COMBINATION OF A GLYCINE TRANSPORTER (GLYTl) INHIBITOR AND AN
ANTIPSYCHOTIC FOR THE TREATMENT OF SYMPTOMS OF SCHIZOPHRENIAAS WELLAS ITS PREPARATION AND USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to Application No. 60/636,575 filed on
December 16, 2004.
FIELD OF THE INVENTION
The present invention relates to the prevention and treatment of symptoms of
schizophrenia. More particularly, the invention relates to pharmaceutical
compositions and treatments comprising an antipsychotic and a GIyTl inhibitor for
treating symptoms of schizophrenia associated with disorders such as schizophrenia,
dementia, depression, Alzheimer's, ADHD, substance abuse and anxiety.
BACKGROUND OF THE INVENTION
Traditional models of schizophrenia and bipolar disorder have focused on
dopaminergic systems. Dopamine antagonists and/or lesions of dopamine pathways reduce the
effects of phencyclidine (PCP) on two behavioral measures that are thought to have relevance
to clinical symptomatology of schizophrenia: cognitive tasks involving work memory and
locomoter activity. (Adams et al., 1998). Adams et al. discloses findings that indicate that
activation of dopamine neurotransmission is not sufficient to sustain PCP-induced locomotion
and impairment of working memory and that glutamatergic hyperstimulation may account for
psychotomimetic and cognitive-impairing effects of PCP. PCP induces a psychotic state that
closely resembles schizophrenia by blocking neurotransmission mediated at N-methyl-D-
aspartate (NMDA)-type glutamate receptors. (Javitt et al., 1997). PCP-like agents uniquely
reproduce negative, cognitive and positive symptoms of schizophrenia. Positive symptoms are
behavioral excesses generally considered psychotic (e.g., hallucinations, delusions, bizarre
behavior), whereas negative symptoms denote a deficiency from normal behavior (e.g., a lack
of normal social responsiveness, flat affect). Cognitive dysfunctions include impairment in
working memory, executive functions, sustained attention, basic processing of sensory stimuli,
verbal episodic memory and smooth pursuit eye movements. More recent models of
schizophrenia now postulate that schizophrenia is associated with dysfunction or dysregulation
of neurotransmission mediated at brain NMDA-type glutamate receptors. The NMDA model
of schizophrenia raised the possibility that agents which augment NMDA receptor-mediated
neurotransmission might be therapeutically beneficial in schizophrenia. The primary
neurotransmitter acting at NMDA receptors is glutamate. However, NMDA receptor activity
is also modulated by the amino acid glycine which binds to a selective modulatory site that is
an integral component of the NMDA receptor complex. U.S. Patent No.5,854,286 discloses
the use of orally administered glycine, in dietary quantities, for the treatment of schizophrenia.
Glycine is considered a full agonist at the NMDA-associated glycine binding site
(McBain et al., 1989). D-Serine, like glycine, is present in brain in high concentration and may
serve as an endogenous ligand for the glycine binding site of the NMDA receptor complex
(Schell et al., 1995). U.S. Patent No. 6,162,827 discloses the use of D-serine in the treatment of
symptoms of psychosis.
Although the findings with glycine and D-serine support the use of full glycine-site
agonists, others have proposed that partial agonists at the glycine site, such as the drug D-
cycloserine, should be more effective than full agonists in the treatment of schizophrenia (see,
e.g., U.S. Patent No. 5,187,171). Partial agonists bind to the same site as full agonists (i.e.,
glycine recognition site of the NMDA receptor complex), but potentiate channel opening to a
smaller percent (typically 40-70% of the activation seen with full agonists, McBain et al.,
1989). Clinical studies with D-cycloserine have provided support for the concept that partial
glycine-site antagonists may be effective in the treatment of schizophrenia (reviewed in
D'Souza et al., 1995), and, the degree of improvement seen in studies of D-cycloserine
(reviewed in D'Souza et al., 1995) has been comparable in some circumstances to the degree of
improvement observed following studies with glycine (reviewed in D'Souza et al., 1995) or D-
serine (Tsai et al., 1998).
A second potential approach to augmentation of NMDA receptor-mediated
neurotransmission is the administration of agents that inhibit glycine transporters in brain,
thereby preventing glycine removal from active sites within the CNS. It has been known for
many years that the brain contains active transport systems for glycine that may regulate brain
levels (D'Souza, 1995). More recent studies demonstrated that glycine transporters are
differentially expressed in different brain regions (Ou et al, 1993; Zafra et al., 1995) and may be
co-localized with NMDA receptors (Smith et al., 1992). However, it has also been known for
many years that extracellular glycine levels are beyond the level needed to saturate the NMDA-
associated glycine binding site, making it unclear whether glycine transporters are, in fact, able
to maintain subsaturating glycine levels in the immediate vicinity of NMDA receptors. This is
a crucial issue in that, if glycine levels were already at or above saturating levels, additional
glycine would not, on theoretical grounds, be able to stimulate NMDA functioning (Wood,
1995). As glycine is an obligatory co-agonist along with glutamate, at the NMDA receptor, it
has been suggested that GIy-Tl functions to maintain subsaturation levels at the synaptic cleft.
(Bergeron et al. 1998). If under physiological conditions, the glycine binding site of the
NMDA receptor is indeed unsaturated, then modulation of synaptic glycine concentrations
using a GIy-Tl inhibitor would be a method of potentiating NMDA receptor function. (Slassi
et al. 2004). This approach is attractive because it could avoid the toxic effects of agonists that
directly act on the NMDA receptor.
Schizophrenia is a cognitive and behavioral disorder that affects up to 1 % of the
human population. Other disease states exhibit symptoms also seen in schizophrenia. Current
understanding of the etiology of the symptoms of schizophrenia and similar disease states
remains vague, but points to a combination of genetic and environmental factors. The search
for medications to treat schizophrenia and similar disease states has traditionally focused on
dopamine receptor antagonists, and more recently on drugs that combine dopamine receptor
blockade with antagonist/agonist actions at other receptors. Based upon work with animal
models, and the fact that blockade of NMDA glutamate receptors in normal humans produces
schizophrenia-like symptoms, it has been postulated that hypofunction of the glutamate system,
specifically at the NMDA receptor, underlies some symptoms in schizophrenia (Goff and
Coyle, 2001). However, drugs that directly inhibit or stimulate NMDA receptors have proven
unsafe for routine patient care in animal models and early clinical trials. Fortunately, the
NMDA receptor is a complex heteromeric channel that can be pharmacologically modulated in
more subtle ways than simply blocking or stimulating the glutamate binding site (Nakanishi et
al., 1998).
Glycine is an obligatory co-agonist at the NRl subunit of the NMDA type glutamate
receptor complex. Thus, increasing tone on the glycine binding site (i.e., increasing
extracellular glycine) potentiates the capacity of glutamate to open the NMDA channel. Given
the proposed reduction in NMDA activity in schizophrenia, it has been postulated that
elevating extracellular glycine may increase NMDA conductances and thereby relieve some
symptoms of schizophrenia and similar disease states. Parsons et al. (1998) and Danysz et al.
(1998) provide reviews of data related to the role of the NMDA receptor in a wide range of
CNS disorders.
U.S. Patent No. 6,355,681 discloses the use of glycine and precursors in the treatment
of symptoms of psychosis.
U.S. Application No. 20020161048 discloses the use of glycine substitutes and
precursors in the treatment of symptoms of psychosis.
U.S. Application No. 20020183390 discloses a method and composition for
augmenting NMDA receptor mediated transmission involving the use of a D-serine transport
inhibitor. U.S. Application No. 20020183390 discloses that the method and composition may
be used in the treatment of neuropsychiatric disorders such as schizophrenia.
One mechanism for increasing extracellular glycine is to prevent its elimination from
the extracellular space by uptake through the glycine transporter 1 (GIyTl) (Javitt and
Frusciante, 1997). Aragon et al. (2003) is a review on the localization, transport mechanism,
structure, regulation and pharmacology of glycine transport inhibitors. Javitt (2002) discloses
that NMDA receptor dysfunction may play a role in the pathophysiology of schizophrenia.
Javitt (2002) also discloses that GIyTl inhibitors may represent a "next generation" approach to
the treatment of the persistent negative and cognitive symptoms of schizophrenia.
U.S. Patent No.5,837,730 discloses that a glycine transport inhibitor,
glycyldodecylamide (GDA), is able to exert glycine-like behavioral effects in rodents.
None of the references cited disclose or suggest the GIyTl inhibitor and
antipsychotic combination of the invention.
SUMMARY OF THE INVENTION
An object of the present invention is a pharmaceutical composition
comprising an antipsychotic and a GIyTl inhibitor.
Another object of the present invention is a method for treating symptoms of
schizophrenia which comprises administration of a combination of an antipsychotic
and a GIyTl inhibitor.
Another object of the present invention is a method for increasing
extracellular glycine levels in a mammal, which comprises administration of an
antipsychotic in combination with a GIyTl inhibitor.
Yet another object of the present invention is a method for increasing
extracellular dopamine levels in a mammal which comprises administration of an
antipsychotic in combination with a GIyTl inhibitor.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1A-1D: Experiment #1, Effects of risperidone on extracellular glycine
(upper panels) and dopamine (lower panels) in the rat striatum. Doses were given in
ascending order as indicated by the arrows. Data in the right panels were normalized
to the percent change from the average of the three baseline values (i.e., the data
obtained before the first arrow). Data are shown as mean ± sem, and were statistically
evaluated using a one-way ANOVA with repeated measures over time.
Figures 2A-2D: Experiment #2, Effects of COMPOUND NO. 1 on
extracellular glycine (upper panels) and dopamine (lower panels) in the rat striatum.
Doses were given in ascending order as indicated by the arrows. Data in the right
panels were normalized to the percent change from the average of the three baseline
values (i.e., the data obtained before the first arrow). Data are shown as mean ± sem,
and were statistically evaluated using a one-way ANOVA with repeated measures
over time.
Figures 3A-3D: Experiment 3: Effects of a combination of risperidone and
COMPOUND NO. 1 on extracellular glycine in the striatum. Drug co-administration
was made at the arrow. Data in the right panels were normalized to the percent
change from the average of the three baseline values (i.e., the data obtained before the
first arrow). Data are shown as mean ± sem, and were statistically evaluated using a
one-way ANOVA with repeated measures over time. Effects of a combination of
risperidone and COMPOUND NO. 1 on extracellular dopamine in the striatum. Drug
co-administration was made at the arrow. Data in the right panels were normalized to
the percent change from the average of the three baseline values (i.e., the data
obtained before the first arrow). Data are shown as mean ± sem, and were statistically
evaluated using a one-way ANOVA with repeated measures over time.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
It has now been found that combining an antipsychotic with a GIyTl inhibitor
in the treatment of symptoms of schizophrenia results in an unexpected increase in
extracellular glycine.
The present invention is directed to an antipsychotic/GlyTl inhibitor
combination. The antipsychotic and the GIyTl inhibitor of the combination may each
be administered separately or may be together in a single pharmaceutical composition.
The antipsychotic/GlyTl inhibitor combination may be used in the treatment of
disorders such as schizophrenia, dementia, depression, Alzheimer's, ADHD,
substance abuse and anxiety.
A number of compounds, including COMPOUND NO. 1, Compound No. 2,
Compound No. 3, bind to and inhibit glycine uptake through GIyTl. GIyTl inhibitors
that may be used in accordance with the invention therefore include:
Compound No. 1, which is disclosed in U.S. Patents Nos. 6,426,364; 6,525,085; and
6,579,987.
(C24H20NNaO3 (MW 393.42))
Compound No. 2, which is disclosed in U.S. Patents Nos. 6,426,364; 6,525,085; and
6,579,987.
(C24H21NO3 (MW 371.44))
Compound No. 3
Additional GIyTl inhibitors that may be used in accordance with the invention are
disclosed in U.S. Patents Nos. 6,426,364; 6,525,085; and 6,579,987, the entire
contents of which are hereby incorporated by reference.
Antipsychotics may be used in accordance with the invention include atypical
and typical antipsychotics. Atypical antipsychotics include, but are not limited to:
Olanzapine,2-methyl-4-(4-methyl-l-piperazinyl)-10H-thieno[2,3-b][l,5] benzodiazepine, is a known compound and is described in U.S. Pat. No. 5,229,382 as
being useful for the treatment of schizophrenia, schizophreniform disorder, acute
mania, mild anxiety states, and psychosis. U.S. Pat. No. 5,229,382 is herein
incorporated by reference in its entirety; Clozapine, 8-chloro-ll-(4-methyl-l-
piperazinyl)-5H-dibenzo[b,e][l,4]diazepine, is described in U.S. Pat. No.. 3,539,573,
which is herein incorporated by reference in its entirety. Clinical efficacy in the
treatment of schizophrenia is described (Hanes, et al., Psychopharmacol. Bull., 24, 62
(1988)); Risperidone, 3-[2-[4-(6-fluoro-l,2-benzisoxazol-3-yl)piperidino]ethyl]-2-
methyl-6,7,8,9 -tetrahydro-4H-pyrido-[l,2-a]pyrimidin-4-one, and its use in the
treatment of psychotic diseases are described in U.S. Pat. No. 4,804,663, which is
herein incorporated by reference in its entirety; Sertindole, l-[2-[4-[5-chloro-l-(4-
fluorophenyl)-lH-indol-3-yl ]-l-piperidinyl]ethyl]imidazolidin-2-one, is described in
U.S. Pat. No. 4,710,500. Its use in the treatment of schizophrenia is described in U.S.
Pat. Nos. 5,112,838 and 5,238,945. U.S. Pat. Nos. 4,710,500; 5,112,838; and
5,238,945 are herein incorporated by reference in their entirety; Quetiapine, 5-[2-(4-
dibenzo[b,f][l,4]thiazepin-ll-yl -l-piperazinyl)ethoxy]ethanol, and its activity in
assays which demonstrate utility in the treatment of schizophrenia are described in
U.S. Pat. No. 4,879,288, which is herein incorporated by reference in its entirety.
Quetiapine is typically administered as its (E)-2-butenedioate (2:1) salt; and
Ziprasidone, 5-[2-[4-(l ,2-benzoisothiazol-3-yl)-l-piperazinyl] ethyl] -6-chloro-l ,3- dihyd ro-2H-indol-2-one, is typically administered as the hydrochloride monohydrate.
The compound is described in U.S. Pat. Nos. 4,831,031 and 5,312,925. Its activity in
assays which demonstrate utility in the treatment of schizophrenia are described in
U.S. Pat. No. 4,831,031. U.S. Pat. Nos. 4,831,031 and 5,312,925 are herein
incorporated by reference in their entirety. Typical antipsychotics are conventional
antipsychotics, including but not limited to, phenothiazine, butryophenones,
thioxantheses, dibenzoxazepines, dihydroindolones, and diphenylbutylpiperidines.
Also included are pharmaceutically acceptable salts thereof, pharmaceutically
acceptable esters thereof, and enantiomeric forms of the atypical or typical
antipsychotics.
EXAMPLES
SUMMARY OF RESULTS
Dopamine transmission microdialysis studies were conducted to determine if
COMPOUND NO. 1 affected dopamine transmission in the brain. Drugs inhibiting
dopamine transmission are to date the most effective medications against
schizophrenia. Likewise, it is known that most drugs effective in schizophrenia
antagonize D2 dopamine autoreceptors and thereby elevate extracellular dopamine
(Ferre et al, 1995). To determine if COMPOUND NO. 1 was synergistic with this
action, COMPOUND NO. 1 was combined with the antipsychotic risperidone and
effects on both glycine and dopamine were quantified in the striatum.
A cumulative dose-response curve for COMPOUND NO. 1 revealed the
expected dose-dependent increase in extracellular glycine levels in the striatum.
While the lowest dose (0.63 mg/kg) was without effect, the highest dose of
COMPOUND NO. 1 (10 mg/kg) caused a 2.5-fold increase in glycine. Although
without affect on glycine, the lowest dose of COMPOUND NO. 1 produced a
significant reduction in extracellular dopamine, and following administration of 2.5
mg/kg the levels of dopamine were normalized and remained unaltered even by the
highest dose of COMPOUND NO. 1.
Risperidone produced a dose-dependent elevation in both extracellular
dopamine and glycine. While the effect on dopamine was expected due to blockade
of D2 autoreceptors, the marked rise in glycine was unexpected. Similar to
dopamine, the elevation in glycine occurred at a threshold dose of 0.16 mg/kg
risperidone. Indeed risperidone was equally effective at producing a rise in
extracellular glycine as COMPOUND NO. 1, as indicated by a 2.5-fold increase in
glycine after 2.5 mg/kg risperidone.
The effects of each drug alone were additive when the drugs were given
together. That is to say, the combination of COMPOUND NO. 1 and risperidone
produced a greater increase in glycine than either drug alone, reaching a 6-fold
increase following a combination of 1 mg/kg risperidone and 10 mg/kg COMPOUND
NO. 1. Also, commensurate with the biphasic reduction in dopamine by
COMPOUND NO. 1, it was found that the combination of low dose COMPOUND
NO. 1 (0.63 mg/kg) tended to inhibit the increase produced by 1 mg/kg risperidone,
while the higher dose of COMPOUND NO. 1 did not alter risperidone-induced
elevations in dopamine. EXPERIMENTAL PROCEDURES
Compounds (as a Na salt) were stored, dissolved, and administered according
to detailed instructions accompanying each compound. Compounds were dissolved
in a solvent consisting of 10% BCD (beta cyclodextrin) and administered
subcutaneously.
Male Sprague Dawley rats weighing 275-325 g at the start of the experiment
were individually housed in a temperature-controlled colony room with a 12-h
light/dark cycle. Food and water was available ad libitum throughout the experiment.
The housing conditions and care of the animals were in accordance with the "Guide
for the Care and Use of Laboratory Animals" (Institute of Laboratory Animal
Resources on Life Sciences, National Research Council, 1996).
Guide cannula were stored in 95% ETOH prior to surgery, whereas surgical
tools underwent heat sterilization (2500C) immediately before each surgery. Rats
were anesthetized using a ketamine hydrochloride (100 mg/kg, IP) xylazine (12
mg/kg) mixture. After adequate anesthesia had been determined (using toe and tail
pinch procedures), rats were placed into a stereotaxic instrument. The skull region
was wiped with a 2% Betadine solution and a rostrocaudal incision was made to
expose the surface of the skull. Bilateral guide cannula (20 gauge; Plastics One) were
chronically implanted over the medial striatum (A/P: +0.5, MfL: +2.5, D/V: -2.0;
Paxinos & Watson, 1998) and secured using four skull screws and cranioplastic
cement. The cannula need to be implanted at an angle to obtain the minimum inter-
cannula distance needed for our probe leash used during microdialysis sampling.
Following surgery, body temperature was maintained using a heating pad and the rats
were monitored until fully conscious. Rats were then individually housed and
assessed daily by monitoring general activity, body weight, and feces. Rats were
monitored for signs of an infection and cefazolin (100 mg/kg; intramuscular) was
available as needed. Notation was made of any animal administered antibiotic.
Rats were given at least five days to recover prior to microdialysis sampling.
Approximately 18 hr prior to sampling, a microdialysis probe (24 gauge; 2-3 mm
exposed membrane; 13000 MWCO) encased in a spring leash and attached to a liquid
swivel connected to a balancing arm was inserted into the guide cannula of an awake
rat. The probe was secured in place by screwing a threaded portion of the probe leash
onto the guide cannula. The rat was then placed into a behavioral chamber
(Omnitech, Columbus OH) equipped with a fan and house light (10W), and food and
water was available ad libitum. On the day of the experiment, dialysis buffer
consisting of 5 mM glucose, 140 mM NaCl, 1.4 mM CaCl2, 1.2 mM MgCl2, and
0.15% phosphate buffer saline, pH 7.4, was perfused through the probe (2.0 μl/min) at
least two hr prior to sample collection. Twenty-min dialysis samples were then
collected for two hr to determine basal glycine levels. Rats were then injected
(intraperitoneal) with vehicle or one dose of the test compound, and 30-min samples
were collected for up to 10 hr. Samples were split for separate chromatographic
evaluation of glycine and dopamine, and frozen (-800C) until analyzed.
Rats, with the dialysis probes in place, were given an overdose of
pentobarbital, and the brains fixed by intracardiac infusion of PBS-formalin. Coronal
brain sections were 100 μM thick and stained with cresyl violet to verify probe
placements. Probes were left in place as the animal was perfused in order to check for
the presence of blood in the probe tract.
The concentrations of glycine and dopamine in dialysate samples was
determined using a Waters Alliance 2690 HPLC system with fluorometeric detection
or an ESA coulometric electrochemical detection HPLC system, respectively. Dialyis
samples were split between the two systems enabling both dopamine and glycine to
be measured in each sample. A Waters Spherisorb ODS2 column (5 μM, 4.6 x 250
mm) was used to separate the amino acids. Glycine was detected using a Waters 474
Fluorescence Detector with an excitation wavelength of 320 nm and an emission
wavelength of 400 nm. The mobile phase consisted of 80% H2O, 20% acetonitrile,
0.1 M Na2HPO4, and 0.1 mM ethylenediamine-tetraacetic acid (pH to 5.8 with
phosphoric acid; 0.2 μm filter) with a flow rate of 0.75 ml/min. Samples were placed
into the refrigerated autosampler (40C) and precolumn derivatization of the amino
acids with o-phthaldehyde was performed using the Waters Alliance System. A total
of 15 μl (5 μl sample plus 15 μl OPA) was injected onto the column. All samples
collected 2 hr before and after treatment were analyzed. For the samples collected
during post-treatment hrs 2-24, one 20-min sample/hr was analyzed; the other two
samples were retained for analysis of compound within the sample. Glycine peak
heights were compared to an external standard curve for quantification. A new
standard curve was generated each day.
For dopamine analysis, samples were placed in an ESA (Chelmsford, MA)
Model 540 autosampler connected to an HPLC system with electrochemical
detection. Separation was achieved by pumping the samples through a 15 cm C18
reversed phase column (ESA, Inc.) and then samples were reduced/oxidized using
coulometric detection. Three electrodes were used: a guard cell (+400 mV), a
reduction analytical electrode (-150 mV) and an oxidation analytical electrode (+250
mv). Peaks were recorded and the area under the curve measured by a computer
running ESA Chromatography Data System. These values were normalized by
comparison to an internal standard curve for isoproteronol and quantified by
comparison to an external standard curve.
Peak heights were compared to an external standard curve for quantification.
The data was normalized to percent change from baseline (mean of 3 30-min samples
prior to treatment). In addition, raw data was furnished and differences were reported
along with the normalized data. All data was evaluated using a one-way ANOVA
with repeated measures over time using the Statview program on a G4 Macintosh.
Experiment #1. Extracellular levels of dopamine and glycine in the striatum
of the rat (N=5) were assessed following administration of risperidone at three (3)
doses (ascending; 0.16, 0.63 and 2.5 mg/kg). Dialysis samples were obtained every
30 min, each dose being assessed for 2 hours (8 hours total). FIGURES IA- ID show
the effect of risperidone on glycine and dopamine. The data are shown both as
amount of analyte per sample, as well as normalized to the percent change from the
average of the baseline values (i.e., samples obtained before the first drug injection).
Risperdone produced the expected elevation in extracellular dopamine, with the
lowest dose eliciting a threshold elevation of approximately 50%, and the two higher
doses producing a 3-4 fold increase in dopamine. Surprisingly, a similar elevation in
extracellular glycine was observed following risperidone. Although the lowest dose
was without effect, the two higher doses of risperidone elicited a dose-dependent
elevation in glycine up to a maximum 2.5-fold increase.
Experiment #2. Extracellular levels of dopamine and glycine in the striatum
of the rat (N=5) were assessed following administration of COMPOUND NO. 1 at
three (3) doses (ascending; 0.63, 2.5 and 10 mg/kg). Dialysis samples were obtained
every 30 min, each dose being assessed for 2 hours (8 hours total). FIGS 2A-2D
show the results of this experiment. As expected, COMPOUND NO. 1 elicited a
dose-dependent elevation in extracellular glycine. A threshold effect was seen after
0.63 mg/kg, and 10 mg/kg produced a 2.5-fold elevation in glycine. The effect of
COMPOUND NO. 1 on extracellular dopamine was biphasic with respect to dose
(N=7). The lowest dose of COMPOUND NO. 1 produced a nearly 50% reduction in
extracellular dopamine. The levels of dopamine returned to normal following
injection an injection of 2.5 mg/kg and remained unaltered by the highest dose of
COMPOUND NO. 1.
Experiment #3. The data generated from Experiments #1 and #2 was assessed
to determine the best combination of doses of COMPOUND NO. 1 and risperidone in
order to determine synergism or antagonism between the two compounds. Two
dosing regimens were identified. In order to evaluate the effect of the low dose of
COMPOUND NO. 1 on dopamine (see FIGURES 2A-2D), a combination of 0.63
mg/kg COMPOUND NO. 1 and 1.0 mg/kg risperidone was administered in a single
bolus injection. In order to examine for a potential synergism between COMPOUND
NO. 1 and risperidone in elevating extracellular glycine (see FIGURES 1A-1D and
2A-2D), 10 mg/kg COMPOUND NO. 1 and 1.0 mg/kg risperidone was administered
in a single bolus injection.
Effects on glycine: FIGURES 3A-3D illustrates the effect of both drug
combinations on extracellular glycine in the striatum. The lower dose of
COMPOUND NO. 1 (0.63 mg/kg) and the dose of risperidone examined each
produce a modest rise in glycine when given alone (see FIGURES 1 A-ID and 2A-2D,
respectively). When the two doses of drug were co-administered (N=5) there was an
approximate doubling of extracellular glycine that was consistent with the effect of
risperidone alone. However, combining risperidone with the higher dose of
COMPOUND NO. 1 (10 mg/kg) caused a clear additive effect (N=6). Thus, while
each drug alone produced a 2-3 fold elevation in glycine, combined there was a 6-fold
increase in extracellular glycine.
Effects on dopamine: FIGURES 3A-3D illustrate the effect of both drug
combinations on extracellular dopamine in the striatum. .The upper panels illustrate
the effect of combining the lower dose of COMPOUND NO. 1 (0.63 mg/kg) with
risperidone (N=6). This dose of COMPOUND NO. 1 reduced extracellular
dopamine, and it can be seen that, although a significant increase was measured,
COMPOUND NO. 1 partly antagonized the increase in dopamine expected following
1.0 mg/kg risperidone. Thus, the expected 300% increase in dopamine following this
dose of risperidone (see FIGURES 1C- ID) was reduced to 150% when given in
combination with COMPOUND NO. 1 (0.63 mg/kg). In contrast, the higher dose of
COMPOUND NO. 1 (10 mg/kg) alone was without effect on dopamine (FIGURES
2C-2D), and when co-administered did not alter the capacity of risperidone to elevate
extracellular dopamine (N=6).
Discussion and Conclusions
These data reaffirm the capacity of the GIyTl antagonist COMPOUND NO. 1
to produce a dose-dependent elevation in extracellular glycine, and demonstrate that
at lower doses, this drug reduces extracellular dopamine. The present data also affirm
the findings of others that risperidone elevates extracellular dopamine, and makes the
surprising and important observation that risperidone produces a dose-dependent
elevation in extracellular glycine. Moreover, in combination, the two drugs appear
additive in their effects on glycine, indicating separate mechanisms of action.
COMPOUND NO. 1 and dopamine. It was surprising that COMPOUND NO.
1 reduced extracellular dopamine. The fact that this was observed only at lower doses
may indicate a separate mechanism of action than GIyTl blockade. Regardless of the
mechanism, this effect is synergistic with known therapeutic actions of antipsychotic
medications. Thus, reducing dopamine transmission in the striatum may be indicative
of a mechanism for reducing dopamine receptor tone that is distinct from the classic
D2 receptor blockade associated with most antipsychotic drugs. While this effect in
the striatum (especially ventral striatum) is thought to be an important therapeutic
action of antipsychotic drugs, reducing dopamine transmission in the prefrontal cortex
would be expected to exacerbate the cognitive impairment associated with
schizophrenia. However, there are known instances where pharmacological and
environmental challenges differentially affect prefrontal cortical and striatal dopamine
transmission, notably in relation to NMDA receptor blockade (Cabib and Puglisi-
Allegga, 1996; Moghaddam and Adams, 1998), and the effects of COMPOUND NO.
1 on extracellular dopamine in striatum may not predict effects in the prefrontal cortex. This would be especially true if the effects on dopamine were indirect since
the synaptic organization of the prefrontal cortex differs markedly from the striatum.
The slight antagonism by low dose COMPOUND NO. 1 (0.63 mg/kg) of the
effect of risperidone to elevate dopamine in the striatum is potentially important,
especially if the effect of COMPOUND NO. 1 is distinct in the cortex and striatum.
Thus, antagonism of risperidone in the striatum, but not in the cortex could have
therapeutically beneficial impact, given that risperidone actions in the striatum are
thought to mediate untoward motor side-effects.
The elevation in glycine by risperidone was unexpected. The mechanism
remains unclear. Given that elimination of glycine from the extracellular space is
primarily by glycine uptake, antagonism of the transporter is one option. While it is
unlikely that risperidone binds directly to GIyT (Goff and Coyle, 2001), it is possible
that blockade of dopamine (or serotonin) receptors may regulate GIyT.
Regardless of the mechanism by which risperidone elevates glycine, there is a
clear additive effect between COMPOUND NO. 1 and risperidone with regard to
elevating extracellular glycine. Inasmuch as schizophrenia may in part result from
reduced NMDA conductance, the additive effect on extracellular glycine may provide
therapeutic benefit by indirectly potentiating NMDA conductances. Thus if in fact
elevating glycine is of therapeutic benefit, combining COMPOUND NO. 1 with
risperidone may permit the use of lower doses of risperidone.
This study identified two novel actions of COMPOUND NO. 1 and
risperidone. Low doses of COMPOUND NO. 1 reduced dopamine levels and
risperidone produced a dose-dependent elevation in glycine. While the cellular
mechanisms mediating these actions remain unclear, they result in potentially
important interactions between the two drags. Thus, COMPOUND NO. 1 (0.63
mg/kg) slightly antagonized the capacity of risperidone to elevate dopamine, while the
capacity of both drugs to elevate glycine was additive. Inasmuch as dopamine and
glutamate are involved in the etiology or symptomatology of schizophrenia the
interactions of COMPOUND NO. 1 with the known antipsychotic risperidone is
therapeutically relevant.
Although only particular embodiments of the invention are specifically
described above, it will be appreciated that modifications and variations of the
invention are possible without departing from the spirit and intended scope of the
invention.
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