U.S. patent application number 13/603150 was filed with the patent office on 2012-12-27 for dopamine transporter inhibitors for use in treatment of movement disorders and other cns indications.
This patent application is currently assigned to Prexa Pharmaceuticals, Inc.. Invention is credited to James R. Hauske.
Application Number | 20120329829 13/603150 |
Document ID | / |
Family ID | 36693103 |
Filed Date | 2012-12-27 |
View All Diagrams
United States Patent
Application |
20120329829 |
Kind Code |
A1 |
Hauske; James R. |
December 27, 2012 |
Dopamine Transporter Inhibitors for Use in Treatment of Movement
Disorders and Other CNS Indications
Abstract
The invention provides a class of dopamine transporter
inhibitors of formula (I) (DAS inhibitors), packaged
pharmaceuticals comprising such inhibitors, and their uses
treating, or the manufacturing medicaments for treating disease
conditions, including Parkinson's disease, Hoehn and Yahr Staging
of Parkinson's Disease. Unified Parkinson Disease Rating Scale
(UPDRS), and Schwab and England Activities of Daily Living Scale.
Related business methods such as marketing the inhibitors to
healthcare providers are also provided.
Inventors: |
Hauske; James R.; (La Jolla,
CA) |
Assignee: |
Prexa Pharmaceuticals, Inc.
Boston
MA
|
Family ID: |
36693103 |
Appl. No.: |
13/603150 |
Filed: |
September 4, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11884965 |
Sep 5, 2008 |
8258305 |
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PCT/US06/06338 |
Feb 21, 2006 |
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13603150 |
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60655978 |
Feb 23, 2005 |
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Current U.S.
Class: |
514/312 ;
514/317 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 3/04 20180101; A61P 25/14 20180101; A61P 25/20 20180101; G06Q
99/00 20130101; A61P 43/00 20180101; C07D 211/22 20130101; A61P
25/30 20180101; A61P 25/28 20180101; A61P 15/00 20180101; A61P
25/24 20180101; A61P 25/16 20180101 |
Class at
Publication: |
514/312 ;
514/317 |
International
Class: |
A61K 31/445 20060101
A61K031/445; A61P 25/00 20060101 A61P025/00; A61P 25/14 20060101
A61P025/14; A61P 3/04 20060101 A61P003/04; A61P 25/30 20060101
A61P025/30; A61P 25/16 20060101 A61P025/16; A61K 31/4709 20060101
A61K031/4709; A61P 25/24 20060101 A61P025/24 |
Claims
1. A method of treating a CNS disorder selected from the group
consisting of a movement disorder, a depressive disorder, a sleep
disorder, obesity, attention deficit disorder (ADD), attention
deficit hyperactivity disorder (ADHD), sexual dysfunction, and
substance abuse; the method comprising administering to a subject
in need thereof a therapeutically effective amount of a compound
represented by Formula I, or a pharmaceutically acceptable salt
thereof ##STR00009## wherein, as valence and stability permit, Ar,
independently for each occurrence, represents a substituted aryl or
heteroaryl ring, and Ar is substituted with one or more groups
selected from halogen, cyano, alkyl, alkenyl, alkynyl, aryl,
hydroxyl, alkoxy, silyloxy, amino, nitro, thiol, imino, amido,
phosphoryl, phosphonate, carboxyl, carboxamide, silyl, thioether,
alkylsulfonyl, arylsulfonyl, sulfoxide, selenoether, ketone,
aldehyde, ester, and --(CH.sub.2).sub.mR.sub.2, wherein in is an
integer from 0 to 4 and R.sub.2, independently for each occurrence,
represents H or substituted or unsubstituted lower alkyl,
cycloalkyl, heterocyclyl heteroaralkyl, aryl, or heteroaryl; X
represents --H or --OR; Y represents --O--; R, independently for
each occurrence, represents --H or lower alkyl; R.sub.1 represents
one or more substituents positioned at the 4- and/or 6-position of
the piperidine ring; n is an integer from 0 to 2; p is 0 or 1; and
q is 1.
2. The method of claim 1, wherein R.sub.1, independently for each
occurrence, represents halogen, amino, acylamino, amidino, cyano,
nitro, azido, ether, thioether, sulfoxido, -J-R.sub.2, -J-OH,
-J-lower alkyl, -J-lower alkenyl, -J-R.sub.2, -J-SH, -J-NH.sub.2,
or substituted or unsubstituted lower alkyl, lower alkenyl,
cycloakyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, aryl,
heteroaryl, aralkyl, or heteroaralkyl, or protected forms of the
above; R.sub.2, independently for each occurrence, represents H or
substituted or unsubstituted lower alkyl, cycloalkyl, heterocyclyl,
aralkyl, heteroaralkyl, aryl, or heteroaryl; and J represents,
independently for each occurrence, a Chain having from 0-8 units
selected from --C(R).sub.2--, --N(R)--, --O--, and --S--.
3. The method of claim 1, wherein Ar is substituted with at least
one halogen, cyano, hydroxyl, alkoxy, alkenyl, aryl, nitro, thiol,
imino, amido, carboxyl, thioether, alkylsulfonyl, arylsulfonyl,
ketone, aldehyde, or ester group.
4. The method of claim 1, wherein Ar is substituted with at least
one halogen, cyano, alkyl, alkenyl, nitro, amido, carboxyl,
alkylsulfonyl, ketone, aldehyde, or ester group.
5. The method of claim 1, wherein Ar is substituted at the para
position.
6. The method of claim 1, wherein each occurrence of Ar is
phenyl.
7. The method of claim 1, wherein each occurrence of Ar is a phenyl
substituted by one or more electron-withdrawing substituents.
8. The method of claim 7, Wherein the electron-withdrawing
substituent is halogen, cyano, nitro, perfluoroalkyl or acyl
group.
9. The method of claim 1, wherein R.sub.1, independently for each
occurrence, represents a lower alkyl group.
10. The method of claim 1, wherein the movement disorder is
Parkinson's disease, restless leg syndrome, or tardive
dyskinesia/dystonia.
11. The method of claim 1, wherein the CNS disorder is a sleep
disorder.
12. The method of claim 1, further comprising administering,
conjointly With the compound represented by Formula I or a
pharmaceutically acceptable salt thereof, a second therapeutic
agent, a physical therapy, an occupational therapy, or a
speech/language therapy.
13. The method of claim 1, wherein the compound is ##STR00010##
##STR00011##
14. A method of treating a CNS disorder selected from the group
consisting of a movement disorder, depression, a sleep disorder,
obesity, attention deficit disorder (ADD), attention deficit
hyperactivity disorder (ADHD), sexual dysfunction, and substance
abuse; the method comprising administering to a subject in need
thereof a therapeutically effective amount of a compound
represented by Formula 1, or a pharmaceutically acceptable salt
thereof: ##STR00012## wherein, as valence and stability permit, Ar,
independently for each occurrence, represents a substituted aryl or
heteroaryl ring, and Ar is substituted with one or more groups
selected from halogen, cyano, alkenyl, alkynyl, aryl, hydroxyl,
alkoxy, silyloxy, amino, nitro, thiol, imino, amido, phosphoryl,
phosphonate, carboxyl, carboxamide, silyl, thioether, alkyl
sulfonyl, arylsulfonyl, sulfoxide, selenoether, ketone, aldehyde,
ester, and --(CH.sub.2).sub.mR.sub.2, wherein m is an integer from
0 to 4 and R.sub.2, independently for each occurrence, represents H
or substituted or unsubstituted lower alkyl, cycloalkyl,
heterocyclyl, aralkyl, heteroaralkyl, aryl, or heteroaryl; X
represents --H or --OR; Y represents --O--; R, independently for
each occurrence, represents --H or lower alkyl; R.sub.1 represents
one or more lower alkyl groups positioned at the 4- and/or
6-position of the piperidine ring; n is an integer from 0 to 2; p 0
or 1; and q is 1.
15. The method of claim 1, wherein Ar is substituted with at least
one halogen, cyano, hydroxyl, alkoxy, alkenyl, alkynyl, aryl,
nitro, thiol, imino, amido, carboxyl, thioether, alkylsulfonyl,
arylsulfonyl, ketone, aldehyde, or ester group.
16. The method of claim 1, wherein Ar is substituted with at least
one halogen, cyano, alkenyl, alkynyl, nitro, amido, carboxyl,
alkylsulfonyl, ketone, aldehyde, or ester group.
17. The method of claim 1, wherein Ar is substituted at the para
position.
18. The method of claim 1, wherein each occurrence of Ar is
phenyl.
19. The method of claim 1, wherein each occurrence of Ar is a
phenyl substituted by one or more electron-withdrawing
substituents.
20. The method of claim 1, wherein the electron-withdrawing
substituent is halogen, cyano, nitro, perfluoroalkyl or acyl group.
Description
BACKGROUND OF THE INVENTION
[0001] A movement disorder is a neurological disturbance that
involves one or more muscles or muscle groups. Movement disorders
affect a significant portion of the population, causing disability
as well as distress. Movement disorders include Parkinson's
disease, Huntington's chorea, progressive supranuclear palsy,
Wilson's disease, Tourette's syndrome, epilepsy, tardive
dyskinesia, and various chronic tremors, tics and dystonias.
Different clinically observed movement disorders can be traced to
the same or similar areas of the brain. For example, abnormalities
of basal ganglia (a large cluster of cells deep in the hemispheres
of the brain) are postulated as a causative factor in diverse
movement disorders.
[0002] Parkinson's disease is a movement disorder of increasing
occurrence in aging populations. Parkinson's disease is a common
disabling disease of old age affecting about one percent of the
population over the age of 60 in the United States. The incidence
of Parkinson's disease increases with age and the cumulative
lifetime risk of an individual developing the disease is about 1 in
40. Symptoms include pronounced tremor of the extremities,
bradykinesia, rigidity and postural change. A perceived
pathophysiological cause of Parkinson's disease is progressive
destruction of dopamine producing cells in the basal ganglia which
comprise the pars compartum of the substantia nigra, basal nuclei
located in the brain stem, Loss of dopamineric neurons results in a
relative excess of acetylcholine. Jellinger, K. A., Post Mortem
Studies in Parkinson's Disease--Is It Possible to Detect Brain
Areas For Specific Symptoms?, J Neural Transm 56 (Supp); 1-29:1999.
Parkinson's disease is a progressive disorder which can begin with
mild limb stiffness and infrequent tremors and progress over a
period of ten or more years to frequent tremors and memory
impairment, to uncontrollable tremors and dementia.
[0003] Tardive dyskinesia (TD) is a chronic disorder of the nervous
system, characterized by involuntary, irregular rhythmic movements
of the mouth, tongue, and facial muscles. The upper extremities
also may be involved. These movements may be accompanied, to a
variable extent, by other involuntary movements and movement
disorders. These include rocking, writhing, or twisting movements
of the trunk (tardive dystonia), forcible eye closure (tardive
blepharospasm), an irresistible impulse to move continually
(tardive akathisia), jerking movements of the neck (tardive
spasmodic torticollis), and disrupted respiratory movements
(respiratory dyskinesia). The vast majority of TD cases are caused
by the prolonged use of antipsychotic drugs (neuroleptics). A
relatively small number are caused by the use of other medications,
such as metoclopramide, that, like neuroleptics, block dopamine
receptors. TD often manifests or worsens in severity after
neuroleptic drug therapy is discontinued. Resumption of neuroleptic
therapy will temporarily suppress the involuntary movements, but
may aggravate them in the long run.
[0004] Tardive dyskinesia affects approximately 15-20% of patients
treated with neuroleptic drugs (Khot et al., Neuroleptics and
Classic Tardive Dyskinesia, in Lang A E, Weiner W J (eds.): Drug
Induced Movement Disorders, Futura Publishing Co., 1992, pp
121466). Therefore, the condition affects hundreds of thousands of
people in the United States alone. The cumulative incidence of TD
is substantially higher in women, in older people, and in those
being treated with neuroleptics for conditions other than
schizophrenia, such as bipolar disorder (manic-depressive illness)
(see, e.g., Hayashi et al., Clin. Nemopharmacol, 19:390, 1996;
Jeste et al., Arch. Gen. Psychiatry, 52:756, 1995). Unlike the
acute motor side effects of neuroleptic drugs, TD does not respond
in general to antiparkinson drugs (Decker et al., New Eng. J Med.,
October 7, p. 861, 1971).
[0005] Focal dystonias are a class of related movement disorders
involving the intermittent sustained contraction of a group of
muscles. The prevalence of focal dystonias in one US county was
estimated as 287 per million (Monroe County Study); this suggests
that at least 70,000 people are affected in the US alone. The
spasms of focal dystonia can last many seconds at a time, causing
major disruption of the function of the affected area. Some of the
focal dystonias are precipitated by repetitive movements; writer's
cramp is the best known example. Focal dystonia can involve the
face (e.g., blepharospasm, mandibular dystonia), the neck
(torticollis), the limbs (e.g., writer's cramp), or the trunk.
Dystonia can occur spontaneously or can be precipitated by exposure
to neuroleptic drugs and other dopamine receptor blockers (tardive
dystonia). No systemic drug therapy is generally effective, but
some drugs give partial relief to some patients. Those most often
prescribed are anticholinergics, baclofen, benzodiazepines, and
dopamine agonists and antagonists. The most consistently effective
treatment is the injection of botulinum toxin into affected
muscles.
[0006] The various focal dystonias tend to respond to the same
drugs (Chen, Clin. Orthop. June, 102-6, 1998; Esper et al; Term.
Med, January, 90:18-20, 1997; De Mattos et al., Arq.
Neuropsychiatry, March 54:30-6, 1996) This suggests that a new
treatment helpful for one focal dystonia would be likely to be
helpful for another. Furthermore, the common symptoms, signs and
responses to medication of spontaneous (idiopathic) dystonia and
neuroleptic-induced dystonia suggest that an effective treatment
for a drug-induced focal dystonia will be effective for the same
dystonia occurring spontaneously.
[0007] A tic is an abrupt repetitive movement, gesture, or
utterance that often mimics a normal type of behavior. Motor tics
include movements such as eye blinking, head jerks or shoulder
shrugs, but can vary to more complex purposive appearing behaviors
such as facial expressions of emotion or meaningful gestures of the
arms and head. In extreme cases, the movement can be obscene
(copropraxia) or self injurious. Phonic or vocal tics range from
throat clearing sounds to complex vocalizations and speech,
sometimes with coprolalia (obscene speech) (Leckman et al., supra).
Tics are irregular in time, though consistent regarding the muscle
groups involved. Characteristically, they can be suppressed for a
short time by voluntary effort.
[0008] Tics are estimated to affect 1% to 13% of boys and 1% to 11%
of girls, the male-female ratio being less than 2 to 1.
Approximately 5% of children between the ages of 7 and 11 years are
affected with tic behavior (Leckman et al., Neuropsychiatry of the
Bas. Gang, December, 20(4): 839-861, 1997). The estimated
prevalence of multiple tics with vocalization, i.e. Tourette's
syndrome, varies among different reports, ranging from 5 per 10,000
to 5 per 1,000. Tourette's syndrome is 3-4 times more common in
boys than girls and 10 times more common in children and
adolescents than in adults (Leckman et al., supra; Esper et al,
Tenn. Med. 90:18-20, 1997).
[0009] Gilles de la Tourette syndrome (TS) is the most severe tic
disorder. Patients with TS have multiple tics, including at least
one vocal (phonic) tic. TS becomes apparent in early childhood with
the presentation of simple motor tics, for example, eye blinking or
head jerks. Initially, tics may come and go, but in time tics
become persistent and severe, and begin to have adverse effects on
the child and the child's family. Phonic tics manifest, on average,
1 to 2 years after the onset of motor tics. By the age of 10, most
children have developed an awareness of the premonitory urges that
frequently precede a tic. Such premonitions may enable the
individual to voluntary suppress the tic, yet premonition
unfortunately adds to the discomfort associated with having the
disorder. By late adolescence/early adulthood, tic disorders can
improve significantly in certain individuals. However, adults who
continue to suffer from tics often have particularly severe and
debilitating symptoms. (Leckman et al., supra.
[0010] Although the present day pharmacopeia offers a variety of
agents to treat movement disorders, none of these agents can
prevent or cure these conditions. Furthermore, the most effective
treatments are often associated with intolerable side effects.
There remains a clear-cut need for new treatments for movement
disorders that have greater efficacy and fewer side effects than
those currently available.
SUMMARY OF THE INVENTION
[0011] The present invention relates to the discovery of certain
dopamine transporter inhibitors (collectively referred to herein as
the subject "DAT inhibitors"), and the use of those inhibitors in
methods of treatment, and the production of packaged
pharmaceuticals and pharmaceutical preparations. The subject DAT
inhibitors are represented by Formula I, or are a pharmaceutically
acceptable salt, solvate, metabolite or pro-drug thereof:
##STR00001##
[0012] wherein, as valence and stability permit, [0013] Ar,
independently for each occurrence, represents a substituted or
unsubstituted aryl or heteroaryl ring; [0014] X represents --H or
--OR; [0015] Y represents --O--, --S--, --C(R).sub.2--, or
--N(R)--; [0016] R, independently for each occurrence, represents
--H or lower alkyl; [0017] R.sub.1 represents one or more
substituents to the ring to which it is attached, such as halogen,
amino, acylamino, amidino, cyano, nitro, azido, ether, thioether,
sulfoxido, -J-R.sub.2, -J-OH, -J-lower alkyl, -J-lower alkenyl,
-J-R.sub.2, -J-SH, -J-NH.sub.2, or substituted or unsubstituted
lower alkyl, lower alkenyl, cycloalkyl, heterocyclyl,
cycloalkylalkyl, heterocyclylalkyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl, or protected forms of the above; [0018] R.sub.2,
independently for each occurrence, represents H or substituted or
unsubstituted lower alkyl, cycloalkyl, heterocyclyl, aralkyl,
heteroaralkyl, aryl, or heteroaryl; [0019] J represents,
independently for each occurrence, a chain having from 0-8
(preferably from 0-4) units selected from --C(R).sub.2--, --N(R)--,
--O--, and --S--; [0020] n is an integer from 0 to 2; [0021] p is 0
or 1; and [0022] q is an integer from 0 to 2, preferably 1.
[0023] In another embodiment, the invention provides a packaged
pharmaceutical comprising: a DAT inhibitor of any of the invention
in an amount sufficient to treat or prevent a movement disorder and
formulated in a pharmaceutically acceptable carrier; and
instructions (written and/or pictorial) describing the use of the
formulation for treating the patient. The movement disorder may be
selected from ataxia, corticobasal ganglionic degeneration (CBGD),
dyskinesia, dystonia, tremors, hereditary spastic paraplegia,
Huntington's disease, multiple system atrophy, myoclonus,
Parkinson's disease, progressive supranuclear palsy, restless legs
syndrome, Rett syndrome, spasticity, Sydenham's chorea, other
choreas, athetosis, ballism, stereotypy, tardive
dyskinesia/dystonia, tics, Tourette's syndrome,
olivopontocerebellar atrophy (OPCA), diffuse Lewy body disease,
hemibalismus, hemi-facial spasm, restless leg syndrome, Wilson's
disease, stiff man syndrome, akinetic mutism, psychomotor
retardation, painful legs moving toes syndrome, a gait disorder, a
drug-induced movement disorder, or other movement disorder. The DAT
inhibitor may be provided in an amount sufficient to treat or
prevent a movement disorder in a patient by a statistically
significant amount when assessed by one or more of Hoehn and Yahr
Staging of Parkinson's Disease, Unified Parkinson Disease Rating
Scale (UPDRS), and Schwab and England Activities of Daily Living
Scale. The DAT inhibitor may be provided in an amount sufficient to
treat or prevent a movement disorder in a patient by a
statistically significant amount when assessed by a standardized
test in combination with an empirical test selected from computer
tomography (CT), magnetic resonance imaging (MRI), and positron
emission tomography (PET).
[0024] In some embodiments, the packaged pharmaceutical may further
comprise another medication selected from dopamine precursors,
dopaminergic agents, dopaminergic and anti-cholinergic agents,
anti-cholinergic agents, dopamine agonists, MAO-B (monoamine
oxidase B) inhibitors, COMT (catechol O-methyltransferase)
inhibitors, muscle relaxants, sedatives, anticonvulsant agents,
dopamine reuptake inhibitors, dopamine blockers, .beta.-blockers,
carbonic anhydrase inhibitors, narcotic agents, GABAergic agents,
or alpha antagonists.
[0025] In another embodiment, the packaged pharmaceutical is
provided in an escalating dose which produces an escalating serum
concentration of said DAT inhibitor(s) over a period of at least 4
hours.
[0026] In another embodiment, the invention provides for the use of
a DAT inhibitor of the invention in the manufacture of a
pharmaceutical composition for prophylaxis or treatment of a
patient susceptible to or suffering from a movement disorder. The
movement disorder may be selected from ataxia, corticobasal
ganglionic degeneration (CBGD), dyskinesia, dystonia, tremors,
hereditary spastic paraplegia, Huntington's disease, multiple
system atrophy, myoclonus, Parkinson's disease, progressive
supranuclear palsy, restless legs syndrome, Rett syndrome,
spasticity, Sydenham's chorea, other choreas, athetosis, ballism,
stereotypy, tardive dyskinesia/dystonia, tics, Tourette's syndrome,
olivopontocerebellar atrophy (OPCA), diffuse Lewy body disease,
hemibalismus, hemi-facial spasm, restless leg syndrome, Wilson's
disease, stiff man syndrome, akinetic mutism, psychomotor
retardation, painful legs moving toes syndrome, a gait disorder, a
drug-induced movement disorder, or other movement disorder. The use
may be for treatment of a human patient.
[0027] In some embodiments of the invention, the packaged
pharmaceutical or use may be for oral administration. In some
embodiments of the packaged pharmaceutical or use, the DAT
inhibitor may be formulated as a transdermal patch.
[0028] In another embodiment, the invention provides a method for
treating a movement disorder comprising administering to the
patient a composition of a DAT inhibitor of the invention in an
amount sufficient to treat the movement disorder in the animal as
evaluated by a standardized test. The movement disorder may be
selected from ataxia, corticobasal ganglionic degeneration (CBGD),
dyskinesia, dystonia, tremors, hereditary spastic paraplegia,
Huntington's disease, multiple system atrophy, myoclonus,
Parkinson's disease, progressive supranuclear palsy, restless legs
syndrome, Rett syndrome, spasticity, Sydenham's chorea, other
choreas, athetosis, ballism, stereotypy, tardive
dyskinesia/dystonia, tics, Tourette's syndrome,
olivopontocerebellar atrophy (OPCA), diffuse Lewy body disease,
hemibalismus, hemi-facial spasm, restless leg syndrome, Wilson's
disease, stiff man syndrome, akinetic mutism, psychomotor
retardation, painful legs moving toes syndrome, a gait disorder, a
drug-induced movement disorder, or other movement disorder. The DAT
inhibitor may be provided in an amount sufficient to treat a
movement disorder in a patient by a statistically significant
amount when assessed by one or more of Hoehn and Yak Staging of
Parkinson's Disease, Unified Parkinson Disease Rating Scale
(UPDRS), and Schwab and England Activities of Daily Living Scale.
The DAT inhibitor may be provided in an amount sufficient to treat
or prevent a movement disorder in a patient by a statistically
significant amount when assessed by a standardized test in
combination with an empirical test selected from computer
tomography (CT), magnetic resonance imaging (MRI), and positron
emission tomography (PET).
[0029] In some embodiments, the method may comprise coadministation
of the DAT inhibitor with one or more of a dopamine precursor, a
dopaminergic agent; a dopaminergic and anti-cholinergic agent, an
anti-cholinergic agent, a dopamine agonist, a MAO-B (monoamine
oxidase B) inhibitor, a COMT (catechol O-methyltransferase)
inhibitor, a muscle relaxant, a sedative, an anticonvulsant agent,
a dopamine reuptake inhibitor, a dopamine blocker, a
.beta.-blocker, a carbonic anhydrase inhibitor, a narcotic agent, a
GABAergic agent, or an alpha antagonist.
[0030] In another embodiment, the invention provides a method for
conducting a pharmaceutical business, comprising: (a) manufacturing
the packaged pharmaceutical of the invention; and (b) marketing to
healthcare providers the benefits of using the package or
preparation to treat patients suffering from a movement
disorder.
[0031] In another embodiment, the invention provides a method for
conducting a pharmaceutical business, comprising: (a) providing a
distribution network for selling the packaged pharmaceutical of the
invention; and (b) providing instruction material to patients or
physicians for using the package or preparation to treat patients
suffering from a movement disorder.
[0032] In another embodiment, the invention provides a method for
conducting a pharmaceutical business, comprising: (a) determining
an appropriate dosage of an DAT inhibitor of the invention to
enhance function performance in a class of patients suffering from
a movement disorder; (b) conducting therapeutic profiling of one or
more formulations of the DAT inhibitor identified in step (a), for
efficacy and toxicity in animals; and (e) providing a distribution
network for selling a the formulations identified in step (b) as
having an acceptable therapeutic profile. The method may include an
additional step of providing a sales group for marketing the
preparation to healthcare providers.
[0033] In another embodiment, the invention provides a method for
conducting a medical assistance reimbursement program, comprising:
(a) providing a reimbursement program which permits, for
prescription of a DAT inhibitors of the invention for treating a
movement order, at least partial reimbursement to a healthcare
provider or patient, or payment to a drug distributor; (b)
processing one or more claims for prescription of an DAT inhibitors
for treating a movement order; and (c) reimbursing the healthcare
provider or patient, or paying a drug distributor, at least a
portion of the cost of said prescription.
[0034] In another embodiment, the invention provides a method for
treating depression, a sleep disorder, obesity, attention deficit
disorder (ADD), attention deficit hyperactivity disorder (ADHD),
sexual dysfunction, or substance abuse comprising administering to
the patient a composition of a DAT inhibitor of the invention in an
amount sufficient to treat the movement disorder in the animal as
evaluated by a standardized test.
[0035] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of synthetic
chemistry, organic chemistry, inorganic chemistry, organometallic
chemistry, pharmaceutical chemistry, and behavioral science, which
are within the skill of the art. Such techniques are described in
the literature. See, for example, Advanced Organic Chemistry:
Reactions, Mechanisms, And Structure by J. March (John Wiley and
Sons, New York, 1992); The Chemist's Companion: A Handbook Of
Practical Data, Techniques, And References by A. J. Gordon and R.
A. Ford (Wiley, NY, 1972); Synthetic Methods Of Organometallic And
Inorganic Chemistry by W. A. Herrmann and Brauer (Georg Thieme
Verlag, New York, 1996); Experimental Organic Chemistry by D. Todd
(Prentice-Hall, New Jersey, 1979); Experimental Organic Chemistry:
Standard And Microscale by L. M. Harwood (Blackwell Science,
Massachusetts, 1999); Experimental Analysis Of Behavior by I. H.
Iversen and K. A. Lattal (Elsevier, N.Y., 1991); A Practical Guide
To Behavioral Research: Tools And Techniques by R. Sommer and B.
Sommer (Oxford University Press, New York, 2002); Advances In Drug
Discovery Techniques by A. L. Harvey (Chichester, N.Y., 1998);
Quantitative Calculations In Pharmaceutical Practice And Research
by T. P. Hadjiioannou (VCH, New York, 1993); Drug Fate And
Metabolism: Methods And Techniques by E. R. Garrett and J. L. Hirtz
(M. Dekker, New York, 1977); Behavioral Science Techniques: An
Annotated Bibliography For Health Professionals by M. K. Tichy
(Praeger Publishers, New York, 1975).
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 shows a few illustrative dopamine transporter
inhibitors, CNS-27100, CNS-27200, CNS-28100, CNS-28200, CNS-28001,
and CNS-28002.
[0037] FIGS. 2-4 show in vivo efficacy of four illustrative
dopamine transporter inhibitors, CNS-27,100, -28,002, -28,100, and
-28,200, as measured by forced swim test using rats.
DETAILED DESCRIPTION OF TILE INVENTION
I. Overview
[0038] The present invention relates to the discovery of certain
dopamine transporter inhibitors (collectively referred to herein as
the subject "DAT inhibitors") which can be used to prevent or
reduce conditions associated with a movement disorder. In certain
preferred embodiments, the movement: disorder is Parkinson's
disease.
[0039] The subject DAT inhibitors can also be effective as part of
a therapy for treating depression, sleep disorders, obesity,
attention deficit disorder (ADD), attention deficit hyperactivity
disorder (ADHD), certain sexual dysfunctions, and substance abuse
(such as for the treatment of cocaine abuse).
[0040] One aspect of the invention features a pharmaceutical
package comprising one or more of the subject DAT inhibitor(s) in
an amount sufficient to treat or prevent a movement disorder in a
patient, a pharmaceutically acceptable carrier, and instructions
(written and/or pictorial) describing the use of the formulation
for treating the patient, wherein the patient suffers from ataxia,
corticobasal ganglionic degeneration (CBGD), dyskinesia, dystonia,
tremors, hereditary spastic paraplegia, Huntington's disease,
multiple system atrophy, myoclonus, Parkinson's disease,
progressive supranuclear palsy, restless legs syndrome, Rett
syndrome, spasticity, Sydenham's chorea, other choreas, athetosis,
ballism, stereotypy, tardive dyskinesia/dystonia, tics. Tourette's
syndrome, olivopontocerebellar atrophy (OPCA), diffuse Lewy body
disease, hemibalismus, hemi-facial spasm, restless leg syndrome,
Wilson's disease, stiff man syndrome, akinetic mutism, psychomotor
retardation, painful legs moving toes syndrome, a gait disorder, a
drug-induced movement disorder, or other movement disorder.
[0041] In certain preferred embodiments, the invention features a
pharmaceutical preparation comprising one or more of the subject
DAT inhibitors provided as a single oral dosage formulation in an
amount sufficient to treat or prevent a movement disorder in a
patient.
[0042] In other preferred embodiments, the invention features a
pharmaceutical preparation comprising one or more DAT inhibitors
provided in the form of a transdermal patch and formulated for
sustained release of the amphetamine(s) in order to administer an
amount sufficient to treat or prevent a movement disorder in a
patient.
[0043] In many preferred embodiments of the packages, preparations,
compositions, and methods, the invention features one or more DAT
inhibitor(s) provided in an amount sufficient to treat or prevent a
movement disorder in a patient by a statistically significant
amount when assessed by a standardized performance test. For
instance, the subject DAT inhibitor(s) are provided in an amount
sufficient to treat or prevent a movement disorder in a patient by
a statistically significant amount when assessed by one or more of
Hoehn and Yahr Staging of Parkinson's Disease, Unified Parkinson
Disease Rating Scale (UPDRS), and Schwab and England Activities of
Daily Living Scale.
[0044] In certain embodiments of the packages, preparations,
compositions, and methods, the invention features one or more DAT
inhibitor(s) provided in an amount sufficient to treat or prevent a
movement disorder in a patient by a statistically significant
amount when assessed by a standardized test in combination with an
empirical test selected from computer tomography (CT), magnetic
resonance imaging (MRI), and positron emission tomography
(PET).
[0045] Another aspect of the invention features the use of the
pharmaceutical composition of DAT inhibitors in the manufacture of
a medicament for prophylaxis or treatment of an animal susceptible
to or suffering from ataxia, corticobasal ganglionic degeneration
(CBGD), dyskinesia, dystonia, tremors, hereditary spastic
paraplegia, Huntington's disease, multiple system atrophy,
myoclonus, Parkinson's disease, progressive supranuclear palsy,
restless legs syndrome, Rett syndrome, spasticity, Sydenham's
chorea, other choreas, athetosis, ballism, stereotypy, tardive
dyskinesia/dystonia, tics, Tourette's syndrome,
olivopontocerebellar atrophy (OPCA), diffuse Lewy body disease,
hemibalismus, hemi-facial spasm, restless leg syndrome, Wilson's
disease, stiff man syndrome, akinetic mutism, psychomotor
retardation, painful legs moving toes syndrome, a gait disorder, a
drug-induced movement disorder, or other movement disorder, which
DAT inhibitor is represented by Formula I, or a pharmaceutically
acceptable salt, solvate, metabolite, or pro-drug thereof.
[0046] Another aspect of the invention relates to a method for
conducting a pharmaceutical business, which includes: (a)
manufacturing the packages, preparations, and compositions of the
present invention; and (b) marketing to healthcare providers the
benefits of using the packages, preparations, and compositions of
the present invention to treat or prevent a movement disorder of
treated patients.
[0047] Another aspect of the invention relates to a method for
conducting a pharmaceutical business, comprising: (a) providing a
distribution network for selling the packages, preparations, and
compositions of the present invention; and (b) providing
instruction material to patients or physicians for using the
packages, preparations, and compositions of the present invention
to treat or prevent a movement disorder of treated patients.
[0048] Yet another aspect of the invention relates to a method for
conducting a pharmaceutical business, comprising: (a) determining
an appropriate dosage of an DAT inhibitor to treat or prevent a
movement disorder in a class of patients; (b) conducting
therapeutic profiling of one or more formulations of the DAT
inhibitor identified in step (a), for efficacy and toxicity in
animals; and (c) providing a distribution network for selling the
formulations identified in step (b) as having an acceptable
therapeutic profile, wherein the patient suffers from ataxia,
corticobasal ganglionic degeneration (CBGD), dyskinesia, dystonia,
tremors, hereditary spastic paraplegia, Huntington's disease,
multiple system atrophy, myoclonus, Parkinson's disease,
progressive supranuclear palsy, restless legs syndrome, Rett
syndrome, spasticity, Sydenham's chorea, other choreas, athetosis,
ballism, stereotypy, tardive dyskinesia/dystonia, tics, Tourette's
syndrome, olivopontocerebellar atrophy (OPCA), diffuse Lewy body
disease, hemibalismus, hemi-facial spasm, restless leg syndrome,
Wilson's disease, stiff man syndrome, akinetic mutism, psychomotor
retardation, painful legs moving toes syndrome, a gait disorder, a
drug-induced movement disorder, or other movement disorder.
[0049] For instance, the subject business method can include an
additional step of providing a sales group for marketing the
preparation to healthcare providers.
[0050] Another aspect of the invention relates to a method for
conducting a medical assistance reimbursement program, comprising:
(a) providing a reimbursement program which permits, for
prescription of an DAT inhibitors for treating a movement order, at
least partial reimbursement to a healthcare provider or patient, or
payment to a drug distributor; (b) processing one or more claims
for prescription of an DAT inhibitors for treating a movement
order; and (c) reimbursing the healthcare provider or patient, or
paying a drug distributor, at least a portion of the cost of said
prescription.
[0051] Another aspect of the invention relates to a method for
conducting a pharmaceutical business, comprising: (a) determining
an appropriate dosage of an DAT inhibitor to treat or prevent a
movement disorder function in a class of patients; and (b)
licensing, to a third party, the rights for further development and
sale of the DAT inhibitor for treating or preventing a movement
disorder, Wherein the patient suffers from ataxia, corticobasal
ganglionic degeneration (CBGD), dyskinesia, dystonia, tremors,
hereditary spastic paraplegia, Huntington's disease, multiple
system atrophy, myoclonus, Parkinson's disease, progressive
supranuclear palsy, restless legs syndrome, Rett syndrome,
spasticity, Sydenham's chorea, other choreas, athetosis, ballism,
stereotypy, tardive dyskinesia/dystonia, tics, Tourette's syndrome,
olivopontocerebellar atrophy (OPCA), diffuse Lewy body disease,
hemibalismus, hemi-facial spasm, restless leg syndrome, Wilson's
disease, stiff man syndrome, akinetic mutism, psychomotor
retardation, painful legs moving toes syndrome, a gait disorder, a
drug-induced movement disorder, or other movement disorder.
II. Definitions
[0052] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0053] As used herein, the term "movement disorders" includes
akinesias and akinetic-rigid syndromes, dyskinesias and
medication-induced parkinsonism (such as neuroleptic-induced
parkinsonism, neuroleptic malignant syndrome, neuroleptic-induced
acute dystonia, neuroleptic-induced acute akathisia,
neuroleptic-induced tardive dyskinesia and medication-induced
postural tremor). Examples of "akinetic-rigid syndromes" include
Parkinson's disease, drug-induced parkinsonism, postencephalitic
parkinsonism, progressive supranuclear palsy, multiple system
atrophy, corticobasal degeneration, parkinsonism-ALS dementia
complex and basal ganglia calcification. Examples of "dyskinesias"
include tremor (including rest tremor, postural tremor and,
intention tremor), chorea (such as Sydenham's chorea, Huntington's
disease, benign hereditary chorea, neuroacanthocytosis, symptomatic
chorea, drug-induced chorea and hemiballism), myoclonus (including
generalised myoclonus and focal myoclonus), tics (including simple
tics, complex tics and symptomatic tics), and dystonia (including
generalised dystonia such as iodiopathic dystonia, drug-induced
dystonia, symptomatic dystonia and paroxymal dystonia, and focal
dystonia such as blepharospasm, oromandibular dystonia, spasmodic
dysphonia, spasmodic torticollis, axial dystonia, dystonic writer's
cramp and hemiplegic dystonia). Another "movement disorder" which
may be treated according to the present invention is Gilles de la
Tourette's syndrome, and the symptoms thereof.
[0054] As used herein, the term "depression" includes depressive
disorders, for example, single episodic or recurrent major
depressive disorders, and dysthymic disorders, depressive neurosis,
and neurotic depression; melancholic depression including anorexia,
weight loss, insomnia and early morning waking, and psychomotor
retardation; atypical depression (or reactive depression) including
increased appetite, hypersomnia, psychomotor agitation, or
irritability, seasonal affective disorder, or bipolar disorders or
manic depression, for example, bipolar I disorder, bipolar II
disorder and cyclothymic disorder.
[0055] An "effective amount" of, e.g., an DAT inhibitor, with
respect to the subject method of treatment, refers to an amount of
the inhibitor in a pharmaceutical preparation which, when applied
as part of a desired dosage regimen, brings about improved state
according to clinically acceptable standards.
[0056] The term "treat," "treating," or "treatment" as used herein
means to counteract a medical condition (e.g., a movement disorder)
to the extent that the medical condition is improved according to
clinically acceptable standard(s). For example, "to treat a
movement disorder" means to improve the movement disorder or
relieve symptoms of the particular movement disorder in a patient,
wherein the improvement and relief are evaluated with a clinically
acceptable standardized test (e.g., a patient self-assessment
scale) and/or an empirical test (e.g., PET scan).
[0057] The term "amelioration" in the case of a movement disorder
refers to a decrease in the abnormal involuntary movements
characterizing these two types of dyskinesia, as can be determined
for example, by using the Abnormal Involuntary Movement Scale
(AIMS) as will be specified hereinbelow.
[0058] The term "prevent," "preventing," or "prevention" as used
herein means reducing the probability/risk of developing a
condition in a subject (e.g., a human), or delaying the onset of a
condition in the subject, or lessening the severity of one or more
symptoms of a condition (e.g., a movement disorder) that may
develop in the subject, or any combination thereof.
[0059] A "patient" or "subject" to be treated by the subject method
can mean either a human or non-human animal.
[0060] The term "prodrug" represents compounds which are rapidly
transformed in vivo, for example, by hydrolysis in blood into the
therapeutically active agents of the present invention. A common
method for making a prodrug is to include selected moieties which
are converted under physiologic conditions (enzymatic or
nonenzymatic) to reveal the desired molecule. A thorough discussion
is provided in T. Higuchi and V. Stella, Pro-drugs as Novel
Delivery Systems. Vol. 14 of the A.C.S. Symposium Series, and in
Edward B. Roche, ed., Bioreversible Carriers in Drug Design,
American Pharmaceutical Association and Pergamon Press, 1987, both
of which are incorporated herein by reference.
[0061] By "transdermal patch" is meant a system capable of delivery
of a drug to a patient via the skin, or any suitable external
surface, including mucosal membranes, such as those found inside
the mouth. Such delivery systems generally comprise a flexible
backing, an adhesive, and a drug-retaining matrix, the backing
protecting the adhesive and matrix, and the adhesive holding the
whole on the skin of the patient. On contact with the skin, the
drug-retaining matrix delivers drug to the skin, the drug then
passing through the skin into the patient's system.
[0062] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described below, but that contain at least one double or
triple bond respectively.
[0063] The terms "alkoxyl" or "alkoxy" as used herein refer to an
alkyl group, as defined below, having an oxygen radical attached
thereto. Representative alkoxy groups include methoxy, ethoxy,
propyloxy, tert-butoxy, and the like. An "ether" is two
hydrocarbons covalently linked by an oxygen. Accordingly, the
substituent of an alkyl that renders that alkyl an ether is or
resembles an alkoxyl, such as can be represented by one of
--O-alkyl, --O-alkenyl, --O-alkynyl, --O--(CH.sub.2)m-R.sub.8,
where R.sub.8 represents an aryl, a cycloalkyl, a cycloalkenyl, a
heterocycle, or a polycycle, and m is zero or an integer in the
range of 1 to 8.
[0064] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups,
alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted
alkyl groups.
[0065] En preferred embodiments, a straight chain or branched chain
alkyl has 8 or fewer carbon atoms in its backbone (e.g., C1-C8 for
straight chains, C3-C8 for branched chains), and more preferably 5
or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon
atoms in their ring structure, and more preferably have 5, 6, or 7
carbons in the ring structure.
[0066] Moreover, the term "alkyl" "lower alkyl") as used throughout
the specification, examples, and claims is intended to include both
"unsubstituted alkyls" and "substituted alkyls," the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, a halogen, a hydroxyl, a
carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an
acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a
thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate,
a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a
nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a
sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl,
an aralkyl, or an aromatic or heteroaromatic moiety. It will be
understood by those skilled in the art that the moieties
substituted on the hydrocarbon chain can themselves be substituted,
if appropriate. For instance, the substituents of a substituted
alkyl may include substituted and unsubstituted forms of amino,
azido, imino, amido, phosphoryl (including phosphonate and
phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl,
and sulfonate), and silyl groups, as well as ethers, alkylthios,
carbonyls (including ketones, aldehydes, carboxylates, and esters),
--CF.sub.3, --CN, and the like. Exemplary substituted alkyls are
described below. Cycloalkyls can be further substituted with
alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls,
carbonyl-substituted alkyls, --CF.sub.3, --CN, and the like.
[0067] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to eight carbons, more preferably from one to five
carbon atoms, in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Throughout the
application, preferred alkyl groups are lower alkyls. In preferred
embodiments, a substituent designated herein as alkyl is a lower
alkyl. The term "aralkyl," as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or heteroaromatic
group).
[0068] The term "aryl" as used herein includes 5-, and 6-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, benzene, pyrrole, furan, thiophene,
imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,
pyrazine, pyridazine, and pyrimidine, and the like. Those aryl
groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles," "heteroaryls," or
"heteroaromaties." The aromatic ring can be substituted at one or
more ring positions with such substituents as described above, for
example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,
amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl,
silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde,
ester, heterocyclyl, aromatic or heteroaromatic moieties,
--CF.sub.3, --CN, or the like. The term "aryl" also includes
polycyclic ring systems having two or more cyclic rings in which
two or more carbons are common to two adjoining rings (the rings
are "fused rings") wherein at least one of the rings is aromatic,
e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls and/or heterocyclyls.
[0069] The term "carbocycle" or "cyclic alkyl," as used herein,
refers to an aromatic or non-aromatic ring in which each atom of
the ring is carbon.
[0070] The term "heteroatom," as used herein, means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
nitrogen, oxygen, and sulfur.
[0071] The terms "heterocyclyl" or "heterocyclic group" refer to 3-
to 10-membered ring structures, more preferably 3- to 7-membered
rings, whose ring structures include one to four heteroatoms.
Heterocycles can also be polycycles. Heterocyclyl groups include,
for example, thiophene, thianthrene, furan, pyran, isobenzofuran,
chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole,
isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline,
quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,
phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine,
oxolane, thiolane, oxazole, piperidine, piperazine, morpholine,
lactones, lactams such as azetidinones and pyrrolidinones, sultams,
sultones, and the like. The heterocyclic ring can be substituted at
one or more positions with such substituents as described above,
for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,
phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,
alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an
aromatic or heteroaromatic moiety, --CF.sub.3, --CN, or the
like.
[0072] The term "metabolites" refers to active derivatives produced
upon introduction of a compound into a biological milieu, such as a
patient.
[0073] As used herein, the term "nitro" means --NO.sub.2; the term
"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl"
means --SH; the term "hydroxyl" means --OH; and the term "sulfonyl"
means --SO.sub.2--.
[0074] The terms "polycyclyl" or "polycyclic group" refer to two or
more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,
and/or heterocyclyls) in which two or more carbons are common to
two adjoining rings, e.g., the rings are "fused rings." Rings that
are joined through non-adjacent atoms are termed "bridged" rings.
Each of the rings of the polycycle can be substituted with such
substituents as described above, for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,
sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone,
aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic
moiety, --CF.sub.3, --CN, or the like.
[0075] The phrase "protecting group" as used herein means temporary
substituents which protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed (Greene, T W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 2nd ed.; Wiley: New York, 1991).
[0076] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
herein above. The permissible substituents can be one or more and
the same or different for appropriate organic compounds. For
purposes of this invention, heteroatoms such as nitrogen may have
hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This invention is not intended to be limited in
any manner by the permissible substituents of organic
compounds.
[0077] It will be understood that "substitution", or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc.
[0078] As used herein, the definition of each expression, e.g.,
alkyl, m, n, etc., when it occurs more than once in any structure,
is intended to be independent of its definition elsewhere in the
same structure.
[0079] Contemplated equivalents of the compounds described above
include compounds which otherwise correspond thereto, and which
have the same general properties thereof (e.g., the ability to
affect movement disorders), wherein one or more simple variations
of substituents are made which do not adversely affect the efficacy
of the compound. In general, the compounds of the present invention
may be prepared by the methods described below, or by modifications
thereof, using readily available starting materials, reagents, and
conventional synthesis procedures. In these reactions, it is also
possible to make use of variants which are in themselves known, but
are not mentioned here.
[0080] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover. Also for purposes of this invention, the term
"hydrocarbon" is contemplated to include all permissible compounds
having at least one hydrogen and one carbon atom. In a broad
aspect, the permissible hydrocarbons include acyclic and cyclic,
branched and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic organic compounds which can be substituted or
unsubstituted.
III. Exemplary Compounds of the Invention
[0081] The subject DAT inhibitors are represented by Formula I, or
are a pharmaceutically acceptable salt, solvate, metabolite or
pro-drug thereof:
##STR00002##
[0082] wherein, as valence and stability permit, [0083] Ar,
independently for each occurrence, represents a substituted or
unsubstituted aryl or heteroaryl ring; [0084] X represents --H or
--OR; [0085] Y represents --O--, --S--, --C(R).sub.2, or --N(R)--;
[0086] R, independently for each occurrence, represents --H or
lower alkyl; [0087] R.sub.1 represents one or more substituents to
the ring to which it is attached, such as halogen, amino,
acylamino, amidino, cyano, nitro, azido, ether, thioether,
sulfoxido, -J-R.sub.2, -J-OH, -J-lower alkyl, -J-lower alkenyl,
-J-R.sub.2, -J-SH, -J-NH.sub.2, or substituted or unsubstituted
lower alkyl, lower alkenyl, cycloalkyl, heterocyclyl,
cycloalkylalkyl, heterocyclylalkyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl, or protected forms of the above; [0088] R.sub.2,
independently for each occurrence, represents H or substituted or
unsubstituted lower alkyl, cycloalkyl, heterocyclyl, aralkyl,
heteroaralkyl, aryl, or heteroaryl;
[0089] J represents, independently for each occurrence, a chain
having from 0-8 (preferably from 0-4) units selected from
--C(R).sub.2--, --N(R)--, --O--, and --S--; [0090] n is an integer
from 0 to 2; [0091] p is 0 or I; and [0092] q is an integer from 0
to 2, preferably 1.
[0093] In certain embodiments, R.sub.1 comprises one or more lower
alkyl groups, e.g., positioned at the 2-, 4-, and/or 6-position of
the piperidine ring.
[0094] In certain embodiments, substituents on Ar (e.g., other than
hydrogen) are selected from halogen, cyano, alkyl (including
perfluoroalkyl), alkenyl, alkynyl, aryl, hydroxyl, alkoxy,
silyloxy, amino, nitro, thiol, amino, imino, amido, phosphoryl,
phosphonate, carboxyl, carboxamide, silyl, thioether,
alkylsulfonyl, arylsulfonyl, sulfoxide, selenoether, ketone,
aldehyde, ester, or --(CH.sub.2).sub.mR.sub.2, where m is an
integer from 0 to 4.
[0095] In certain embodiments, non-hydrogen substituents are
selected from halogen, cyano, alkyl (including perfluoroalkyl),
hydroxyl, alkoxy, alkenyl, alkynyl, aryl, nitro, thiol, imino,
amido, carboxyl, thioether, alkylsulfonyl, arylsulfonyl, ketone,
aldehyde, and ester. In certain embodiments, non-hydrogen
substituents are selected from halogen, cyano, alkyl (including
perfluoroalkyl), alkenyl, alkynyl, nitro, amido, carboxyl,
alkylsulfonyl, ketone, aldehyde, and ester.
[0096] In certain embodiments, substituents on Ar are located at
the para position.
[0097] In certain embodiments, one or both occurrences of Ar are
phenyl rings. In certain such embodiments, the phenyl rings are
substituted by one or more electron-withdrawing substituents, such
as halogen, cyano, nitro, perfluoroalkyl (e.g., CF.sub.3,
C.sub.2F.sub.5, etc.), acyl, etc.
[0098] Certain representative illustrative dopamine transporter
inhibitors are shown in FIG. 1, including CNS-27100, CNS-27200,
CNS-28100, CNS-28200, CNS-28001, and CNS-28002. These are the
preferred embodiments of the DAT inhibitors. The structures of
these compounds are shown below:
##STR00003## ##STR00004##
[0099] Other embodiments of the DAT inhibitors are listed
below:
##STR00005## ##STR00006##
[0100] In addition to humans, other animal subjects to which the
invention is applicable extend to both domestic animals and
livestock, raised either as pets or for commercial purposes.
Examples are dogs, cats, cattle, horses, sheep, hogs, and
goats.
[0101] Still another aspect of the invention relates to the use of
DAT inhibitors for lessening the severity or prophylactically
preventing the occurrence of movement disorders in an animal, and
thus, altering the mental or physical state of the animal. The
compounds of the present invention may also be useful for treating
and/or preventing memory impairment due to a movement disorder.
[0102] In certain preferred embodiment, the movement disorder is
Parkinson's disease.
A. Synthesis of DAT Inhibitors
[0103] The following section describes in detail the synthesis of
several exemplary DAT inhibitors of the invention. However, these
descriptions/examples are for illustrative purpose only, and should
not be construed to be limiting to only the compounds described. A
skilled artisan could readily synthesize other related compounds of
the invention with (or without) minor modifications of the schemes
described below.
[0104] Unless otherwise noted, reagents and solvents were used as
received from commercial suppliers. Proton and carbon nuclear
magnetic resonance spectra were obtained on a Bruker AV 400 at 400
MHz for proton and 100 MHz for carbon, or on a Bruker AMX 500
spectrometer at 500 MHz for proton and 125 MHz for carbon. Spectra
are given in ppm (.delta.). Tetramethylsilane was used as an
internal standard for proton spectra and the solvent peak was used
as the reference peak for carbon spectra. HPLC analyses were
performed on a Waters 2695 HPLC with an Alltech Platinum C18 column
(53.times.7 mm, 100 .ANG.) with UV detection at 220 nm or 254 nm,
using a standard solvent gradient program (Method A). Mass spectra
were obtained on a Perkin Elmer Sciex API 150EX Turbo Ion Spray
detector.
HPLC Method A:
Column: Alltech Platinum C18 Column, 53.times.7 mm, 100 .ANG., 3
.mu.m;
[0105] Column temperature: 40.degree. C. Mobile phase A: 99.9: 0.1
Water/TFA Mobile phase B: 99.9: 0.1 Acetonitrile/TFA
Detector: 220 nm or 254 nm
[0106] Sample preparation: Dissolve in acetonitrile or 50:50
acetonitrile/water Injection volume: 10 .mu.L
Gradient:
TABLE-US-00001 [0107] Time (Minutes) Flow (mL/min.) % A % B 0 2.5 5
95 1 2.5 5 95 8 2.5 95 5 10 2.5 5 95 12 2.5 5 95
Collection A: Preparation of 2-Me-2
[0108] A 400-mL Fisher-Porter reactor was charged with absolute
ethanol (225 mL), concentrated hydrochloric acid (13.0 g), 10% Pd/C
(4.0 g) and ethyl-2-methylnicotinate (15.0 g, 90.8 mmol). The
mixture was heated to 80.degree. C. and placed under 60 psi
hydrogen pressure. The mixture was then stirred for 16 hour under
these conditions. The mixture was cooled and filtered. The filtrate
was evaporated under reduced pressure to give a tacky solid. This
solid was dissolved in water (25 mL) and the pH was adjusted to pH
8.2 using saturated sodium bicarbonate. The solution was
freeze-dried to give 2-Me-2 (12.6 g, 81%). The .sup.1H NMR spectrum
was consistent with the assigned structure.
Collection A: Preparation of 2-Me-4
[0109] A 1-L, three-neck, round-bottomed flask, fitted with a
mechanical stirrer and placed under an argon atmosphere, was
charged with 2-Me-2 (10.5 g, 51.0 mmol) and methylene chloride (630
mL). While stirring at ambient temperature, triethylamine (22.7 g,
224 mmol) was added. Next, 1-(4-chlorophenyl)-cyclobutanecarboxylic
acid (17.2 g, 82.0 mmol) was added, followed by
bromotris(pyrrolidino)phosphonium hexafluorophosphate ("PyBroP,"
39.2 g, 84.0 mmol). The mixture was stirred under argon at ambient
temperature for 16 h. A solution of 10% potassium hydroxide (700
mL) was added to the reaction mixture. Ethyl acetate (350 mL) was
then added and the mixture was stirred for 5 min. The layers were
separated and the aqueous layer was re-extracted with ethyl acetate
(300 mL). The ethyl acetate extracts were combined and dried over
anhydrous magnesium sulfate. This mixture was filtered and the
filtrate was evaporated under reduced pressure to give crude
product (57.2 g). The crude product was split in two equal
portions. Each portion was placed on a 100 mm diameter flash
column, packed with silica gel (750 g) using 60:30:1
chloroform/ethyl acetate/MeOH. Each column was eluted with 60:30:1
chloroform/ethyl acetate/MeOH. The fractions containing the purest
product were combined and the solvents evaporated to dryness under
reduced pressure. After column chromatography, purified 2-Me-4
(17.8 g) was isolated in two lots: (9.4 g, 50.5%), HPLC (Method A)
58.1% (AUC), t.sub.R=6.11 min (see Attachment 2); and (8.7 g,
46.9%), HPLC (Method A) 76.5% (AUC), t.sub.R=6.10 min.
Collection A: Preparation of 2-Me-5
[0110] A 1-1, three-neck, round-bottomed flask placed under argon
was charged with tetrahydrofuran (210 mL), then was cooled to
0.degree. C. Lithium aluminum hydride (27.9 g) was added slowly at
0.degree. C. In a separate flask, 2-Me-4 (8.2 g, 22.5 mmol) was
dissolved in tetrahydrofuran (150 mL). This solution of 2-Me-4 was
added to the cold slurry at 0.degree. C. Additional tetrahydrofuran
(50 mL) was added to rinse in residues. The mixture was stirred for
16 h under argon, allowing the mixture to warm to ambient
temperature. The mixture was cooled to 0.degree. C. and water (200
mL) was cautiously added. Next, 15% sulfuric acid was added, which
dropped the pH to pH 3.3. Saturated sodium bicarbonate was added to
adjust pH to pH 8.0. The solids were filtered through paper in a
Buchner funnel in portions (very sluggish). The filter-cake was
washed with ethyl acetate (1.times.500 mL, 2.times.800 mL). These
washes were each used to re-extract the aqueous layer. The ethyl
acetate extracts were combined and dried over anhydrous magnesium
sulfate, then the mixture was filtered. The filtrate was evaporated
under reduced pressure to give a crude product (6.1 g). The crude
product was combined with other crops (7.1 g) and placed on a 100
mm diameter flash column, packed with silica gel (800 g) using
60:30:1 chloroform/ethyl acetate/meOH. The column was eluted with
60:30:1 chloroform/ethyl acetate/meOH. The fractions containing the
purest product were combined and the solvents evaporated to dryness
under reduced pressure to give purified 2-Me-5 (3.4 g, 22.5%): HPLC
(Method A) 92.8% (ALP, t.sub.R=4.79 min.
Collection A: Preparation of 2-Me-5 Mesylate Intermediate
[0111] A 100-mL, one-neck, round-bottomed flask was charged with
2-Me-5 (3.4 g, 11.0 mmol) and methylene chloride (47 mL). Next,
diisopropylethylamine (3.6 g, 27.6 mmol) was added to the flask,
followed by the addition of mesyl chloride (1.4 g, 12.2 mmol). The
reaction mixture had warmed to a gentle reflux. The mixture was
stirred for 1 h, while it cooled toward ambient temperature. The
reaction mixture was evaporated to dryness under reduced pressure
to give crude product (7.3 g). The crude product was placed on a 40
mm diameter flash column, packed with silica gel (185 g) using
230:30:3 chloroform/ethyl acetate/2 M ammonia in methanol The
column was eluted with 230:30:3 chloroform/ethyl acetate/2 M
ammonia in methanol. The fractions containing the purest product
were combined and the solvents evaporated to dryness under reduced
pressure to give purified 2-Me-5 mesylate intermediate (3.4 g,
79.8%): HPLC (Method A) 98.8% (AUC), t.sub.R=5.15 min.
Collection A: Preparation of 2-Me-6
[0112] A 200-mL, one-neck, round-bottomed flask was charged with
2-Me-5 mesylate intermediate (3.4 g, 8.8 mmol and dimethylformamide
(50 mL). To the reaction mixture,
.alpha.,.alpha.,.alpha.-trifluoro-p-cresol (1.4 g, 8.8 mmol) was
added, followed by cesium carbonate (7.2 g, 22.1 mmol). The mixture
was stirred in a preheated oil bath (75.degree. C.) for 4 h, then
was stirred for 16 h with no heating, while cooling toward ambient
temperature. Ethyl acetate (140 mL) was added and the mixture was
washed with brine (3.times.100 mL). The ethyl acetate layer was
dried over anhydrous magnesium sulfate, then filtered. The filtrate
was evaporated under reduced pressure to dryness to give a crude
product (4.0 g). The crude product was placed on a 40 mm diameter
flash column, packed with silica gel (220 g) using 460:60:3
chloroform/ethyl acetate/2 M ammonia in methanol. The column was
eluted with 460:60:3 chloroform/ethyl acetate/2 M ammonia in
methanol. The fractions containing the purest product were combined
and the solvents evaporated to dryness under reduced pressure to
give purified 2Me-6 (2.1 g, 52.5%): LC/MS (Ion spray) m/z 452
[C.sub.25H.sub.29ClF.sub.3NO+H].sup.+, HPLC (Method A)>99%
(AUC), t.sub.R=6.56 min. The .sup.1H NMR and .sup.13C NMR spectra
were consistent with the assigned structure.
Collection A: Preparation of 4-Me-1
[0113] A 500-mL, three-neck, round-bottomed flask was charged with
4-methylnicotinic acid hydrochloride (7.4 g, 42.8 mmol) and
hydrochloric acid in methanol (200 mL; 200 mg/mL). The mixture was
heated at a gentle reflux for 5 h, then it was stirred for 16 h,
while cooling to ambient temperature. An in-process HPLC was run
after stirring for 15 h at ambient temperature [HPLC (Method A):
95.0% (AUC), t.sub.R=1.84 min]. The solution was evaporated under
reduced pressure to dryness to give 4-Me-1 (10.9 g,
quantitative).
Collection A: Preparation of 4-Me-2
[0114] A 400-mL Fisher-Porter reactor was charged with methanol
(115 mL), concentrated hydrochloric acid (4.8 g), 10% Pd/C (1.5 g)
and 4-Me-1 (10.9 g, 42.8 mmol). The mixture was heated to
80.degree. C. and placed under 60 psi hydrogen pressure. The
mixture was then stirred for 16 h under these conditions. The
mixture was cooled and filtered through a bed of diatomaceous
earth. The filtrate was evaporated under reduced pressure to give
4-Me-2 (9.6 g, quantitative). The .sup.1H NMR spectrum was
consistent with the assigned structure.
Collection A: Preparation of 4-Me-4
[0115] A 2-L, three-neck, round-bottomed flask, fitted with a
mechanical stirrer and placed under an argon atmosphere, was
charged with 4-Me-2 (9.6 g, 42.8 mmol) and methylene chloride (535
mL). While stirring at ambient temperature, triethylamine (19.1 g,
188 mmol) was added. Next, 1-(4-chlorophenyl)-cyclobutanecarboxylic
acid (14.5 g, 68.8 mmol) was added, followed by
bromotis(pyrrolidino)phosphonium hexafluorophosphate (PyBroP, 32.9
g, 70.5 mmol). The mixture was stirred under argon at ambient
temperature for 16 h. A solution of 10% potassium hydroxide (650
mL) was added to the reaction mixture. Ethyl acetate (400 mL) was
then added and the mixture was stirred for 5 min. The layers were
separated and the aqueous layer was re-extracted with ethyl acetate
(400 mL). The ethyl acetate extracts were combined and dried over
anhydrous magnesium sulfate. This mixture was filtered and the
filtrate was evaporated under reduced pressure to give crude
product (47.1 g). The crude product was split in two equal
portions. The first portion was placed on a 100 mm diameter flash
column, packed with silica gel (700 g) using 60:30:1
CHCl.sub.3/EtOAc/MeOH This column was eluted with 60:30:1
CHCl.sub.3/EtOAc/MeOH. The second portion was placed on a 100 mm
diameter flash column, packed with silica gel (700 g) using
160:40:1 CHCl.sub.3/EtOAc/MeOH. This column was eluted with
160:40:1 CHCl.sub.3/EtOAc/MeOH. The fractions containing the purest
product were combined and the solvents evaporated to dryness under
reduced pressure. Less pure fractions were combined and the
solvents evaporated under reduced pressure to give material (3.2 g)
for a third smaller column. After column chromatography, purified
4-Me-4 (11.4 g) was isolated in three lots: (9.4 g, 35.4%), HPLC
(Method A) 83.8% (AUC), t.sub.R=5.90 min. (4.5 g, 30.2%), HPLC
(Method A) 93.4% (AUC), t.sub.R=5.88 min; and (1.6 g, 10.4%).
Collection A: Preparation of 4-Me-5
[0116] A 2-L, three-neck, round-bottomed flask placed under argon
was charged with tetrahydrofuran (300 mL), then was cooled to
0.degree. C. Lithium aluminum hydride (383 g) was added slowly at
0.degree. C. In a separate flask, 4-Me-4 (11.4 g) in three lots
(5.3 g, 15.1 mmol; 4.5 g, 12.9 mmol; and 1.6 g, 4.4 mmol) was
dissolved in tetrahydrofuran (250 mL). The solution of 4-Me-4 was
added to the cold slurry of LAH at 0.degree. C. Additional
tetrahydrofuran (25 mL) was added to rinse in residues. The mixture
was stirred for 16 h under argon, allowing the mixture to warm to
ambient temperature. The mixture was cooled to 0.degree. C. and
water (300 mL) was cautiously added. Next, 15% sulfuric acid was
added, which dropped the pH to pH 3.5. Solid sodium bicarbonate was
added to adjust pH to pH 7.6. The solids were filtered through
diatomaceous earth/paper in a Buchner funnel in portions (very
sluggish). The filter cake was washed with ethyl acetate
(1.times.250 mL, 2.times.400 mL). These washes were each used to
re-extract the aqueous layer. The ethyl acetate extracts were
combined and dried over anhydrous magnesium sulfate, then the
mixture was filtered. The filtrate was evaporated under reduced
pressure to give a crude product (8.1 g). The crude product was
placed on a 100 mm diameter flash column, packed with silica gel
(800 g) using 160:40:1 CHCl.sub.3/EtOAc/MeOH. The column was eluted
with 60:30:1 CHCl.sub.3/EtOAc/MeOH. The fractions containing the
purest product were combined and the solvents evaporated to dryness
under reduced pressure to give purified 4-Me-5 (2.8 g, 28.1%): HPLC
(Method A) 77.6% (AUC), t.sub.R=5.13 min.
Collection A: Preparation of 4-Me-5 Mesylate Intermediate
[0117] A 100-mL, one-neck, round-bottomed flask was charged with
4-Me-5 (2.8 g, 9.1 mmol) and methylene chloride (40 mL). Next;
diisopropylethylainine (2.9 g, 22.7 mmol) was added to the flask,
followed by the addition of mesyl chloride (1.2 g, 10.0 mmol). The
reaction mixture had warmed to a gentle reflux. The mixture was
stirred for 1 h, while it cooled toward ambient temperature. The
reaction mixture was evaporated to dryness under reduced pressure
to give crude product (5.7 g). The crude product was placed on a 40
mm diameter flash column, packed with silica gel (200 g) using
230:30:3 chloroform/ethyl acetate/2 M ammonia in MeOH. The column
was eluted with 230:30:3 chloroform/ethyl acetate/2 M ammonia in
MeOH. The fractions containing the purest product were combined and
the solvents evaporated to dryness under reduced pressure to give
purified 4-Me-5 mesylate intermediate (2.2 g, 62.7%): HPLC (Method
A) 91.6% (AUC), t.sub.R=5.26 min.
Collection A: Preparation of 4-Me-6
[0118] A 200-mL, one-neck, round-bottomed flask was charged with
4-Me-5 mesylate intermediate (2.2 g, 5.7 mmol) and
dimethylformamide (35 mL). To the reaction mixture,
.alpha.,.alpha.,.alpha.-trifluoro-p-cresol-(0.9 g, 5.7 mmol) was
added, followed by cesium carbonate (4.7 g, 14.3 mmo). The mixture
was stirred in a preheated oil bath (75.degree. C.) for 5 h, then
was stirred for 16 h with no heating while cooling toward ambient
temperature. Ethyl acetate (100 mL) was added and the mixture was
washed with brine (3.times.70 mL). The ethyl acetate layer was
dried over anhydrous magnesium sulfate, then filtered. The filtrate
was evaporated under reduced pressure to dryness to give a crude
product (3.8 g). The crude product was placed on a 40 mm diameter
flash column, packed with silica gel (215 g) using 460:60:3
chloroform/ethyl acetate/2 M ammonia in methanol. The column was
eluted with 460:60:3 chloroform/ethyl acetate/2 M ammonia in
methanol. The fractions containing the purest product were combined
and the solvents evaporated to dryness under reduced pressure to
give purified 4-Me-6 (1.1 g, 43.1%): LC/MS (Ion spray) m/z 452
[C.sub.25H.sub.29ClF.sub.3NO+H].sup.+; HPLC (Method A) 93.8% (AUC),
t.sub.R=6.64 min. The .sup.1H NMR and .sup.13C NMR spectra were
consistent with the assigned structure.
Collection A: Preparation of 6-Me-2
[0119] A 400-mL Fisher-Porter reactor was charged with methanol
(300 mL), concentrated hydrochloric acid (13.0 g), 10% Pd/C (4.0 g)
and methyl-6-methylnicotinate (20.0 g, 132 mmol). The mixture was
heated to 80.degree. C. and placed under 60 psi hydrogen pressure.
The mixture was then stirred for 21 h under these conditions. The
mixture was cooled and filtered. The filtrate was evaporated under
reduced pressure to give 6-Me-2 (27.0 g, quantitative). The .sup.1H
NMR spectrum was consistent with the assigned structure.
Collection A: Preparation of 6-Me-4
[0120] A 2-L, three-neck, round-bottomed flask, fitted with a
mechanical stirrer and placed under an argon atmosphere, was
charged with 6-Me-2 (14.0 g, 72.3 mmol) and methylene chloride (900
mL). While stirring at ambient temperature, triethylamine (32.2 g,
318 mmol) Was added, Next, 1-(4-chlorophenyl)-cyclobutanecarboxylic
acid. (24.5 g, 116.2 mmol) was added, followed by
bromotris(pyrrolidino)phosphonium hexafluorophosphate (PyBroP, 55.5
g, 119.1 mmol). The mixture was stirred under argon at ambient
temperature for 16 h. A solution of 10% potassium hydroxide (1.0 L)
was added to the reaction mixture. Ethyl acetate (500 mL) was then
added and the mixture was stirred for 5 min. The layers were
separated and the aqueous layer was re-extracted with ethyl acetate
(500 mL). The ethyl acetate extracts were combined and dried over
anhydrous magnesium sulfate. This mixture was filtered and the
filtrate was evaporated under reduced pressure to give crude
product (74.3 g). The crude product was split in two equal
portions. Each portion was placed on a 100 mm diameter flash
column, packed with silica gel (800 g) using 60:30:1
CHCl.sub.3/EtOAc/MeOH. Each column was eluted with 60:30:1
CHCl.sub.3/EtOAc/MeOH. The fractions containing the purest product
were combined and the solvents evaporated to dryness under reduced
pressure. Less pure fractions were combined and the solvents
evaporated under reduced pressure to give material (8.0 g) for a
third smaller column. After column chromatography, purified 6-Me-4
(17.8 g) was isolated in three lots: (7.5 g, 29.7%), HPLC (Method
A) 82.0% (AUC), t.sub.R=5.83 min.; (7.4 g, 29.3%), HPLC (Method A)
78.3% (AUC), t.sub.R=5.83 min.; (2.9 g, 11.5%), HPLC (Method A)
80.0% (AUC), t.sub.R=5.82 min.
Collection A: Preparation of 6-Me-5
[0121] A 3-L, three-neck, round-bottomed flask placed under argon
was charged with tetrahydrofuran (450 mL), then was cooled to
0.degree. C. Lithium aluminum hydride (60.6 g) was added slowly at
0.degree. C. In a separate flask, 6-Me-4 (17.8 g) from three lots
(7.5 g, 21.4 mmol; 7.4 g, 21.2 mmol; 2.9 g, 8.3 mmol) was dissolved
in tetrahydrofuran (400 mL). This solution of 6-Me-4 was added to
the cold slurry of LAH at 0.degree. C. Additional tetrahydrofuran
(50 mL) was added to rinse in residues. The mixture was stirred for
16 h under argon, allowing the mixture to warm to ambient
temperature. The mixture was cooled to 0.degree. C. and water (350
mL) was cautiously added. Next, 1 N sulfuric acid (350 mL) was
added, which dropped the pH to pH 7.7. The solids were filtered
through paper in a Buchner funnel in portions (very sluggish).
Additional water (800 mL) and ethyl acetate (400 mL) were added, to
facilitate stirring. The filter cakes were each washed with ethyl
acetate (1.times.100 mL). These washes were each used to re-extract
the aqueous layer. The ethyl acetate extracts were combined and
dried over anhydrous magnesium sulfate, then the mixture was
filtered. The filtrate was evaporated under reduced pressure to
give a crude product (13.7 g). The crude product was placed on a
100 mm diameter flash column, packed with silica gel (800 g) using
60:30:1 CHCl.sub.3/EtOAc/MeOH. The column was eluted with 60:30:1
CHCl.sub.3/EtOAc/MeOH. The fractions containing the purest product
were combined and the solvents evaporated to dryness under reduced
pressure to give purified 6-Me-5 (4.2 g) in three lots: (1.7 g,
10.7%), HPLC (Method A) 86.6% (AUC), t.sub.R=4.88 min.; (1.9 g,
12.2%), HPLC (Method A) 85.4% (AUC), t.sub.R=4.92 min.; and (0.6 g,
3.8%), HPLC (Method A) 81.3% (AUC), t.sub.R=4.83 min.
Collection A: Preparation of 6-Me-5 Mesylate Intermediate
[0122] A 200-mL, one-neck., round-bottomed flask was charged with
6-Me-5 (4.2 g) from three lots (1.7 g, 5.5 mmol); 1.9 g, 6.2 mmol;
and 0.6 g, 1.9 mmol) and methylene chloride (70 mL). Next,
diisopropylethylamine (4.4 g, 33.8 mmol) was added to the flask,
followed by the addition of mesyl chloride (1.7 g, 14.9 mmol). The
reaction mixture had warmed to a gentle reflux. The mixture was
stirred for 1 h, while it cooled toward ambient temperature. The
reaction mixture was evaporated to dryness wider reduced pressure
to give crude product (8.5 g). The crude product was placed on a 40
mm diameter flash column, packed with silica gel (230 g) using
230:30:2 chloroform/ethyl acetate/2 M ammonia in MeOH. The column
was eluted with 230:30:2 chloroform/ethyl acetate/2 M ammonia in
MeOH. The fractions containing the purest product were combined and
the solvents evaporated to dryness under reduced pressure to give
purified 6-Me-5 mesylate intermediate (2.4 g, 45.2%): HPLC (Method
A) 87.2% (AUC), t.sub.R=5.17 min.
Collection A: Preparation of 6-Me-6
[0123] A 100-mL, one-neck, round-bottomed flask was charged with
6-Me-5 mesylate intermediate (2.4 g, 6.1 mmol) and
dimethylformamide (38 mL). To the reaction mixture,
.alpha.,.alpha.,.alpha.-trifluoro-p-cresol (1.0 g, 6.1 mmol) was
added, followed by cesium carbonate (5.0 g, 15.3 mmol). The mixture
was stirred in a preheated oil bath (75'C) for 4 h, then was
stirred for 16 h with no heating, while cooling toward ambient
temperature. Ethyl acetate (100 mL) was added and the mixture was
washed with brine (3.times.70 mL). The ethyl acetate liquors were
dried over anhydrous magnesium sulfate, then filtered. The filtrate
was evaporated under reduced pressure to dryness to give a crude
product (3.9 g). The crude product was placed on a 40 mm diameter
flash column, packed with silica gel (230 g) using chloroform (460
parts), ethyl acetate (60 parts) and 2M ammonia in methanol (3
parts). The column was eluted with a solvent mixture of chloroform
(460 parts), ethyl acetate (60 parts) and 2M ammonia in methanol (3
parts). The fractions containing the purest product were combined
and the solvents evaporated to dryness under reduced pressure to
give purified 6-Me-6 (2.1 g, 76.2%). LC/MS (Ion spray) m/z 452
[C.sub.25H.sub.29ClF.sub.3NO+H].sup.+. HPLC (Method A) 96.3% (AUC),
t.sub.R=6.41 min. The .sup.1H NMR and .sup.13C NMR spectra were
consistent with the assigned structure.
Collection B: Preparation of 6-Me-2
[0124] A 400-mL Fisher-Porter reactor was charged with methanol
(300 mL), concentrated hydrochloric acid (13.0 g), 10% Pd/C (4.0 g)
and methyl-6-methylnicotinate (20.0 g, 132 mmol). The mixture was
heated to 80.degree. C. and placed under 60 psi hydrogen pressure.
The mixture was then stirred for 21 h under these conditions. The
mixture was cooled and filtered. The filtrate was evaporated under
reduced pressure to give 6-Me-2 (27.0 g, quantitative). The .sup.1H
NMR spectrum was consistent with the assigned structure.
Collection B: Preparation of 6-Me-3
[0125] A 250-mL, four-neck, round-bottomed flask, fitted with a
magnetic stirrer was charged with 6-Me-2 (13.3 g, 68.4 mmol),
tetrahydrofuran (60 mL), water (60 mL) and sodium bicarbonate (14.4
g, 171 mmol). The reaction mixture was cooled to 5.degree. C. While
keeping the pH between pH 8 to pH 9, benzylchloroformate (12.0 g,
70.4 mmol) was added slowly over 90 min. The mixture was stirred at
ambient temperature for 1 h. The reaction mixture was placed under
reduced pressure to remove most of the tetrahydrofuran. Ethyl
acetate (50 mL) was then added and the mixture was stirred for 5
min. The layers were separated and the organic layer was washed
with water (20 mL). The ethyl acetate layer was dried over
anhydrous magnesium sulfate. This mixture was filtered and the
filtrate was evaporated under reduced pressure to give crude
product (16.5 g). The crude product was split in two equal
portions. One portion was placed on a 40 mm diameter flash column,
packed with silica gel (225 g) using 230:30:3
CHCl.sub.3/EtOAc/MeOH. The column was eluted with 230:30:3
CHCl.sub.3/EtOAc/MeOH. The fractions containing the purest product
were combined and the solvents evaporated to dryness under reduced
pressure. Other fractions were combined and the solvents evaporated
under reduced pressure to dryness as a minor product (possible
separation of diastereomers). After column chromatography, there
were two products: 6-Me-3 (major product), (3.4 g, 16.8%), HPLC
(Method A) 94.0% (AUC), t.sub.R=5.43 min.; and 6-Me-3 (minor
product), (0.4 g, 2.0%), HPLC (Method A) 98.1% (AUC), t.sub.R=5.28
min.
Collection B: Preparation of 6-Me-3 Acid
[0126] A 100-mL, one-neck, round-bottomed flask was charged with
tetrahydrofuran (15 mL), water (30 mL), lithium hydroxide (0.35 g,
14.6 mmol) and 6-Me-3 (3.25 g, 11.2 mmol). The mixture was stirred
at ambient temperature for 16 hours. The mixture was treated with
HCl to adjust the pH to pH 2.7. The liquors were extracted with
ethyl acetate (2.times.50 mL). The ethyl acetate extracts were
combined and dried over anhydrous magnesium sulfate, then the
mixture was filtered. The filtrate was evaporated under reduced
pressure to give a 6-Me-3 acid (3.0 g, 95.5%): HPLC (Method A)
90.7% (AUC), t.sub.R=4.78 min.
Collection B: Preparation of 6-Me-4
[0127] A 50-mL, one-neck, round-bottomed flask under argon was
charged with 6-Me-3 acid (2.9 g, 10.5 mmol) and tetrahydrofuran (15
mL). Next, 5.0 M borane-methyl sulfide (2.32 mL, 11.6 mmol) was
added slowly to the flask over 45 min., initially at ambient
temperature. As the addition progressed, the mixture was heated to
a gentle reflux and this was maintained during the remainder of the
addition. After the addition, the mixture was refluxed for 30 min.
The mixture was then stirred for 18 hour under inert atmosphere,
while cooling to ambient temperature. The reaction mixture was
added slowly to cold (5.degree. C.) methanol (35 mL). Gas evolution
was seen. The reaction mixture was evaporated to dryness under
reduced pressure to give 6-Me-4 (2.7 g, 97.6%): HPLC (Method A)
89.9% (AUC), t.sub.R=4.87 min.
Collection B: Preparation of SC-2
[0128] A 200-mL, one-neck, round-bottomed flask under argon was
charged with 1-(4-chlorophenyl)-1-cyclobutanecarbonitrile (10.0 g,
52.2 mmol) and dry toluene (60 mL). To the reaction mixture, 3.0 M
methylmagnesium bromide (52.2 mL, 157 mmol) was added slowly over
20 min. The mixture was heated to 95.degree. C. for 12 hours. A
solution of 6 N HCl (40 mL) was added to the mixture (gas evolution
seen, exothermic). The mixture was heated at reflux for 60 min.,
then cooled to ambient temperature. The liquors were extracted with
ethyl acetate (2.times.400 mL). The ethyl acetate extracts were
combined and washed with brine (80 mL). The ethyl acetate liquors
were dried over anhydrous magnesium sulfate, then filtered. The
filtrate was evaporated under reduced pressure to dryness to give
SC-2 (11.1 g, quantitative HPLC (Method A) 97.3% (AUC),
t.sub.R=5.64 min.
Collection B: Preparation of SC-3
[0129] A 100-mL, one-neck, round-bottomed flask under argon was
charged with SC-2 (11.0 g, 52.7 mmol) and methylene chloride (40
mL). The mixture was cooled to 20'C, then liquid bromine (8.4 g,
52.7 mmol) was added slowly over 20 min. The mixture was stirred at
ambient temperature for 30 min., then was poured over ice water (55
g). The liquors were separated and the aqueous layer was
re-extracted with methylene chloride (20 mL). The methylene
chloride extracts were combined and were dried over anhydrous
magnesium sulfate, then filtered. The filtate was evaporated under
reduced pressure to dryness to give SC-3 (14.5 g, 95.3%): HPLC
(Method A) 71.2% (AUC), t.sub.R=5.88 min.
##STR00007##
[0130] A mixture of 1 (0.942 g, 4.48 mmol) and thionyl chloride (2
L) were heated at reflux for 3 hours. The reaction mixture was
concentrated, diluted with THF (2 mL), and concentrated in vacuo to
give an oil. The oil was dissolved in THF (15 mL) and then cooled
to 0.degree. C. Next, diazomethane (generated at 0.degree. C. from
2 g 1-methyl-1-nitro-1-nitrososguanidine in 15 mL diethyl ether and
1.36 g sodium hydroxide in 15 mL water) was added. The resulting
solution was maintained at 0.degree. C. overnight. Hydrochloric
acid (5 mL; 4 M) was carefully added. The reaction mixture was
maintained at 0.degree. C. for 1 hour, and then concentrated to an
oil. The oil was purified by column chromatography on silica gel
eluting with hexane/ethyl acetate (90:10) to give 2 as a colorless
oil.
[0131] To a solution of 2 (96 mg, 0.393 mmol) in acetone (0.5 mL)
was added sodium iodide (59 mg, 0.393 mmol). After 5 minutes at
room temperature, the mixture was added to a mixture of 3 (127 mg,
0.328 mmol) and potassium carbonate (226 mg) in acetone (0.5 mL).
The resulting mixture was heated to 50.degree. C. for 18 hours. The
reaction mixture was poured into water (20 mL) and extracted with
ethyl acetate (2.times.20 mL). The organic extracts were combined,
washed with brine (15 mL), dried over anhydrous sodium sulfate,
filtered, and concentrated to a yellow oil. The oil was purified by
column chromatography on silica gel eluting with hexane/ethyl
acetate/2 N ammonia in ethanol (80:16:4) to give 4 as a colorless
oil.
##STR00008##
[0132] To a solution of 4 (67.5 mg, 0.141 mmol) in methanol (1 mL)
at 0.degree. C. was added sodium borohydride (11 mg, 0.282 mmol).
The reaction mixture was maintained at room temperature for 2
hours. The reaction mixture was poured into water (10 mL) and
extracted with ethyl acetate (2.times.15 mL). The organic extracts
were combined, washed with brine (10 mL), dried over anhydrous
sodium sulfate, filtered, and concentrated to a colorless oil. The
oil was purified by column chromatography on silica gel eluting
with hexane/ethyl acetate/2 N ammonia in ethanol (80:16:4) to give
5 as a colorless oil.
[0133] Other compounds of similar structure be synthesized with
minor modification.
B. Combinations Including DAT Inhibitors
[0134] In certain embodiments, the method includes administering,
conjointly with the pharmaceutical preparation, one or more of
physical therapy, occupational therapy, or speech/language
therapy.
[0135] An agent to be administered conjointly with a subject
compound may be formulated together with a subject compound as a
single pharmaceutical preparation, e.g., as a pill or other
medicament including both agents, or may be administered as a
separate pharmaceutical preparation.
[0136] In certain embodiments of the packages, preparations,
compositions, and methods for the treatment of a movement disorder,
the invention further comprises one or more therapeutic agents for
treating Parkinson's disease selected from a dopamine precursor,
such as L-dopa; a dopaminergic agent, such as Levodopa-carbidopa
(Sinemet.RTM., Sinemet CR.RTM.) or Levodopa-benzerazide
(Prolopa.RTM., Madopar.RTM., Madopar HBS.RTM.); a dopaminergic and
anti-cholinergic agent, such as amantadine (Symmetryl.RTM.,
Symadine.RTM.); an anti-cholinergic agent, such as trihexyphenidyl
(Artane.RTM.), benztropine (Cogentin.RTM.), ethoproprazine
(Parsitan.RTM.), or procyclidine (Kemadrin.RTM.); a dopamine
agonist, such as apomorphine, bromocriptine (Parlodel.RTM.),
cabergoline (Dostinexe), lisuride (Dopergine.RTM.), pergolide
(Permax.RTM.), pramipexole (Mirapex.RTM.), or ropinirole
(Requip.RTM.); a MAO-B (monoamine oxidase B) inhibitor, such as
selegiline or deprenyl (Atapryl.RTM., Carbex.RTM., Eldepryl.RTM.);
a COMT (catechol O-methyltransferase) inhibitor, such as tolcapone
(Tasmar.RTM.) or entacapone (Comtan.RTM.); pr other therapeutic
agents, such as baclofen (Lioresal.RTM.), domperidone
(Motilium.RTM.), fludrocortisone (Florinef.RTM.), midodrine
(Amatine.RTM.), oxybutinin (Ditropan.RTM.), propranolol
(Inderal.RTM., Inderal-LA.RTM.), clonazepam (Rivotril.RTM.), or
yohimbine.
[0137] In certain embodiments of the packages, preparations,
compositions, and methods for the treatment of a movement disorder,
the invention further comprises one or more therapeutic agents for
treating dystonia selected from an anti-cholinergic agent, such as
trihexyphenidyl (Artane.RTM.), benztropine (Cogentin.RTM.),
ethoproprazine (Parsitan.RTM.), or procyclidine (Kemadrin.RTM.); a
dopaminergic agent, such as Levodopa-carbidopa (Sinemet.RTM.,
Sinemet CR.RTM.) or Levodopa-benzerazide (Prolopa.RTM.,
Madopar.RTM., Madopar HBS.RTM.); a muscle relaxant, such as
baclofen (Lioresal.RTM.); a sedative, such as Clonazepam
(Rivotril.RTM.); an anticonvulsant agent, such as carbamazepine
(Tegretol.RTM.); a dopamine reuptake inhibitor, such as
tetrabenazine (Nitoman.RTM.); or a dopamine blocker, such as
haloperidol (Haldol.RTM.).
[0138] In certain embodiments of the packages, preparations,
compositions, and methods for the treatment of a movement disorder,
the invention further comprises one or more therapeutic agents for
treating tremor selected from a .beta.-blocker, such as propranolol
(Inderal.RTM., Inderal-LA.RTM.); an anticonvulsant agent, such as
primidone (Mysoline.RTM.); or a carbonic anhydrase inhibitor, such
as acetalzolamide (Diamox.RTM.) or methazolamide
(Neptazane.RTM.).
[0139] In certain embodiments of the packages, preparations,
compositions, and methods for the treatment of a movement disorder,
the invention further comprises one or more therapeutic agents for
treating myoclonus selected from a sedative, such as clonazepam
(Rivotril.RTM.); or an anticonvulsant agent, such as valproic acid
(Epival.RTM.).
[0140] In certain embodiments of the packages, preparations,
compositions, and methods for the treatment of a movement disorder,
the invention further comprises one or more therapeutic agents for
treating chorea selected from a dopamine blocker, such as
haloperidol (Haldol.RTM.); or a dopamine reuptake inhibitor, such
as tetrabenazine (Nitoman.RTM.).
[0141] In certain embodiments of the packages, preparations,
compositions, and methods for the treatment of a movement disorder,
the invention further comprises one or more therapeutic agents for
treating restless leg syndrome selected from a dopaminergic, such
as Levodopa-carbidopa (Sinemet.RTM., Sinemet CR.RTM.) or
Levodopa-benzerazide (Prolopa.RTM., Madopar.RTM., Madopar
HBS.RTM.); a sedative, such as clonazepam (Rivotril.RTM.); a
dopamine agonists, such as bromocriptine (Parlodel.RTM.), pergolide
(Permax.RTM.), pramipexole (Mirapex.RTM.), or ropinirole
(Requip.RTM.); a narcotic agent, such as codeine (Tylenol #3.RTM.);
or a GABAergic, such as gabapentin (Neurontin.RTM.).
[0142] In certain embodiments of the packages, preparations,
compositions, and methods for the treatment of a movement disorder,
the invention further comprises one or more therapeutic agents for
treating tics selected from a sedative, such as clonazepam
(Rivotril.RTM.); an alpha antagonist, such as clonidine
(Catapress.RTM.); a dopamine reuptake inhibitor, such as
tetrabenazine (Nitoman.RTM.); or a dopamine blocker, such as
haloperidol (Haldol.RTM.) or perphenazine.
[0143] In certain embodiments of the packages, preparations,
compositions, and methods for the treatment of a movement disorder,
the invention further comprises one or more
cyclooxygenase-2-selective inhibitors.
C. Pharmaceutical Preparations of DAT Inhibitors
[0144] In another aspect, the present invention provides
pharmaceutical preparations comprising the subject DAT inhibitors.
The DAT inhibitors for use in the subject method may be
conveniently formulated for administration with a biologically
acceptable, non-pyrogenic, and/or sterile medium, such as water,
buffered saline, polyol (for example, glycerol, propylene glycol,
liquid polyethylene glycol, and the like) or suitable mixtures
thereof. The optimum concentration of the active ingredient(s) in
the chosen medium can be determined empirically according to
procedures well known to behavioral scientists. As used herein,
"biologically acceptable medium" includes any and all solvents,
dispersion media, and the like which may be appropriate for the
desired route of administration of the pharmaceutical preparation.
The use of such media for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the activity of the DAT inhibitors, its use in
the pharmaceutical preparation of the invention is contemplated.
Suitable vehicles and their formulation inclusive of other proteins
are described, for example, in the book Remington's Pharmaceutical
Sciences (Remington's Pharmaceutical Sciences. Mack Publishing
Company, Easton, Pa., USA. 1985). These vehicles include injectable
"deposit formulations,"
[0145] Pharmaceutical formulations of the present invention can
also include veterinary compositions, e.g., pharmaceutical
preparations of the DAT inhibitors suitable for veterinary uses,
e.g., for the treatment of livestock or domestic animals, e.g.,
dogs.
[0146] Methods of introduction may also be provided by rechargeable
or biodegradable devices. Various slow release polymeric devices
have been developed and tested in vivo in recent years for the
controlled delivery of drugs. A variety of biocompatible polymers
(including hydrogels), including both biodegradable and
non-degradable polymers, can be used to form an implant for the
sustained release of an DAT inhibitor at a particular target site.
In accordance with the practice of this invention, it has been
found that a dosage form and a method can be provided that
administers an DAT inhibitor in a program that substantially
lessens or completely compensates for tolerance in a patient.
Tolerance, as defined in Pharmacology in Medicine, by Brill, p. 227
(1965) McGraw-Hill, is characterized as a decrease in effect
followed by administering a drug. When tolerance develops following
a single dose or a few doses over a very short time, it is referred
to as acute tolerance. When the drug is administered over a more
protracted period of time to show a demonstrable degree of
tolerance, it is referred to as chronic tolerance. The medical
literature, as exemplified in The Pharmacological Bases of
Therapeutics, by Goodman and Gilman, 8th Ed., p, 72 (1990) Pergamon
Press, reported tolerance may be acquired to the effects of many
drugs and this literature classifies tolerance as acute or chronic
based on when it is acquired. That is, acute tolerance develops
during a dosing phase of one dose or on one day, and chronic
tolerance is acquired due to chronic administration, typically
weeks, months, and years.
[0147] In certain embodiments, particularly where the selected DAT
inhibitor is one which may produce tolerance, e.g., acute
tolerance, in the patient, it may be desirable to formulate the
compound for variable dosing, and preferably for use in a
dose-escalation regimen. In preferred embodiments, the subject DAT
inhibitors are formulated to deliver a sustained and increasing
dose, e.g., over at least 4 hours, and more preferably, over at
least 8 or even 16 hours.
[0148] In certain embodiments, representative dosage forms include
hydrogel matrix containing a plurality of tiny pills. The hydrogel
matrix comprises a hydrophilic polymer, such as a polysaccharide,
agar, agarose, natural gum, alkali alginate including sodium
alginate, carrageenan, fucoidan, furcellaran, laminaran, hypnea,
gum arabic, gum ghatti, gum karaya, gum tragacanth, locust bean
gum, pectin, amylopectin, gelatin, and a hydrophilic colloid. The
hydrogel matrix comprises a plurality of tiny pills (such as 4 to
50), each tiny pill comprising an increasing dose population of
from 100 ng ascending in dose, such as 0.5 mg, 1 mg, 1.2 mg, 1.4
mg, 1.6 mg, 1.8 mg, etc. The tiny pills comprise a release rate
controlling wall of 0.0 mm to 10 mm thickness to provide for the
timed ascending release of drug. Representative wall-forming
materials include a triglyceryl ester selected from glyceryl
tristearate, glyceryl monostearate, glyceryl dipalmitate, glyceryl
laureate, glyceryl didecenoate, and glyceryl tridecenoate. Other
wall forming materials comprise polyvinyl acetate phthalate,
methylcellulose phthalate, and microporous vinyl olefins.
Procedures for manufacturing tiny pills are disclosed in U.S. Pat.
Nos. 4,434,153; 4,721,613; 4,853,229; 2,996,431; 3,139,383, and
4,752,470, which are incorporated by reference herein.
[0149] In certain embodiments, the drug releasing beads are
characterized by a dissolution profile wherein 0 to 20% of the
beads undergo dissolution and release the drug in 0 to 2 hours, 20
to 40% undergo dissolution and release the drug in 2 to 4 hours, 40
to 60% exhibit dissolution and release in 4 to 6 hours, 60 to 80%
in 6 to 8 hours, and 80 to 100% in 8 to 10 hours. The drug
releasing beads can include a central composition or core
comprising a drug and pharmaceutically acceptable composition
forming ingredients including a lubricant, antioxidant, and buffer.
The beads comprise increasing doses of drug, for example, 1 mg, 2
mg, 5 mg, and so forth to a high dose, in certain preferred
embodiments, of 15 to 100 mg. The beads are coated with a release
rate controlling polymer that can be selected utilizing the
dissolution profile disclosed above. The manufacture of the beads
can be adapted from, for example, Liu et al. (1994) Inter. J. of
Pharm., 112:105-116; Liu et al. (1994) Inter, J. of Pharm.,
112:117-124; Pharm. Sci., by Remington, 14th Ed. pp. 1626-1628
(1970); Fincher et al. (1968) J. Pharm. Sci., 57:1825-1835; and
U.S. Pat. No. 4,083,949.
[0150] Another exemplary dosage form provided by the invention
comprises a concentration gradient of DAT inhibitor from 1 mg to
15-600 mg coated from the former low dose to the latter high dose
on a polymer substrate. The polymer can be an erodible or a
nonerodible polymer. The coated substrate is rolled about itself
from the latter high dose at the center of the dosage form, to the
former low dose at the exposed outer end of the substrate. The
coated substrate is rolled from the high dose to the low dose to
provide for the release of from low to high dose as the substrate
unrolls or erodes. For example, 1 mg to 600 mg of amphetamine is
coated onto an erodible polymer such as an polypeptide, collagen,
gelatin, or polyvinyl alcohol, and the substrate rolled
concentrically from the high dose rolled over and inward to adapt a
center position, and then outward towards the low dose to form an
outer position. In operation, the dosage form erodes dispensing an
ascending dose of amphetamine that is released over time.
[0151] Another dosage form provided by the invention comprises a
multiplicity of layers, wherein each layer is characterized by an
increasing dose of drug. The phrase "multiplicity of layers"
denotes 2 to 6 layers in contacting lamination. The multiplicity of
layers are positioned consecutively, that is, one layer after
another in order, with a first exposed layer, the sixth layer in
contact with the fifth layer and its exposed surface coated with a
drug impermeable polymer. The sixth layer is coated with a drug
impermeable polymer to insure release of the DAT inhibitor from the
first layer to the sixth layer. The first layer comprises, for
example, 1 to 50 mg of drug and each successive layer comprises an
additional 1 to 50 mg of drug. The biodegradable polymers undergo
chemical decomposition to form soluble monomers or soluble polymer
units. The biodegradation of polymers usually involves chemically
or enzymatically catalyzed hydrolysis. Representative of
biodegradable polymers acceptable for an increase drug loading in
each layer of from 5 to 50 wt % over the first and successive
layers Wherein the first layer comprises 100 ng. Representative
biodegradable polymers comprise biodegradable poly(amides),
poly(amino acids), poly(esters), polylactic acid), poly(glycolic
acid), poly(orthoesters), poly(anhydridcs), biodegradable
poly(dehydropyrans), and poly(dioxinones). The polymers are known
to the art in Controlled Release of Drugs, by Rosoff, Ch. 2, pp.
53-95 (1989); and in U.S. Pat. Nos. 3,811,444; 3,962,414;
4,066,747; 4,070,347; 4,079,038; and 4,093,709.
[0152] In still other embodiments, the invention employs a dosage
form comprising a polymer that releases a drag by diffusion, flux
through pores, or by rupture of a polymer matrix. The drug delivery
polymeric system comprises a concentration gradient, wherein the
gradient is an ascent in concentration from a beginning or initial
concentration to a final, or higher concentration. The dosage form
comprises an exposed surface at the beginning dose and a distant
nonexposed surface at the final dose. The nonexposed surface is
coated with a pharmaceutically acceptable material impermeable to
the passage of drug. The dosage form structure provides for a flux
increase delivery of drug ascending from the beginning to the final
delivered dose.
[0153] The dosage form matrix can be made by procedures known in
the polymer art. In one manufacture, 3 to 5 or more casting
compositions are independently prepared wherein each casting
composition comprises an increasing dose of drug with each
composition overlayered from a low to the high dose. This provides
a series of layers that come together to provide a unit polymer
matrix with a concentration gradient. In another manufacture, the
higher dose is cast first followed by laminating with layers of
decreasing dose to provide a polymer matrix with a drug
concentration gradient. An example of providing a dosage form
comprises blending a pharmaceutically acceptable carrier, like
polyethylene glycol, with a known dose of an DAT inhibitor and
adding it to a silastic medical grade elastomer with a
cross-linking agent, like stannous octanoate, followed by casting
in a mold. The step is repeated for each successive layer. The
system is allowed to set, e.g., for 1 hour, to provide the dosage
form. Representative polymers for manufacturing the dosage form
comprise olefin and vinyl polymers, condensation polymers,
carbohydrate polymers, and silicon polymers as represented by
poly(ethylene), poly(propylene), polyvinyl acetate), poly(methyl
acrylate), poly(isobutyl methacrylate), poly(alginate),
poly(amide), and poly(silicone). The polymers and manufacturing
procedures are known in Polymers, by Coleman et al., Vol. 31, pp.
1187-1230 (1990); Drug Carrier Systems, by Roerdink et al., Vol. 9,
pp. 57-109 (1989); Adv. Drug Delivery Rev., by Leong et al., Vol.
1, pp. 199-233 (1987); Handbook of Common Polymers, compiled by
Roff et al., (1971) published by CRC Press; and U.S. Pat. No.
3,992,518.
[0154] in still other embodiments, the subject formulations can be
a mixture of different prodrug forms of one or more different DAT
inhibitors, each prodrug form having a different hydrolysis rate,
and therefore activation rate, to provide an increasing serum
concentration of the active DAT inhibitors.
[0155] In other embodiments, the subject formulations can be a
mixture of different DAT inhibitors, each compound having a
different rate of adsorption (such as across the gut or epithelia)
and/or serum half-life.
[0156] The dose-escalation regimen of the present invention can be
used to compensate for the loss of a therapeutic effect of an DAT
inhibitor, if any, by providing a method of delivery that
continually compensates for the development of acute tolerance, by
considering the clinical effect (E) of a drug at time (t) as a
function of the drug concentration (C) according to Equation 1:
Effect=f(t,C)
[0157] In addition, the rate of drug delivered (A), in mg per hour,
is inversely proportional to the concentration times the clearance
of the drug. As the effect varies with time and the functionality
is expressed, then, according to this invention, (A) can be
governed to ensure the therapeutic effect is maintained at a
clinical value. If the effect from a drug is found clinically to
decrease with time, this decline could be linear as expressed by
Equation 2:
Effect(t)=Effect(ini)-.sup.keffect*t
[0158] wherein, Effect(ini) is the clinical effect observed
initially at the start of drug administration and Effect(t) is the
effect observed at time (t) hours, keffect is a proportionality
constant ascertained by measuring the clinical effect (E1) at time
(t1) hours and (E2) at time (t2) hours while maintaining a constant
plasma concentration followed by dividing (E1) minus (E2) by (t1)
minus (t2). In order to maintain a constant effect, (A) must be
adjusted with the same functionality according to Equation 3:
A(t)=A(ini)+keffect*t
[0159] wherein A(ini) is the initial drug input in mg per hour at
the start of the therapy and A(t) is the drug input at time (t)
hours, and keffect is the proportionality constant presented above.
If the therapeutic effect is found to decline exponentially with
time, this relationship is expressed by Equation 4:
Effect(t).times.Effect(ini)*exp(-keffect*t)
[0160] wherein Effect(ini) and Effect(t) are as defined before,
keffect is a rate constant (h.sup.-1), a unit of reciprocal hours,
ascertained by measuring the clinical effect (E1) at time (t1)
hours and (E2) at time (t2) hours while maintaining a constant
plasma concentration followed by dividing natural log of (E1) minus
natural log of (E2) by (t1) minus (t2). To maintain a constant
effect, (A) must be adjusted according to Equation 5:
A(t)=A(ini)*exp(keffect*t)
[0161] wherein A(ini) and A(t) are as defined before, keffect is
the rate constant (h.sup.-1) presented above. The equations are
presented in Holford et al. (1982) Pharmac. Ther., 16:143-166.
[0162] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are of course
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
infusion, inhalation, rectal suppository, or controlled release
patch. Oral and controlled release patch administrations are
preferred.
[0163] In certain preferred embodiments, the subject therapeutic is
delivered by way of a transdermal patch. A patch is generally a
flat hollow device with a permeable membrane on one side and also
some form of adhesive to maintain the patch in place on the
patient's skin, with the membrane in contact with the skin so that
the medication can permeate out of the patch reservoir and into and
through the skin. The outer side of the patch is formed of an
impermeable layer of material, and the membrane side and the outer
side are joined around the perimeter of the patch, forming a
reservoir for the medication and carrier between the two
layers.
[0164] Patch technology is based on the ability to hold an active
ingredient in constant contact with the epidermis. Over substantial
periods of time, drug molecules, held in such a state, will
eventually find their way into the bloodstream. Thus, patch
technology relies on the ability of the human body to pick up drug
molecules through the skin. Transdermal drug delivery using patch
technology has recently been applied for delivery of nicotine in an
effort to assist smokers in quitting, the delivery of
nitroglycerine to angina sufferers, the delivery of replacement
hormones in post menopausal women, etc. These conventional drug
delivery systems comprise a patch with an active ingredient such as
a drug incorporated therein, the patch also including an adhesive
for attachment to the skin so as to place the active ingredient in
close proximity to the skin. Exemplary patch technologies are
available from Ciba-Geigy Corporation and Alza Corporation. Such
transdermal delivery devices can be readily adapted for use with
the subject DAT inhibitors.
[0165] The flux of the subject DAT inhibitors across the skin can
be modulated by changing either (a) the resistance (the diffusion
coefficient), or (b) the driving force (the solubility of the drug
in the stratum corneum and consequently the gradient for
diffusion). Various methods can be used to increase skin permeation
by the subject DAT inhibitors, including penetration enhancers, use
of pro-drug versions, superfluous vehicles, iontophoresis,
phonophoresis, and thermophoresis. Many enhancer compositions have
been developed to change one or both of these factors. See, for
example, U.S. Pat. Nos. 4,006,218; 3,551,154; and 3,472,931, which
respectively describe the use of dimethylsulfoxide (DMSO), dimethyl
formamide (DMF), and N,N-dimethylacetamide (DMA) for enhancing the
absorption of topically applied drugs through the stratum corneum.
Combinations of enhancers consisting of diethylene glycol monoethyl
or monomethyl ether with propylene glycol monolaurate and methyl
laurate are disclosed in U.S. Pat. No. 4,973,468. A dual enhancer
consisting of glycerol monolaurate and ethanol for the transdermal
delivery of drugs is shown in U.S. Pat. No. 4,820,720. U.S. Pat.
No. 5,006,342 lists numerous enhancers for transdermal drug
administration consisting of fatty acid esters or fatty alcohol
ethers of C2 to C4 alkanediols, where each fatty acid/alcohol
portion of the ester/ether is of about 8 to 22 carbon atoms. U.S.
Pat. No. 4,863,970 shows penetration-enhancing compositions for
topical application comprising an active permeant contained in a
penetration-enhancing vehicle containing specified amounts of one
or more cell-envelope disordering compounds such as oleic acid,
oleyl alcohol, and glycerol esters of oleic acid; a C2 or C3
alkanol; and an inert diluent such as water. Other examples are
included in the teachings of U.S. Pat. No. 4,933,184 which
discloses the use of menthol as a penetration enhancer; U.S. Pat.
No. 5,229,130 which discloses the use of vegetable oil (soybean
and/or coconut oil) as a penetration, enhancer; and U.S. Pat. No.
4,440,777 which discloses the use of eucalyptol as a penetration
enhancer.
[0166] The phrases "parenteral administration" and "administered
parenterally" as used herein mean modes of administration other
than enteral and topical administration, usually by injection, and
include, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0167] The phrases "systemic administration," "administered
systemically," "peripheral administration," and "administered
peripherally" as used herein mean the administration of a compound,
drug, or other material other than directly into the central
nervous system, such that it enters the patient's system and, thus,
is subject to metabolism and other like processes, for example,
subcutaneous administration.
[0168] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally, and topically, as
by powders, ointments or drops, including buccally and
sublingually.
[0169] Regardless of the route of administration selected, the
compounds of the present invention, Which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms such as described below or by other conventional
methods known to those of skill in the art.
[0170] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration without being toxic to the
patient.
[0171] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular DAT inhibitors employed,
the age, sex, weight, condition, general health and prior medical
history of the patient being treated, and like factors well known
in the medical arts.
[0172] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0173] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
Generally, intravenous, intracerebroventricular, and subcutaneous
doses of the compounds of this invention for a patient will range
from about 0.0001 to about 100 mg per kilogram of body weight per
day.
[0174] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six, or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms.
[0175] The term "treatment" is intended to encompass also
prophylaxis, therapy, and cure.
[0176] The patient receiving this treatment is any animal in need,
including primates, in particular, humans and other mammals such as
equines, cattle, swine, and sheep; and poultry and pets in
general.
[0177] The compound of the invention can be administered as such or
in admixtures with pharmaceutically acceptable carriers and can
also be administered in conjunction with other drugs such as
dopamine precursors, dopaminergic agents, dopaminergic and
anti-cholinergic agents, anti-cholinergic agents, dopamine
agonists, MAO-B (monoamine oxidase B) inhibitors, COMT (catechol
O-methyltransferase) inhibitors, muscle relaxants, sedatives,
anticonvulsant agents, dopamine reuptake inhibitors, dopamine
blockers, 3-blockers, carbonic anhydrase inhibitors, narcotic
agents, GABAergic agents, or alpha antagonists. Conjunctive therapy
thus includes sequential, simultaneous and separate administration
of the active compound in a way that the therapeutic effects of the
first one administered are not entirely absent when the subsequent
is administered.
[0178] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical formulation (composition). The DAT
inhibitors according to the invention may be formulated for
administration in any convenient way for use in human or veterinary
medicine.
[0179] Thus, another aspect of the present invention provides
pharmaceutically acceptable compositions comprising a
therapeutically effective amount of one or more of the compounds
described above, formulated together with one or more
pharmaceutically acceptable carriers (additives) and/or diluents.
As described in detail below, the pharmaceutical compositions of
the present invention may be specially formulated for
administration in solid or liquid form, including those adapted for
the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets,
boluses, powders, granules, or pastes for application to the
tongue; (2) parenteral administration, for example, by
subcutaneous, intramuscular, or intravenous injection as, for
example, a sterile solution or suspension; (3) topical application,
for example, as a cream, ointment, or spray applied to the skin; or
(4) intravaginally or intrarectally, for example, as a pessary,
cream, or foam. However, in certain embodiments, the subject
compounds may be simply dissolved or suspended in sterile
water.
[0180] The phrase "pharmaceutically acceptable carrier" as used
herein means a pharmaceutically acceptable material, composition,
or vehicle, such as a liquid or solid filter, diluent, excipient,
solvent, or encapsulating material, involved in carrying or
transporting the subject regulators from one organ or portion of
the body to another organ or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as pharmaceutically
acceptable carriers include (1) sugars, such as lactose, glucose,
and sucrose; (2) starches, such as corn starch and potato starch;
(3) cellulose and its derivatives, such as sodium carboxymethyl
cellulose, ethyl cellulose, and cellulose acetate; (4) powdered
tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such
as cocoa butter and suppository waxes; (9) oils, such as peanut
oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn
oil, and soybean oil; (10) glycols, such as propylene glycol; (11)
polyols, such as glycerin, sorbitol, mannitol, and polyethylene
glycol; (12) esters such as ethyl oleate and ethyl laurate; (13)
agar; (14) buffering agents, such as magnesium hydroxide and
aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water;
(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol;
(20) phosphate buffer solutions; and (21) other non-toxic
compatible substances employed in pharmaceutical formulations.
[0181] As set out above, certain embodiments of the present DAT
inhibitors may contain a basic functional group, such as amino or
alkylamino, and are, thus, capable of forming pharmaceutically
acceptable salts with pharmaceutically acceptable acids. The term
"pharmaceutically acceptable salts" in this respect, refers to the
relatively non-toxic, inorganic and organic acid addition salts of
compounds of the present invention. These salts can be prepared in
situ during the final isolation and purification of the compounds
of the invention, or by separately reacting a purified compound of
the invention in its free base form with a suitable organic or
inorganic acid and isolating the salt thus formed. Representative
salts include but are not limited to following:
2-hydroxyethanesulfonate, 2-naphthalenesulfonate,
3-hydroxy-2-naphthoate, 3-phenylpropionate, acetate, adipate,
alginate, amsonate, aspartate, benzenesulfonate, benzoate,
besylate, bicarbonate, bisulfate, bitartrate, borate, butyrate,
calcium edetate, camphorate, camphorsulfonate, camsylate,
carbonate, citrate, clavulariate, cyclopentanepropionate,
digluconate, dodecylsulfate, edetate, edisylate, estolate, esylate,
ethanesulfonate, fumarate, gluceptate, glucoheptanoate, gluconate,
glutamate, glycerophosphate, glycollylarsanilate, hemisulfate,
heptanoate, hexafluorophosphate, hexanoate, hexylresorcinate,
hydrabamine, hydrobromide, hydrochloride, hydroiodide,
hydroxynaphthoate, iodide, isothionate, lactate, lactobionate,
laurate, laurylsulphonate, malate, maleate, mandelate, mesylate,
methanesulfonate, methylbromide, methylnitrate, methylsulfate,
mucate, naphthylate, napsylate, nicotinate, nitrate,
N-methylglucamine ammonium salt, oleate, oxalate, palmitate,
pamoate, pantothenate, pectinate, persulfate, phosphate,
phosphate/diphosphate, picrate, pivalate, polygalacturonate,
propionate, p-toluenesulfonate, salicylate, stearate, subacetate,
succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate,
teoclate, thiocyanate, tosylate, triethiodide, undecanoate, and
valerate salts, and the like, (See, for example, Berge et al.
(1977) "Pharmaceutical Salts," J. Pharm. Sci. 66:1-19).
[0182] In certain embodiments, the pharmaceutically acceptable
salts of the subject compounds include the conventional non-toxic
salts of the compounds, e.g., from non-toxic organic or inorganic
acids. Particularly suitable are salts of weak acids. For example,
such conventional non-toxic salts include those derived from
inorganic acids such as hydrochloric, hydrobromic, hydriodic,
cinnamic, gluconic, sulfuric, sulfamic, phosphoric, nitric, and the
like; and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, maleic, tartaric,
citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isothionic, and the like,
[0183] In other cases, the compounds of the present invention may
contain one or more acidic functional groups and, thus, are capable
of forming pharmaceutically acceptable salts with pharmaceutically
acceptable bases. The term "pharmaceutically acceptable salts" in
these instances refers to the relatively non-toxic, inorganic and
organic base addition salts of compounds of the present invention.
These salts can likewise be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form with a
suitable base, such as the hydroxide, carbonate, or bicarbonate of
a pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts,
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine, and the
like. (See, for example, Berge et al., supra).
[0184] Wetting agents, emulsifiers, and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives, and antioxidants can also be present in the
compositions.
[0185] Examples of pharmaceutically acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite, and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0186] Formulations of the present invention include those suitable
for oral, nasal, topical (including buccal and sublingual), rectal,
vaginal, and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the host
being treated and the particular mode of administration. The amount
of active ingredient which can be combined with a carrier material
to produce a single dosage form will generally be that amount of
the compound which produces a therapeutic effect. Generally, out of
one hundred percent, this amount will range from about 1 percent to
about ninety-nine percent of active ingredient, preferably from
about 5 percent to about 70 percent, most preferably from about 10
percent to about 30 percent.
[0187] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0188] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia), and/or as mouth washes, and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary,
or paste.
[0189] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules, and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as carboxymethylcellulose,
alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia;
(3) humectants, such as glycerol; (4) disintegrating agents, such
as agar-agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain silicates, and sodium carbonate; (5) solution
retarding agents, such as paraffin; (6) absorption accelerators,
such as quaternary ammonium compounds; (7) wetting agents, such as
cetyl alcohol and glycerol monostearate; (8) absorbents, such as
kaolin and bentonite clay; (9) lubricants, such a talc, calcium
stearate, magnesium stearate, solid polyethylene glycols, sodium
lauryl sulfate, and mixtures thereof; and (10) coloring agents. In
the case of capsules, tablets, and pills, the pharmaceutical
compositions may also comprise buffering agents. Solid compositions
of a similar type may also be employed as fillers in soft and
hard-filled gelatin capsules using such excipients as lactose or
milk sugars, as well as high molecular weight polyethylene glycols
and the like.
[0190] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), or surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0191] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills, and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes, and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0192] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups, and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as water or
other solvents, solubilizing agents, and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0193] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, and sweetening, flavoring, coloring, perfuming,
and preservative agents.
[0194] Suspensions, in addition to the active compounds, may
contain suspending agents, such as ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar, and
tragacanth, and mixtures thereof.
[0195] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax, or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active DAT inhibitor.
[0196] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams, or spray formulations containing such carriers
as are known in the art to be appropriate.
[0197] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches, and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0198] The ointments, pastes, creams, and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc, and zinc oxide, or mixtures
thereof.
[0199] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates, and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0200] In certain embodiments, the subject compound(s) are
formulated as part of a transdermal patch. Transdermal patches have
the added advantage of providing controlled delivery of a compound
of the present invention to the body. Such dosage forms can be made
by dissolving or dispersing the DAT inhibitors in the proper
medium. Absorption enhancers can also be used to increase the flux
of the DAT inhibitors across the skin. The rate of such flux can be
controlled by either providing a rate-controlling membrane or
dispersing the compound in a polymer matrix or gel.
[0201] The "free base form" of the subject compound relates to a
form in which the compound is not complexed with an acid, e.g., is
not an ammonium salt. Such forms may be incorporated into a patch.
It will be appreciated that the DAT inhibitors may be complexed,
for example, with elements of the drug-retaining matrix of the
patch and, as such, the DAT inhibitors may not necessarily be in
the form of the free base, when actually retained by the patch.
[0202] The patch preferably comprises a drug-impermeable backing
layer. Suitable examples of drug-impermeable backing layers which
may be used for transdermal or medicated patches include films or
sheets of polyolefins, polyesters, polyurethanes, polyvinyl
alcohols, polyvinyl chlorides, polyvinylidene chloride, polyamides,
ethylene-vinyl acetate copolymer (EVA), ethylene-ethylacrylate
copolymer (EEA), vinyl acetate-vinyl chloride copolymer, cellulose
acetate, ethyl cellulose, metal vapour deposited films or sheets
thereof, rubber sheets or films, expanded synthetic resin sheets or
films, nonwoven fabrics, fabrics, knitted fabrics, paper, and
foils. Preferred drug-impermeable, elastic backing materials are
selected from polyethylene tereplithalate (PET), polyurethane,
ethylene-vinyl acetate copolymer (EVA), plasticized
polyvinylchloride, and woven and non-woven fabric. Especially
preferred is non-woven polyethylene tereplithalate (PET). Other
backings will be readily apparent to those skilled in the art.
[0203] The term "block copolymer," in the preferred adhesives of
the invention, refers to a macromolecule comprised of two or more
chemically dissimilar polymer structures, terminally connected
together (Block Copolymers: Overview and Critical Survey, Noshay
and McGrath, 1977). These dissimilar polymer structures, sections
or segments, represent the "blocks" of the block copolymer. The
blocks may generally be arranged in an A-B structure, an A-B-A
structure, or a multi-block-(A-B)n-system, wherein A and B are the
chemically distinct polymer segments of the block copolymer.
[0204] It is generally preferred that the block copolymer is of an
A-B-A structure, especially wherein one of A and B is an
acrylic-type polymeric unit. It will be appreciated that the
present invention is also applicable using block copolymers which
possess three or more different blocks, such as an A-B-C block
copolymer. However, for convenience, reference hereinafter to block
copolymers will assume that there are only A and B sub-units, but
it will be appreciated that such reference also encompasses block
copolymers having more than two different sub-units, unless
otherwise specified.
[0205] It will be appreciated that the properties of block
copolymers are very largely determined by the nature of the A and B
blocks. Block copolymers commonly possess both `hard` and `soft`
segments. A `hard` segment is a polymer that has a glass transition
temperature (Tg) and/or a melting temperature (Tm) that is above
room temperature, while a `soft` segment is a polymer that has a Tg
(and possibly a Tm) below room temperature. The different segments
are thought to impart different properties to the block copolymer.
Without being constrained by theory, it is thought that association
of the hard segments of separate block copolymer units result in
physical cross-links within the block copolymer, thereby promoting
cohesive properties of the block copolymer. It is particularly
preferred that the hard segments of the block copolymers form such
physical close associations.
[0206] The block copolymers useful in the present invention
preferably are acrylic block copolymers. In acrylic block
copolymers, at least one of the blocks of the block copolymer is an
acrylic acid polymer or a polymer of an acrylic acid derivative.
The polymer may be composed of just one repeated monomer species.
However, it will be appreciated that a mixture of monomeric species
may be used to form each of the blocks, so that a block may, in
itself, be a copolymer. The use of a combination of different
monomers can affect various properties of the resulting block
copolymer. In particular, variation in the ratio or nature of the
monomers used allows properties such as adhesion, tack, and
cohesion to be modulated, so that it is generally advantageous for
the soft segments of the block copolymer to be composed of more
than one monomer species.
[0207] It is preferred that alkyl acrylates and alkyl methacrylates
are polymerized to form the soft portion of the block copolymer.
Alkyl acrylates and alkyl methacrylates are thought to provide
properties of tack and adhesion. Suitable alkyl acrylates and alkyl
methacrylates include n-butyl acrylate, n-butyl methacrylate, hexyl
acrylate, 2-ethylbutyl acrylate, isooctyl acrylate, 2-ethylhexyl
acrylate, 2-ethylhexyl methacrylate, decyl acrylate, decyl
methacrylate, dodecyl acrylate, dodecyl methacrylate,
tridecylacrylate, and tridecyl methacrylate, although other
suitable acrylates and methacrylates will be readily apparent to
those skilled in the art. It is preferred that the acrylic block
copolymer comprises at least 50% by weight of alkyl acrylate or
alkyl methacrylate(co)polymer.
[0208] Variation in the components of the soft segment affects the
overall properties of the block copolymer, although the essential
feature remains the cross-linking of the soft segments. For
example, soft segments essentially consisting of diacetone
acrylamide with either butyl acrylate and/or 2-ethylhexyl acrylate,
in approximately equal proportions, work well, and a ratio by
weight of about 3:4:4 provides good results. It is preferred that
diacetone acrylamide or other polar monomer, such as
hydroxyethylmethacrylate or vinyl acetate, be present in no more
than 50% w/w of the monomeric mix of the soft segment, as this can
lead to reduced adhesion, for example. The acrylate component may
generally be varied more freely, with good results observed with
both 2-ethylhexyl acrylate and butyl acrylate together or
individually.
[0209] As noted above, ratios of the various monomers are generally
preferred to be approximately equal. For adhesives, this is
preferred to be with a polar component of 50% or less of the soft
segment, with the apolar portion forming up to about 85% w/w, but
preferably between about 50 and 70% w/w. In the example above, this
is about 72% (4+4) polar to about 18% (3) polar.
[0210] In general, it is particularly preferred that any apolar
monomer used does not confer acidity on the adhesive. Adhesives of
the invention are preferably essentially neutral, avoiding any
unnecessary degeneration of the DAT inhibitors.
[0211] Limiting active functionalities, especially those with
active hydrogen, is generally preferred, in order to permit wide
use of any given formulation of adhesive without having to take
into account how it is likely to interact chemically with its
environment. Thus, a generally chemically inert adhesive is
preferred, in the absence of requirements to the contrary.
[0212] As discussed above, polymers suitable for use as the hard
portion of the block copolymer possess glass transition
temperatures above room temperature. Suitable monomers for use in
forming the hard segment polymer include styrene, x-methylstyrene,
methyl methacrylate, and vinyl pyrrolidone, although other suitable
monomers will be readily apparent to those skilled in the art.
Styrene and polymethylmethacrylate have been found to be suitable
for use in the formation of the hard segment of the block
copolymers. It is preferred that the hard portion of the block
copolymer forms from 3-30% w/w of the total block copolymer,
particularly preferably from 5-15% w/w.
[0213] The block copolymer is further characterized in that the
soft portions contain a degree of chemical cross-linking. Such
cross-linking may be effected by any suitable cross-linking agent.
It is particularly preferable that the cross-linking agent be in
the form of a monomer suitable for incorporation into the soft
segment during polymerization. Preferably the cross-linking agent
has two or more radically polymerizable groups, such as a vinyl
group, per molecule of the monomer, at least one tending to remain
unchanged during the initial polymerization, thereby permitting
cross-linking of the resulting block copolymer.
[0214] Suitable cross-linking agents for use in the present
invention include divinylbenzene, methylene bis-acrylamide,
ethylene glycol di(meth)acrylate, ethyleneglycol
tetra(meth)acrylate, propylene glycol di(meth)acrylate, butylene
glycoldi(meth)acrylate, or trimethylolpropane tri(meth)acrylate,
although other suitable cross-linking agents will be readily
apparent to those skilled in the art. A preferred cross-linking
agent is tetraethylene glycol dimethacrylate. It is preferred that
the cross-linking agent comprises about 0.01-0.6% by weight of the
block copolymer, with 0.1-0.4% by weight being particularly
preferred.
[0215] Methods for the production of block copolymers from their
monomeric constituents are well known. The block copolymer portions
of the present invention may be produced by any suitable method,
such as step growth, anionic, cationic, and free radical methods
(Block Copolymers, supra). Free radical methods are generally
preferred over other methods, such as anionic polymerization, as
the solvent and the monomer do not have to be purified.
[0216] Suitable initiators for polymerization include polymeric
peroxides with more than one peroxide moiety per molecule. An
appropriate choice of reaction conditions is well within the skill
of one in the art, once a suitable initiator has been chosen.
[0217] The initiator is preferably used in an amount of 0.005-0.1%
by weight of the block copolymer, with 0.01-0.05% by weight being
particularly preferred, although it will be appreciated that the
amount chosen is well within the skill of one in the art. In
particular, it is preferred that the amount should not be so much
as to cause instant gelling of the mix, nor so low as to slow down
polymerization and to leave excess residual monomers. A preferred
level of residual monomers is below 2000 ppm.
[0218] It will also be appreciated that the amount of initiator
will vary substantially, depending on such considerations as the
initiator itself and the nature of the monomers.
[0219] The block copolymers are adhesives, and preferably are
pressure sensitive adhesives. Pressure sensitive adhesives can be
applied to a surface by hand pressure and require no activation by
heat, water, or solvent. As such, they are particularly suitable
for use in accordance with the present invention.
[0220] The block copolymers may be used without tackifiers and, as
such, are particularly advantageous. However, it will be
appreciated that the block copolymers may also be used in
combination with a tackifier, to provide improved tack, should one
be required or desired. Suitable tackifiers are well known and will
be readily apparent to those skilled in the art.
[0221] Without being constrained by theory, it is thought that the
combination of chemical cross-links between the soft segments of
the copolymer combined with the, generally, hydrophobic
interaction, or physical cross-linking, between the hard portions
results in a "matrix-like" structure, Copolymers having only
physical cross-linking of the hard segments are less able to form
such a matrix. It is believed that the combination of both forms of
cross-linking of the block copolymers provides good internal
strength (cohesion) and also high drug storage capacity.
[0222] More particularly, it is believed that the hard segments
associate to form "islands," or nodes, with the soft segments
radiating from and between these nodes.
[0223] There is a defined physical structure in the "sea" between
the islands, where the soft segments are cross-linked, so that
there is no necessity for extensive intermingling of the soft
segments. This results in a greater cohesion of the whole block
copolymer while, at the same time, allowing shortened soft segment
length and still having as great, or greater, distances between the
islands, thereby permitting good drug storage capacity.
[0224] block copolymer preferably cross-links as the solvent is
removed, so that cross-linking can be timed to occur after coating,
this being the preferred method.
[0225] Accordingly, not only can the block copolymer easily be
coated onto a surface, but the complete solution can also be stored
for a period before coating. Accordingly, in the manufacturing
process of the patches, the process preferably comprises
polymerizing the monomeric constituents of each soft segment in
solution, then adding the constituents of the hard segment to each
resulting solution and polymerizing the resulting mix, followed by
cross-linking by removal of any solvent or solvent system, such as
by evaporation. If the solution is to be stored for any length of
time, it may be necessary to keep the polymer from precipitating
out which may be achieved by known means, such as by suspending
agents or shaking. It may also be necessary to select the type of
polymers that will be subject to substantially no cross-linking
until the solvent is evaporated.
[0226] In general, it is preferred that the adhesive possesses a
minimum number of functionalities having active hydrogen, in order
to avoid undesirable reactions/interactions, such as with any drug
that it is desired to incorporate into the adhesive material. It
will be appreciated that this is only a preferred restriction, and
that any adhesive may be tailored by one skilled in the art to suit
individual requirements.
[0227] Suitable monomers for use in forming the hard segment
include styrene, methylstyrene, methyl methacrylate, and vinyl
pyrrolidone, with the preferred proportion of the hard segment
being between 5 and 15% w/w. In particular, it is advantageous to
use the compounds of WO 99/02141, as it is possible to load over
30% of drug into such a system.
[0228] Thus, in the patches of the present invention, it is
generally possible to calculate the amount of drug required and
determine the appropriate patch size with a given drug loading in
accordance with a patient's body weight which can be readily
calculated by those skilled in the art.
[0229] In certain embodiments, small amounts of plasticizer, such
as isopropyl myristate (IPM), are incorporated. This has the
advantage of helping solubilize the DAT inhibitor(s) as well as
rendering the adhesive less rough on the skin. Levels of between 2
and 25%, by weight, are generally useful, with levels of between 3
and 20% being more preferred and levels of 5 to 15%, especially
about 10%, being most preferred. Other plasticizers may also be
used, and suitable plasticizers will be readily apparent to those
skilled in the art.
[0230] Plasticizers generally take the form of oily substances
introduced into the adhesive polymer. The effect of the
introduction of such oily substances is to soften the physical
structure of the adhesive whilst, at the same time, acting at the
interface between the adhesive and the skin, thereby helping to
somewhat weaken the adhesive, and to reduce exfoliation.
[0231] The free base oil may be obtained by basifying salts of the
subject compounds, or any other suitable salt, with a suitable
base, in the presence of a hydrophilic solvent, especially water,
and an organic solvent. For instance, water and ethyl acetate, in
approximately equal proportions, work well, with ammonia serving as
the basifying agent. The water may then be removed and the
preparation washed with further water, or other aqueous
preparation, after which the preparation may be suitably extracted
with ether, for example, after having removed the ethyl acetate. It
is preferred to keep the preparation under an inert atmosphere,
especially after completion.
[0232] Whilst it will be appreciated that patches of the present
invention may be removed from the patient at any time once it is
desired to terminate a given dose, this can have the disadvantage
of providing an opportunity for potential drug abuse of the
partially discharged patch. Abuse of the subject compounds is
highly undesirable.
[0233] In certain embodiments, it may be advantage to use a patch
tailored to have delivered, by about 8 hours after application, the
majority of the subject compound that it is capable of delivering
in a 24 hour period, so that a patch can be left in place, and
levels of drug still diminish appreciably. It is advantageous that
the drug delivery profile has first order kinetics, so that the
majority of the drug is delivered during the main part of the day
and, even if the patient omits to remove the patch, the amount of
drug is moving towards exhaustion by the end of the day, and the
amount of drug is dropping rapidly.
[0234] It will be appreciated that patches of the invention may be
constructed in any suitable manner known in the art for the
manufacture of transdermal patches. The patches may simply comprise
adhesive, drug, and backing, or may be more complex, such as having
edging to prevent seepage of drug out of the sides of the patch.
Patches may also be multi-layered.
[0235] Ophthalmic formulations, eye ointments, powders, solutions,
and the like, are also contemplated as being within the scope of
this invention.
[0236] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more pharmaceutically
acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient, or suspending or thickening
agents.
[0237] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0238] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents, and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0239] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0240] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0241] When the compounds of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0242] The addition of the active compound of the invention to
animal feed is preferably accomplished by preparing an appropriate
feed premix containing the active compound in an effective amount
and incorporating the premix into the complete ration.
[0243] Alternatively, an intermediate concentrate or feed
supplement containing the active ingredient can be blended into the
feed. The way in which such feed premixes and complete rations can
be prepared and administered are described in reference books (such
as "Applied Animal Nutrition," W.H. Freedman and Co., San
Francisco, U.S.A., 1969 or "Livestock Feeds and Feeding" O and B
books, Corvallis, Ore., U.S.A., 1977).
IV Biochemical Activity at Cellular Receptors, and Assays to Detect
that Activity
[0244] Assaying processes are well known in the art in which a
reagent is added to a sample, and measurements of the sample and
reagent are made to identify sample attributes stimulated by the
reagent. For example, one such assay process concerns determining
in a chromogenic assay the amount of an enzyme present in a
biological sample or solution. Such assays are based on the
development of a colored product in the reaction solution. The
reaction develops as the enzyme catalyzes the conversion of a
colorless chromogenic substrate to a colored product.
[0245] Another assay useful in the present invention concerns
determining the ability of a ligand to bind to a biological
receptor utilizing a technique well known in the art referred to as
a radioligand binding assay. This assay accurately determines the
specific binding of a radio-ligand to a targeted receptor through
the delineation of its total and nonspecific binding components.
Total binding is defined as the amount of radio-ligand that remains
following the rapid separation of the radio-ligand bound in a
receptor preparation (cell homogenates or recombinate receptors)
from that which is unbound. The nonspecific binding component is
defined as the amount of radio-ligand that remains following
separation of the reaction mixture consisting of receptor,
radio-ligand and an excess of unlabeled ligand. Under this
condition, the only radio-ligand that remains represents that which
is bound to components other that receptor. The specific
radio-ligand bound is determined by subtracting the nonspecific
from total radioactivity bound. For a specific example of
radio-ligand binding assay for .mu.-opioid receptor, see Wang, J.
B. et al. FEBS Letters 1994, 338, 217.
[0246] Assays useful in the present invention concern determining
the activity of receptors the activation of which initiates
subsequent intracellular events in which intracellular stores of
calcium ions are released for use as a second messenger. Activation
of some G-protein-coupled receptors stimulates the formation of
inositol triphosphate (IP3, a O-protein-coupled receptor second
messenger) through phospholipase C-mediated hydrolysis of
phosphatidylinositol, Berridge and Irvine (1984). Nature
312:315-21. IP3 in turn stimulates the release of intracellular
calcium ion stores.
[0247] A change in cytoplasmic calcium ion levels caused by release
of calcium ions from intracellular stores is used to determine
G-protein-coupled receptor function. This is another type of
indirect assay. Among G-protein-coupled receptors are muscarinic
acetylcholine receptors (mAChK), adrenergic receptors, sigma
receptors, serotonin receptors, dopamine receptors, angiotensin
receptors, adenosine receptors, bradykinin receptors, metabotropic
excitatory amino acid receptors and the like. Cells expressing such
G-protein-coupled receptors may exhibit increased cytoplasmic
calcium levels as a result of contribution from both intracellular
stores and via activation of ion channels, in which case it may be
desirable although not necessary to conduct such assays in
calcium-free buffer, optionally supplemented with a chelating agent
such, as EGTA, to distinguish fluorescence response resulting from
calcium release from internal stores. Another type of indirect
assay involves determining the activity of receptors which, when
activated, result in a change in the level of intracellular cyclic
nucleotides, e.g., cAMP, cGMP. For example, activation of some
dopamine, serotonin, metabotropic glutamate receptors and
muscarinic acetylcholine receptors results in a decrease in the
cAMP or cGNIP levels of the cytoplasm.
[0248] Furthermore, there are cyclic nucleotide-gated ion channels,
e.g., rod photoreceptor cell channels and olfactory neuron channels
[see, Altcnhofen, W. et al. (1991) Proc. Natl. Acad. Sci U.S.A.
88:9868-9872 and Malian et al. (1990) Nature 347:184-187] that are
permeable to cations upon activation by binding of cAMP or cGMP. A
change in cytoplasmic ion levels caused by a change in the amount
of cyclic nucleotide activation of photo-receptor or olfactory
neuron channels is used to determine function of receptors that
cause a change in CAMP or cGMP levels when activated. In cases
where activation of the receptor results in a decrease in cyclic
nucleotide levels, it may be preferable to expose the cells to
agents that increase intracellular cyclic nucleotide levels, e.g.,
forskolin, prior to adding a receptor-activating compound to the
cells in the assay. Cell for this type of assay can be made by
co-transfection of a host cell with DNA encoding a cyclic
nucleotide-gated ion channel and a DNA encoding a receptor (e.g.,
certain metabotropic glutamate receptors, muscarinic acetylcholine
receptors, dopamine receptors, serotonin receptors and the like,
which, when activated, causes a change in cyclic nucleotide levels
in the cytoplasm.
[0249] Any cell expressing a receptor protein which is capable,
upon activation, of directly increasing the intracellular
concentration of calcium, such as by opening gated calcium
channels, or indirectly affecting the concentration of
intracellular calcium as by causing initiation of a reaction which
utilizes Ca.sup.2+ as a second messenger (e.g., G-protein-coupled
receptors), may form the basis of an assay. Cells endogenously
expressing such receptors or ion channels, and cells which may be
transfected with a suitable vector encoding one or more such cell
surface proteins are known to those of skill in the art, or may be
identified by those of skill in the art. Although essentially any
cell which expresses endogenous ion channel and/or receptor
activity may be used, it is preferred to use cells transformed or
transfected with heterologous DNAs encoding such ion channels
and/or receptors so as to express predominantly a single type of
ion channel or receptor. Many cells that may be genetically
engineered to express a heterologous cell surface protein are
known. Such cells include, but are not limited to, baby hamster
kidney (BHK) cells (ATCC No. CCL10), mouse L cells (ATCC No.
CCLI.3), DG44 cells [see, Chasin (1986) Cell. Moles. Genet. 12:555]
human embryonic kidney (HEK) cells (ATCC No. CRL1573), Chinese
hamster ovary (CHO) cells (ATCC Nos. CRL9618, CCL61, CRL9096), PC12
cells (ATCC No. CRL1721) and COS-7 cells (ATCC No. CRL1651).
Preferred cells for heterologous cell surface protein expression
are those that can be readily and efficiently transfected.
Preferred cells include HEK 293 cells, such as those described in
U.S. Pat. No. 5,024,939.
[0250] Any compound which is known to activate ion channels or
receptors of interest may be used to initiate an assay. Choosing an
appropriate ion channel- or receptor-activating reagent depending
on the ion channel or receptor of interest is within the skill of
the art. Direct depolarization of the cell membrane to determine
calcium channel activity may be accomplished by adding a potassium
salt solution having a concentration of potassium ions such that
the final concentration of potassium ions in the cell-containing
well is in the range of about 50-150 mM (e.g., 50 mM KCI). With
respect to ligand-gated receptors and ligand-gated ion channels,
ligands are known which have affinity for and activate such
receptors. For example, nicotinic acetyloholine receptors are known
to be activated by nicotine or acetylcholine; similarly, muscarinic
and acetylcholine receptors may be activated by addition of
muscarine or carbamylcholine.
[0251] Agonist assays may be carried out on cells known to possess
ion channels and/or receptors to determine what effect, if any, a
compound has on activation or potentiation of ion channels or
receptors of interest. Agonist assays also may be carried out using
a reagent known to possess ion channel- or receptor-activating
capacity to determine whether a cell expresses the respective
functional ion channel or receptor of interest.
[0252] Contacting a functional receptor or ion channel with agonist
typically activates a transient reaction; and prolonged exposure to
an agonist may desensitize the receptor or ion channel to
subsequent activation. Thus, in general, assays for determining ion
channel or receptor function should be initiated by addition of
agonist (i.e., in a reagent solution used to initiate the
reaction). The potency of a compound having agonist activity is
determined by the detected change in some observable in the cells
(typically an increase, although activation of certain receptors
causes a decrease) as compared to the level of the observable in
either the same cell, or substantially identical cell, which is
treated substantially identically except that reagent lacking the
agonist (i.e., control) is added to the well. Where an agonist
assay is performed to test whether or not a cell expresses the
functional receptor or ion channel of interest, known agonist is
added to test-cell-containing wells and to wells containing control
cells (substantially identical cell that lacks the specific
receptors or ion channels) and the levels of observable are
compared. Depending on the assay, cells lacking the ion channel
and/or receptor of interest should exhibit substantially no
increase in observable in response to the known agonist. A
substantially identical cell may be derived from the same cells
from which recombinant cells are prepared but which have not been
modified by introduction of heterologous DNA. Alternatively, it may
be a cell in which the specific receptors or ion channels are
removed. Any statistically or otherwise significant difference in
the level of observable indicates that the test compound has in
some manner altered the activity of the specific receptor or ion
channel or that the test cell possesses the specific functional
receptor or ion channel.
[0253] In an example of drug screening assays for identifying
compounds which have the ability to modulate ion channels or
receptors of interest, individual wells (or duplicate wells, etc)
contain a distinct cell type, or distinct recombinant cell line
expressing a homogeneous population of a receptor or ion channel of
interest, so that the compound having unidentified activity may be
screened to determine whether it possesses modulatory activity with
respect to one or more of a variety of functional ion channels or
receptors. It is also contemplated that each of the individual
wells, may contain the same cell type so that multiple compounds
(obtained from different reagent sources in the apparatus or
contained within different wells) can be screened and compared for
modulating activity with respect to one particular receptor or ion
channel type.
[0254] Antagonist assays, including drug screening assays, may be
carried out by incubating cells having functional ion channels
and/or receptors in the presence and absence of one or more
compounds, added to the solution bathing the cells in the
respective wells of the microtiter plate for an amount of time
sufficient (to the extent that the compound has affinity for the
ion channel and/or receptor of interest) for the compound(s) to
bind to the receptors and/or ion channels, then activating the ion
channels or receptors by addition of known agonist, and measuring
the level of observable in the cells as compared to the level of
observable in either the same cell, or substantially identical
cell, in the absence of the putative antagonist.
[0255] The assays are thus useful for rapidly screening compounds
to identify those that modulate any receptor or ion channel in a
cell. In particular, assays can be used to test functional
ligand-receptor or ligand-ion channel interactions for cell
receptors including ligand-gated ion channels, voltage gated ion
channels, G-protein-coupled receptors and growth factor
receptors.
[0256] Those of ordinary skill in the art will recognize that
assays may encompass measuring a detectable change of a solution as
a consequence of a cellular event which allows a compound, capable
of differential characteristics, to change its characteristics in
response to the cellular event. By selecting a particular compound
which is capable of differential characteristics upon the
occurrence of a cellular event, various assays may be performed.
For example, assays for determining the capacity of a compound to
induce cell injury or cell death may be carried out by loading the
cells with a pH-sensitive fluorescent indicator such as BCECF
(Molecular Probes, Inc., Eugene, Oreg. 97402, Catalog #B1150) and
measuring cell-injury or -cell-death as a function of changing
fluorescence over time.
[0257] In a further example of useful assays, the function of
receptors whose activation results in a change in the cyclic
nucleotide levels of the cytoplasm may be directly determined in
assays of cells that express such receptors and that have been
injected with a fluorescent compound that changes fluorescence upon
binding cAMP. The fluorescent compound comprises
cAMPdependent-protein kinase in which the catalytic and regulatory
subunits are each labelled with a different fluorescent-dye [Adams
et al. (1991) Nature 349:694-697]. When cAMP binds to the
regulatory subunits, the fluorescence emission spectrum changes;
this change can be used as an indication of a change in cAMP
concentration.
[0258] The function of certain neurotransmitter transporters which
are present at the synaptic cleft at the junction between two
neurons may be determined by the development of fluorescence in the
cytoplasm of such neurons when conjugates of an amine acid and
fluorescent indicator (wherein the fluorescent indicator of the
conjugate is an acetoxymethyl ester derivative e.g.,
5-(aminoacetamido)fluorescein; Molecular Probes, Catalog #A1363)
are transported by the neurotransmitter transporter into the
cytoplasm of the cell where the ester group is cleaved by esterase
activity and the conjugate becomes fluorescent.
[0259] In practicing an assay of this type, a reporter gene
construct is inserted into an eukaryotic cell to produce a
recombinant cell which has present on its surface a cell surface
protein of a specific type. The cell surface receptor may be
endogenously expressed or it may be expressed from a heterologous
gene that has been introduced into the cell. Methods for
introducing heterologous DNA into eukaryotic cells are-well known
in the art and any such method may be used. In addition, DNA
encoding various cell surface proteins is known to those of skill
in the art or it may be cloned by any method known to those of
skill in the art.
[0260] The recombinant cell is contacted with a test compound and
the level of reporter gene expression is measured. The contacting
may be effected in any vehicle and the testing may be by any means
using any protocols, such as serial dilution, for assessing
specific molecular interactions known to those of skill in the art.
After contacting the recombinant cell for a sufficient time to
effect any interactions, the level of gene expression is measured.
The amount of time to effect such interactions may be empirically
determined, such as by running a time course and measuring the
level of transcription as a function of time. The amount of
transcription may be measured using any method known to those of
skill in the art to be suitable. For example, specific mRNA
expression may be detected using Northern blots or specific protein
product may be identified by a characteristic stain. The amount of
transcription is then compared to the amount of transcription in
either the same cell in the absence of the test compound or it may
be compared with the amount of transcription in a substantially
identical cell that lacks the specific receptors. A substantially
identical cell may be derived from the same cells from which the
recombinant cell was prepared but which had not been modified by
introduction of heterologous DNA. Alternatively, it may be a cell
in which the specific receptors are removed. Any statistically or
otherwise significant difference in the amount of transcription
indicates that the test compound has in some manner altered the
activity of the specific receptor.
[0261] If the test compound does not appear to enhance, activate or
induce the activity of the cell surface protein, the assay may be
repeated and modified by the introduction of a step in which the
recombinant cell is first tested for the ability of a known agonist
or activator of the specific receptor to activate transcription if
the transcription is induced, the test compound is then assayed for
its ability to inhibit, block or otherwise affect the activity of
the agonist.
[0262] The transcription based assay is useful for identifying
compounds that interact with any cell surface protein whose
activity ultimately alters gene expression. In particular, the
assays can be used to test functional ligand-receptor or ligand-ion
channel interactions for a number of categories of cell
surface-localized receptors, including: ligand-gated ion channels
and voltage-gated ion channels, and G protein-coupled
receptors.
[0263] Any transfectable cell that can express the desired cell
surface protein in a manner such the protein functions to
intracellularly transduce an extracellular signal may be used. The
cells may be selected such that they endogenously express the cell
surface protein or may be genetically engineered to do so. Many
such cells are known to those of skill in the art. Such cells
include, but are not limited to Ltk.sup.- cells, PC12 cells and
COS-7 cells.
[0264] Any cell surface protein that is known to those of skill in
the art or that may be identified by those of skill in the art may
be used in the assay. The cell surface protein may be endogenously
expressed on the selected cell or it may be expressed from cloned
DNA. Exemplary cell surface proteins include, but are not limited
to, cell surface receptors and ion channels. Cell surface receptors
include, but are not limited to, muscarinic receptors (e.g., human
M2 (GenBank accession #M16404); rat M3 (GenBank accession #M16407);
human M4 (GenBank accession #M16405); human M5 (Bonner et al.
(1988) Neuron 1:403-410); and the like); neuronal nicotinic
acetylcholine receptors (e.g., the alpha 2, alpha 3 and beta 2
subtypes disclosed in U.S. Ser. No. 504,455 (filed Apr. 3, 1990),
hereby expressly incorporated by reference herein in its entirety);
the rat alpha 2 subunit (Wada et al. (1988) Science 240:330-334);
the rat alpha 3 subunit (Boulter et al. (1986) Nature 319:368-374);
the rat alpha 4 subunit (Goldman et al. (1987) cell 48:965973); the
rat alpha 5 subunit (Boulter et al. (1990) J. Biol. Chem.
265:4472-4482); the rat beta 2 subunit (Deneris et al. (1988)
Neuron 1:45-54); the rat beta 3 subunit (Deneris et al. (1989) J.
Biol. Chem. 264: 6268-6272); the rat beta 4 subunit (Duvoisin et
al. (1989) Neuron 3:487-496); combinations of the rat alpha
subunits, beta subunits and alpha and beta subunits; GABA receptors
(e.g., the bovine alpha 1 and beta 1 subunits (Schofield et al.
(1987) Nature 328:221227); the bovine alpha 2 and alpha 3 subunits
(Levitan et al. (1988) Nature 335:76-79); the gamma-subunit
(Pritchett et al. (1989) Nature 338:582-585); the beta 2 and beta 3
subunits (Ymer et alo (1989) EMBO J. 8:1665-1670); the delta
subunit (Shivers, B. D. (1989) Neuron 3:327-337); and the like);
glutamate receptors (e.g., receptor isolated from rat brain
(Hollmann et al. (1989) Nature 342:643-648); and the like);
adrenergic receptors (e.g., human beta I (Frielle et al. (1987)
Proc. Natl. Acad. Sci. 84:7920-7924); human alpha 2 (Kobilka et al.
(1987) Science 238:650-656); hamster beta 2 (Dixon et al. (1986)
Nature 321:75-79); and the like); dopamine receptors (e.g., human
D2 (Stormann et al. (1990) Molec. Pharm. 37:1-6); rat (Bunzow et
al. (1988) Nature 336:783-787); and the like); NGF receptors (e.g.,
human NGF receptors (Johnson et al. (1986) Cell 47:545-554); and
the like); serotonin receptors (e.g., human 5HTla (Kobilka et al.
(1987) Nature 329:75-79); rat 5HT2 (Julius et al. (1990) PNAS
87:928-932); rat 5HTlc (Julius et al. (1988) Science 241:558-564);
and the like).
[0265] Reporter gene constructs are prepared by operatively linking
a reporter gene with at least one transcriptional regulatory
element. If only one transcriptional regulatory element is
included, it must be a regulatable promoter. At least one of the
selected transcriptional regulatory elements must be indirectly or
directly regulated by the activity of the selected cell-surface
receptor whereby activity of the receptor can be monitored via
transcription of the reporter genes.
[0266] The construct may contain additional transcriptional
regulatory elements, such as a FIRE sequence, or other sequence,
that is not necessarily regulated by the cell surface protein, but
is selected for its ability to reduce background level
transcription or to amplify the transduced signal and to thereby
increase the sensitivity and reliability of the assay.
[0267] Many reporter genes and transcriptional regulatory elements
are known to those of skill in the art and others may be identified
or synthesized by methods known to those of skill in the art.
[0268] A reporter gene includes any gene that expresses a
detectable gene product, which may be RNA or protein. Preferred
reporter genes are those that are readily detectable. The reporter
gene may also be included in the construct in the form of a fusion
gene with a gene that includes desired transcriptional regulatory
sequences or exhibits other desirable properties.
[0269] Examples of reporter genes include, but are not limited to
CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979),
Nature 282: 864-869) luciferase, and other enzyme detection
systems, such as beta-galactosidase; firefly luciferase (deWet et
al. (1987), Mot. Cell. Biol. 7:725-737); bacterial luciferase
(Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al.
(1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et
al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J.
Mol. Appl. Gen. 2: 101).
[0270] Transcriptional control elements include, but are not
limited to, promoters, enhancers, and repressor and activator
binding sites, Suitable transcriptional regulatory elements may be
derived from the transcriptional regulatory regions of genes whose
expression is rapidly induced, generally within minutes, of contact
between the cell surface protein and the effector protein that
modulates the activity of the cell surface protein. Examples of
such genes include, but are not limited to, the immediate early
genes (see, Sheng et al. (1990) Neuron 4: 477-485), such as c-fos,
Immediate early genes are genes that are rapidly induced upon
binding of a ligand to a cell surface protein. The transcriptional
control elements that are preferred for use in the gene constructs
include transcriptional control elements from immediate early
genes, elements derived from other genes that exhibit some or all
of the characteristics of the immediate early genes, or synthetic
elements that are constructed such that genes in operative linkage
therewith exhibit such characteristics. The characteristics of
preferred genes from which the transcriptional control elements are
derived include, but are not limited to, low or undetectable
expression in quiescent cells, rapid induction at the
transcriptional level within minutes of extracellular simulation,
induction that is transient and independent of new protein
synthesis, subsequent shut-off of transcription requires, new
protein synthesis, and mRNAs transcribed from these genes have a
short half-life. It is not necessary for all of these properties to
be present.
V. Exemplary Uses of the Compounds of the Invention
[0271] In various embodiments, the present invention contemplates
modes of treatment and prophylaxis which utilize one or more of the
subject DAT inhibitors. These agents may be useful for decreasing
or preventing the effects of defects in an animal which cause a
movement disorder.
[0272] In various other embodiments, the present invention
contemplates modes of treatment and prophylaxis which utilize one
or more of the subject DAT inhibitors to alter defects which cause
a movement disorder. The improvement and/or restoration of mental
or physical state in an organism has positive behavioral, social,
and psychological consequences.
[0273] In certain embodiments, the subject method can be used to
treat patients who have been diagnosed as having or at risk of
developing movement disorders.
[0274] Parkinson's disease is the second most common
neurodegenerative disorder, affecting nearly 1 million people in
North America. The disease is characterized by symptoms such as
muscle rigidity, tremor and bradykinesia.
[0275] Early studies of Parkinson's disease showed unusual
inclusions in the cytoplasm of neurons (i.e., Lewy bodies),
occurring predominantly in the substantia nigra, which innervate
the striatal region of the forebrain. Although Lewy bodies were
also found in other neurodegenerative conditions, the presence of
Lewy bodies in Parkinson's disease is accompanied by cell loss in
the substantia nigra. This cell loss is considered to be the
defining pathological feature of Parkinson's disease.
[0276] Epidemiological studies have reported geographic variation
in Parkinson's disease incidence, leading to the search for
environmental factors (Olanow and Tatton, Ann. Rev. Neurosci.,
22:123-144 [1998]). The recent discovery that
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin causes a
Parkinson's-like syndrome indistinguishable from the idiopathic
disease suggests that Parkinson's disease may be caused by
environmental factors (e.g., toxins and causative agents). (See
e.g., Langston, Ann. Neurol., 44:S45-S52 [1998]).
[0277] Recent research has also identified genes associated with
Parkinson's disease (Mizuno et al., Biomed. Pharmacother.,
53(3):109-116 [1999]; Dunnett and Bjorklund, Nature 399 (6738
Suppl):A32-A39 [1999]); namely, the .alpha.-synuclein gene
(Polymeropouos et al., Science 276:2045-2047 [1997]), the parkin
gene (Kitada et al., Nature 392:605-608 [1998]), and the UCH-L1
thiol protease gene (Leroy et al., Nature 395:451-452 [1998]).
Although additional chromosomal loci associated with the disease
state have been identified, these chromosomal loci have not been
analyzed at the molecular level. At present, the biochemical roles
played by these gene products in both normal cells and in diseased
neurons remain ambiguous, and no gene therapy protocols involving
their use have been developed.
[0278] Furthermore, Parkinson's disease is associated with the
progressive loss of dopamine neurons in the ventral mesencephalon
of the substantia nigra (Shoulson, Science 282: 1072-1074 [1998]),
which innervates the major motor-control center of the forebrain,
the striatum. Although a gradual decline in the number of neurons
and dopamine content of the basal ganglia is normally associated
with increasing age, progressive dopamine loss is pronounced in
people suffering from Parkinson's disease, resulting in the
appearance of symptoms when about 70-80% of striatal dopamine and
50% of nigral dopamine neurons are lost (Dunnett and Bjorklund,
supra). This loss of dopamine-producing neurons resulting in a
dopamine deficiency is believed to be responsible for the motor
symptoms of Parkinson's disease.
[0279] Although the cause of dopaminergic cell death remains
unknown, it is believed that dopaminergic cell death is affected by
a combination of necrotic and apoptotic cell death. Mechanisms and
signals responsible for the progressive degeneration of nigral
dopamine neurons in Parkinson's disease have been proposed (Olanow
et al., Ann. Neurol., 44:S1-S196 [1998]), and include oxidative
stress (from the generation of reactive oxygen species),
mitochondrial dysfunction, excitotoxicity, calcium imbalance,
inflammatory changes and apoptosis as contributory and
interdependent factors in Parkinson's disease neuronal cell
death.
[0280] Apoptosis (i.e., programmed cell death) plays a fundamental
role in the development of the nervous system (Oppenheim, Ann. Rev.
Neurosci., 14: 453-501 [1991]), and accelerated apoptosis is
believed to underlie many neurodegenerative diseases, including
Parkinson's disease (Barinaga, Science 281: 1303-1304 [1998];
Mochizuki et al., J. Neurol. Sci., 137: 120-123 [1996]; and Oo et
al., Neuroscience 69: 893-901 [1995]). In living systems, apoptotic
death can be initiated by a variety of external stimuli, and the
biochemical nature of the intracellular apoptosis effectors is at
least partially understood.
[0281] Drugs used to treat Parkinson's disease include L-dopa,
selegiline, apomorphine and anticholinergics. L-dopa
(levo-dihydroxy-phenylalanine) (Sinemet) is a dopamine precursor
which can cross the blood-brain barrier and be converted to
dopamine in the brain. Unfortunately, L-dopa has a short half life
in the body and it is typical after long use (i.e., after about 4-5
years) for the effect of L-dopa to become sporadic and
unpredictable, resulting in fluctuations in motor function,
dyskinesias and psychiatric side effects. Additionally, L-dopa can
cause B vitamin deficiencies to arise.
[0282] Selegiline (Deprenyl, Eldepryl) has been used as an
alternative to L-dopa, and acts by reducing the breakdown of
dopamine in the brain. Unfortunately, selegiline becomes
ineffective after about nine months of use. Apomorphine, a dopamine
receptor agonist, has been used to treat Parkinson's disease,
although is causes severe vomiting when used on its own, as well as
skin reactions, infection, drowsiness and some psychiatric side
effects.
[0283] Systemically administered anticholinergic drugs (such as
benzhexol and orphenedrine) have also been used to treat
Parkinson's disease and act by reducing the amount of acetylcholine
produced in the brain and thereby redress the
dopamine/acetylcholine imbalance present in Parkinson's disease.
Unfortunately, about 70% of patients taking systemically
administered anticholinergics develop serious neuropsychiatric side
effects, including hallucinations, as well as dyskinetic movements,
and other effects resulting from wide anticholinergic distribution,
including vision effects, difficulty swallowing, dry mouth, and
urine retention. See e.g. Playfer, J. R., Parkinson's Disease,
Postgrad Med J, 73; 257-264:1997 and Nadeau, S. E., Parkinson's
Disease, J Am Ger Soc, 45; 233-240:1997.
[0284] Newer drug refinements and developments include
direct-acting dopamine agonists, slow-release L-dopa formulations,
inhibitors of the dopamine degrading enzymes
catechol-O-methyltransferase (COMT) and monoamine oxidase B
(MAO-B), and dopamine transport blockers. These treatments enhance
central dopaminergic neurotransmission during the early stages of
Parkinson's disease, ameliorate symptoms associated with
Parkinson's disease, and temporarily improve the quality of life.
However, despite improvements in the use of L-dopa for treating
Parkinson's disease, the benefits accorded by these dopaminergic
therapies are temporary, and their efficacy declines with disease
progression. In addition, these treatments are accompanied by
severe adverse motor and mental effects, most notably dyskinesias
at peak dose and "on-off" fluctuations in drug effectiveness (Poewe
and Granata, in Movement Disorders. Neurological Principles and
Practice (Watts and Koller [eds]) McGraw-Hill, New York [1997]; and
Marsden and Parkes, Lancet 1:345-349 [1977]). No drug treatments
are currently available that lessen the progressive pace of
nigrostriatal degeneration, postpone the onset of illness, or that
substantively slow disability (Shoulson, supra).
[0285] Other methods for the treatment of Parkinson's disease
involve neurosurgical intervention, such as thalamotomy,
pallidotomy, and deep brain stimulation. The thalamic outputs of
the basal ganglia are an effective lesion target for the control of
tremor (i.e., thalamotomy). Thalamotomy destroys part of the
thalamus, a brain region involved in movement control. Unilateral
stereotactic thalamotomy has proven to be effective for controlling
contralateral tremor and rigidity, but carries a risk of
hemiparesis. Bilateral thalamotomy carries an increased risk of
speech and swallowing disorders resulting.
[0286] Stereotactic pallidotomy, surgical ablation of part of the
globus pallidus (a basal ganglia), has also be used with some
success. Pallidotomy is performed by inserting a wire probe into
the globus pallidus and heating the probe to destroy nearby tissue.
Pallidotomy is most useful for the treatment of peak-dose
diskinesias and for dystonia that occurs at the end of a dose.
[0287] Aside from surgical resection, deep brain stimulation, high
frequency stimulating electrodes placed in the ventral
intermedialis nucleus, has been found to suppress abnormal
movements in some cases. A variety of techniques exist to permit
precise location of a probe, including computed tomography and
magnetic resonance imaging. Unfortunately, the akinesia, speech and
gait disorder symptoms of Parkinson's disease, are little helped by
these surgical procedures, all of which result in destructive brain
lesions. Despite the development of modem imaging and surgical
techniques to improve the effectiveness of these neurosurgical
interventions for the treatment of Parkinson's disease tremor
symptoms, the use of neurosurgical therapies is not widely
applicable. For example, thalamotomy does not alleviate the
akinetic symptoms which are the major functional disability for
many people suffering from Parkinson's disease (Marsden et al.,
Adv. Neurol., 74:143-147 [1997]).
[0288] Therapeutic methods aimed at controlling suspected causative
factors associated with Parkinson's disease (e.g., therapies which
control oxidative stress and excitotoxicity) have also been
developed. Clinical trials have shown that administration of
antioxidative agents vitamin E and deprenyl provided little or no
neuroprotective function (Shoulson et al., Ann. Neurol., 43:318-325
[1998]). Glutamate-receptor blockers and neuronal nitric oxide
synthase (NOS) inhibitors have been proposed as therapies for
Parkinson's disease, however, no experimental results from human
studies have yet been published (Rodriguez, Ann. Neurol.,
44:S175-S188 [1998]).
[0289] The use of neurotrophic factors to stimulate neuronal
repair, survival, and growth in Parkinson's disease has also been
studied, particularly the use of glial cell line-derived
neurotrophic factor (GDNF). Although GDNF protein protects some
dopamine neurons from death, it is difficult to supply GDNF protein
to the brain. Furthermore, the use of such protein therapies in
general is problematic, since protein molecules show rapid in vivo
degradation, are unable to penetrate the blood-brain barrier, and
must be directly injected into the ventricles of the patient's
brain (Palfi et al., Soc. Neurosci. Abstr., 24:41 [1998]; Hagg,
Exp. Neural., 149:183-192 [1998]; and Dunnett and Bjorklund,
supra). Other neurotrophic factors which may have therapeutic value
have been proposed based on in vitro and animal model systems,
including neurturin, basic fibroblast growth factor (bFGF),
brain-derived neurotrophic factor (BDNF), neurotrophins 3 and 4/5,
ciliary neurotrophic factor and transforming growth factor .beta.
(TGF-.beta.). However, the effectiveness of these therapies in
humans remains unknown. At present, no single chemical compound or
peptide has been reported to completely protect dopamine neurons
from death by tropic factor withdrawal or neurotoxin exposure.
[0290] Cell replacement therapies have also received much attention
as potential methods for treating Parkinson's disease (Freed et
al., Arch. Neural., 47:505-512 [1990]; Freed et al., N. Engl. J.
Med., 327:1549-1555 [1992]; Lindvall et al., Science 247:574-577
[1990]; Spencer et al., N. Engl. 3. Med., 327:1541-1548 [1992];
Widner et al., N. Engl. S. Med., 327:1556-1563 [1992]; Lindvall,
NeuroReport 8:iii-x [1997]; Olanow et al., Adv. Neural., 74:249-269
[1997]; and Lindvall, Nature Biotechn., 17:635-636 [1999]). These
neural grafting therapies use dopamine supplied from cells
implanted into the striatum as a substitute for nigrostriatal
dopaminergic neurons that have been lost due to neurodegeneration.
Although animal models and preliminary human clinical studies have
shown that cell replacement therapies may be useful in the
treatment of Parkinson's disease, the failure of the transplanted
neurons to survive in the striatum is a major impediment in the
development of cell replacement therapies.
[0291] Various sources of dopaminergic neurons for use in the
transplantation process have been tried in animal experiments,
including the use of mesencephalic dopamine neurons obtained from
human embryo cadavers, immature neuronal precursor cells (i.e.,
neuronal stem cells), dopamine secreting non-neuronal cells,
terminally differentiated teratocarcinoma-derived neuronal cell
lines (Dunnett and Bjorkland, supra), genetically modified cells
(Raymon et al., Exp. Neural., 144:82-91 [1997]; and Kang, Mov.
Dis., 13:59-72 [1998]), cells from cloned embryos (Zawada et al.,
Nature Medicine 4:569-573 [1998]) and xenogenic cells (Bjorklund et
al., Nature 298:652-654 [1982]; Huffaker et al., Exp. Brain Res.,
77:329-336 [1989]; Galpem et al., Exp. Neurol., 140:1-13 [1996];
Deacon et al., Nature Med., 3:350-353 [1997]; and Zawada et al.,
Nature Med., 4:569-573 [1998]). Nonetheless, in current grafting
protocols, no more than 5-20% of the transplanted dopamine neurons
survive.
[0292] Additional therapies are also available, such as physical
therapy, occupational therapy, or speech/language therapy.
Exercise, diet, nutrition, patient/caregiver education, and
psychosocial interventions have also been shown to have a positive
effect on the mental and/or physical state of a person suffering
from Parkinson's disease.
[0293] Various methods of evaluating Parkinson's disease in a
patient include Hoehn and Yahr Staging of Parkinson's Disease,
Unified Parkinson Disease Rating Scale (UPDRS), and Schwab and
England Activities of Daily Living Scale.
[0294] A person suffering from Parkinson's disease should avoid
contraindicated and potentially contraindicated drugs such as
antipsychotic drugs, Haloperidol (Haldol), Perphenazine (Trilafon),
Chlorpromazine (Thorazine), Trifluoperazine (Stelazine),
Flufenazine (Prolixin, Permitil) Thiothixene (Navane), Thioridazine
(Mellaril); antidepressant drug, combination of Perphenazine and
Amitriptyline (Triavil); anti-vomiting drugs, Prochlorperazine
(Compazine), Metoclopramide (Reglan, Maxeran), Thiethylperazine
(Torecan), Reserpine (Serpasil), Tetrabenazine (Nitoraan); blood
pressure drug, Alpha-methyldopa (Aldomet); anti-seizure drug,
Phenyloin (Dilantin); mood stabilizing drug, lithium; and
anti-anxiety drug, Buspirone (Buspar).
EXEMPLIFICATION
[0295] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
Example 1
Antagonism of Dopamine Receptors or Transporters & Functional
Activity
[0296] Functional activity of the compounds was determined in vitro
in cellular assays using recombinant human cell lines. Measurements
of functional activity for serotonine uptake inhibition was
determined in human HEK-293 cell lines according to the procedures
of Cu et al. (J. Biol. Chem. 269: 27124, 1994) using fluoxetine
(EC.sub.50=57 nM) as the reference compound. Determination of
functional activity for norephinephrine uptake inhibition was
accomplished using an MDCK cell line according to the methods of
Galli et al. (J. Exp. Biol. 198: 2197, 1995) with desipramine
(EC.sub.50=7 nM) as a reference compound. For determination of
dopamine functional activity, a hDAT cell line was used as
described by Giros et al. (Mol. Pharmacol. 42: 383, 1992) with
nomifensine (EC.sub.50=11 nM) as a reference compound.
TABLE-US-00002 TABLE I In Vitro Selectivity - Functional Uptake
Profiles CNS- CNS- CNS- R- 28,100 27,100 28,001 R-DDMS DMS HUMAN
DAT (nM) 1 1 5 100 30 EC.sub.50 NET (nM) 200 1000 5000 200 300
EC.sub.50 5-HT (nM) 825 5000 5000 1500 1500 EC.sub.50 RAT DAT (nM)
50 80 300 100 70 EC.sub.50 NET (nM) 300 1000 5000 180 180 EC.sub.50
5-HT (nM) 5000 5000 5000 1500 1500 EC.sub.50
[0297] Table I above listed the representative results obtained
from several subject compounds, demonstrating superb in vitro
selectivity for inhibiting functional uptake of DAT, as compared to
uptake of related ligands (NET and 5-HT). For comparison, the
results for two control compounds, R-DDMS and R-DMS, are also
listed.
[0298] It is evident, based on these results, that the subject
compounds are quite selective inhibitors of DAT uptake. For
example, CNS-28,100 is a 200-fold and 825-fold more selective
inhibitor for DAT than for NET and 5-HT, respectively. The
selectivity for CNS-27,100 is 1000-fold (NET) and 5000-fold (5-HT),
respectively. The selectivity for CNS-28,001 is 1000-fold (NET) and
1000-fold (5-HT), respectively. In contrast, R-DDMS is only 2-fold
more selective for DAT over NET, and 15-fold more selective for DAT
over 5-HT. Similarly, R-DMS is 10-fold more selective for DAT over
NET, and 50-fold more selective for DAT over 5-HT.
[0299] The ability of the compounds of the invention to displace
norephinephrine ligands in vitro was determined by the methods of
Galli et al. (J. Exp. Biol. 198: 2197, 1995) using desipramine
(IC.sub.50=920 nM) as a reference compound. The displacement of
dopamine, and serotonine ligands in vitro was determined by the
methods of Gu et al. (J. Biol. Chem. 269: 7124, 1994) using
GBR-12909 (IC.sub.50(DA uptake)=490 nM, IC.sub.50(5-HT uptake)=110
nM) as a reference compound. Other similar methods are also
available in the art.
[0300] For example, in a typical uptake assay for measuring
IC.sub.50 of DAT, the assay is performed at room temperature in
Krebs-Ringer's--HEPES (KRH) buffer (125 mM NaCl, 4.8 mM KCl, 1.2 mM
MgSO.sub.4, 1.2 mM KH.sub.2PO.sub.4, 1.3 mM CaCl.sub.2, and 25 mM
HEPES, pH 7.4), supplemented with 0.1% D-glucose, 1 mM ascorbic
acid, 1 mM tropolone [catechol-O-methyltransferase (EC
2.1.1.6)-inhibitor] and 10 .mu.M pargyline (monoamine oxidase-B
inhibitor). Before the assay, cells expressing DAT are washed once
with KRH and equilibrated for 5 min. The cells may be assayed in
24-well plates and incubated for 2-5 min. with tritiated amines.
Nontransported inhibitors were preincubated for 5 min, and
substrates were applied together with the tritiated substrate. The
uptake assay is terminated with two washes of ice-cold KRH, and the
accumulated radioactivity is recovered by lysing the cells in 0.2%
SDS and 0.1 N NaOH and counting on a Liquid Scintillation Analyzer
1900 TR (Packard, Meriden, Conn.). Nonspecific uptake can be
determined in the presence of 10 .mu.M GBR12909 (for hDAT).
[0301] Experiments to determine the ionic requirements for
DAT-mediated uptake are done in KRH buffer, substituting LiCl or
choline Cl for NaCl (sodium-dependence) or substituting
D-gluconates for NaCl and KCl, and Ca(NO.sub.3).sub.2 for
CaCl.sub.2 (chloride dependence). Cells are washed twice with
sodium- or chloride-free KRH before the assay (each wash step at
least S min). In all transport assays, incubation periods and
substrate concentrations are chosen such that uptake obeyed
first-order rate kinetics.
[0302] V.sub.max values for amine uptake in stable transfected
DAT-cells are determined in parallel assays for at least two amines
per experiment and expressed as relative values.
[0303] Table II represents a typical result in table form.
Specifically, the IC.sub.50 for CNS-28,100 against DAT (SLC6A3) is
1 nM, while the IC.sub.50 for the related NET (norepinephrine
transporter or SLC6A2) and 5-HT receptors are 150 nM and 550 nM,
respectively, indicating that the inhibitory effect of CNS-28,100
against DAT is not only highly effective, but also very specific
(over 150-550 fold selectivity against related receptors).
[0304] Similar results were also obtained for CNS-27,100, where the
IC.sub.50 for DAT is also 1 nM, and the IC.sub.50 for the related
NET and 5-HT receptor are 175 nM and 1200 nM, respectively
(175-1200 fold selectivity against related receptors).
[0305] Similar results were also obtained for CNS-28,001, where the
IC.sub.50 for DAT is 5 nM, and the IC.sub.50 for the related NET
and 5-HT receptor are 870 nM and 10,000 nM, respectively (174-2,000
fold selectivity against related receptors).
TABLE-US-00003 TABLE II In Vitro Selectivity - Inhibition Profiles
CNS-28, In vitro 100 CNS-27,100 CNS-28,001 DAT (nM) IC.sub.50 1 1 5
NET (nM) IC.sub.50 150 175 870 5-HT (nM) IC.sub.50 550 1,200
10,000
[0306] In these experiments, CNS-27,100, CNS-28,001, and CNS-28,100
were all tested as racemic mixtures of enriched diastereomers.
[0307] The in vitro selectivity profile of two representative
subject compounds, CNS-28,100 and CNS-27,100, are also tested
against a panel of other receptors, including the M.sub.1 receptor,
Histamine H.sub.1 receptor, sigma-1 (.sigma..sub.1) receptor,
.beta..sub.1-adrenergic receptor, and dopamine D.sub.2 receptor.
Representative results are listed below in Table III:
TABLE-US-00004 TABLE III In Vitro Selectivity Profiles for Other
Receptors In vitro CNS-28,100 CNS-27,100 M.sub.1 (nM).sub.h 5000
5000 Histamine H.sub.1 (nM).sub.h 5000 5000 Sigma .sigma.1
(nM).sub.h 5000 5000 .beta..sub.1-adrenergic (nM).sub.h 5000 5000
D.sub.2 (nM).sub.h 1000 1000
[0308] The results indicate that neither of these subject compounds
are very selective for these other non-related or more distantly
related receptors.
Example 2
In Vivo Efficacy of Several Illustrative Dopaminc Transporter
Inhibitors
[0309] In vivo efficacy of several illustrative DAT inhibitors of
the instant invention, CNS-27,100, CNS-28,100, and CNS-28,200, were
measured using standard forced swim test model using rat. The
objective of this study was to assess the antidepressant effects of
test compounds in the behaviral despair assay in rats using a
modification of a method described by Porsolt R. D. et al. in
Behaviroural despair in rats: a new model sensitive to
antidepressant treatment, Eur. J. Pharmacol., 47: 379-391, 1978;
Porsolt et al., Nature 266: 730-732, 1977; and Porsolt et al., in
Psychopharmacology, Olivier, Mos, and Slangen (eds) Birkhauser
Verlag, Basel, pp. 137-159, 1991. Briefly, when mice (or rats) are
forced to swim in a cylinder from which no escape is possible, they
readily adopt a characteristic immobile posture and make no further
attempts to escape except for small movements needed to keep
floating. The immobility is considered by some to reflect a
"depressive mood" (Porsolt et al., Nature 266: 730-732, 1977) in
which animals cease to struggle to escape the aversive situation.
The immobility induced by the procedure is influenced by a wide
variety of antidepressants (Porsolt et al., in Psychopharmacology,
Olivier, Mos, and Slangen (eds) Birkhauser. Verlag, Basel, pp.
137-159, 1991) and has a good predictive validity in that it
detects antidepressants with different mechanisms of action (TCAs,
SSRIs, MAOIs, and other atypical ones). The test is sensitive to
muscle-relaxant (benzodiazepines) and sedative (neuroleptics)
effects, leading to enhanced immobility (Porsolt et al.,
supra).
[0310] In a typical experiment, animals are placed singly into a
cylinder (e.g. 46.times.30 cm) containing fresh water at about
20.degree. C. for 6 minutes. The activity (or immobility) of the
animal is measured by an observer minute by minute. In more detail,
the animals were preconditioned in a pretest session, where the
rats were individually forced to swim inside a vertical plexiglass
cylinder containing water maintained at 19-20.degree. C. After 15
minutes in the water, they were allowed to dry for 15 minutes in a
heated enclosure, Twenty four hours later, the compounds were
administered either intraperitoneally or orally to the animals. One
hour after administration of the test compound, animals were put
back into the cylinder containing water. The total duration of
immobility was measured during the last 4 minutes of a 6 minute
test.
[0311] The results are expressed as the percentage of variation of
the total duration of immobility calculated from the mean value of
the vehicle-treated group (% variation=[(immobility duration of
vehicle-immobility duration of test compound)/(immobility duration
of vehicle)].times.100%). Only compounds which exibit a
statistically significant variation (e.g. >30%) are considered
effective in this in vivo model.
[0312] In order to measure the in vivo efficacy of inhibiting DAT
in rats using the DAT inhibitor of the instant invention, one test
inhibitor (CNS-27,100, CNS-28,100, or CNS-28,200) was injected i.p.
as racemic mixtures of diastereamers into the animals, at various
doses (e.g. 7.5 and 15 mg/kg). Sibutramine (2.0 and 2.5 mg/kg),
Bupropion (7.5 and 10 mg/kg), and Imipramine (30 mg/kg) were
similarly administered as controls. FIG. 2 indicates that at the
doses tested, these DAT inhibitors performed equally well, if not
better, than the commercial drugs Sibutramine, Bupropion, and
imipramine. Asterisks indicate highly statistical significant
results.
[0313] A fourth DAT inhibitor, CNS-28,002 was administered p.o. as
a racemic mixture of diastereomers at either 35 or 75 mg/kg.
Sibutramine (5.0 and 3.75 mg/kg), Bupropion (30 and 40 mg/kg), and
Imipramine (100 mg/kg) were similarly administered as controls.
FIG. 4 indicates that at the doses tested, CNS-28,002 performed
equally well, if not better, than the commercial drugs Sibutramine,
Bupropion, and Imipramine.
[0314] Similarly, CNS-27,100 was also administered p.o. as a
racemic mixture of enriched (95:5) diastereomer at either 35 or 75
mg/kg. Sibutramine (5.0 and 335 mg/kg), Bupropion (30 and 40
mg/kg), and imipramine (100 mg/kg) were similarly administered as
controls. FIG. 3 indicates that at the doses tested, CNS-27,100
performed equally well, if not better, than the commercial drugs
Sibutramine, Bupropion, and imipramine.
[0315] Asterisks indicate highly statistical significant
results.
[0316] Other in vitro profiles of the representative compounds
CNS-28,100 and CNS-27,100 are listed below in Table IV.
TABLE-US-00005 TABLE IV In Vivo Profiles for Representative
Compounds In Vivo (Rat) CNS-28,100 CNS-27,100 T.sub.1/2 (i.v.) 500
minutes 200 minutes Oral Bioavailability 70% 40% Volume of
Distribution 10 L/kg 6 L/kg
Example 3
Toxicological Profiles of Illustrative Dopamine Transporter
Inhibitors
[0317] An in vivo evaluation was carried out to determine the
maximum tolerated dose of numerous test compounds in rat. The
compounds were administered i.v., and the animals were then
observed for 72 hours.
[0318] Table V summarizes the acute single-dose toxicological
profile data for three DAT inhibitors of the instant invention,
CNS-27,100, CNS-28,002, and CNS-28,200.
TABLE-US-00006 TABLE V Acute Single-Dose Toxicological Profiles
Acute Single Dose Toxicology CNS-27, 100 CNS-28,002 CNS-28,200 RAT
30 mg/kg No No No (n = 5) Significant Significant Significant
Symptoms Symptoms Symptoms 90 mg/kg No No No Significant
Significant Significant Symptoms Symptoms Symptoms 120 mg/kg No No
Decrease grip strength Significant Significant and limb tone and
Symptoms Symptoms convulsions 200 mg/kg Decrease No Convulsions
grip strength. Significant Slight Symptoms depression.
[0319] Briefly, experimental rats, in groups of 5 animals, were
administered with various doses of respective DAT inhibitors (e.g.
30, 90, 120, and 200 mg/kg), and the observed toxicological effects
were recorded.
[0320] As is shown in Table V, rats tolerate doses below 120 mg/kg
of CNS-27,100 well, with no significant observed symptoms
associated with drug administration. At 200 mg/kg, animals showed
decreased grip strength, and slight depression. Animals tolerates
CNS-28,002 rather well, with no observed symptoms at the highest
dose of 200 mg/kg. However, rats administered with CNS-28,200
showed decreased grip strength and limb tone and convulsions at 120
mg/kg, and convulsions at 200 mg/kg. But this dose is about 10
times the effective dose as shown in FIG. 2.
[0321] Multidose toxicology study was also conducted for CNS-27,100
(administered as enantiomerically enriched diastereomer), with
Sibutramine as a control. Briefly, over the span of 7 days, 6
Sprague-Dawley rats (3 males and 3 females) were orally
administered various doses of representative compound CNS-27,100,
or the control compound Sibutramine at a dose volume of about 10
mL/kg body weight. The oral doses tested are 50 mg/kg/day, 100
mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day. The representative
results are listed below in Table VI.
TABLE-US-00007 TABLE VI Multidose Toxicological Profiles CNS-27,100
(enantiomerically 7-Day Multidose Oral enriched Dosing Toxicology
Study diastereomer) Sibutramine Sprague-Dawley 50 mg/kg/day No
Decreased grip Rats significant strength. (n = 6; 3M/3F) symptoms.
Slight depression Dose Volume: 100 mg/kg/day No Decreased grip 10
mL/kg significant strength. symptoms. depression; 3 self mutilation
200 mg/kg/day Decreased grip Decreased grip strength. strength.
Slight depression; Convulsions; 1 self mutilation 6 self mutilation
2 deaths 400 mg/kg/day Decreased grip Convulsions; strength. 4
deaths Slight depression; 3 self mutilation
[0322] The results indicate that experimental animals tolerate
CNS-27,100 better than Sibutramine at similar doses. For example,
at 100 mg/kg/day, rats treated by CNS-27,100 did not display any
significant symptoms. In contrast, rats treated by Sibutramine
showed decreased grip strength, depression, and even 3
self-mutilation. Such symptoms were not seen in CNS-27,100-treated
rats until the dose was raised 4-times higher to 400 mg/kg/day. At
that dose, however, treatment with Sibutramine resulted in
convulsions, and 4 deaths in 6 experimental animals.
EQUIVALENTS
[0323] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0324] All patents, publications, and other references cited above
are hereby incorporated by reference in their entirety.
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