Patent Application: US-32044699-A

Abstract:
process for treating psychosis such as schizophrenia using a glycine substitute or a precursor thereof to potentiate nmda receptormediated neurotransmission .

Description:
study 1 — relative effectiveness of a glycine binding site full agonist ( glycine ) and partial agonist ( d - cycloserine ) in the treatment of schizophrenia . endogenous dysfunction or dysregulation of neurotransmission mediated at nmda - type glutamate receptors may contribute significantly to the pathophysiology of negative and cognitive symptoms of schizophrenia . nmda receptor activation is regulated not only by glutamate but also by glycine , which mediates its action at a strychnine - insensitive binding site associated with the nmda receptor complex . glycinergic agents , therefore , may be therapeutically beneficial in schizophrenia . two potential agents have been suggested as potential agents for stimulation of nmda receptor - mediated neurotransmission in schizophrenia : glycine and d - cycloserine ( reviewed in d &# 39 ; souza et al ., 1995 ). glycine is a naturally occurring amino acid and a normal dietary constituent . glycine acts as a full agonist at the nmda - associated glycine binding site , but must be given at large (& gt ; 30 g / day ) doses because of its poor permeability across the blood brain barrier . d - cycloserine is an anti - tubercular drug that also functions as a partial agonist at the nmda - associated glycine site . d - cycloserine readily crosses the blood - brain barrier . however , d - cycloserine is only 40 % as effective as glycine in potentiating nmda receptor - mediated neurotransmission . thus , at high doses , d - cycloserine may act as a functional glycine antagonist . both glycine ( the present inventor ) and d - cycloserine ( d &# 39 ; souza et al ., 1995 ) are reported to significantly ameliorate neuroleptic - resistant negative symptoms in schizophrenia . the relative effectiveness of these two agents , however , has not been compared . seven subjects were identified who participated in each of two separate studies investigating effects of nmda augmenting agents in schizophrenia . the first investigated effects of 0 . 8 g / kg / day ( approx . 60 g / day ) glycine . the second study investigated effects of 50 mg / day d - cycloserine . separate informed consent was obtained for each study . subjects met dsm - iv criteria for schizophrenia and were free of other axis i diagnoses ( including substance abuse ) or concurrent medical illness . all met criteria for neuroleptic - resistance , and manifested moderate - to - severe symptoms despite continuous neuroleptic treatment for at least 3 months . mean age of the patients upon study entry was 39 . 9 ± 15 . 7 yrs ( table 1 ). patients had been ill , on average , for 20 . 1 ± 13 . 4 yrs . at the time of study entry . their duration of most recent hospitalization was 8 . 5 ± 7 . 4 yrs . both studies were conducted using a double blind , placebo - controlled crossover design . total study length was 16 weeks . patients underwent an initial 2 week stabilization period , following by 6 weeks of treatment with either active medication or placebo . they then underwent a 2 - week washout followed by 6 weeks of crossover treatment . antipsychotic dose was held constant throughout each trial . symptom ratings were performed using the positive and negative syndrome scale ( panss ). one patient discontinued during week 4 of d - cycloserine treatment due to symptom exacerbation , and was not available for the placebo treatment arm . statistical analyses ( two - tailed ) were accomplished using the spss computer program . pre - vs . post - treatment comparisons were performed using paired t - tests . treatment vs . placebo effects were evaluated using repeated measures anova . values in text represent mean ± standard deviation . at entry into the glycine treatment study , the baseline negative symptom score for the 7 subjects was 39 . 0 ± 6 . 6 . during glycine treatment , subjects experienced an 11 . 3 ± 3 . 6 point reduction in negative symptoms ( t = 8 . 21 , df = 6 , p & lt ; 0 . 0001 ), corresponding to a mean 28 . 5 % reduction in symptoms ( fig1 ). in contrast , negative symptoms increased by 0 . 1 ± 1 . 7 points during treatment with placebo . the treatment by time interaction was highly significant ( f [ 1 , 6 ]= 83 . 5 , p & lt ; 0 . 001 ). positive symptoms did not change significantly from beginning ( 25 . 7 ± 7 . 0 points ) to end ( 22 . 0 ± 4 . 0 points ) of glycine treatment . however , a significant , 13 . 0 ± 7 . 7 point change in general psychopathology from 56 . 0 ± 19 . 4 to 43 . 0 ± 13 . 8 points was observed ( t = 4 . 44 , df = 6 , p & lt ; 0 . 005 ). this reduction corresponded to a 23 . 2 % decrease in symptoms . following completion of the double blind glycine treatment study , subjects included in this report were treated with antipsychotics alone for a mean duration of 15 . 6 ± 5 . 9 mo . ( range : 6 - 23 mo .) before being entered into the d - cycloserine study . the mean negative symptom score for these subjects upon entry into the d - cycloserine study , 39 . 0 ± 7 . 8 , was highly similar to the score that had been observed prior to glycine treatment . medications and doses used during the d - cycloserine trial were similar to those used during the prior glycine trial ( table 1 ). during d - cyloserine treatment , subjects experienced a significant , 3 . 6 ± 3 . 7 point reduction in negative symptoms ( t = 2 . 56 , p & lt ; 0 . 05 ), corresponding to a mean 9 . 2 % decrease in symptoms . in contrast , the degree of negative symptom reduction during placebo treatment for these subjects , 1 . 0 ± 3 . 1 points , was not significant . however , the treatment by time interaction was not significant ( f [ 1 , 5 ]= 1 . 87 , p = 0 . 23 ). positive symptoms and general psychopathology scores did not change significantly during treatment with either d - cycloserine or placebo . in order to assess relative effectiveness of glycine and d - cycloserine for treatment of negative symptoms in these subjects , negative symptom change scores were compared across the two trials . the degree of reduction in negative symptoms observed during glycine treatment was 3 - fold greater than that observed during d - cycloserine treatment . the difference was highly significant ( t = 8 . 2 , df = 6 , p & lt ; 0 . 0001 ). these data provide the first direct comparison of effects of glycine and d - cycloserine in the same group of patients , and the first demonstration that the full agonist glycine is significantly more effective in the treatment of negative symptoms of schizophrenia than the partial agonist d - cycloserine . the finding that the full agonist , glycine , is superior to the partial agonist d - cycloserine suggests that other full agonists of the glycine site ( or precursors thereof ) should also be therapeutically beneficial in schizophrenia . other potential agents to be used would include d - serine , d - alanine , glycineamide , threonine and poplypeptide precursors of such compounds . earlier double blind , placebo - controlled trials of glycine in schizophrenia support the idea that nmda augmenting agents will be beneficial in the treatment of schizophrenia . see u . s . pat . no . 5 , 854 , 286 . the clinical utility of this agent is limited , however , by the relatively large doses that are required to significantly elevate cns glycine levels ( toth and lajtha , 1986 ; d &# 39 ; souza et al ., 1995 ). the large doses are required because glycine permeates across the blood - brain barrier slowly by passive diffusion , and , once in the cns , is sequestered intracellularly by glycine transporters . two families of glycine transporters have been identified glycine type 2 ( glyt2 ) transporters are co - localized with inhibitory ( strychnine - sensitive ) glycine receptors in hindbrain and spinal cord and maintain low glycine levels within the synaptic cleft in those brain regions . in contrast , type 1 transporters ( glyt1 ) are co - localized with nmda receptors in forebrain and hippocampus , and may serve to maintain low intrasynaptic glycine levels specifically in the local region around nmda receptors . it is possible , therefore , that inhibition of glyt1 transporters would lead to elevations of glycine levels in the immediate vicinity of nmda receptors and augmentation of nmda receptor - mediated neurotransmission without requiring administration of exogenous glycine . support for such a hypothesis comes from a recent study in which co - expression of glyt1 transporters along with nmda receptors in xenopus oocytes led to significant inhibition of nmda receptor responsiveness ( supplison and bergman , 1996 ). blockade of glyt1 transporters would thus theoretically be expected to exert an opposite effect . the use of glycine transport blockers to augment nmda receptor - mediated neurotransmission would be analogous to the use of noradrenaline / serotonin reuptake inhibitors ( rather than precursors such as tyrosine or tryptophan ) to enhance monoaminergic neurotransmission . in early preclinical studies investigating effects of glycine on pcp - induced hyperactivity , it was noted that a specific glycine derivative , gda , was significantly more potent than glycine itself in reversing pcp - induced hyperactivity ( toth et al ., 1986 ). although the mechanism of action of gda was unknown at the time of those initial studies , more recent research has demonstrated that gda acts as a glycine transport inhibitor at doses similar to those used in behavioral studies ( u . s . pat . no . 5 , 837 , 730 ); javitt and frusciante , 1997 ; javitt et al ., 1997 ). to the extent that the behavioral effects of gda are due to its inhibition of glycine transport , the ability of gda to antagonize pcp - induced hyperactivity supports the concept that glycine transport inhibitors , particularly those acting at glyt1 transporters , may be useful in the treatment of schizophrenia . however , because gda may have idiosyncratic effects unrelated to its effects at the glycine transport site , it is important to test additional compounds with known affinity for glycine transporters . for this study , several novel gda - related compounds were synthesized and their effects on pcp - induced hyperactivity and in vitro glycine transport were characterized . these compounds were structurally similar to gda , but differed in the length of the carbon sidechain that was joined to the glycine backbone via the amide linkage . the length of carbon chain varied from 6 to 13 carbon atoms . the 6 - carbon analog ( g6a ) was found in one early study not to significantly inhibit pcp - induced hyperactivity ( toth et al ., 1986 ). the other agents ( g8a , g10a , g11a and g13a ) had not been synthesized previously . glycineamide derivatives were synthesized in house according to the approach of toth et al ( 1986 ). the specific agents used for study were glycylhexylamide ( g6a ), glycyloctylamide ( g8a ), glycyldecylamide ( g10a ), glycylundecylamide ( g11a ) and glycyltriscadecylamide ( g13a ). behavioral studies were performed using balb / c mice ( 25 g ) of either sex . rodent activity was monitored using a photocell - based activity meter ( columbus instruments auto - track system , columbus , ohio ). animals were placed in test cages and allowed to accommodate overnight . on the day of experiment , animals , in their home cages , were placed on the activity monitors and baseline activity was monitored for 20 min ., after which time animals were pretreated with either saline or a specified glycineamide derivative ( 0 . 1 g / kg i . p .). 30 min . after pretreatment , animals were injected with either pcp ( 5 mg / kg i . p .) or amphetamine ( 5 mg / kg s . c .). activity was then monitored for an additional 90 minutes . for statistical analyses , summed activity over the 90 min . following pcp / amphetamine injection was used as a measure of drug - induced hyperactivity . synaptosomal p 2 fractions were prepared from cerebral cortex + hippocampus of sprague - dawley rats ( 200 - 250 g ). brain tissue was homogenized in 0 . 32 m sucrose in tris - hcl buffer ( ph 7 . 4 ), and centrifuged at 1 , 000 × g for 10 min at 4 ° c . in a sorvall 5c centrifuge . the supernatant was then centrifuged at 14 , 000 × g for 10 min and the pellet resuspended in artificial csf having the following composition ( mm ): nacl , 125 ; kcl , 3 ; mgso 4 , 1 . 2 ; cacl 2 , 1 . 2 ; nah 2 po 4 , 1 ; nahco 3 , 22 ; glucose , 10 . homogenate was aerated with 95 % o 2 / 5 % co 2 until use . 1 ml aliquots of homogenate were preincubated with specific concentrations of glycineamide derivatives , following which incubation was initiated by the addition of 10 μl of 10 μm [ 3 h ] glycine ( dupont / nen or sigma , spec . act .≈ 45 ci / mmol ) to obtain a final concentration of 100 nm . nonspecific binding was determined in the presence of 10 mm sarcosine . following 5 min , incubation was terminated by filtration under reduced pressure using a brandel 24 - well cell harvester and whatman gf - b filters . radioactivity was determined by scintillation counter with approximate efficiency of 50 %. in prior studies , gda has been found to inhibit pcp - induced hyperactivity at doses of 50 - 200 mg / kg i . p . for the present study , an initial experiment evaluated the specificity of the gda effect . rats were pre - treated with gda ( 50 mg / kg i . p .) after which they received either pcp ( 5 mg / kg i . p .) or amphetamine ( 5 mg / kg i . p .). both pcp and amphetamine induced significant levels of hyperactivity . pretreatment with gda did not significantly affect basal activity . however , gda significantly reduced the degree of hyperactivity induced by pcp ( t = 4 . 09 , p & lt ; 0 . 001 ) ( fig2 ). in contrast , the level of hyperactivity induced by amphetamine was not significantly altered ( t = 0 . 59 , p & gt ; 0 . 5 ). gda - induced inhibition of pcp - induced hyperactivity was significantly dose - dependent with 50 mg / kg gda inducing a reduction of hyperactivity to below 70 % of control ( pcp alone ) levels , and 100 mg / kg gda inducing a reduction to below 50 % of control ( t = 5 . 04 , p & lt ; 0 . 001 ) ( fig3 ). because it was assumed that the majority of glycineamide derivatives tested would be less potent that gda in reversing pcp - induced hyperactivity , a dose of 100 mg / kg i . p . was chosen for comparative testing . as has been reported previously ( toth et al ., 1986 ), glycylhexylamide ( g6a ) did not significantly inhibit pcp - induced hyperactivity . significant reductions in activity were , however , observed following pretreatment with g10a , gda ,. and g13a , with the degree of reduction increasing with increasing length of the carbon sidechain ( fig3 ). the potency with which the identified glycineamide derivatives inhibited glycine transport in vitro was analyzed using a synaptosomal assay system . all agents tested induced significant , dose - dependent inhibition of synaptosomal sarcosine - sensitive glycine uptake ( fig4 ). agents with longer side chains showed greater potency for inhibition of glycine transport with ic50 values in the low micromolar range , whereas shorter chain derivatives showed ic50 values in the low to mid millimolar range . in order to compare the potency of these agents at a concentration relevant to the dose used for behavioral testing , all agents were re - tested for inhibitory potency at a single fixed concentration of 100 μg / ml . significant variation was seen across compounds , with gda , ( t = 18 . 6 , p & lt ; 0 . 0001 ), g13a ( t = 15 . 3 , p & lt ; 0 . 0001 ), g11a ( t × 6 . 79 , p & lt ; 0 . 001 ) and g10a ( t = 4 . 64 , p & lt ; 0 . 01 ) leading to significant inhibition of glyt1 - mediated transport , g8a having no significant effect ( t = 1 . 37 , p & gt ; 0 . 2 ), and g6a leading to significant potentiation of transport ( t = 4 . 2 , p & lt ; 0 . 1 ). the potency with which the glycineamide derivatives inhibited glyt1 - mediated synaptosomal glycine transport correlated significantly ( p & lt ; 0 . 05 ) with their potency in antagonizing pcp - induced hyperactivity ( fig5 ). other agents which should be useful for treating schizophrenia through inhibition of glycine transport include other glycylakylamides in which the alkyl group contains 3 to 30 carbon atoms , and structures containing branched , cyclic or polycyclic side chains . davis k l , kahn r s , ko g , davidson m ( 1991 ): dopamine in schizophrenia : a review and reconceptualization . am j psychiatry 148 : 1474 - 1486 . debler e a , lajtha a ( 1987 ): high - affinity transport of n - aminobutyric acid , glycine , taurine , l - aspartic acid , and l - glutamatic acid in synaptosomal ( p2 ) tissue : a kinetic and substrate specificity analysis . j neurochem 48 : 1851 - 6 . d &# 39 ; 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