Patent Application: US-36588999-A

Abstract:
determination of a synaptosomal d - serine transporter and use of an assay method for discovering inhibitors thereof to be used in the treatment of psychotic disorders .

Description:
current state - of - the - art brain is known to contain multiple amino acid transport systems , including system “ gly ”, which is specialized for uptake of glycine , system which is specialized for uptake of alanine , system which 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 . 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 . 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 . 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 . membranes were suspended in oxygenated artificial csf and incubated in the presence of l - or d -[ 3 h ] serine , as appropriate . incubation was terminated by filtration under reduced pressure through whatman gf / b filters . for initial studies , uptake was measured over a 30 min . period ( fig1 ). uptake of l - and d -[ 3 h ] 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 ( 2 - aminobicyclo ( 2 , 2 , 1 ) heptane - 2 carboxylic acid , 10 mm ). effects of the system asc substrates alanine , cysteine and serine were evaluated at concentrations between 0 . 03 and 30 mm ( fig2 ). complete inhibition of serine uptake was obtained with either l - or d - serine . in both cases , l - serine showed greater potency that d - serine in inducing inhibtion . 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 . 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 ( fig3 ). 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 . in summary , a prior patent u . s . pat . no . 5 , 854 , 286 described efficacy of glycine and other nmda agonist in the treatment of schizophrenia . a prior patent u . s . pat . no . 5 , 837 , 730 , described use of glycine transport inhibitors as potentiators of nmda receptor - mediated neurotransmission and potential treatments for persistent symptoms of schizophrenia . the present study discloses use of d - serine - transport inhibitors for a similar purpose . an assay system for identifying d - serine - transport inhibitors is described . bergeron r , meyer t m , coyle j t , greene r w . modulation of n - methyl - d - aspartate receptor function by glycine transport . proc natl acad sci u s a . 1998 ; 95 : 15730 - 4 . danysza w , parsons c g . glycine and n - methyl - d - aspartate receptors : physiological significance and possible therapeutic applications . pharmacol . rev . 1998 ; 50 : 597 - 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