Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TREATMENT OF NEGATIVE AND COGNITIVE SYMPTOMS
OF SCHIZOPHRENIA WITH GLYCINE UPTAKE ANTAGONISTS
BACKGROIJND OF THE INVENTION
For the past 30 years, the dopamine hypothesis has been the leading
neurochemical
model of schizophrenia. The dopamine hypothesis is based upon observations
that
amphetamine-like dopamine releasing agents induce a psychotomimetic state that
closely
resembles schizophrenia and that agents that block dopamine receptors (e.g.,
chlorpromazine,
haloperidol) are clinically beneficial in the treatment of schizqhrenia. The
dopamine
hypothesis posits that symptoms of schizophrenia reflect functional
hyperactivity of brain
dopamineigic symptoms, primarily in the mesolimbic and mesocortical brain
regions.
Despite its heuristic value, however, there are several limitations of the
dopamine hypothesis
that have contributed to limitations in clinical treatment in schizophrenia.
First, amphetamine
psychosis provides an accurate model only for the positive symptoms of
schizophrenia (e.g.,
hyperactivity, hallucinations). In contrast, amphetamine administration does
not lead to the
development of negative symptoms (e.g., blunted affect, emotional withdrawal)
or cognitive
dysfunction similar to that observed in schizophrenia. A significant
percentage (20 - 50%)
of schizophrenic patients continue to show prominent negative symptoms and
thought disorder
despite optimal treatment with dopamine-blocking agents, indicating that new
treatment
approaches are necessary. Second, for the majority of schizophrenic patients
no clear
disturbances of dopaminergic neurotransmission have been demonstrated. Thus,
to the extent
that functional dopaminergic . hyperactivity does exist, it may be secondary
to a more
fundamental disturbance in other neurotransmitter systems. Antidopaminergic
treatment,
therefore, while controlling symptoms may not address underlying
pathophysiology.
A potential direction for the development of a new treatment approach first
became
available in the late 1950's with the development of phencyclidine (PCP,
"angel dust"). PCP
was initially developed for use as a general anesthetic. In early clinical
trials, PCP and
related agents (e.g., ketamine) were found to induce psychotic symptoms that
closely
resembled those of schizophrenia. As opposed to amphetamine psychosis, PCP
psychosis
incorporated both negative and positive symptoms of schizophrenia. Moreover,
PCP
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uniquely reproduced the type of cognitive dysfunction seen in schizophrenia.
Mechanisms
underlying PCP-induced psychosis remained largely unknown until the initial
description of
brain PCP receptors in 1979. Subsequent research in the early 1980s
demonstrated that the
PCP receptor constitutes a binding site located within the ion channel
associated with N-
methyl-D-aspartate (NMDA)-type glutamate receptors, and that PCP and related
agents induce
their psychotogenic effects by blocking NMDA receptor-mediated
neurotransmission. This
finding led to the suggestion (Reference 14; Reference 5) that endogenous
dysfunction or
dysregulation of NMDA receptor-mediated neurotransmission might contribute
significantly
to the etiology of schizophrenia, and, in particular, might lead to the
expression of
neuroleptic-resistant negative and cognitive symptoms. Further, it raised the
possibility that
medications that could potentiate NMDA receptor-mediated neurotransmission
might be
beneficial in the treatment of neuroleptic-resistant signs and symptoms of
schizophrenia.
Prior to discovery of the glycine binding site in 1987, it was found that
administration
of oral glycine to rodents at high doses similar to those used later by the
present inventor
leads to reversal of behavioral effects induced by PCP (Reference 13),
indicating that that
behavioral assay may be sensitive to the anti-psychotic effects of NMDA
augmenting agents.
NMDA receptors are primarily activated by glutamate, which serves as the major
excitatory neurotransmitter in cortex. Exogenous glutamate cannot be
administered
effectively, however, because (1) glutamate does not cross the blood-brain
barrier, (2)
glutamate activates several types of receptors other than NMDA receptors, and
(3) activation
by glutamate analogs that cross the blood-brain barrier may lead to
overexcitation of cortical
neurons, resulting in neuronal degeneration (excitotoxicity). A potential
alternate approach
for potentiating NMDA receptor-mediated neurotransmission became available in
1987 with
the demonstration that glycine acts as an allosteric modulator at the NMDA
receptor complex
(Reference 7). This finding raised the possibility that exogenously
administered glycine
might selectively potentiate NMDA receptor-mediated potentiation and might,
therefore, lead
to clinical improvement in schizophrenic patients with prominent neuroleptic-
resistant
symptomatology. Limitations to the use of glycine were (1) it was unknown to
what extent
exogenously administered glycine might permeate the CNS, (2) it was unknown to
what
extent glycine regulation of NMDA receptor-mediated neurotransmission would be
of
physiological relevance in vivo, and (3) it was unknown to what extent
augmentation of NMDA receptor-mediated neurotransmission might, in fact, lead
to clinical improvement.
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Subsequent to the discovery of the glycine binding site in 1987, several small
clinical
trials were attempted which were suggestive of possible beneficial clinical
effects but which
failed to demonstrate efficacy using standard statistical approaches. Waziri
in 1988
(Reference 12) published regarding the treating of 11 schizophrenic patients
with doses of
5 - 25 g/day in an open study which lasted 9 months. They reported improvement
in 4 of
the 11 patients, but failed to provide a control group or statistical analysis
of their results.
Costa et al., in 1990 (Reference 2) published their work on treating 6
patients with doses of
g/day of glycine in a 5 week open design, and observed positive responses in 2
patients,
as reflected in a greater than 30% decrease in symptoms as measured by the
Brief Psychiatric
10 Rating Scale (BPRS). However, overall statistical analysis was not
performed, and
independent analysis of their published data does not reveal a statistically
significant effect
(t=1.89, p=.12). A subsequent study (Reference 19) of 18 patients in a double-
blind study
of 15 g/day of glycine vs. placebo showed significant improvement in Clinical
Global
Impression (CGI), but did not show significant improvement in either the BPRS
or a scale
15 developed specifically for the assessment of negative symptoms, the
Schedule for Negative
Symptoms (SANS). Although it was concluded by these authors that use of higher
doses of
glycine might be required to demonstrate efficacy, no follow-up studies were
conducted.
Rosse et al., (Reference 11, 1989) administered 10.8 g/day glycine to 6
chronic schizophrenic
subjects for periods of 4 days to 8 weeks in an open-design but failed to
observe overall
clinical efficacy. These authors also concluded that this treatment approach
was limited by
the poor CNS permeation of glycine. Until 1994, no clinical studies were
performed by any
group with doses greater than 25 g/day, and the practicality of using glycine
at higher doses
was not determined.
The first study to be performed with higher doses of glycine was initiated in
8/89 and
involved the work of the present inventor. In this study, 14 chronic
schizophrenic subjects
with neuroleptic-resistant symptomatology were treated with 0.4 g/Kg/day
(approx. 30 g/day)
in a double-blind placebo-controlled fashion and positive and negative
symptoms were
monitored using the Positive and Negative Symptom Scale (PANSS). This study
validated
the use of high doses of glycine in that the medication was well tolerated.
Moreover,
preliminary encouraging results were obtained such that significant
improvement in negative
symptoms was observed in the glycine-treated subjects, whereas no similar
improvement was
observed in those treated with placebo. However, the study remained
inconclusive in that
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no significant difference was observed between the glycine- and placebo
groups. Results of
this study were published in August, 1994 (Javitt et al., 1994, Reference 6).
The present inventor and another disclose in their co-pending application a
treatment
with ultra-high (> 30 g/day) doses of glycine for effective augmentation of
NMDA receptor-
-mediated neurotransmission and for treatment of illness associated with
psychosis and
psychosis associated with drug intoxication, especially schizophrenia in vivo.
Two recently
completed studies validated this concept. In the first study (Leiderman et
al., Reference 9),
5 schizophrenic subjects who had participated in the above-noted original 30
g/day glycine
study at Bronx Psychiatric Center were rechallenged with a dose of 60 g/day.
Glycine levels
were monitored along with positive and negative symptoms, which were rated
using both the
SANS and PANSS. Treatment with 60 g/day of glycine was found to lead to a 6.3-
fold
increase in serum glycine levels Such a rise in serum levels has been shown by
others
(D'Souza et al., 1995, Reference 3), to lead to an approximate doubling of CNS
glycine
levels. Thus, doses in excess of those used in prior studies (i.e., in excess
of 30 g/day) may
- be required to significantly affect CNS glycine levels. No significant side
effects were
observed during treatment with 60 g/day of glycine. Thus, this study provided
the first
evidence of the practicality of clinical treatment with high-dose glycine.
Finally, despite the
small number of subjects significant improvement was observed on SANS negative
symptoms
(<.05) and a trend toward significant improvement was observed on the PANSS,
indicating
potential efficacy of ultra-high dose glycine.
A second recently completed study provided more definitive evidence for the
effectiveness of 60 g/day in the treatment of neuroleptic-resistant negative
symptoms. This
study was conducted by a former Bronx Psychiatric Center Schizophrenia
Research Fellow,
Dr. Uri Heresco-Levy, at the Sarah Herzog Hospital in Israel, in collaboration
with the
inventor and using the protocol developed by the inventor. Subjects were
treated with 60
g/day of glycine vs. placebo in a double-blind crossover design. Results from
the first 11
subjects were transmitted for analysis. These results demonstrate
significantly greater
reduction in PANSS negative symptoms in schizophrenic patients during the
glycine-treatment
phase than during the placebo-treatment phase. Thus, this study provides the
first double
blind, placebo-controlled evidence for efficacy of high-dose glycine
treatment. Significant
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improvement was also observed in other aspects of schizophrenic symptomatology
including
general psychopathology and cognitive functioning. No significant side-effects
were observed
in any of the treated subjects.
Although the concept that treatment with oral glycine might be of significant
clinical
benefit in schizophrenia had been discussed in prior papers, the above-noted
two recently
completed studies (BPC and Israel) provided the first definitive evidence that
a high dose
glycine treatment is safe, practical and efficacious.
Up to 50% of schizophrenic subjects continue to show prominent negative and
cognitive symptoms following treatment with neuroleptic medications. Newly
developed
agents, such as clozapine and risperidone, may show some improved efficacy
compared to
standard neuroleptics. Despite the introduction of such medications, however,
significant
numbers of schizophrenic patients remain chronically hospitalized. Treatment
of such patients
with glycine at doses of 30 g/day or above will lead to significant clinical
improvement, and
would thus address a clinical need that is not presently targeted by other
available
medications.
It was found treatment of psychotic conditions such as schizophrenic subjects
with
high (> 30 g/day)-doses of oral glycine or agents which induce elevations in
overall CNS
glycine levels by serving as glycine precursors or which would substitute for
glycine at the
glycine site of the NMDA receptor complex (such as glycinamide, threonine and
D-serine)
lead to significant improvement in negative symptoms, depression and cognitive
dysfunction
without affecting positive symptoms or excitement. The glycine dose (0.8
g/Kg/day or
approx. 60 g/day) that was used for the studies is substantially higher than
the doses used in
any prior study. Moreover, the serum glycine levels that resulted from the
administration
of 0.8 g/Kg/day of glycine are within the range of levels that are known to be
associated with
significant elevations of CNS glycine levels. The dosage range for the
administration of
glycine in accordance with the invention claimed in the above-identified co-
pending patent
application is above 0.4 g/Kg/day to about 2.0 g/Kg/day.
The precursor is administered in an amount sufficient for providing an
equivalent
elevation of extracellular glycine in the brain.
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SUMMARY OF THE IWVENTION
Limitations in the use of glycine and/or precursors are that (1) large doses
must -be
administered, and (2) systems exist in the brain which serve to limit the
degree to which
exogenously administered glycine can increase glycine levels at critical
sites:within the brain.
This application describes an invention in which- glycine uptake antagonists
(also
known as "glycine reuptake antagonists" and/or "glycine transport inhibitors")
are used to
augment NMDA receptor-mediated neurotransmission.
Glycine levels in brain are regulated via the action of glycine transporters
(AKA
uptake or reuptake pumps) which maintain lo.w glycine levels in the vicinity
of NMDA
receptors (Reference 15; Reference 18). Blockade of glycine uptake, therefore,
would
increase glycine levels in the vicinity of NMDA receptors without, of
necessity, increasing
whole brain or net extracellular levels. Recent studies have been conducted
with
glycyldodecylamide (GDA), a compound which the applicant has recently shown to
be an
effective antagonist of glycine uptake in brain homogenate (Javitt DC,
Frusciante M.
Glycyldodecylamide, a phencyclidine behavioral antagonist, blocks. cortical
glycine uptake:
implications for schizophrenia and substance abuse. Psychopharmacology (Berl)
1997;129:96-8).
These studies investigated the effects of GDA on PCP-induced hyperactivity in
rodents, an
assay system that has been shown to be sensitive to the effects of glycine
(Reference 20) and
other agents which potentiate NMDA receptor-mediated neurotransmission
(Reference 19).
Glycine (Figure 5) and GDA (Figure 6) showed similar profiles of activity in
that both drugs
inhibited PCP-induced hyperactivity without affecting baseline activity. GDA,
however, was
significantly more potent than glycine as evidenced by the fact that a dose of
0.05 g/kg GDA
inhibited PCP-induced hyperactivity to the same degree as a dose of 0.8 g/kg
glycine
(equivalent to the dose of glycine used in clinical studies). Other GDA-like
drugs also
inhibited PCP-induced hyperactivity (Figure 8) and their potency in blocking
PCP-induced
hyperactivity varied in proportion to their potency in inhibiting glycine
uptake (Figure 9).
These findings indicate that glycine uptake antagonists will be as or more
effect'ive than
glycine in treating PCP, psychosis-like symptoms of schizophrenia (e.g.,
negative and
cognitive symptoms). In this embodiment of the invention, human subjects would
be treated
with glycine uptake antagonists at doses which are effective in blocldng
glycine uptake in
vitro and reversing PCP-induced hyperactivity in rodents in vivo. Two types of
glycine
uptake systems have been described in brain: GLYT1 transporters which are
expressed in
highest concentration in spinal cord, brainstem, diencephalon, and retina and
to a lesser
degree in olfactory bulb and cerebral heinispheres; and GLYT2 transporters
which are
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restricted to spinal cord, brainstem and cerebellum (Reference 21). Moreover,
GLYT1
transporters may exist in multiple isoforms which may arise, in part, through
differential
splicing (Reference 17). This embodiment of the invention would include
inhibitors of either
GLYT1- or GLYT2-mediated glycine uptake, including inhibitors of any isoform
of said
transporters.
In still another embodiment of the invention, psychosis associated with other
psychiatric conditions including drug-induced (phencyclidine, ketamine and
other dissociative
anesthetics, amphetamine and other psychostimulants and cocaine) psychosis,
psychosis
associated with affective disorders, brief reactive psychosis, schizoaffective
psychosis, and
psychosis NOS, "schizophrenia-spectrum" disorders such as schizoid or
schizotypal
personality disorders, or illness associated with psychosis (such as major
depression, manic
depressive (bipolar) disorder, Alzheimers disease and post-traumatic stress
syndrome) is
treated.
In another embodiment of this application glycine uptake antagonists would be
administered parenterally.
Other objects of the invention will be apparent to the skilled artisan from
the detailed
description of the invention herein.
DESCRIPTION OF THE DRAWING
Figure 1 of the drawing depicts effect of 0.8 g/Kg/day of oral glycine on
serum glycine
levels (scatter plot) and negative symptoms (bar plot) as determined using the
Positive and
Negative Symptom Scale (PANSS) (Kay et al., 1987, Reference 8) which includes
such items
as blunted affect, emotional or withdrawal, and difficulty in abstract
thinking from Study #1.
All statistics were performed using paired, two tailed t-tests (* p < 0.1 vs.
baseline (week 0),
** p<0.05 vs. baseline, *** p<0.01 vs. baseline).
Figure 2 (a)-(d) of the drawing depicts, from Study #2, three-factor and total
PANSS change
scores during double-blind adjunctive treatment with glycine and placebo (*p
<.05, **
p<.01, *** p<.001).
Figure 3 of the drawing depicts, from Study #2, three-factor and total PANSS
scores during
double-blind adjunctive treatment with glycine and during the subsequent
placebo period in
7 subjects who received glycine during the first treatment arm.
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Figure 4 (a)-(e) of the drawing depicts, from Study #2, five-factor PANSS
change scores
during double-blind adjunctive treatment with glycine and placebo (*p <.05, **
p<.01, ***
p < .001).
.
Figure 5 of the drawing depicts, the effect of glycine on PCP-induced
hyperactivity. Male
BALB/c mice were pretreated with either glycine (0.8 g/kg) or placebo at time
= 30 min (first arrow). PCP (5 mg/kg sc.) was administered at time = 50 min
(second arrow) and
ambulatory counts were monitored using an automated rodent activity chamber.
Pretreatment
with glycine led to an approximately 25 % reduction in PCP-induced
hyperactivity.
Figure 6 of the drawing depicts, the effect of glycyldodecylamide (GDA) on PCP-
induced
hyperactivity. Male BALB/c mice were pretreated with either GDA (0.1 g/kg) or
placebo
at time = 30 min (first arrow). PCP (5 mg/kg i.p.) was administered at time =
50 min
(second arrow) and ambulatory counts were monitored using an automated rodent
activity
chamber. Pretreatment with this dose of GDA led to an approximately 50%
reduction in
PCP-induced hyperactivity, with a similar pattern of effect observed
previously for glycine.
Figure 7 of the drawing depicts, the effect of glycine and GDA on PCP-induced
hyperactivity
across the indicated number of experiments. A dose of 0.05 g/kg GDA led to a
similar
degree of behavioral inhibition as a dose of 0.8 g/kg glycine. A dose of 0.1
g/kg GDA was
approximately twice as effective. *p <.05 vs. PCP alone (CTL). *** p<.001 vs.
CTL
Figure 8 of the drawing depicts, the effect of several GDA-like compounds on
PCP-induced
hyperactivity. The rank order of potency for these agents was
glycyltriscadecylamide (GTA)
> glycyldodecylamide (GDA) > glycylundecylamide (GUA).
Figure 9 of the drawing depicts, the relationship between potency for
inhibition of PCP-
induced hyperactivity in rodents and potency for inhibition of [3H]glycine
uptake in brain
homogenate. The rank order of potency for inhibiting PCP-induced hyperactivity
(% of PCP
alone level) was the same as the rank order of potency for inhibiting
j3HJglycine uptake.
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Figure 10 of the drawing depicts, the kinetics of [3H]glycine uptake into P2
synaptosomes
and inhibition by GDA and the known glycine uptake antagonist sarcosine.
Points represent
means s. e. m. of three separate experiments, each performed in triplicate.
Figure 11 of the drawing depicts, the inhibition of [3H]glycine uptake by
indicated glycine
derivatives. Points represent means s.e.m. of three separate experiments,
each performed
in triplicate.
Figure 12 of the drawing depicts, the inhibition of [3H]glycine uptake by
glycylhexyiamide
(GHA) relative to GDA. GHA, which is known to be ineffective in antagonizing
PCP-
induced hyperactivity at doses of up to 150 mg/kg, also shows very low potency
for
inhibition of [3H]glycine uptake.
Figure 13 of the drawing depicts, the relative inhibition of PCP- and
amphetamine-induced
hyperactivity by GDA (0.05 g/kg). Whereas GDA significantly antagonized PCP-
induced
hyperactivity (p=.001, ***), it did not significantly affect amphetamine-
induced
hyperactivity. In the absence of PCP and amphetamine, integrated activity was
<500 counts.
DETAILED DESCRIPTION OF THE INVENTION
Administration can be through the use of liquid and solid formulations and
also
through the use of injectables, such as intravenous injectables, wherein
conventional
pharmaceutical carriers would be employed. Suitable pharmaceutical
preparations include
tablets, capsules, oral liquids and parenteral injectables. Tablet and capsule
formulations can
be employed utilizing conventional diluents, excipients, and the like such as
lactose in
conventional capsule and tablet-making procedures. When administered as an
oral liquid,
some compounds may be made more palatable through the use of pleasant tasting
diluents.
The compound for the present invention is to be administered at a dose
sufficient to
block total or partial glycine uptake. Glycine is effective above 0.4
g/Kg/day, for example,
0.5 g/Kg/day or above in one to several doses, preferably in a dose of 0.8
g/Kg/day divided
into three equal doses in treating schizophrenia. The dose of the glycine
uptake antagonist
can be roughly determined by its inhibitory activity on PCP-induced
hyperactivity compared
with glycine. A convenient assay for evaluating augmentation of NMDA receptor-
mediated
transmission in vivo is the rodent assay used in study #3 hereinafter. At
present, it is
believed that GDA would be administered at a dose of about .025 g/Kg/day to
.50 g/Kg/day,
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with doses of other compounds determined in relationship to GDA. The glycine
uptake
antagonist is given as the sole treatment for the psychotic-related condition,
or is used
adjunctively to conventional antipsychotic drugs such as haloperidol (Haldol),
fluphenazine
(Prolixin), chlorpromazine (Thorazine) or thioridazine (Mellaril), to atypical
antipsychotic
drugs such as clozapine (Clozaril~ and risperidone (Risperidal), to
medications used for the
control of antipsychotic medication side effects, and to other medications
commonly used for control of symptoms in conditions and illnesses such as
schizophrenia.
When given in doses as herein, the glycine uptake antagonists would exert a
clinically
beneficial effect on symptoms of schizophrenia, in particular on negative
symptoms and
cognitive dysfunction. The beneficial effects of glycine uptake antagonists on
negative
symptoms occur in the absence of deterioration in any other aspects of
schizophrenia, such
as positive symptoms or excitement. In one embodiment of the invention,
glycine uptake
antagonist administration would be continued indefinitely for control of
symptoms that do not
respond adequately to traditional classes of medication.
The present invention contemplates the employment of any biologically
acceptable
glycine uptake antagonist for treatment of the noted disorders and diseases.
Certain of the
glycine uptake inhibitors are glyclyalkylamides, such as glycyldodecylamide
and others used
in study #3, are glycine alkyl esters. Other glycine uptake antagonists are
known such as
sarcosine. Once the concept of this invention is understood, the skilled
artisan can employ
any pharmacologically acceptable glycine uptake antagonist.
The following examples are provided to illustrate the effectiveness of glycine
and
glycine uptake antagonists for the treatment of schizophrenia.
Study #1 (Leiderman et al., supra.)
Methods: This study was conducted at the Bronx Psychiatric Center in the
Bronx, NY. Five
DSM-IV schizophrenic patients chosen because of participation in a prior
double-blind study
with 0.4 g/Kg/day of glycine entered this study after providing informed
consent. Their
mean age was 45.0 7.6 years old and their mean chronicity of illness 24.2
5.9 years.
All were considered markedly to severely ill (CGI > 4). All patients were
receiving
antipsychotics (2 clozapine, 2 risperidone and 1 haloperidol), on which they
had been 30 maintained for at least 4 weeks prior to the trial.
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Oral glycine was added to their neuroleptic regimen at a dose of 10 g/day (-
0.14
g/Kg/day), and incremented to 0.2 g/Kg/day (- 14g/day) at day 3. Glycine dose
was
increased by 0.2 g/Kg/day every 2 days until a dose of 0.8 g/Kg/day was
reached, and was
then maintained for the remainder of the 8 weeks treatment period. Biweekly
ratings were
performed using the Positive and Negative Syndrome Scale (PANSS) (Kay et al.,
1987,
Reference 8) and the Scale for the Assessment of Negative Symptoms (SANS)
(Andreasen,
1989, Reference 1).
The Extrapyramidal Rating Scale (ERS) and the Abnormal Involuntary Movement
Scale (AIMS) were used to measure motoric side effects. All ratings were
performed by a
single individual who was blind to outcome of the prior glycine treatment
study. Glycine and
neuroleptic blood levels for haloperidol and clozapine were obtained every two
weeks.
Plasma glycine was determined by a liquid chromatographic procedlre (Harihan
et al., 1993,
Reference 4) for plasma amino acids and optimized for glycine using O-
methylserine as an
internal standard.
Values in text represent mean standard deviation. Treatment effects were
determined using two-tailed, paired t-tests.
Results: Treatment with oral glycine led to a significant, 6.3-fold increase
in glycine
blood levels that remained stable from week 2 to week 6 (Figure 1). There was
an apparent
decrease in glycine level between weeks 6 and 8, although the difference did
not reach
statistical significance. No adverse effects, including weakness, nausea or
sedation were seen
in any patient during the 8 weeks of the trial.
A significant improvement in negative symptoms was found using the SANS
(baseline:
75.8 7.2 vs. end of study: 72.2 8.6, t=2.79, p=0.049) and a trend towards
improvement, using the PANSS negative symptom scale (baseline: 31.0 2.3 vs.
end of the
study: 27.4 3.2, t=2.21, p=0.092). Two of the 5 subjects experienced a
greater than
20% reduction in negative symptoms. Treatment response was not significantly
correlated
with glycine level either across subjects or across time within individual
subjects.
Of the patients included in this study, those who showed the greatest
treatment
response to glycine were those who had shown the greatest response to prior
double-blind
treatment with 0.4 g/Kg/day of glycine (Javitt et al., 1994, supra. ). There
was thus a
significant across-subject correlation between change in total PANSS score
observed in the
present study and that observed in the prior study (r=0.82, p=.045). As in the
prior study,
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there were no significant changes in PANSS positive symptoms (t= 1.68, df=4,
p=0.17) or
general psychopathology (t=0.72, df=4, p= 0.5) in the present study. There was
a
significant reduction in extrapyramidal (t =4.81, df = 4 , p=0.009), but not
dyskinetic
(t=0.91, df=4, p=0.4), symptoms during glycine treatment. However, there was
no
correlation between improvement in extrapyramidal symptoms and clinical
response. Glycine
treatment did not significantly affect serum neuroleptic levels.
Study #2 - Heresco-Levy, et al., above
Methods: Subjects consisted of inpatients drawn from the research unit of the
Sarah
Herzog Memorial Hospital, Jerusalem, Israel. Subjects were diagnosed with
schizophrenia
according to DSM-III-R (American Psychiatric Association, 1987). Subjects,
moreover, were
considered to be treatment resistant on the basis of poor response to prior
neuroleptics. Prior
to study entry, subjects had been treated with stable, clinically determined
optimal oral doses
of conventional neuroleptics or clozapine for at least 3 months. Schizophrenic
patients who
met the criteria of additional DSM-III-R diagnoses, were receiving additional
psychotropic
medications or had a concurrent medical or neurological illness were excluded.
Twelve
patients were enrolled in the study. All subjects gave written informed
consent to participate
and the study was approved by the institutional review board.
After a 2 week (week -2 to week 0) baseline assessment period, subjects were
randomly assigned to receive, under double-blind conditions, either glycine
powder or
placebo solution for six weeks (week 0 - week 6). Medication was administered
under
double blind conditions. Glycine powder was administered dissolved in water.
The placebo
solution consisted of glucose. Each patient then underwent a 2 week adjunctive
treatment
washout period after which he/she crossed over to the alternate substance for
another 6 weeks
(week 8 - week 14). Glycine administration was initiated at a dose of 4 g/day
and was
increased by 4 g/day until a fixed daily dose equivalent to 0.8 g/Kg body
weight was
reached. Daily glycine treatment was administered in three divided doses. The
only other
medications allowed during the study were trihexyphenidyl (2 - 5 mg/day) for
treatment of
extrapyramidal symptoms and chloral hydrate (250 - 750 mg/day on PRN basis)
for treatment
of insomnia or agitation. For patients needing antiparkinsonian medication,
trihexyphenidyl 30 dose was kept constant throughout the study.
Symptoms and extrapyramidal side effects were assessed starting from week -2,
biweekly throughout the study, using the PANSS, the Simpson-Angus Scale for
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Extrapyramidal Symptoms (SAS) and the Abnormal Involuntary Movement Scale
(AIMS).
Patients requiring, at any point during the study, neuroleptic dose increases
were withdrawn
from the study and appropriate treatment was instituted. Withdrawal decisions
were based
on clinical evaluations and coincided with an increase of at least 30% on the
PANSS score.
Physical complaints and status were monitored daily. Hematology, blood
chemistry,
liver and kidney function, laboratory measures were assessed biweekly. Blood
samples for
the assessment of glycine serum levels were obtained at baseline and at the
end of study
weeks 6 and 14. Blood drawings were performed before breakfast and first daily
administration of medication. Serum glycine levels were determined on a Perkin
Elmer-
Pickering Amino Acid Analyzer using a lithium pH gradient and postcolumn
derivation with
ninhydrin. Quantification was carried out using a UV detector at 570 mn.
Calculations were
based on a nor-leucine internal standard. Statistical analyses (two-tailed)
were performed
using the SPSS/PC computer program.
Results: Of the 12 patients who entered the study, 11 completed. The one early
termination occurred at week 4 of placebo treatment. Of the patients who
completed, 7 had
been randomized to receive glycine during the first phase of the study, while
the remaining
4 had been randomized to receive placebo. All patients showed stable
pretreatment baselines
as evidenced by a lack of change in positive and negative symptoms during the
two weeks
prior to double-blind treatment (Table 1). Pretreatment baselines did not
differ among those
subjects who received glycine during the first double-blind treatment phase
and those who
received placebo.
In order to assess treatment response to glycine relative to placebo, rmANOVA
were
performed across all subjects with within-subject factors of treatment phase
(glycine/placebo)
and treatment week (0, 2, 4 or 6). Highly significant between-treatment
differences were
observed for negative symptoms and general psychopathology, as reflected in
significant
treatment by time interactive effects, with no corresponding worsening of
positive symptoms
(Table 2). However, when changes in general psychopathology and total PANSS
score were
covaried for changes in negative symptom score, no significant treatment or
treatment by
time effects were observed, indicating that the changes in general
psychopathology might
have been secondary to changes in negative symptoms. Significant effects of
glycine on total
PANSS score was also observed. As with the general psychopathology effects,
changes in
total PANSS score were not significant following covariation for changes in
negative
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symptoms. In order to assess the possibility that treatment order affected
overall results,
rmANOVA of negative symptoms by treatment phase and week were covaried for
treatment
order. Significance of the treatment by time effect F(3, 8) =42.6, p < .0001),
indicating that
results were not significantly affected by treatment order.
Analysis of symptom change scores revealed that significant reductions in
negative
symptoms were apparent by week 2 of the glycine treatment phase and increased
progressively until termination of glycine treatment after week 6 (Figure 2).
The mean
percentage reduction in negative symptoms at 6 weeks was 36.2 7.3 % compared
to
preglycine treatment values t=.22, df=10, p<.0001). Reductions in general
psychopathology were first apparent after 4 weeks of glycine treatment and
increased
progressively thereafter. Mean reduction in general psychopathology was 23.5
10.5 %
(t=7.41, df=10, p<.0001). A small reduction in positive symptims was also
observed in
the glycine treatment group (12.6 t 18_3 %). Although this effect was
significant when
compared to preglycine treatment values (t=2.29, df=10, p<.05), changes in
positive
symptoms at the end of 6 weeks of glycine treatment were not significantly
greater than
changes following 6 weeks of placebo (Figure 2). 8 of the 11 subjects had
PANSS negative
symptoms decreases of 30% or more and PANSS total score decreases of 25% or
more
during treatment with glycine. No reductions in symptoms of any type were
apparent during
the placebo treatment phase, and a small but significant increase in general
psychopathology
was observed at week 4 of the placebo treatment period.
Because 7 of the 11 subjects received glycine during the initial double blind,
it was
possible to evaluate the degree to which symptom improvement was maintained
throughout
the subsequent placebo treatment period (Figure 3). No change in positive
symptoms
occurred in these 7 subjects during any phase of the study, whereas negative
symptoms
improved significantly during the glycine treatment phase (F(3,4)=45.7,
p=.001) and
remained stable thereafter, with no significant worsening occurring during the
subsequent
placebo phase (F(3,4)=1.86, p=.28). Similarly general psychopathology improved
significantly during the glycine treatment phase (F(3,4) = 19.2, p<.01) and
remained stable
thereafter (F(3,4) =2.52, p =.20), indicating that the improvements observed
during glycine
treatment were maintained during the subsequent 8 weeks of the study period.
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5-factor analysis of the PANSS
Although traditional analysis of the PANSS divides symptoms into positive,
negative
and general clusters, alternative analyses have been proposed that incorporate
either 5 or 7
factors. The 5 factor model divides symptoms into clusters that are labeled
positive,
negative, cognitive, depression and excitement. In order to determine the
degree to which
{ glycine affected dimensions of schizophrenia other than positive and
negative, a secondary
analysis of the data was performed using the 5-factor components (Figure 4).
As in the 3-
factor analysis, no significant reduction in PANSS positive symptoms were
observed during
either glycine or placebo treatment, while significant, progressive
improvement was observed
during the glycine-, but not placebo-, treatment period (treatment by time
F(3, 8) =19.5,
p<.0001). Using the 5-factor analysis, however, significant reductions were
also observed
for depression (F(3, 8) =7.23, p<.02) and cognitive symptoms (F(3, 8) =4.74,
p<.05).
Improvements in depression (F(3,5)=2.13, p=.22) and cognitive impairment
(F(3,5)=0.89,
p=.51) did not remain significant following covariation for changes in
negative symptoms.
In contrast, the effect of glycine on negative symptoms remained significant
even following
covariation for changes in cognitive impairment or depression (F(3,5)=6.8,
p=.032). The
percentage reduction was greatest for negative symptoms (41.0 15.4% decrease
vs.
preglycine levels, p<.0001), followed by depression (23.0 17.9%, p=.002) and
cognitive
impairment (15.2 13.5%, p=.004). Reductions in excitement (11.9 26.3%,
p=0.17)
and positive symptoms (9.4 20.5%, p=.16) did not reach statistical
significance.
Table 1- 3 Mean (sd) PANSS factor scores during prestudy baseline
Factor Week -2 Week 0
Positive symptoms 23.1 (3.6) 23.6(3.2)
Negative symptoms 35.6 (3.2) 37.0 (6.5)
General psychopathology 44.9 (13.2) 45.5 (13.4)
Total PANSS score 103.6 (20.6) 105.5 (18.9)
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du~ri double-blind treatment with g1Ycme (60 g/day) O
Tb =
A S n
a le 2 3-factor P N S scores
~
PANSS Factor Scores - mean (sd) Statistical (rmANOVA) Results
PANSS Treatment Treatment wce = I'reatment I'ime I'reat. time
Positive Glycine 24.6 23.7 22.6 21.0 F= 3.38 F= 2.25 F= 0.85 w
Placebo 20.6 20.2 21.5 20.9
Negative Glycine 37.0 33.5 28.7 24.2 F= 2.20 F 41.1 F = 42.5
Placebo 27.8 27.1 27.4 26.9
General Glycine 46.5 44.6 40.2 35.4 F= 1.22 F= 4.55 F= 12.1
Placebo 38.1 38.6 41.8 40.5
Total PA\SS Glycine 108.1 101.5 91.7 80.6 F= 2.93 F= 16.6 F= 13.4
Placebo 86.6 S5.9 90.6 88.3
c/) >
C
W N
m 4~,
C/)
m
C
m 4~,
-~+
N
! a
CA 02239624 1998-06-04
WO 97/20553 PCTIUS96/19142
Study #3, Javitt and Frusciante, as above
Glycyldodecylamide (GDA) is a glycine derivative that was first described in
1986
(Reference 20). It was shown at that time to be significantly more potent than
glycine in
reversing PCP-induced hyperactivity. Further, it was shown that GDA
administration did
not lead to increased whole brain glycine levels, indicating that GDA did not
act as glycine
precursor. At the time, no mechanism for GDA-induced inhibition of PCP-induced
hyperactivity was postulated.
This study was undertaken to investigate the possibility that GDA reverses PCP-
induced hyperactivity by blocking glycine reuptake in brain, thereby
increasing glycine levels
in the immediate vicinity of NMDA receptors. This hypothesis, while based upon
the
observation that GDA inhibits PCP-induced hyperactivity without increasing
whole brain
glycine levels, is not obvious from the prior literature. For glycine uptake
studies,
synaptosomal P2 fractions were prepared from cortex of adult Sprague-Dawley
rats and
suspended in artificial CSF. Uptake of 100 nM PH]glycine was measured at 25 C
for the
indicated time period in the presence of indicated ligands. Incubation was
terminated by
filtration under reduced pressure. Uptake was linear for at least the first 10
min of incubation
(Figure 10). Apparent plateau was reached at 30 min, with no significant
increase in binding
between 30 and 60 min. Kinetic binding parameters were determined by nonlinear
regression. All uptake curves were determined to be first order. The mean t1/2
value was
9.7 0.7 min. The IC50 value for inhibition of [3H]glycine uptake by cold
glycine was 78.7
37.8 M. Specific [3H]glycine uptake was abolished in the absence of added
Na+/Cl-.
Inhibition studies were conducted following 5 min incubation in the absence
and
presence of GDA concentrations between 0.1 and 10 mM (Figure 11). Several
comparison
agents were also tested including (1) sarcosine, a known high potency glycine
uptake
antagonist, and (2) glycine ethyl ester (GEE) and glycine methyl ester (GME),
agents with
known lower affinity for the cortical glycine uptake site (Reference 18). The
rank order of
potency for inhibition of glycine uptake was glycine > sarcosine > GDA > GEE >
GME.
Both glycinamide and n-serine, which function as glycine precursors, showed
ICso values of
= > 10 mM. Effects of GDA and sarcosine remained significant throughout the 30
min
incubation period. 1 mM concentrations of GDA and sarcosine significantly (p
<.05)
reduced the maximal level of [3H]glycine uptake by 29 7 and 72 3 %,
respectively
(Figure 11). 5 mM GDA decreased maximal [3H]glycine uptake by 51 13.3.
Effects of
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these agents on the rate constant of glycine uptake were not significant,
although t,h values
in the presence of both GDA (7.9 2.9 min-1) and sarcosine (6.3 1.9 miri 1)
were
somewhat lower than under baseline conditions. Given that the effective
concentration of
GDA in behavioral studies is approximately 0.3 mmol/kg, these studies
demonstrate that
GDA acts as a glycine uptake antagonist at a concentration similar to what may
be obtained
in behavioral studies.
Potential direct agonist-like effects of GDA at the glycine site of the NMDA
receptor
complex were excluded using PCP receptor binding as a functional probe of NMDA
receptor
activation. In this assay, glycine-like agents stimulate [3H]MK-801 (available
from Merck)
binding in the presence, but not absence, of NMDA agonist (GLU, which is an
abbreviation
for glutamate). Assays were performed using crude synaptic membranes prepared
from rat
cortex and hippocampus and incubated for 15 min in 5 mM TRIS-acetate buffer
(pH 7.4) in
the presence of 1 nM [3H]MK-801 and indicated ligands. Incubation was
terminated by
filtration under reduced pressure through Whatman GF/B filters. Nonspecific
binding was
determined in the presence of 10 M MK-801. Incubation with GLU alone led to a
9-fold
increase in binding compared to control conditions (Javitt and Frusciante, in
press).
Incubation with GLU and glycine led to a highly significant 22-fold increase
compared to
control conditions and a significant 2.5-fold increase compared to GLU alone.
Binding in
the presence of combined GLU and GDA was comparable to binding in the presence
of GLU
alone.
Specificity of glycine uptake antagonism by GDA was examined by evaluating the
effects of glycine, GCA and GDA on uptake of GABA (gamma-aminobutyric acid)
and GLU.
Methods were the same as in the glycine uptake assay except that 10 nM
concentrations of
[3H]GABA or L-[3H]GLU were substituted for [3H]glycine. L-trans-pyrollidine-
2,4-
dicarboxyllic acid (L-PDC) and nipecotic acid were used as active controls for
the GLU and
GABA uptake assays, respectively. As opposed to its effects on [3H]glycine
uptake, GDA
significantly increased uptake of [3H]GLU and [3H]GABA. Given that activation
of the
glycine transporter in brain leads presynaptic GLU and GABA release,
potentiation of GLU
and GABA uptake is an expected in vitro consequence of glycine reuptake
inhibition (Javitt
and Frusciante, in press).
A subsequent study has investigated the rank order of potency of several GDA-
like
compounds for (1) inhibiting glycine uptake and (2) antagonizing PCP-induced
hyperactivity
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in rodents. For this study, several additional compounds were synthesized,
including
glycyltriscadecylamide (GTA), glycylundecylamide (GUA), and glycylhexylamide
(GHA).
These compounds are glycinamide derivates which contain 13, 11 and 6 carbon
atoms linked
to the amide, respectively, as opposed to GDA which contains 12 carbons. GTA
was found
to be more potent that GDA both in inhibiting PCP-induced hyperactivity and in
inhibiting
glycine uptake (Figure 8,9). GUA was less effective in both assays. GHA, which
was most
recently synthesized, was found to be approximately 20-fold less potent that
GDA in
inhibiting glycine uptake (Figure 12).
This compound has previously been shown to be ineffective in blocking PCP-
induced
hyperactivity at doses of up to 0.15 g/kg (Reference 20).
In a recent experiment also, the effect of GDA on amphetamine-induced
hyperactivity
was evaluate as a control condition. Currently available antipsychotic agents,
which exert
their clinical effects primarily by blocking dopamine receptors, may reverse
PCP-induced
hyperactivity in rodents. However, they are less effective in blocking PCP-
induced
hyperactivity than hyperactivity induced by amphetamine (Reference 16). This
study thus
evaluated the effects of GDA on amphetamine-induced hyperactivity. A dose of
GDA (0.05
g/kg) that potently and significantly (t=3.30, p=.001) inhibited PCP-induced
hyperactivity,
did not significantly inhibit hyperactivity induced by amphetamine (t=0.59,
NS) (Figure 13).
Glycine has previously been shown not to inhibit amphetamine-induced
hyperactivity
(data not shown). These findings thus support the concept that GDA inhibits
PCP-induced
hyperactivity via a mechanism different from that of currently available
antipsychotic agents.
In summary, this example demonstrates that GDA inhibits glycine uptake, and
that
a dose of 0.05 g/kg GDA induces a similar degree of inhibition of PCP-induced
hyperactivity
as a dose of 0.8 g/kg glycine, even though this dose of GDA is known not to
lead to
increases in whole brain glycine levels (although it may increase glycine
levels in select brain
compartments). At doses which are effective in blocking glycine uptake, GDA
does not bind
to NMDA receptors, or inhibit uptake of other amino acid neurotransmitters.
GDA-like
compounds inhibit PCP-induced hyperactivity in proportion to their potency in
blocking
cortical glycine uptake, and a GDA-like compound that is known to be
relatively ineffective
in blocking PCP-induced hyperactivity (glycylhexylamide) is also relatively
ineffective in
blocking glycine uptake. GDA also does not block amphetamine-induced
hyperactivity at a
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dose that is effective in blocking PCP-induced hyperactivity. These findings
provide the first
demonstration that agents which block glycine uptake lead to behaviorally
significant
augmentation of brain NMDA receptor-mediated neurotransmission. Given the
recent clinical
fmding that glycine leads to significant amelioration in symptoms of
schizophrenia when -
given at doses similar to those which inhibit PCP-induced hyperactivity in
rodents, the
present findings indicate that GDA and other compounds which inhibit brain
glycine uptake
will be effective in the treatment of schizophrenia and other psychotic
disorders.
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Psychopharmacol 10:71-72.
3. D'Souza DC, Morrissey K, Abi-Saab D, Damon D, Gil R, Bennett A, Krystal JH
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4. Hariharan M, Naga S, VanNoord T (1993): Systematic approach to the
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5. Javitt DC and Zukin SR (1991): Recent advances in the phencyclidine model
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6. Javitt DC, Zylberman I, Zukin SR, Heresco-Levy U, Lindenmayer JP (1994):
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7. Johnson JW. Ascher P. Glycine potentiates the NMDA response in cultured
mouse
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8. Kay SR, Fiszbein A, Opler LA (1987): The positive and negative syndrome
scale
(PANSS) for schizophrenia. Schiz Bull 13:261-276
9. Leiderman Eduardo, Zylberman Ilana, Zukin Stephen R., Cooper Thomas B,
Javitt
Daniel C. (1996): Preliminary Investigation of High-Dose Oral Glycine on Serum
Levels
and Negative Symptoms in Schizophrenia: An Open-Label Trial. Biol Psychiatry
39:213-
215.
10. Potkin SG, Costa J, Roy S, Sramek J, Jin Y, Gulasekaram B (1992): Glycine
in the
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treatment of schizophrenia - theory and preliminary results, in Novel
Antipsychotic Drugs
(pp. 179-188). Edited by Meltzer HY. New York, Raven Press.
11. Rosse RB, Theut SK, Banay-Schwartz M, Leighton M, Scarcella E, Cohen CG,
Deutsch SI (1989): Glycine adjuvant therapy to conventional neuroleptic
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schizophrenia; an open-label, pilot study. Clin Neuropharmacol 12:416-24.
12. Waziri R (1989): Glycine therapy of schizophrenia. Biol Psychiatry 1988,
23:210-
211 [letter].
13. Toth Eugene, Weiss Benjamine, Banay-Schwartz Miriam, Lajtha Abel (1986):
Effect
of Glycine Derivatives on Behavioral Changes induced by 3-Mercaptopropionic
Acid or
Phencyclidine in Mice. 11:1-8.
14. Javitt, D.C. (1987): Negative Schizophrenia Symptomatology and the PCP
Model
of Schizophrenia, Hillside Journal of Clinical Psychiatry, 9, 12-35.
15. Guastella J, Brecha N, Weigmann C, Lester HA, Davidson N (1992) Cloning,
expression, and localization of a rat brain high-affinity glycine transporter.
Proc Natl Acad
Sci USA 89:7189-7193.
16. Jackson DM, Johansson C, Lindgren L-M, Bengtsson A (1994) Dopamine
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antagonists block amphetamine- and phencyclidine-induced motor stimulation in
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Pharmacol Biochem Behav 48:465-471.
17. Liu Q-R, Lopez-Corcuera B, Mandiyan S, Nelson H, Nelson, N (1993) Cloning
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18. Smith KE, Borden LA, Hartig PR, Branchek T, Weinshank RL (1992) Cloning
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927-935.
- 19. Tanii Y, Nishikawa T, Hashimoto A, Takahashi K (1994) Stereoselective
antagonism
by enantiomers of alanine and serine of phencyclidine-induced hyperactivity,
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20. Toth E, Lajtha A (1986) Antagonism of phencyclidine-induced hyperactivity
by
glycine in mice. Neurochem Res 11: 393-400.
21. Zafra F, Aragon C, Olivares L, Danbolt NC, Gimenez C, Storm-Mathisen
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Glycine transporters are differentially expresses among CNS cells. J Neurosci
15:3952-3969.
From the above, the effectiveness of this invention for treating symptoms of
schizophrenia can be seen.
The skilled artisan will be able to select other naturally occurring and
synthetic, i. e. ,
of presently known and to be discovered chemical structures, glycine uptake
antagonists for
use in providing an antipsychotic effect.
Variations of the invention will be apparent to the skilled artisan.
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