U.S. patent application number 11/080551 was filed with the patent office on 2005-07-21 for d-serine transport antagonist for treating psychosis.
This patent application is currently assigned to DR. DANIEL JAVITT. Invention is credited to Javitt, Daniel.
Application Number | 20050159488 11/080551 |
Document ID | / |
Family ID | 23440810 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050159488 |
Kind Code |
A1 |
Javitt, Daniel |
July 21, 2005 |
D-serine transport antagonist for treating psychosis
Abstract
Method and composition for augmenting NMDA receptor mediated
neurotransmission involving use of a D-serine transport
inhibitor.
Inventors: |
Javitt, Daniel; (Riverdale,
NY) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
DR. DANIEL JAVITT
|
Family ID: |
23440810 |
Appl. No.: |
11/080551 |
Filed: |
March 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11080551 |
Mar 16, 2005 |
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10066657 |
Feb 6, 2002 |
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10066657 |
Feb 6, 2002 |
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09365889 |
Aug 3, 1999 |
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6361957 |
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Current U.S.
Class: |
514/563 ;
514/475 |
Current CPC
Class: |
A61K 31/16 20130101;
A61K 45/06 20130101; G01N 33/5082 20130101; A61K 31/198 20130101;
A61P 25/18 20180101; A61P 25/22 20180101; A61P 25/28 20180101; A61P
25/24 20180101; A61K 31/00 20130101 |
Class at
Publication: |
514/563 ;
514/475 |
International
Class: |
A61K 031/198; A61K
031/336 |
Claims
1-26. (canceled)
27. A D-serine transport inhibitor compound comprising a serine or
alanine compound having a hydrophobic group linked to either the
C-or N-terminus, wherein the hydrophobic group is selected from the
group consisting of a C1-13 alkyl group optionally substituted with
a halogen atom, a phenyl group optionally substituted with a C1-13
alkyl group, a cyano group and a halogen atom.
28. The D-serine transport inhibitor compound according to claim
27, wherein the serine or alanine compound is selected from
D-serine, L-serine, D-alanine and L-alanine.
29. The D-serine transport inhibitor compound according to claim
27, wherein the compound is a selective D-serine transport
inhibitor.
30. The D-serine transport inhibitor compound according to claim
27, which is selective for glycine uptake inhibition.
31. The D-serine transport inhibitor compound according to claim
30, wherein the inhibitor compound is D-serine dodecylamide.
32. The D-serine transport inhibitor compound according to claim
29, which is selective for D-serine uptake inhibition.
33. The D-serine transport inhibitor compound according to claim
32, wherein the inhibitor compound is D-alanine dodecylamide.
34. A process for augmentation of N-methyl-D-aspartate
receptor-mediated neurotransmission in vivo which comprises
administration of an effective amount of a selective D-serine
transport inhibitor.
35. The process of claim 34, wherein a psychotic disorder
associated with decreased N-methyl-D-aspartate receptor-mediated
neurotransmission is treated.
36. The process of claim 34, wherein schizophrenia is treated.
37. The process of claim 34, wherein a neuropsychiatric disorder
selected from the group consisting of Alzheimer's disease, bipolar
illness, depression and an anxiety disorder is treated.
38. The process of claim 34, wherein the inhibitor compound is a
serine or alanine compound having a hydrophobic group linked to
either the C-or N-terminus, wherein the hydrophobic group is
selected from the group consisting of a C1-13 alkyl group
optionally substituted with a halogen atom, a phenyl group
optionally substituted with a C1-13 alkyl group, a cyano group and
a halogen atom.
39. The process according to claim 38, wherein the serine or
alanine compound is selected from D-serine, L-serine, D-alanine and
L-alanine.
40. The process according to claim 38, wherein the inhibitor is
selective for D-serine uptake inhibition.
41. The process according to claim 40, wherein the inhibitor is
D-alanine dodecylamide.
42. The process according to claim 38, wherein the inhibitor is
selective for glycine uptake inhibition.
43. The process according to claim 42, wherein the inhibitor is
D-serine dodecylamide.
44. A composition for treating schizophrenia comprising an
effective amount of a D-serine transport inhibitor according to
claim 27 and a pharmaceutically acceptable carrier.
Description
RELATED APPLICATION
[0001] The present application is a continuation-in-part of U.S.
application Ser. No. 09/365,889 filed Aug.3, 1999.
BACKGROUND
[0002] Schizophrenia is a psychotic disorder associated with
positive, negative and cognitive symptoms, neuropsychological
deficits and poor social functioning. Traditional theories of
schizophrenia have focused on abnormal dopaminergic
neurotransmission. Mainstay treatments for schizophrenia including
use of typical (e.g., thorazine, haloperidol) or atypical (e.g.
clozapine, risperidone, olanzapine, quetiapine, ziprasidone,
aripiprazol, sertindole) antipsychotics (also called neuroleptics
or major tranquilizers). Antipsychotics may be administered orally,
parenterally and in depot formulations. In addition, patients may
be treated with mood stabilizers (e.g., lithium, valproic acid),
antidepressants (e.g., tricyclics, SSRIs), or antianxiety agents
(e.g., benzodiazepines, barbiturates). Recent theories postulate
that schizophrenia is associated with dysfunction or dysregulation
of neurotransmission mediated at brain N-methyl-D-aspartate
(NMDA)-type glutamate receptors (Javitt, 1987; Javitt and Zukin,
1991). PCP and other psychotomimetic drugs mediate their effects by
blocking NMDA receptor-mediated neurotransmission. NMDA agonists,
including glycine and D-serine, reverse the behavioral effects of
PCP in rodents (Toth et al., 1986; Javitt and Frusciante, 1997;
Javitt et al., 1997; Tanii et al., 1994, 1991; Nilsson et al.,
1997), and induce significant improvement in negative and cognitive
symptoms in remitted schizophrenics (Javitt et al., 1994;
Heresco-Levy et al, 1999; Tsai et al., 1998), supporting the
PCP/NMDA model of schizophrenia.
[0003] Both glycine and D-serine serve as endogenous modulators of
NMDA receptors (Hashimoto et al., 1995; Hashomoto and Oka, 1997).
In general, amino acid levels in brain are regulated by endogenous
transporters that limit CNS levels. A potential alternate approach
to increasing glycine or D-serine levels in brain, therefore, would
be the use of agents that inhibit either glycine or D-serine
transport in brain. This approach has been well described in the
case of glycine. Glycine (GlyTl) transporters in brain are well
described and have been shown to be colocalized with NMDA receptors
(Smith et al., 1992; 1992; Liu et al., 1993). Further, modulation
of glycine transporters has been shown to modulate NMDA
receptor-mediated neurotransmission both in vitro and in vivo
(Javitt and Frusciante, 1997; Javitt et al., 1997; Supplison and
Bergman, 1998; Bergeron et al, 1998; Berger et al, 1998; Danysz and
Parsons, 1998), supporting the physiological relevance of amino
acid transport processes.
[0004] The present invention relates to the use of D-serine
transport inhibitors (also referred to as uptake antagonists) in
the treatment of schizophrenia. D-Serine, like glycine, has been
shown to be effective in treatment of persistent negative symptoms
of schizophrenia (Tsai et al., 1998). However, as with glycine,
sufficient concentrations of D-serine are already present in brain
that NMDA/glycine sites (the molecular target of D-serine) would be
saturated under normal circumstances (Hashimoto et al., 1995,
Hashimoto and Oka, 1997; Matsui et al., 1995). If the NMDA/glycine
site were saturated by endogenous D-serine, then neither exogenous
glycine or exogenous D-serine would have significant neurochemical
or behavioral effects since both these agents share a common target
(i.e., the NMDA/glycine site). The fact that glycine and serine do
potentiate NMDA receptor-mediated neurotransmission suggests that
for D-serine, as with glycine, there must be an endogenous process
that "protects" NMDA receptors from extracellular D-serine. At
present, D-serine transport processes in brain are poorly
understood. The present application describes a novel brain
D-serine transport system that is relatively selective for D-serine
over other amino acids, and provides the first demonstration that
agents which block D-serine transport in vitro also stimulate NMDA
receptor in vivo and are effective in animal models of
schizophrenia.
[0005] The next section describes state-of-the-art regarding
existence of D-serine transport systems in brain. Example 1
describes the existence of multiple high affinity D-serine
transport systems in synaptosomal membranes that can be
differentiated based upon sensitivity to inhibition by alanine.
Example 2 describes the relative ability of three amino acid
derivates, glycine dodecylamide (GDA), D-serine dodecylamide (D-Ser
DA) and D-alanine dodecylamide (D-Ala DA) on amphetamine- and
PCP-induced locomotor activity in rodents. These findings
demonstrate likely effectiveness of D-serine transport inhibitors
in treatment of persistent negative and cognitive symptoms in
schizophrenia and treatment of related symptoms in other
neurological and psychiatric disorders associated with reduced NMDA
function including but not limited to Alzheimers disease, age
associated memory impairment, autism, attention-deficit deficit
disorder, bipolar disorder, depression, Parkinsons disease,
Huntingtons disease, and recovery from stroke, neurological insult
or closed head injury. Example 3 demonstrates effects of the amino
acid glycine and the amino acid derivative D-Ala DA on in vivo
[3H]MK-801 binding, an in vivo model of NMDA receptor
activation.
DETAILED DESCRIPTION OF THE INVENTION
[0006] Brain is known to contain multiple amino acid transport
systems, including system "Gly", which is specialized for uptake of
glycine, system "A" which is specialized for uptake of Alanine,
system "L" which is specialized for uptake of Leucine, and system
"ASC" which is specialized for uptake of Alanine, Serine and
Cysteine (Sershen and Lajtha, 1979; Hashimoto and Oka, 1997).
Serine transport, including transport of both L- and D-isomers of
serine, is generally considered to occur via system ASC (Hashimoto
and Oka, 1997), although transport may also occur though system L
(Sershen and Lajtha, 1979). The hallmark of this system is high
affinity for alanine. Two ASC-like transporters have recently been
cloned and have been termed ASCT1 (Arriza et al., 1993) and ASCT2
(Utsunomiya-Tate et al., 1996). Studies with cloned transporters
have confirmed that ASC-family transporters show highest affinity
for L-alanine, along with high affinity for L-cysteine and
L-serine, and stereoselectivity for L- vs. D-amino acids. A related
transporter, termed SATT was found to have differential affinity
for serine and cysteine. However, this transporter was found not to
be sensitive to D-serine (Shafqat et al., 1993). Based on the
relatively low affinity of these transporters for D-amino acids,
Hashimoto et al. (1997) concluded that "further study is needed to
clarify a specific transport system for D-serine in
mammals."D-Serine transport has also been studied in glioma cells
(Hayashi et al., 1997) and astocyte cultures (Schell et al., 1995).
Glia have also been shown to accumulate exogenously administered
D-serine in vivo (Wako et al., 1995; Schell et al., 1995).
Transport in these cells, like transport through cloned receptors,
was found to be inhibited most strongly by L-cysteine, L-alanine,
and L-serine. D-Serine was transported, but affinity for D-serine
was approximately 20-fold lower than affinity for L-serine. This
finding is consistent with glial D-serine uptake being mediated by
system ASC transporters. The relative insensitivity of these
transporters to D-serine makes it unlikely that they regulate
synaptic D-serine levels in vivo.
[0007] Further suggestion that additional D-serine transporters are
present in brain comes from a study by Tanii et al. (1994). In that
study, they observed that intracerebroventricularly administered
D-alanine was significantly more potent in reversing PCP-induced
hyperactivity than was intracerebroventricularly administered
D-serine, even though D-serine is more potent in binding to the
NMDA/glycine site. This finding suggests the existence of a brain
transporter with higher affinity for serine and alanine. Such a
pattern would be opposite to the known selectivity pattern of
system ASC. In discussing relative potency of D-serine to other
amino acids, Tanii et al. (1994) postulated the existence of
"specific metabolizing systems" for D-serine, but did not
specifically postulate the existence of a selective transporter.
Moreover, despite the recognition that D-serine serves as an
endogenous agonist of NMDA receptors, use of selective D-serine
transport antagonists in the treatment of schizophrenia has not
been previously suggested.
[0008] For this study, two novel amino acid derivatives were
synthesized--D-serine dodecylamide (D-Ser-DA) and D-alanine
dodecylamide (D-Alanine-DA). Synthesis was performed by addition of
an alkyl group to the C-terminus (COOH or equivalent) of the amino
acid by amide linkage. Variations of this approach, wherein other
hydrophobic groups are linked to either the C- or N-terminus
(NH.sub.2 or equivalent) of glycine, D-serine, D-alanine, L-serine
or L-alanine, are obvious extensions of this invention. Such groups
would include but not be limited to alkyls (C1-C13) such as methyl,
phenyls or phenylalkyls (C1-C 13), cyano, halogen such as fluoro or
halalkyl (C1-C13) such as fluoromethyl groups.
EXAMPLE 1
[0009] Based upon the observation that glycine is effective in the
treatment of schizophrenia (Javitt and Zukin, U.S. Pat. No.
5,854,286), it can be concluded that glycine sites are not
saturated under normal physiological conditions in schizophrenia.
Extracellular concentrations of D-serine in brain are known to be
above those necessary to saturate NMDA/glycine sites. These
findings raise the possibility that brain may contain a D-serine
transporter that protects NMDA receptors from extracellular
D-serine concentrations. Actions of such a transporter would be
analogous to the role played by glycine transporters in protecting
NMDA receptors from extracellular glycine levels. Use of glycine
transport inhibitors in treatment of schizophrenia were described
in a separate application (Javitt, U.S. Pat. No. 5,837,730). The
present application demonstrates the existence of a novel D-serine
transporter, supporting the feasibility of use of D-serine
transport inhibitors in treatment of schizophrenia.
[0010] In order to investigate the existence of a synaptosomal
D-serine transporter, synaptosomal (P2) preparations were prepared
from rodent brain. This preparation permits identification of
transport mechanisms on pre- and post-synaptic terminals and so is
crucial for identifying systems that may be co-localized with NMDA
receptors which are located on synaptic terminals. In contrast, the
majority of transport studies are performed using either cloned
transporters or brain slices, which provide less specificity for
identifying perisynaptic transport mechanisms. An obvious extension
of the present invention, however, is use of an assay system in
which the D-serine transporter is cloned and expressed in a
suitable expression system. Methods for cloning would include but
not be limited to derivation of mRNA from rodent or human brain,
including postmortem human brain tissue, or from other suitable
brain tissue or from other tissues expressing D-serine
transporters, and progressive fractionation of such mRNA until
relevant sequences are obtained, or from expression libraries
derived from rodent, primate or other appropriate sources, or from
cell lines expressing D-serine transporters. Cloned transporters
would be expressed in a suitable expression system including, but
not limited to, Xenopus oocytes, or immortalized cells derived from
mammalian, reptilian or avian sources. Obvious extensions would
also be use of brain slices, use of cell culture lines which
express D-serine transporters or primary culture of neurons.
[0011] Synaptosomal (P2) preparations were prepared from brains of
Sprague-Dawley rats (200-250 g) using the method of Debler and
Lajtha (1987). Rodents were decapitated and brains
(cortex+hippocampus) were homogenized in 0.32 M sucrose buffered to
pH 7.4 with Tris-HCl. Homogenate was centrifuged at 1000 g for 12
min at 4.degree. C. The supernatant was centrifuged at 14,000 g 12
min and the P2 pellet was resuspended in Krebs solution (pH 7.4)
containing 124 mM NaCl, 26 mM NaHCO.sub.3, 10.5 mM glucose, 5 mM
KCl, 1.3 mM MgSO.sub.4, 1.2 mM KH.sub.2PO.sub.4, and 2.4 mM
CaCl.sub.2. For uptake studies, membranes were incubated at
37.degree. C. in the presence of L- or D-[.sup.3H]serine, as
appropriate. L- and D-[.sup.3H]Serine were obtained from DuPont-NEN
(Natick, Mass.) and had specific activities of 25.4 and 19.7 Ci/mM,
respectively. Incubation was terminated by filtration under reduced
pressure through Whatman GF/B filters, rinsing twice with 5 ml
ice-cold buffer.
[0012] For initial kinetic studies, incubations were conducted at 6
time points between 1 and 30 min. Non-specific binding was
determined in the presence of 30 mM L- or D-serine. For inhibition
studies, L-alanine, L-cysteine, L-serine , and D-serine were tested
at 6 concentrations between 0.03 and 10 mM. Control uptake levels
were defined as uptake levels in the absence of added L-alanine,
L-cysteine, L-serine , or D-serine. % control binding was defined
as level of uptake in the presence of specified concentrations of
antagonist divided by uptake under control conditions, expressed as
a percent. 1 mM concentrations of HA-966,
L-trans-pyrollidine-2,4-dicarboxyllic acid (L-PDC), nipecotic acid,
2-aminobicyclo (2,2,1)heptane-2 carboxylic acid (BCH) and
methylaminoisobutyric acid (MeAIB) and 10 mM sarcosine were added
to the homogenate to prevent binding to NMDA-associated glycine
binding sites, and potential uptake via glutamate, GABA, system L,
system A and system GLY transporters, respectively. Incubations
were terminated following 30 min. For both kinetic and inhibition
studies, non-specific binding was determined in the presence of 30
mM D- or L-serine, as appropriate.
[0013] For saturation studies, L- and D-serine were added at 12
concentrations between 0.01 and 5 mM. Assays were conducted at
37.degree. C. Non-specific binding was determined at 0.degree. C.
Incubations were terminated following 5 min. Assays were conducted
in the presence of 30 mM L-alanine to prevent uptake through system
ASC, along with 1 mM concentrations of HA-966, L-PDC, nipecotic
acid, BCH and MeAIB. Km values were determined by non-linear
regression to a single rectangular, 3 parameter hyperbolic function
using Sigmaplot 2000 (SPSS Inc., Chicago, Ill.). Data in text
represent mean .+-.sem. Statistical comparisons were performed
using two-tailed Student's t statistic.
[0014] For initial studies, uptake was measured over a 30 min.
period (FIG. 1). Uptake of L- and D-[.sup.3H]serine was linear over
the first 10 min. with a tendency for plateau by 30 min. Uptake was
unaffected by incubation with the selective system L antagonist BCH
(10 mM). Effects of the system ASC substrates alanine, cysteine and
serine were evaluated at concentrations between 0.03 and 30 mM
(FIG. 2). Complete inhibition of serine uptake was obtained with
either L- or D-serine. In both cases, L-serine showed greater
potency than D-serine in inducing inhibition. Inhibition was also
obtained with cysteine, although potency of cysteine was
significantly less than that of either L- or D-serine. In contrast,
only partial inhibition was observed with alanine, even at doses as
high as 30 mM. This pattern of inhibition is opposite to that of
system ASC, indicating that the observed L- and D-serine uptake is
mediated primarily by a system other than system ASC. This system
has not been previously described.
[0015] Finally, in order to characterize kinetics of uptake,
saturation studies were conducted following 5 min. incubation with
concentrations of L- and D-serine between 0.01 and 5 mM (FIG. 3).
Studies were conducted in the presence of 30 mM L-alanine to
prevent uptake through system ASC. Even in the presence of alanine,
significant uptake of L- and D-serine was observed. Saturation of
D-serine binding was observed between 3 and 5 mM, with half-maximal
binding occurring between 1-2 mM. A Michaelis-Menton constant (Km)
of 3.33 mM was obtained by non-linear regression. An Eadie-Hofstee
plot demonstrated linear uptake, supporting the concept that this
uptake occurs via a discrete, alanine-insenstive D-serine transport
system with approximately equal affinity for D- and L-serine. The
presence of such a system in synaptosomal tissue from rodent
forebrain indicates that it may play a crucial role in regulation
of D-serine concentrations in the vicinity of NMDA receptors.
Inhibition of this system would be expected to increase local
D-serine concentrations in brain, leading to augmentation of NMDA
receptor-mediated neurotransmission. Inhibition of selective serine
uptake would thus constitute a novel mechanism for stimulation of
NMDA receptor-mediated neurotransmission in vivo.
[0016] In summary, the present example demonstrates the existence
of multiple transport systems for D-serine in P2 synaptosomal
membranes which can be distinguished based upon sensitivity to
inhibition by alanine.
EXAMPLE 2
[0017] We have previously reported ability of the glycine
derivative glycyldodecylamide (GDA) to inhibit synaptosomal glycine
transport. This study evaluated the ability of two novel amino acid
derivatives D-serine dodecylamide (D-Ser-DA) and D-alanine
dodecylamide (D-Ala-DA) to inhibit glycine and D-serine uptake in
vitro and to affect rodent activity in vivo. Effects of these
agents on synaptosomal glycine and D-serine transport were
determined using assay methods described previously in Javitt and
Frusciante, 1997 and in Example 1, above. The amino acid
derivatives were then evaluated in a rodent behavioral model that
assesses locomotor hyperactivity following administration of either
the dopamine releasing agent amphetamine or the NMDA antagonist
PCP. Atypical antipsychotics and agents that potentiate NMDA
receptor mediated neurotransmission are more effective in
modulating effects of PCP than of amphetamine. The relationship
between ability to antagonize D-serine transport in vitro and to
modulate PCP-induced hyperactivity in vivo was then assessed.
[0018] Effects of amino acid derivatives on glycine and D-serine
uptake are shown in Table 1. As shown previously (Javitt and
Frusciante, 1997), GDA significantly inhibited glycine uptake into
synaptosomes. Similar effects were shared by dSDA whereas dADA was
ineffective. GDA also significantly inhibited D-serine uptake into
synaptosomes at concentrations similar to those used to inhibit
glycine transport. Similar effects were shared by dADA but not
dSDA.
[0019] To assess effectiveness of these agents in modulating NMDA
receptor-mediated neurotransmission, amphetamine and PCP-induced
was evaluated in absence and presence of these agents. Assays were
conducted using methods of Javitt et al. (1997). Mice (C57) were
acclimated to automatic test chambers. As opposed to BALB/c mice
which were used in prior studies, C57 mice were used for the
present study because they are known to have relatively little
spontaneous hyperactivity in response to PCP. Such mice are thus
more similar to primates that have a predominant reduction in
activity in response to PCP. Therefore, in C57 mice, as in
primates, it is predicted that the prominent behavioral response to
NMDA agonists would be a decrease in activity following amphetamine
administration and an increase in activity following PCP
administration.
[0020] Baseline activity was monitored for 30 min. Animals were
then pretreated with saline, GDA, D-Ser-DA or D-Ala-DA and activity
was monitored for an additional 15 min. All drugs were administered
at a dose of 1.6 mg/kg. Finally, animals were challenged with
amphetamine (1 mg/kg) or PCP (3 mg/kg). Distance traveled (cm) per
min (DT) was used as the primary dependent measure.
[0021] All agents produced a numerical reduction in
amphetamine-induced activity and increase in PCP-induced activity,
suggesting similar effects by both glycine and D-serine transport
inhibitors. For statistical analysis, data were collapsed across
treatments associated with D-serine transport inhibition (GDA,
dADA) vs. those not associated with inhibition (saline, D-Ser-DA).
A 2.times.2 ANOVA evaluated alteration in amphetamine vs. PCP
induced hyperactivity by agents associated with D-serine transport
inhibition vs. those not associated with serine transport
inhibition. A significant amphetamine/PCP X serine transport
inhibitor interaction effects was observed (F=4.82, df=1/60,
p=0.03), reflecting ability of D-serine transport inhibitors to
both decrease amphetamine-induced hyperactivity and reverse
PCP-induced hypoactivity. The ability of D-serine transport
inhibitors to modulate amphetamine and PCP induced effects suggests
effectiveness of these agents in treatment of neuropsychiatric
illness.
EXAMPLE 3
[0022] An additional assay system that has been shown to be
sensitive to effects of NMDA agonists is in vivo [3H]MK-801 binding
(Murray et al., 2000). Consequently, the in vivo [3H]MK-801 binding
system, therefore, was used to evaluate effects of glycine site
agonists and antagonists on NMDA activation in vivo. To implement
this protocol, mice (C57) were pretreated with either saline,
glycine (1.6 g/kg) or D-Ala-DA (16 mg/kg) administered i.p. Animals
used for "nonspecific" in vivo binding were then administered
unlabelled MK-801 (3 mg/kg) by retroorbital injection whereas
animals used for "total binding" were administered saline. Both
"nonspecific" and "total binding" animals then received [3H]MK-801
(200 .mu.Ci/kg, 28.9 Ci/mmol) by retroorbital injection. "Specific"
binding to NMDA receptors was defined as the difference between
"total binding" and "nonspecific." Following 10 min, animals were
sacrificed. Brains were removed and homogenized in 40 vol/w buffer.
500 .mu.l aliquots were then filtered in triplicate under reduced
pressure through Whatman GF/B filters. [3H]MK-801 levels were
determined by liquid scintillation. It has previously been
demonstrated by others that treatment with NMDA agonists increases
including D-serine increases specific binding to NMDA receptors in
vivo whereas treatment with glycine site antagonists decreases
specific binding (Murray et al., 2000).
[0023] In this experiment, specific binding under control
conditions was 990.+-.2766 DPM/500 .mu.l homogenate (n=3). Under
control conditions, difference between total and nonspecific was
not significant given the small n, indicating low levels of NMDA
activation under control conditions. Pretreatment with glycine
increased specific binding by 15% to 1133.+-.1455 DPM/500 .mu.l
homogenate. Under these circumstances, the difference between
nonspecific and total binding was specific at trend level (p=0.1,
one tail). However, the level of specific binding following glycine
treatment was not significantly greater than control levels.
Treatment with D-Ala-DA led to a 250% increase in specific binding
to 3888.+-.1734 DPM/500 .mu.l homogenate. In the presence of
D-Ala-DA, total binding was significantly higher than non-specific
indicating substantial NMDA activation (p=0.015, one tail).
Further, there was a significant trend toward increased specific
binding following D-Ala-DA than under control conditions (p=0.1,
one tail). These findings support the concept that agents that
activate the glycine site either directly (e.g., glycine, D-serine
) or by inhibition of glycine or D-serine transport (e.g.,
D-Ala-DA) potentiate NMDA receptor-mediated neurotransmission in
vivo.
1TABLE 1 Inhibition of glycine and D-serine uptake by
glycyldodecylamide (GDA), D-serine dodecylamide (D-Ser-DA) and
D-alanine dodecylamide (D-Ala-DA). Values represent percent control
uptake in the presence of specified concentration of antagonist.
Data are mean of 3 separate experiments each performed in
triplicate Concentration Glycine uptake D-serine uptake T Value
Compound (.mu.M) Average: Std. Dev. T Value P value Average: Std.
Dev. (df = 2) P value GDA 1 99.2 6.1 0.24 0.8 95.1 9.46 0.89 0.5 10
99.7 6.3 0.07 0.9 102.4 5.33 -0.78 0.5 25 88.1 3.2 6.41 0.02 104.0
1.44 -4.83 0.04 50 70.7 9.2 5.52 0.03 97.4 6.62 0.69 0.6 100 41.5
2.6 38.36 0.0007 77.6 3.08 12.6 0.006 200 31.6 1.9 61.95 0.0003
60.9 6.82 9.91 0.01 500 24.4 4.4 29.90 0.001 47.6 1.98 45.9 0.0005
D-SER-DA 1 90.9 4.7 3.33 0.08 95.2 8.0 4.09 0.4 10 83.5 4.8 5.94
0.03 100.6 7.5 0.07 0.9 25 60.2 12.1 5.70 0.03 97.0 8.5 3.64 0.6 50
42.0 17.5 5.73 0.03 96.7 9.5 13.3 0.6 100 24.1 15.4 8.57 0.02 96.4
19.2 7.65 0.8 200 13.7 16.4 9.12 0.02 105.9 44.7 5.30 0.8 500 10.2
12.9 12.09 0.007 83.5 21.7 3.85 0.3 D-ALA-DA 1 101.9 11.0 -0.30 0.8
94.9 4.0 0.41 0.2 10 98.9 11.7 0.16 0.9 101.4 5.9 -0.89 0.7 25 92.5
16.4 0.79 0.5 93.3 8.1 -0.85 0.3 50 83.4 32.3 0.89 0.5 86.2 4.0
0.47 0.03 100 79.3 51.1 0.70 0.6 67.1 5.5 0.43 0.009 200 70.7 51.5
0.99 0.4 51.3 17.2 -0.26 0.04 500 67.8 53.4 1.04 0.4 44.1 2.8 1.51
0.0009
[0024]
2TABLE 2 Effect of amino acid derivatives on rodent activity levels
following amphetamine (1 mg/kg) or PCP (3 mg/kg) challenge. DT =
distance traveled. n = 8 per group Baseline Amph/ DT Pretreatment
PCP challenge Condition (cm) Std dev DT (cm) Std dev DT (cm) Std
dev Amphetamine treatment Saline 69.0 46.4 69.0 49.7 211.3 141.4
GDA 52.5 31.1 53.4 52.5 207.9 97.8 D-Ser-DA 60.6 44.6 103.2 91.8
196.2 100.1 D-Ala-DA 48.3 36.6 48.0 35.3 100.3 77.9 PCP treatment
Saline 41.4 27.6 85.6 58.9 91.5 51.0 GDA 51.3 64.2 77.2 72.5 132.6
69.0 D-Ser-DA 59.2 63.0 59.2 27.6 108.2 48.3 D-Ala-DA 64.5 41.1
83.2 47.3 149.2 66.2 Variations of the invention will be apparent
to the skilled artisan.
* * * * *