1-Methyl-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a
neurotoxin that damages nigrostriatal dopaminergic neurons in
several mammalian species, including mice, and produces a
Parkinsonian syndrome in humans and primates. A crucial initial
step in the mechanism of its neurotoxicity involves conversion of
MPTP to its toxic metabolite 1-methyl-4-phenyl pyridinium ion
(MPP+). This reaction is catalyzed by the enzyme MAO-B and probably
takes place outside of dopaminergic neurons, mainly in glia. It is
known that MPTP is both a substrate and an irreversible inhibitor
of MAO-B. Pretreatment of experimental animals with MAO-B
inhibitors such as deprenyl or pargyline protects against and
prevents the MPTP-induced damage to nigrostriatal neurons because
the oxidative conversion of MPTP to MPP+ is blocked. The
progressive nigrostriatal degeneration in Parkinson's may be due to
exposure to environmentally-derived exogenous MPTP-like
neurotoxins. In such cases, there is an additional strong
indication of initiation of sustained treatment with an MAO-B
inhibitor from the very early stages of Parkinson's disease in the
hope that it will neutralize the damaging effects of such yet
putative MPTP-toxins, and thus arrest or slow down the progression
of the illness. A successful MAO-B inhibitor drug is currently
judged by its ability to block MPTP-induced damage to nigrostriatal
dopaminergic neurons in vivo. The (-) and (+) enantiomers of PAI
were therefore tested for their potency in preventing or
attenuating the MPTP-induced striatal dopamine depletions in
mice.
[0205] Experimental Protocol
[0206] Male C57 black mice (20-25 g weight) were (a) injected with
MPTP.HCl (30 mg/kg dissolved in distilled water, s.c.), or vehicle
alone, or one hour after pretreatment with the (-) or (+) isomers
of PAI (2.5 mg/kg, i.p.), or with deprenyl (5 mg/kg, i.p.), and (b)
decapitated 5 days later. Brains were removed and corpora striata
dissected on an ice-cold glass plate and frozen on dry ice.
Striatal tissues were homogenized in 0.1 M perchloric acid, and
deproteinized aliquots containing dihydroxybenzylamine as an
internal standard were assayed for dopamine and its major
metabolite 3,4-dihydroxy-phenylacetic acid (DOPAC) using HPLC with
electrochemical detection.
[0207] Results
[0208] Table 4 shows the results of this experiment. Treatment with
MPTP alone produced marked striatal dopamine (DA) and DOPAC
depletions. Treatment with the (-) and (+) enantiomers of PAI or
with (-) deprenyl did not affect striatal DA concentrations.
Pretreatment with the (-) isomer of PAI did not affect the
MPTP-induced DA and DOPAC levels in the striatum. The (+)-isomer of
PAI given before MPTP completely abolished the reduction in
striatal DA and DOPAC levels produced by the toxin. At a dose of
2.5 mg/kg, (+)PAI was equipotent to (-) deprenyl (5 mg/kg) in its
protective effect. TABLE-US-00019 TABLE 4 EFFECT OF PRETREATMENT
WITH THE (-) AND (+) ENANTIOMERS OF THE MAO-B INHIBITOR PAI ON THE
STRIATAL DA AND DOPAC DEPLETIONS INDUCED BY MPTP IN MICE IN VIVO DA
DOPAC (ng/mg protein) Control 162.8 .+-. 7.2 8.4 .+-. 0.5 MPTP 53.1
.+-. 6.2 3.2 .+-. 0.3 (-)PAI 174.0 .+-. 4.8 7.5 .+-. 0.2 (-)PAI +
MPTP 53.4 .+-. 6.9 7.0 .+-. 0.6 (+)PAI 185.0 .+-. 6.9 3.3 .+-. 0.3
(+)PAI + MPTP 177.8 .+-. 14.4 6.0 .+-. 0.3 (-)Deprenyl 170.6 .+-.
7.1 5.6 .+-. 0.3 (-)Deprenyl + MPTP 197.0 .+-. 8.0 6.4 .+-. 0.5
Above values for DA and DOPAC expressed as Mean .+-. S.E.M. and
number of rats. n = 7-11 in each group.
[0209] These results indicate that the R(+)PAI is an excellent
MAO-B inhibitor in vivo, and is of especially great potential for
the treatment of Parkinson's disease.
[0210] While the invention has been described with reference to the
aforementioned Examples and the accompanying Tables and Figures, it
is not restricted thereto. Various modifications and applications
of the invention are possible. For example, (R)-PAI may be
combined, in a synergistic way, with .alpha.-tocopherol (a vitamin
E derivative) for the treatment of Parkinson's disease.
EXAMPLE 29
Effect of PAT Enantiomers on Amphetamine Induced Stereotype
Behavior in Senescent Rats
[0211] Amphetamine is known to induce stereotypic behavior (Sulser,
F., and Sanders-Bush, E,, Ann. Rev. Pharmacol., 11, 209-230 (1971))
by the mobilization of endogenous dopamine. Amphetamine is not
metabolized by MAO-B. Inhibition of MAO-B by an effective inhibitor
and administration of amphetamine cause release of dopamine which
will not undergo degradation by the inhibited MAO-B. Thus, an
increase of synaptic dopamine is expected after administration of
amphetamine and effective MAO-B inhibitor leading to an increase in
stereotype behavior-potentiation of the amphetamine effect. The
extent of this behavior is rated in accordance with the number of
lateral head movements over a period of 1 minute.
[0212] Experimental Protocol
[0213] The test compound was administered at a dose of 0.5
mg/kg/day in drinking water, 24 hours before the infliction of
hypoxia (92% nitrogen+8% oxygen for 6 hours). Following that,
amphetamine was injected s.c. at a dose of 0.5 mg/kg. 45 minutes
later, lateral head movements were counted.
[0214] Results
[0215] The results of these experiments are shown in Table 5.
TABLE-US-00020 TABLE 5 EFFECT OF PAI ISOMERS ON AMPHETAMINE-INDUCED
STEREOTYPE BEHAVIOR IN SENESCENT RATS (CONTROL AND HYPOXIA
LESIONED) Stereotype Group Treatment Behavior Rating Control (6) --
87 .+-. 10 Control (5) (+)PAI 126 .+-. 16* Control (4) (-)PAI 94
.+-. 18 Hypoxia lesioned (5) -- 93 .+-. 12 Hypoxia lesioned (6)
(+)PAI 143 .+-. 6* Numbers in parentheses are numbers of animals
tested. *P < 0.001 with respect to untreated hypoxia group or
untreated control group correspondingly.
[0216] The results in Table 5 indicate that (+)PAI caused
significant potentiation of the amphetamine-induced stereotype
behavior in both hypoxia-lesioned and control rats. (-)PAI was
totally inactive in this respect. These behavioral in vivo results
corroborate previous biochemical findings that (+)PAI is an active
inhibitor of MAO-B in the brain while (-)PAI is inactive in this
respect.
EXAMPLE 29
[0217] Effect on R(+)-PAT on the Improvement or Restoration of
Memory
[0218] Newborn rat pups subjected to a brief episode of anoxia and
then allowed to resume their growth in a normal way develop a
long-lasting impairment of memory (Speiser, et al., Behav. Brain
Res., 30, 89-94 (1988)). This memory impairment is expressed as an
inferior performance in the passive avoidance test.
[0219] The effect of R(+)-PAI and S(-)-PAI on the improvement or
restoration of memory was investigated in the passive avoidance
test. If the drug is effective, it increases the latency of
response to enter a dark compartment or chamber where an
electroshock has been experienced earlier by the rat being tested.
The latency of the maximal response is 300 seconds.
[0220] Experimental Protocol
[0221] Young rats were subjected to post-natal anoxia as described
in Example 27. R(+)-PAI or S(-)-PAI were administered according to
one of the following protocols.
[0222] Protocol A--Nursing mothers were given a dose of either
isomer of 1-1.5 mg/kg/day in drinking water until weaning at 21
days. Following that, the weaned offsprings were directly treated
with the same dose for 20 days. Treatment was terminated at 40 days
and the test was performed at 60 days, that is 20 days after the
last dose of the drug.
[0223] Protocol B--The dose was reduced to 0.5 mg/kg/day
administered to the nursing mother until weaning at 21 days, then
directly to the young rats to 60 days at which time the test was
performed.
[0224] Passive Avoidance Test--The apparatus consisted of a lit
chamber adjoining a dark chamber and a sliding door separating the
two. At training, a rat was placed in the lit chamber for 30
seconds, and then the door was opened. The rat moved to the dark
chamber with a latency that was recorded. Upon entry of the rat
into the dark compartment, the door was closed and a 0.3 mA
foot-shock was delivered for 3 seconds.
[0225] Retention (memory) after 48 hours was determined by
repeating the test and recording the latency to step through from
light to darkness to an arbitrary maximum of 300 seconds.
[0226] Results
[0227] The results of these experiments are shown in Table 6.
TABLE-US-00021 TABLE 6 EFFECT OF PAI ISOMERS ON PASSIVE AVOIDANCE
RESPONSE IN YOUNG RATS (60-DAYS OLD) Before After Group Treatment
Electroshock Electroshock PROTOCOL A Control - 49 .+-. 13 201 .+-.
111 Control (+)PAI 49 .+-. 19 220 .+-. 100(+9%)* Control (-)PAI 48
.+-. 13 192 .+-. 116 Anoxia-lesioned - 45 .+-. 11 183 .+-. 109
Anoxia-lesioned (+)PAI 49 .+-. 10 239 .+-. 99(19%)* Anoxia-lesioned
(-)PAI 55 .+-. 27 179 .+-. 123 PROTOCOL B Control - 53 .+-. 20 104
.+-. 101 Control (+)PAI 48 .+-. 11 128 .+-. 119(+23%)*
Anoxia-lesioned - 45 .+-. 8 119 .+-. 105 Anoxia-lesioned (+)PAI 52
.+-. 12 137 .+-. 126(+15%)* Anoxia-lesioned (-)PAI 48 .+-. 19 112
.+-. 112 Figures represent the latency in seconds for entering a
dark compartment where an electroshock had been first experienced
by the rat tested. *The indicated percent increases are with
respect to the corresponding anoxia or control groups.
[0228] The experimental results indicated that (+)PAI but not (-)
PAI is effective in improving the memory of anoxia-lesioned and
control rats. Drugs active in this test are considered to be
potentially useful for treatment of various memory impairment
disorders, dementia and especially senile dementia of the
Alzheimer's type.
EXAMPLE 30
Effect of R(+)-PAI on the Anoxia-Induced Hyperactive Syndrome in
Juvenile Rats
[0229] Rats that had been exposed postnatally to anoxia and then
left to grow under normal conditions show increased motor activity
in the open field at the age of 10-42 days (Hertshkowitz, et al.,
Dev. Brain Res., 7, 145-155 (1983)).
[0230] The effect of R(+)PAI and S(-)PAI on such hyperactive
syndrome was investigated.
[0231] Experimental Protocol
[0232] Anoxia was performed on rat pups on the first post-natal
day. They were placed in a glass chamber and exposed to 100%
nitrogen for 25 minutes. They were resuscitated by intermittent
massage softly applied to the chest and then returned to their
respective mothers. Control rats received the same treatment but
with air instead of nitrogen.
[0233] The R(+)-PAI or S(-)-PAI (0.5 mg/kg/day) was administered to
the nursing mothers in drinking water, thereby transferred to the
sucklings through milk.
[0234] Locomotion was measured in 6 fully computerized cages
(28.times.25 cm) by recording the number of crossings over a given
period of time. Crossings of grid infrared beams at 4-cm intervals
initiated electrical impulses which fed a counter. Recordings of
motor activity were made at the ages of 15 and 20 days, over a
period of 15 minutes.
[0235] Results
[0236] The experimental results are given in Table 7.
TABLE-US-00022 TABLE 7 EFFECT OF EACH OF THE TWO ENANTIOMERS ON THE
ANOXIA-INDUCED HYPERACTIVE SYNDROME 15-day old 20-day old Group
Treatment rats rats Control - 414 .+-. 192(11) 808 .+-. 212(12)
Control (+)PAI 254 .+-. 149(11)c 719 .+-. 110(13) Anoxia- - 482
.+-. 119(7) 858 .+-. 96(9) lesioned Anoxia- (+)PAI 276 .+-.
186(15)a 737 .+-. 150(16)c lesioned Anoxia- (-)PAI 334 .+-. 196(5)
778 .+-. 232 (6) lesioned - Numbers in parenthesis are numbers of
animals tested. The figures are the numbers of crossings of
infrared beam grid in the activity cage over a period of 15
minutes. .sup.aP < 0.001 compared to anoxia untreated group.
.sup.bP < 0.05 compared to anoxia untreated group. .sup.cP <
0.05 compared to control group.
[0237] These results indicate that chronic oral treatment with
R(+)-PAI at a dose of 0.5 mg/kg administered to the nursing mother
and reaching the milk-fed offspring significantly improved the
hyperactive syndrome. Consequently, R(+)-PAI is a potentially
useful drug for the treatment of the hyperactive syndrome in
children.
EXAMPLE 31
Stability Differences Among Ten Salts of PAI
[0238] Stability is an important factor in the selection of an
optimal salt as a therapeutic drug. Different salts may alter the
physicochemical and biological characteristics of a drug and can
have a dramatic influence on its overall properties. (Berge, S. M.,
et al., J. Pharm. Sci. 66, 1 (1977); Gould, P. L., Int. J.
Pharmaceutics, 33, 201 (1986)).
[0239] Experimental
[0240] Synthesis of PAT salts
[0241] A solution of an appropriate acid (1 mol-eg.) in 2-propanol
was added to a solution of PAI (1 mol-eq.) while stirring in
2-propanol (Ar, BHT). The salt formed was filtered, washed with
2-propanol and ether, and dried under low pressure. Yields were
between 70 to 90%. An exception in preparing PAI acetate involved
using ether as the solvent.
[0242] Analytical methods
[0243] The chromatographic separations were carried out using a
Lichrosphere 60 RP select B 5.mu. 125.times.4 mm (Merck) column, an
HPLC (Jasco BIP-1) equipped with a L-4200 UV-Vis detector
(Merck-Hitachi) set to 210 nm, and a D-2500 chromato-integrator
(Merck-Hitachi). The eluent and diluent consisted of 80% distilled
water/20% acetonitrile (HPLC grade), and 0.07 M perchloric acid
adjusted to pH 2.5 with aqueous ammonia. The flow rate used was 1
ml/min, the appropriate PA: salt solution concentration was 250
.mu.g/ml, and 20 .mu.l of the solution were injected onto the
chromatographic system.
[0244] The melting range was measured with an automatic apparatus
(Mettler FP 80) and thermo-gravimetric analysis was performed on a
Mettler TA 3000 system at a rate of 10.degree. C./min in the
applicable range. Solubility was determined by an appropriate
dilution of the supernatant from a saturated PAI salt water
solution and measured in a UVIKON 941 (Kontron) UV-Vis
spectrophotometer. The salt form (mono- or di-salt) was obtained by
elemental analysis using standard equipment for C, H, N and S
determination. The pH was measured in a it aqueous solution of the
PAI salts.
[0245] Results
[0246] The characterization of the various salts are summarized in
Table 8. TABLE-US-00023 TABLE 8 PHYSICOCHEMICAL PROPERTIES OF PAI
SALTS Melting Solubility range Salt PAI-salt pH mg/ml (.degree. C.)
% Wt. loss form m.w. tartarate 5.5 33 176.2-177.3 LT 0.1 di 492
mesylate 4.3 635 156.8-157.6 0.1 mono 267 maleate 4.0 NLT 1000
87.2-87.8 0.1 mono 287 sulphate 3.9 485 159.4-161.1 3.2 di 440
chloride 4.2 238 177.0-180.0 LT 0.5 mono 207 tosylate 4.4 60-70
129.3-129.9 LT 0.1 mono 343 fumarate 3.5 95 125.4-126.2 0.2 mono
287 phosphate 7.0 NLT 720 109.5-110.4 n.a. n.a. n.a. esylate 2.4
NLT 300 n.a. n.a. mono 279 acetate 6.1 NLT 720 69.2-69.7 0.4 mono
231 n.a. = not available
[0247] Comparative stability studies were carried out under sets of
several accelerating conditions: I) heating at 80.degree. C. for
72, 96 or 144 hours; and II) reflux in isopropanol or 30 hours. The
degradation products developed were measured by HPLC and confirmed
by TLC. The results are presented in Table 9 with the relative
retention time (relative to the PAI peak; RRT) as an area
percentage relative to to integrated peak area. TABLE-US-00024
TABLE 9 DEGRADATION PRODUCTS DEVELOPED IN PAI SALTS UNDER SHOR TERM
CONDITIONS Reflux in 80 C./72 h 80 C./144 h iPrOH/ Salt RRT.sup.a
%.sup.b RRT % RRT % sulfate ND.sup.c ND ND ND 0.47 0.22 0.60 0.72
phosphate 0.60 0.22 0.60 0.57 0.60 2.62 0.74 0.21 1.84 0.20 1.98
0.73 chloride ND ND ND ND 2.23 0.71 mesylate ND ND ND ND 0.60 0.08
maleate 0.60 0.41 n.a. 0.60 2.17 1.27 0.50 0.65 1.35 1.48 0.33 1.29
0.59 1.81 0.10 1.42 1.30 3.07 1.44 1.50 0.16 4.16 0.10 1.83 0.18
4.84 7.76 1.98 0.23 4.09 0.65 acetate 0.44 0.10 n.a. 0.60 6.74 0.60
2.56 0.74 0.35 0.73 0.13 1.76 0.33 1.29 0.71 1.84 0.16 1.55 1.06
1.99 4.17 1.75 21.85 3.60 0.27 1.96 3.33 2.15 0.08 2.32 0.15 2.83
0.15 3.54 1.82 esylate.sup.d ND ND 0.85 0.26 ND ND 1.96 0.31 limit
of quantitation = 0.08% n.a. = not available .sup.aRelative
retention time (relative to the PAI peak). .sup.bArea percentage
relative to total integrated peak area. .sup.cNo impurities
detected. .sup.dEthyl sulfate salt.
[0248] The salts were submitted to visual inspection of color an
form. The findings are shown in Table 10. TABLE-US-00025 TABLE 10
APPEARANCE OF PAI SALTS UNDER DESTRUCTIVE CONDITIONS reflux in Salt
80.degree. C./72 h 80.degree. C./96 h 80.degree. C./144 h iPrOH/30
h sulfate off white n.a. off white brown powder powder powder
phosphate brownish n.a. brown brown powder powder powder chloride
white n.a. white off white powder powder powder mesylate white n.a.
white white powder powder powder maleate brown brown n.a. brown
melted melted esylate brownish n.a. dark brown dark brown melted
melted melted n.a. = not available
[0249] These studies show that sulphate, esylate and mesylat
possess significant advantages relative to the other salt due to
good solubility and chemical stability. Of thes three salts,
mesylate is preferable due to its excellen stability even under
destructive conditions.
EXAMPLE 32
Reversal of Haloperidol-Induced Catalepsy in Mice
[0250] Male, ICR mice 25-30 g each, were pretreated with either of
the following drugs: Saline, (R)-PAI mesylate, or racemic-PAI
mesylate. All drugs were administered i.p. in a volume of 0.2 mL.
Two hours later, haloperidol was injected s.c. at a dose of 6 mg/kg
in a volume of 0.1-0.2 mL. Motor coordination tests were made at 3
hours after giving haloperidol, that is, 5 hours after
administering the presumed protective drugs.
[0251] Motor coordination tests and rigidity were quantified
according to three different parameters: (a) ability to walk the
length of a horizontal rod, 80 cm-long; (b) ability to climb down,
face down, a vertical rod, 80 cm-long; (c) duration of immobility
in an unnatural sitting posture whereby the abdomen of the mouse is
pressed against a "wall." Full performance as in
haloperidol-untreated mice is given the score of 4 in each test, or
a total of 12 in all tests. Poor performance is given a score from
1 to 3. A key to score ratings is given in Table 9A. The effects of
the various agents in antagonizing haloperidol-induced catalepsy
are given in Table 11. At three hours after haloperidol, (R)-PAI
mesylate conferred protection against haloderidol at 5-15 mg/kg,
reaching a peak after effect at 7.5-10 mg/kg (activity score=94% of
saline control) Racemic PAI mesylate conferred partial protection
in the range of 7.5-15 mg/kg, and was not active at 5 mg/kg. From
FIG. 17, it can been seen that the dose-effect profile of either
(R)-PAI mesylate or racemic PAI is such that an increase in dose
beyond 10 mg/kg entails a decrease in effect, but that the racemic
mixture is less potent throughout. This means that racemic PAI
mesylate at twice the dose of (R)-PAI mesylate will always be less
active than the (R) enantiomer.
[0252] Reversal of .alpha.-MpT-induced hypokinesia in rats
[0253] The drug .alpha.-MpT is assumed to inhibit the formation of
L-DOPA from tyrosine, and consequently the formation of dopamine
itself. Lack of CNS dopamine is expressed as hypoactivity. Six
month-old male Wistar rats (from Harlan Orkack, UK) were pretreated
with saline, (R)-PAI Mesylate or Rac PAI Mesylate, at the indicated
doses. Two hours later they received i.p. .alpha.-MpT at a dose of
100 mg/kg in 0.3-0.5 mL. Controls received saline. Following this,
motor activity was recorded in a computerized activity cage for the
duration of 10 hours. The results are given in Table 12 and FIG.
18. At 2 mg/kg, (R)-PAI Mesylate restored the level of activity to
about 90% of the saline-treated rats, but Rac PAI Mesylate was not
active. In either case, the profile of the dose-effect curve was
bell-shaped, suggesting a decrease in effect with an increase in
dose beyond a peak of 2-5 mg/kg. At 5 mg/kg Rac PAI Mesylate could
not elicit a level of activity comparable to that of (R)-PAI
Mesylate at 2 mg/kg.
[0254] From these measurements, (R)-PAI Mesylate and Rac PAI
Mesylate do not share a similar pattern of activity in the
restoration of normokinesia in haloperidol-treated mice and
.alpha.-Mpt-treated rats. At all doses studied, (R)-PAI Mesylate is
always more potent that Rac PAI Mesylate at the corresponding dose.
Also, peak activity of Rac PAI Mesylate is always lower than peak
activity of (R)-PAI Mesylate. Thus, Rac PAI Mesylate at a given
dose is always less effective than (R)-PAI Mesylate at half the
same dose. Doubling the dose of Rac PAI Mesylate with respect to
(R)-PAI Mesylate does not produce an effect equivalent to that of
(R)-PAI Mesylate.
[0255] Pharmacologically, Rac PAI Mesylate cannot be considered as
consisting of 50% active ingredient which is (R)-PAI Mesylate and
50% inert material as diluent. The presence of (S)-PAI in Rac PAI
Mesylate has an adverse effect on the activity of (R)-PAI,
resulting in a more than two-fold decrease in potency. The decrease
may be due to a direct adverse effect of (S)-PAI on behavioral
parameters. TABLE-US-00026 TABLE 11 REVERSAL OF HALOPERIDOL-INDUCED
CATALEPSY IN MICE WITH (R)-PAI MESYLATE AND RACEMIC MESYLATE Mice
received each of the test drugs i.p. at the indicated doses. Two
hours later they received haloperidol as described in the text. The
doses shown are for the free base. (R)-PAI Mesylate Rac PAI
Mesylate Dose, % of % of mg/kg Score + SE n control Score + SE n
control 1.8 7.2 .+-. 1 6 60 7.0 .+-. 0.6 6 59 3.0 6.4 .+-. 0.5 6 60
5.9 .+-. 0.7 6 49 5.0 8.7 .+-. 0.9* 6 73 6.4 .+-. 0.4 6 53 7.5 11.0
.+-. 0.4*** 5 92 9.4 .+-. 0.8++ 6 78 10 11.3 .+-. 0.3*** 6 94 9.2
.+-. 0.6*** 6 77 15 10.8 .+-. 0.5*** 5 90 8.8 .+-. 0.8* 6 73
Control 12 .+-. 0 12 100 saline Haloperidol 6.6 .+-. 0.3 16 59
alone Statistical significance with respect to haloperidol alone:
*p .ltoreq. 0.05; **p .ltoreq. 0.01; ***p .ltoreq. 0.001 by the
Student's "t" test. The scores for (R)-PAI are significantly
different from those of racemic PAI at 5 mg/kg, p .ltoreq. 0.05; at
10 mg/kg, p .ltoreq. 0.01; and at 15 mg/kg, p .ltoreq. 0.05.
[0256] TABLE-US-00027 TABLE 11A KEY TO SCORE RATING OF
HALOPERIDOL-INDUCED CATALEPSY IN MICE AND ITS REVERSAL BY VARIOUS
AGENTS Vertical Rod: Unable to grasp rod with limbs 1 Able to grasp
but slips down 2 Able to grasp, partly slips, partly climbs down 3
Able to grasp, climbs down using all limbs 4 Horizontal Rod: Unable
to grasp, falls off rod 1 Able to grasp, unable to walk on rod more
than 2 paces 2 Able to grasp, walks half-length of rod 3 Able to
grasp, walks full length of rod 4 Immobility Sitting Against Wall:
Immobility >5 min 1 Immobility 3-5 min 2 Immobility 1-3 min 3
Immobility 0.1 min 4 Fractional scores are assigned, such as 2.5,
when behavior falls between two categories, as between 2 and 3.
[0257] TABLE-US-00028 TABLE 12 RESTORATION OF MOTOR ACTIVITY IN
RATS TREATED WITH .alpha.-METHYL-p-TYROSINE (.alpha.-MpT) AT 100
mg/kg i.p. Rats received the test drugs i.p. at the indicated
doses. After two hours they received .alpha.-MpT and were
immediately placed in activity cages. Total motor activity was
automatically recorded for 10 hours, as described in the text.
(R)-PAI Mesylate Rac PAI Mesylate Dose, % of % of mg/kg Score + SE
n control Score + SE n control 2 14,132** .+-. 1457 7 89 9,035 .+-.
829 6 57 5 12,893* .+-. 1,869 7 81 10,926* .+-. 8 69 820 7.5 6,679
.+-. 414 4 42 9,698 .+-. 557 4 61 Control 15,862 .+-. 1,424 5 100
saline .alpha.-Mpt 8,108*** .+-. 810 6 51 alone Statistical
significance by the Student's "t", *p .ltoreq. 0.01; ***p .ltoreq.
0.001 for Test drugs + .alpha.-Mpt versus .alpha.-MpT alone
.alpha.-Mpt alone versus control saline The scores of (R)-PAI
versus racemic PAI are significantly different at 2 mg/kg, p
.ltoreq. 0.01.
EXAMPLE 33
The Effects of (R)-PAT Mesylate Following Closed Head Injury in
Rats
[0258] Methods
[0259] 1. Induction of trauma
[0260] Head trauma was induced in male rats under ether anesthesia
by a well calibrated weight-drop device that falls over the exposed
skull, covering the left cerebral hemisphere, 1-2 mm lateral to the
midline, in the midcoronal plane.
[0261] 2. Evaluation of motor function
[0262] One hour after induction of trauma, the rats were tested by
a set of criteria which evaluated their neurologic outcome (the
criteria described by Shohami, et al., J. Neurotrauma, 10, 113
(1993)). These criteria, referred to as the Neurological Severity
Score (NSS), consist of a series of reflexes and motor functions.
Points are given based on deficits in these criteria. At 24 h the
rats were re-evaluated.
[0263] 3. Evaluation of brain edema
[0264] The brains were removed after the second evaluation of motor
function (24 h). A piece of tissue (-20 mg) was weighed to yield
wet weight (WW). After drying in a desiccator oven for 24 h at
90.degree. C., it was reweighed to yield dry weight (DW). Water
percentage in the tissue was calculated as
(WW-DW).times.100/WW.
[0265] 4. Drug treatment
[0266] (R)-PAI Mesylate was dissolved in water. The rats were
injected intraperitoneally at a dose of 0.1 mg/kg, 0, 4, 8 and 12 h
after induction of head trauma. Control rats were treated with
water at the same times.
[0267] Results
[0268] The NSS, which measures the "clinical" status of the rats,
was almost identical in the treated and nontreated groups at 1 hour
after the head injury, but significantly lower at 24 hours in the
(R)-PAI mesylate-treated rats (Table 13). These results indicate
that PAI mesylate is effective in improving motor function recovery
following closed head injury in rats.
[0269] At 24 hours after trauma, a major edema was found in the
hemisphere (85.4% water in the brain of control rats vs. 78.5% in
undamaged brain tissue). PAI mesylate was effective in reducing
edema as verified by its effect on the percent of water.
[0270] In conclusion, the results reported herein demonstrate that
(R)-PAI mesylate has neuroprotective properties in a model intended
to mimic human nerve injury and to induce trauma to a closed skull.
TABLE-US-00029 TABLE 13 NSS .DELTA. NSS % H.sub.20 1 h 24 h (1 h-24
h) in the brain control 15.6 12.3 4.3 .+-. 0.5 85.4 .+-. 0.4 (n =
6) (R)-PAI 16.7 10.2 6.5 .+-. 0.7* 82.1 .+-. 0.6** Mesylate (n = 6)
*P < 0.05 (Mann Whitney U-test) **P < 0.005 (t-test)
EXAMPLE 34
Effects of PAT Mesylate on Prevention of NMDA Induced Cell Death of
Cerebellum Cell Cultures
[0271] Results of in vitro assays
[0272] Procedures: Cultures of mechanically dissociated neonatal
rat cerebellum, The cerebella are dissected aseptically from 6 or
7-day-old rat pups and placed in a 15 ml sterile plastic conic tube
containing 3 ml of enriched medium (the medium is made up of
Dulbecco's modified Eagle's medium (DMEM) with high glucose
concentration (1 g/l), 2 mM (v/v) L-glutamine, antibiotic
antimitotic mixture, and enriched with 15% (v/v) heat-inactivated
fetal calf serum). The cerebella are then dissociated after 20-25
passages through a sterile 13 gauge, 10 cm long stainless steel
needle attached to a 5 ml syringe with an inserted 45 .mu.m pore
size nylon sieve. The dissociated cells are centrifuged at 200 g
for 5 minutes, the supernatant discarded and the cells resuspended
in enriched medium. The cell viability is determined by the trypan
blue exclusion test. The cells are then plated at a density of
200/mm.sup.2 on poly-L-lysine-coated surfaces (Poly-L-lysine-coated
glass coverslips are prepared at least 1 hour before plating, by
immersing in a sterile distilled water solution containing 15
.mu.g/ml poly-L-lysine, and just before use, washing with sterile
water and drying), covered with enriched medium and incubated at
37.degree. C. in an atmosphere of 5% CO.sub.2 in air and 100%
humidity. After 4 days in culture, the media are replaced with
media containing the desired test compounds. Experiments are done
in duplicate and repeated 2 or 3 times. After determining the test
compound toxic dose-response, four groups are compared: (I) control
(enriched medium alone), (II) test compound (one subgroup for each
concentration (2 concentrations are tested)), (III)
N-methyl-D-aspartate (NMDA, exposure to a concentration of 1 mM for
3 h) as the cytotoxic challenge, (IV) test compound plus NMDA (one
subgroup for each of the 2 concentrations of test compounds), (V)
control group to test the effect of solvent (in which the test
compound is dissolved), and (VI) an additional "positive control"
group of spermine (0.01 .mu.M dissolved in culture medium) plus
NMDA. Nerve cell survival is evaluated by phase contrast microscopy
and trypan blue staining after 24 h.
[0273] Results
[0274] It is well established that glutamic acid (Glu) possesses
neurotoxic properties which are expressed in several neurological
disorders including epilepsy and stroke, and most likely also in
brain neurodegenerative diseases such as Parkinson's disease,
Alzheimer's disease and traumatic brain injury. The neurotoxic
effects of Glu are mediated by membrane bound Glu receptors, such
as N-methyl-D-asparate (NMDA) receptors.
[0275] The results, as shown in Table 14, demonstrate that 10 .mu.M
of (R)-PAI mesylate increased the survival of cerebellum cells by
27 percent following 1 .mu.M NMDA exposure. These in vitro results
support the in vivo effects of (R)-PAI mesylate presented in
Examples 33 and 35, indicating that this drug has neuroprotective
properties against neurotoxic concentration of NMDA. TABLE-US-00030
TABLE 14 NEUROPROTECTIVE EFFECT OF (R)-PAI MESYLATE ON PREVENTION
OF NMDA-INDUCED CELL DEATH OF CEREBELLUM CELLS Experimental Group
Surviving Cells Percent Protection Cerebellar Cultures (Toxicity
TD.sub.25 = 30 .mu.M; TD.sub.50 = 85 .mu.M; TD.sub.100 = 320 .mu.M)
Control 100 Solvent 97 NMDA 10 Solvent + NMDA 10 0 Compound + NMDA:
1) 0.01 .mu.M + NMDA 12 2 2) 1.00 .mu.M + NMDA 22 12 3) 10.00 .mu.M
+ NMDA 37 27 Spermine + NMDA 75 65
[0276] Values, expressed as the percent of untreated controls,
represent the average of 2 experiments run in duplicate for culture
experiments, and the mean.+-.SEM of 4 animals for ischemia. The
percent protection value is the effect of the test compound after
subtraction of the solvent effect.
EXAMPLE 35
Effects of (R)-PAI Mesylate After Graded Crush of the Rat Optic
Nerve
[0277] Neuroprotective effects of (R)-PAI Mesylate were determined
for application immediately after crush injury of the optic nerve
in the adult rat. Short-term effects were measured metabolically,
and long-term effects electrophysiologically.
[0278] Methods
[0279] 1. Metabolic measurements
[0280] a) General. The method is described by Yoles, et al.,
Investigative Ophthalmology & Visual Science, 33, 3586-91
(1992). At short terms, metabolic measurements were monitored in
terms of the mitochondrial NADH/NAD ratio, which depends on the
activity of the electron transport system, and thus indicate levels
of energy production. Changes in ability of the nerve to produce
energy as a consequence of injury were determined by comparing NADH
levels in response to artificial transient anoxic insult before and
after the injury.
[0281] b) Surface fluorometry--reflectometry. Monitoring of the
intramitochondrial NADH redox state is based on the fact that NADH,
unlike the oxidized form NAD.sup.-, fluoresces when illuminated at
450 nm. A flexible Y-shaped bundle of optic fibers (light guide)
was used to transmit the light to and from the optic nerve. The
light emitted from the nerve was measured at two wavelengths: 366
nm (reflected light) and 450 nm (fluorescent light). Changes in the
reflected light were correlated with changes in tissue absorption
caused by hemodynamic effects and with movements of the optic nerve
secondary to alterations in arterial blood pressure and nerve
volume. The fluorescence measurements were found to be adequately
corrected for NADH redox state measurements by subtraction of the
reflected light (366 nm) from the fluorescent light (1:1 ratio) to
obtain the corrected fluorescence signal.
[0282] c) Animal preparation. Animal utilization was in accord with
the ARVO Resolution on the use of animals in research. Male
Sprague-Dawley (SPD) rats weighing 300-400 g were anesthetized with
sodium pentobarbitone (50 mg/kg intraperitoneally). With the
animal's head held in place by a head holder, a lateral canthotomy
was performed under a binocular operating microscope and the
conjuctiva was incised lateral to the cornea. After separation of
the retractor bulbi muscles, the optic nerve was identified and a
length of 3-3.5 mm was exposed near the eyeball by blunt
dissection. The dura was left intact and care was taken not to
injure the nerve. A special light-guide holder was implanted around
the optic nerve in such a way that the light guide was located on
the surface of the optic nerve 1 mm distal to the injury site.
Animals, while still anesthetized, were allowed to recover for 30
minutes from the surgical procedures and were then exposed to
anoxic conditions. An anoxic state was achieved by having the rat
breathe in an atmosphere of 100% nitrogen for 2 minutes after which
time it was returned to air. In order to evaluate the metabolic
activity of the optic nerve, the relative changes in reflected and
fluorescent light intensities in response to anoxia were measured
before and after crush injury.
[0283] d) Experimental protocol for crush injury and metabolic
measurements. With the aid of calibrated cross-section forceps, a
moderate crush injury was inflicted on the nerve between the eye
and the light guide holder at a pressure corresponding to 120 g for
30 sec. Immediately after injury, animals received intraperitoneal
injections of water with and without (R)-PAI Mesylate (2 mg/kg). To
assess the activity of the energy production system, NADH response
to 2 minutes of anoxia was measured in all animals prior to injury,
30 minutes after injury, and thereafter at hourly intervals up to 4
hours (see FIG. 19).
[0284] 2. Electrophysiologically Measurements. This method is
described by Assia, et al., Brain Res., 476, 205-212 (1989). Animal
preparation and optic nerve injury were preferred as in the
metabolic studies. Immediately after injury, animals received a
single injection of water with or without (R)-PAI Mesylate (0.5
mg/kg). Fourteen days after injury and treatment, the optic nerves
were excised and measured electrophysiologically. Prior to removal
of optic nerves for electrophysiological measurement, the rats were
deeply anesthetized with 70 mg/kg pentobarbitone. The skin was
removed from the skull and the optic nerves were detached from the
eyeballs. Subtotal decapitation was performed and the skull was
opened with a rongeur. The cerebrum was displaced laterally,
exposing the intracranial portion of the optic nerve. Dissection
was at the level of the nerve, which was transferred to vials
containing fresh salt solution consisting of NaCl (126 mM), KCl (3
mM), NaH.sub.2PO, (1.25 mM), NaHCO.sub.3 (26 mM), MgSO.sub.4 (2
mM), CaCl.sub.2 (2 mM), and D-glucose (10 mM), and aerated with 95%
O.sub.2 and 5% CO.sub.2 at room temperature. The nerves were kept
in this solution, in which electrical activity remained stable for
at least 3-4 hours. After 0.5 hours of recovery at room
temperature, electrophysiological recordings were obtained from the
nerve distal to the crush lesion. The nerve ends were then
connected to two suction Ag-AgCl electrodes immersed in a bathing
solution at 37.degree. C. A stimulating pulse was applied through
the electrode at the proximal end and the action potential was
recorded by the distal electrode. A Grass SD9 stimulator was used
for supramaximal electrical stimulation (0.5 pps). The measured
signal was transmitted to a Medelec PA36 preamplifier and then to
an electromyograph (Medelec MS7, AA7T amplifier). The solution,
stimulator, and amplifier had a common ground. The maximum
amplitude of eight averaged compound action potentials (CAPs) was
recorded and photographed with a Polaroid camera. The CAP values
measured in contralateral uninjured nerves served as a
reference.
[0285] Results
[0286] The results demonstrate that (R)-PAI Mesylate applied
immediately after optic nerve injury blocked the injury-induced
reduction in energy production. (R)-PAI Mesylate also has a
long-term effect measured by electrophysiological monitoring.
[0287] The CAP (compound action potentials) amplitude is directly
correlated with the number of conducting fibers in the tested
segment of the nerve.
[0288] (R)-PAI Mesylate significantly attenuated the injury-induced
loss of activity in the distal segment of the injured nerve,
indicating that (R)-PAI Mesylate is a neuroprotective agent or at
least slows down degeneration. TABLE-US-00031 TABLE 15
Electrophysiological Measurements CAP amplitude (.mu.V) Group (Mean
.+-. Std. Error.) Vehicle 441 .+-. 95 N = 13 (R)-PAI 2104 .+-. 313*
Mesylate N = 7
EXAMPLE 36
Comparison of Anticonvulsive Properties of R-PAI end S-PAI
Salts
[0289] Both (R)-PAI and (5)-PAI HCl salts have significant
anticonvulsant activities. In mice (i.p. administration) in the
maximal electroshock test (MES test), (S)-PAI HCl has greater
anticonvulsant activity (ED.sub.50=57 mg/kg) than (R)-PAI HCl
(ED.sub.50=79 mg/kg). Analogous results were observed in rats (p.o.
administration). Four out of four rats were protected from seizures
in the MES test when 50 mg/kg of (S)-PAI HCl was administered,
whereas three out of four mice were protected after the same dose
of (R)-PAI HCl. With respect to efficacy for Parkinson's disease,
the enhanced anticonvulsant activity is a detrimental side effect.
The same trend occurs with the mesylate salts. (S)-PAI Mesylate has
greater anticonvulsant activity than (R)-PAI Mesylate in the MES
test. At doses of 100 mg/kg, (S)-PAI Mesylate protected three out
of three mice, whereas only one out of three mice was protected
with (R)-PAI Mesylate.
[0290] The MES test is a classical model to indicate efficacy for
partial and generalized seizures in humans. The agents' mechanism
of action is via their ability to prevent the spread of seizures.
Some agents, however, that prevent seizure spread have the side
effect of lowering seizure threshold. These agents therefore have
both proconvulsive and anticonvulsive side effects.
[0291] Results herein show that (S)-PAI Mesylate has proconvulsive
activity. In the Timed Intravenous Infusion of Metrazol test, 141
mg/kg of (S)-PAI Mesylate reduces the time, and therefore the
amount of Metrazol, required to induce the appearance of both the
first focal seizure and the onset of clonus. Other agents that are
classically used for partial and generalized seizures, such as
phenytoin and carbamazepine, do not show this effect. (H. J.
Kupferberg, Epilepsia, 30, s51-s56 (1989)). Likewise, (S)-PAI
Mesylate showed a significantly higher acute neurotoxicity than
(R)-PAI Mesylate. At 300 mg/kg, (R)-PAI Mesylate did not show any
neurotoxicity with mice in the rotorod ataxia test. With (S)-PAI
Mesylate, four out of four mice showed neurotoxicity and
spasticity.
[0292] Methods
[0293] TD.sub.50 (median toxic dose). This test measures
neurological deficits by the rotorod ataxia test. A mouse is placed
on a knurled rod rotating at 6 rpm. It is then determined whether a
mouse has the ability to maintain its equilibrium and stay on the
rod for one minute in each of three trials.
[0294] Timed Intravenous Infusion of Metrazol Test. This test
measures the minimal seizure threshold of each animal. Metrazol is
infused at 0.185 mg/ml into the tail veins of mice. The time is
then recorded (sec) from the start of infusion until the appearance
of the first twitch (first focal seizure) and onset of clonus
(clonic seizure). Proconvulsants require less Metrazol to produce
these symptoms and therefore show endpoints at a shorter period of
time.
EXAMPLE 37
Peripheral Effects of (R)-PAI and (S)-PAI on the Contractility of
Intestinal Smooth Muscle Preparations
[0295] Peripheral effects of the hydrochloride salts of the
enantiomers of PAI were determined in isolated rabbit or guinea-pig
small intestine. These observations provide useful information on
their relative peripheral side effects in humans. The first point
of contact of the subject with an orally administered drug is the
gastrointestinal tract where concentrations of the drug are much
higher than after absorption and distribution. In the case of PAI
hydrochloride (MW=208), a 10 mg oral dose contained in a liquid
volume of about 100 ml would be equivalent to a concentration of
about 0.5 mM. In contrast, the therapeutic plasma concentration of
(R)-PAI hydrochloride is in the nanomolar range.
[0296] The effect of the enantiomers of PAI in the isolated rabbit
jejunum and the guinea-pig ileum were determined so as to find out
whether the intake of (S)-PAI together with (R)-PAI (as found in
racemic PAI) would produce side effects absent in the
administration of pure (R)-PAI. (R)-PAI is the preferred enantiomer
for the inhibition of MAO-B in the brain, in view of its potency
and high selectivity towards this form of the enzyme. (S)-PAI is
much less potent than (R)-PAI an this respect and is also not
selective toward MAO-B. In principle, its presence in PAI racemate
might be tolerated or overlooked provided (S)-PAI is inert at the
recommended doses of (R)-PAI. The results provided in Tables 16-19
show that (S)-PAI is not an inert substance. On the contrary, in
the guinea-pig ileum, it is a more potent relaxant than (R)-PAI.
Hence its peripheral effects cannot be discounted as negligible.
These data show that there would be fewer peripheral side effects
in the administration of pure (R)-PAI than in the administration of
racemic PAI containing an equivalent dose of (R)-PAI.
TABLE-US-00032 TABLE 16 TYRAMINE POTENTIATION BY EACH OF THE TWO
ENANTIOMERS OF PAI HCl IN RATE JEJUNUM PREPARATION A stretch of
rabbit jejunum, mounted in an organ bath, displays rhythmic
contractions that are inhibited by norepinephrine but not by
tyramine. If however the jejunum is pretreated with a monoamine
oxidase inhibitor such as PAI, then tyramine causes relaxation of
the spontaneous contractions. The extent of relaxation can be
correlated with the relative potency of the inhibitor. Percent Drug
and concentration (.mu.M) relaxation Tyramine alone 40 0
Norepiniphrine 0.002 100 (R)PAI alone 0.2-4.0 0 (S)PAI alone
0.2-4.0 0 Tyramine 40 after (R)PAI 0.2 67 2 88 40 85-90 after
(S)PAI 0.2 0 2 35 40 33-50
[0297] Results
[0298] (S)-PAI is much less potent than (R)-PAI as an inhibitor of
brain MAO-B. Therefore, (S)-PAI is not a useful agent for the
prevention of brain dopamine degradation, but can potentiate the
tyramine-evoked release of norepinephrine in the small intestine.
Its activity in the small intestine is an undesirable side effect
as it is expected to increase the absorption and action of
undegraded tyramine. Thus, (S)-PAI is not an inert substance when
used together with (R)-PAI as found in racemic PAI. TABLE-US-00033
TABLE 17 ANTAGONISM OF BETHANECHOL-INDUCED CONTRACTIONS OF THE
GUINEA PIG ILEUM PREPARATION IN THE PRESENCE OF 400 .mu.M OF EACH
OF THE TWO ENANTIOMERS OF PAI HCl A stretch of guinea-pig ileum
mounted in a physiological solution in an organ bath contracts
dose-dependently when treated with bethanechol which is an
enzymatically stable analog of the natural gastrointestinal
neurotransmitter acetylcholine. These contractions are attenuated
in the presence of PAI. The data are expressed in gram-tension.
gram-tension Bathenechol (.mu.M) control (R)PAI control (S)PAI) 0.8
0.5 0.2 0.6 0 2 1.5 0.3 2.0 0 4 2.2 0.7 3.0 0 8 4.0 1.0 3.8 0.6 20
5.6 2.0 3.8 1.2 40 6.2 2.8 3.8 1.7 80 6.2 3.1 3.8 2.6 200 6.2 4.3
3.8 2.6
[0299] Results
[0300] (S)-PAI is almost inactive as a MAO-B inhibitor with respect
to (R)-PAI, and hence is not effective in preventing the
degradation of brain dopamine. However, it is more effective than
R(PAI) in the prevention of the bethanechol-induced contraction of
the small intestine. Thus (S)-PAI is not an inert substance when
used with R(PAI) as found in racemic PAI. TABLE-US-00034 TABLE 18
ANTAGONISM OF THE HISTAMINE-INDUCED CONTRACTIONS OF THE GUINEA-PIG
ILEUM PREPARATION BY EACH OF THE TWO ENANTIOMERS OF PAI HCl A fixed
dose of histamine (40 nM) causes a sustain contraction of a stretch
of guinea-pig ileum mounted physiological solution in an organ
bath. Increment addition of each of the two enantiomers of PAI HCl
causes dose-dependent relaxation of the muscle. Results a expressed
as percent relaxation with respect to the bas line before addition
of histamine, which is taken as 10 relaxation. PAI Percent
relaxation concentration .mu.M (R)PAI (S)PAI 2 0 11 4 0 15 10 0 30
20 20 30 31 33 40 37 36 100 81 71 200 90 300 92 400 100 98 700 100
1000 100
[0301] Results
[0302] (S)-PAI is inactive with respect to (R)-PAI as a MA
inhibitor in the brain, and hence useless for preventing
degradation of brain dopamine, but is more active than (R) isomer
in causing relaxation of intestinal smo muscle. Thus, (S)-PAI is
not an inert substance when taken together with the (R)isomer as
found in racemic PAI. TABLE-US-00035 TABLE 19 ANTAGONISM OF THE
BETHANECHOL-INDUCED CONTRACTIONS OF THE GUINEA-PIG ILEUM
PREPARATION BY EACH OF THE TWO ENANTIOMERS OF PAI HCl A fixed dose
of bethanechol (0.8 .mu.M) causes a sustained contraction of a
stretch of guinea-pig ileum mounted in physiological solution in an
organ bath. Incremental addition of each of the two enantiomers of
PAI HCl causes a dose-dependent relaxation of the preparation.
Results are expressed as percent relaxation with respect to the
base- line before addition of histamine, which is taken as 100%
relaxation. PAI Percent relaxation concentration .mu.M (R)PAI
(S)PAI 20 25 40-50 60 25-50 60-70 100 50-70 100 300 100 100
[0303] Results
[0304] (S)-PAI is inactive with respect to (R)-PAI as a MAO-B
inhibitor in the brain, and hence useless for the prevention of the
degradation of brain dopamine, but is more active than the (R)
isomer in causing relaxation of intestinal smooth muscle. Thus,
(S)-PAI is not an inert substance when taken together with the (R)
isomer as found in racemic PAI.
EXAMPLE 38
Some Effects of [R](+)PAI Mesylate in Middle Cerebral Artery
Occlusion in the Rat as a Model for Stroke
[0305] Methods
[0306] 1.1. Middle cerebral artery occlusion (MCAO) in the rat. A
modification of the procedure described by Tamura et al (1981) was
used. Male Wistar rats (Olac England-Jerusalem) 300-400 g each were
anesthetized with a solution of Equitesine administered i.p. at a
dose of 3 mL/kg. Equitesine consists of 13.5 mL sodium pentothal
solution (60 mg/mL), 3.5 g chloral hydrate, 1.75 g MgSO.sub.4, 33
mL propylene glycol. 8.3 mL absolute alcohol made up to 83 mL with
distilled water. Surgery was performed with the use of a high
magnification operating microscope, model SMZ-2B, type 102 (Nikon,
Japan). In order to expose the left middle cerebral artery, a cut
was made in the temporal muscle. The tip of the coronoid process of
mandible was excised as well and removed with a fine rongeur.
Craniectomy was made with a dental drill at the junction between
the median wall and the roof of the inferotemporal fossa. The dura
matter was opened carefully using a 27 gauge needle. The MCA was
permanently occluded by microbipolar coagulation at low power
setting, beginning 2-3 mm medial to the olfactory tract between its
cortical branch to the rhinal cortex and the laterate striate
arteries.
[0307] After coagulation, the MCA was severed with microscissors
and divided to ensure complete occlusion. Following this, the
temporalis muscle was sutured and laid over the craniectomy site.
The skin was closed with a running 3-0 silk suture. A sham
craniectomy operation was performed on a parallel group of rats,
but without cauterization of the MCA. During the entire surgical
operation (20-25 min) in either group, body temperature was
maintained at 37 to 38.degree. C. by means of a body-temperature
regulator (Kyoristsu, Japan) consisting of a self-regulating
heating pad connected to a rectal thermistor. At 24 hours post
surgery a neurological score was taken in order to assess the
severity of the injury in the drug-treated rats with respect to
their untreated controls. At 46 hours, the animals were
anesthetized with Equitesine and the severity of the injury was
visualized by an MRI procedure. The volume of brain tissue
incurring damage following ischemia was determined.
[0308] 1.2. Drug administration
[0309] [R](+)PAI Mesylate was administered as an i.p. injection in
0.3-0.4 mL distilled water, according to the following
schedule:
[0310] 1 mg/kg immediately after surgery.
[0311] 0.5 mg/kg 2 hours after surgery
[0312] 1 mg/kg 20-24 hours after surgery
[0313] 1.3. MRI scan of ischaemic brain lesion
[0314] All experiments were performed using a 4.7T BIOSPEC system
(BRUKER) (See T. Back, et al., "Diffusion Nuclear Magnetic
Resnonance Imaging in Experimental Stroke: Correlation with
Cerebral Metabolites," Stroke (February 1994) 25: 494-500).
Forty-eight hours after MCAO or sham operation, every animal was
subjected to a fast multislices T1 weighted imaging (TR/TE).
(500/25) for positioning. Then multislices T2-weighted images
(3000/80) were acquired (5 contiguous slices, 3 mm thick).
[0315] The size and severity of the infarcted area was estimated
using the hyperintensity observed in the T2 weighted MRI at 48
hours post-occlusion or post sham-operation. The following MRI
parameters were determined for each group of rats:
[0316] c. Ischemic area (in mm.sup.2)
[0317] d. Area of the ischemic hemisphere (in mm.sup.2 )
[0318] e. Area of the unaffected hemisphere (in mm.sup.2)
[0319] The use of contiguous slices allows the conversion of area
units into volume units by simply multiplying the area value by the
slice thickness
[0320] 1.4. Neurological score
[0321] The neurological score consists of the sum total of a series
of ratings assigned to the performance of specific locomotor
activities in a given rat. The scale runs from 0 (fully normal
rats) to 13 (fully incapacitated rats). Most parameters are rated
as either 0 (normal), or 1 (incapacitated); others are graded. The
following tests were used in the present study:
[0322] General observational tests: hypoactivity; sedation;
piloerection
[0323] Motor reflex. Rats were lifted by the tail about 15 cm above
the floor. Normal rats assume a posture in which they extend both
forelimbs towards the floor and spread the hind limbs to the sides
in a trapeze-like manner. MCAO when severe causes consistent
flexion of the contralateral limb.
[0324] Motor ability. This is seen as the ability to grasp a rod 1
cm in diameter by the contralateral limb for 5-15 sec when the rat
is left hanging on the rod through the arm pit.
[0325] Motor coordination. Normal rats are able to walk up and down
a beam 5 cm wide placed at a moderate slant. Failure to walk the
beam in either direction reveals some motor incoordination, lack of
balance and limb weakness.
[0326] Gait. Ability to restore normal position to either hind
contralateral limb when intentionally displaced while on a narrow
beam.
[0327] Balance. Ability to grasp and balance on a narrow beam 2 cm
wide.
[0328] Locomotor activity. Total movements over a period of 15 min
in an automated activity cage.
[0329] Ratings assigned to each of the above parameters are given
in Table 20. TABLE-US-00036 TABLE 20 Neurological scores assigned
to each of 10 parameters of posture and locomotion Parameter Score
a. Activity in the home cage normal = 0 hypoactive = 1 b. Sedation
none = 0 marked = 1 c. Piloeretion none = 0 marked = 1 d. Extension
of contralateral good = 0 forelimb towards floor when flexed limb =
1 lifted by tail e. Spread of contralateral hind good = 0 limb when
lifted by tail flexed limb = 1 (trapezoid posture) f. Grasp rod
with contralateral limb good = 0 poor = 1 for 5-15 sec. when
suspended by the armpit g. Walk on beam 5-cm wide good = 0 poor = 1
h. Restoration of contralateral good = 0 hind and or fore limb to
original poor = 1 (one limb) position when intentionally 2 (two
limbs) displaced i. Grasping and balance on beam good = 0 poor = 1
2-cm wide j. Motor activity with respect to .ltoreq.25% of control
3 control (for 15 min in an automated 26-50% of control 2 activity
cage 51-75% of control 1 76-100% of control 0
[0330] 2. Results
[0331] 2.1. Infarct size
[0332] The results of the MRI study are summarized in Table 21 and
FIG. 20. The infarct size was significantly smaller in [R](+)PAI
Mesylate-treated rats (n=9) than in untreated rats (n=10). In the
former, the infarct size was about 60% of that in the untreated
animals. TABLE-US-00037 TABLE 21 Ischaemic brain lesion evaluation
by MRI T2-SCAN - 48 following MCA-Occlusion and [R](+)PAI Mesylate
treatment Wistar Rats. MCA-O + MCA-O [R](+)PAI Mesylate* Infarct
size Infarct size Animal No. (mm.sup.3) Animal No. (mm.sup.3) 1 252
1 94.4 2 272 2 139 3 314 3 240 4 273 4 137 5 201 5 137 6 221 6 174
7 358 7 164 8 265 8 171 9 341 9 215 10 236 MEAN .+-. SD 273.3 .+-.
50.9 MEAN .+-. SD 163.5 .+-. 43.9 t = 5.0475 f = 17 p < 0.001
[R](+)PAI Mesylate reduces infarct size by 40% significantly
*[R](+)PAI Mesylate administered: Time after MCA-Occlusion: 0 - 1.0
mg/kg ip; 2 hrs - 0.5 mg/kg ip; 24 hrs - 1.0 mg/kg ip.
[0333] 2.2. Neurological score
[0334] The neurological score in five [R](+)PAI Mesylate treated
rats and six untreated rats were determined by a blinded observer.
The results are given in Table 22 where they are compared with the
infarct size in each animal as determined by the MRI test, and also
in FIG. 21. It can be seen that those animals with the least
neurological scores were those treated with [R](+)PAI Mesylate. The
neurological score was reduced by 54% and the infarct size by 36%
in [R](+)PAI Mesylate-treated MCAO rats as compared to untreated
ones. TABLE-US-00038 TABLE 22 Neurological score of Rats submitted
to MCA-Occlusion and [R](+)PAI Mesylate treatment with relation to
their ischaemic infarct size. MCA-O MCA-O + [R](+)PAI Mesylate***
Animal Neurological* Infarct size** Animal Neurological* Infarct
size** No. Score (mm.sup.3) No. Score (mm.sup.3) 1 5.0 201 1 1.0
137 2 5.0 221 2 2.0 174 3 6.0 358 3 4.0 164 4 6.0 265 4 4.0 171 5
8.25 341 5 2.88 215 6 5.75 236 MEAN + SD 6.0 .+-. 1.19 270 .+-. 65
MEAN + SD 2.78 .+-. 1.3 172 .+-. 28 Neurological Score Infarct Size
t = 4.25 t = 3.34 f = 9 f = 9 p < 0.01 p < 0.01 [R](+)PAI
Mesylate reduces the neurological score by 53.7% and infarct size
by 36.3%. *Examined 24 hrs after MCA-Occlusion. **Evaluated by MRI
T2-SCAN 48 hrs after MCA-Occlusion. ***[R](+)PAI Mesylate
administered: Time after MCA-Occlusion: 0 1.0 mg/kg ip; 2 hrs 0.5
mg/kg ip; 24 hrs 1.0 mg/kg ip.
REFERENCES FOR EXAMPLE 38
[0335] Cechetto D F, Wilson J X, Smith K E, Wolski D, Silver M D,
Hachinski V C (1989). Autonomic and myocardial changes in middle
cerebral artery occlusion: stroke models in the rat. Brain Res
502:296-305. [0336] Kolb E, Sutherland R J, Whishaw I Q (1983). A
comparison of the contributions of the frontal and parietal
association cortex to spatial localizations in the rat. Behav.
Neurosci. 97:13-27. [0337] Menzies S A, Hoff J T, Betz A L (1992).
Middle cerebral artery occlusion in rats: A neurological and
pathological evaluation of a reproducible model. Neurosurgery
31:100-106. [0338] Sauer D, Allegrini P R, Cosenti A, Pataki A,
Amaceker X, Fagg G E (1993). Characterization of the
cerebroprotective efficacy of the competitive NMDA receptor
antagonist CGP40116 in a rat model of focal cerebral ischemia: An
in vivo magnetic resonance imaging study. J. Cerebr. Blood Flow and
Metabol. 13:595-602. [0339] Stephanovich C, Editor, Stroke: Animal
Models, Pergamon Press, 1983 [0340] Tamura A, Graham D I, McCulloch
J, Teasdale G H (1981). Focal cerebral ischemia in the rat: 1.
Description of technique and early neuropathological consequences
following MCA occlusion. J. Cereb. Blood Flow and Metab. 1:53-60.
[0341] Teasdale G, Tyson G, Tamura A, Graham D I, McCulloch J.
Focal cerebral ischaemia in the rat: Neuropatholgy, local cerebral
blood flow and cerebrovascular permeability. In Stroke: Animal
Models, Stephanovic C, Editor, Pergamon Press 1983, pp. 83-97.
[0342] Yamamoto M, Tamura A, Kirino T, Shimitzu M, Sano K (1966).
Behavioral changes after focal cerebral ischemia by left middle
cerebral artery occlusion in rats. Brain Re. 452:323-328. [0343]
Yamori Y et al. (1976). Pathogenic similarity of strokes in stroke
prone spontaneously hypertensive rats and humans. Stroke 7:46-53.
[0344] Young W. DeCrescito V, Flamm E S, Hadani M, Rappaport H,
Cornu P (1986), Tissue Na, K, and Ca changes in regional cerebral
ischemia: Their measurement and interpretation. Central Nervous
System Trauma, 3:215-234.
* * * * *