Patent Application: US-44198607-A

Abstract:
the atypical antipsychotic drugs have markedly enhanced the treatment of schizophrenias but their use has been hindered by the major weight gain elicited by some aapds . we found that orexigenic aapds potently and selectively activate hypothalamic amp kinase , an action abolished in mice with deletion of histamine h1 receptors . these findings afford a means of developing better therapeutic agents and provide insight into the hypothalamic regulation of food intake .

Description:
the inventors have discovered that orexigenic aapds potently and selectively stimulate hypothalamic amp kinase ( ampk ). the inventors further demonstrated that orexigenic aapds reverse the actions of the anorexigenic hormone leptin . moreover , the inventors have found that this action involves histamine h1 receptors , as clozapine augmentation of ampk activity is abolished in h1 receptor knockout mice . in addition , orexigenic potencies of neuroleptics have been found to correlate with their affinities for histamine h1 receptors . based on these showings , the inventors have devised methods for testing agents , in particular psychotropic drugs or candidate psychotropic drugs , to predict whether the drugs will be orexigenic . the methods involve testing the agents for their interactions with hypothalamic ampk and hi receptors . the testing can be done in cell lines , in whole animals , and in cell - free systems . psychotropic drugs and candidate psychotropic drugs are those which have proven or suggested psychotropic activity . they can be , for example , approved drugs , under - review drugs , regulated or over - the - counter , neuropleptic or psycholeptic agents , antipsychotic or anti - depressant agents . the testing for orexigenic potential can be performed at any stage in the “ life cycle ” of a drug , from initial indication of in vitro activity to post - approval or marketing . histamine 1 receptors ( h1r ) can be used in purified form , in cellular membrane preparations , in cloned cells , from any mammalian source , including but not limited to human , rat , dog , mouse , sheep , guinea pig , cow , and pig . cells or animals which are deficient in hir , such as knock - out mice can be used as negative controls . one exemplary sequence of hir which can be used is genbank accession no . np — 000852 , the disclosure of which is expressly incorporated herein as it exists on the filing date of this application . hypothalamic ampk activity and / or phosphorylation can be measured in slices of hypothalamus from any mammal , including but not limited to human , rat , dog , mouse , sheep , guinea pig , cow , and pig . for scaling up of such measurements , cells of cell lines from hypothalamic sources can be used . the cell lines can similarly be from any mammalian source . assays for activity or phosphorylation of ampk can be any that are known in the art . see for example , y . minokoshi et al ., nature 428 , 569 ( 2004 ). binding to hir can be determined using any techniques known in the art . direct binding can be determined or inhibition of a known ligand , such as histamine , can be determined . one or both of the binding partners or the known ligand can be labeled , for example with a radiolabel , a fluorescent label , a chromophore , etc . one of the binding partners can be bound to a solid support during or after the binding reaction to facilitate separation from unbound components . suitable solid supports are known in the art and any can be used . these include beads , chromatography resin or matrix , polystyrene microwells , etc . leptin and insulin are known to reduce ampk activity . ampk assays of the present invention can be performed in the presence of leptin and / or insulin . typically , orexigenic aapds are able to reverse the reduction of activity caused by these agents . other hormones and agents which are known to reduce ampk activity can be used similarly . isogenic pairs of hypothalamic cells can be used to assay for effects on ampk . the cells may be in animals , such as knock out mice and their wild - type siblings , or the cells may be cultured cells of a continuous cell line . the cells may be of any mammalian species , including mice , rats , guinea pigs , dogs , human , sheep , cows , pigs , etc . preferably one of the pair of cells is deficient in hir . more preferably one of the pair of cells is homozygously deficient in hir . methods for making homozygous deficient cell lines and knock out mice are well known in the art . see for example , hogan , b ., beddington , r ., costantini , f . and lacy , e . ( 1994 ) manipulating the mouse embryo : a laboratory manual , cold spring harbor laboratory and the webpage at the boston university medical campus for the transgenic / knockout core facility , at the page entitled , “ production of knockout mice .” according to general methods of laboratory investigation , changes in properties such as activity , binding , and phosphorylation are assessed using statistical measures of significance . there are many statistical measures of significance which can be used in the present invention . any which are accepted in the scientific community may be used . one such measure which can be used is the student &# 39 ; s t - test . another is the chi square test . the specific findings described below indicate that the appetite stimulation - weight gain associated with aapds is mediated by activation of hypothalamic ampk following blockade of h1 receptors . ampk stimulation correlates with the orexigenic actions of the drugs , with clozapine and olanzapine producing the most marked effects . the drug actions are very potent with substantial effects evident at 5 nm concentration . they are selective with effects restricted largely to the arcuate and paraventricular nuclei of the hypothalamus . these findings are consistent with studies implicating central histamine ( 23 , 24 ) and ampk ( 11 ) in weight control as well as the orexigenic role of the paraventricular and arcuate nuclei ( 25 , 26 ). weight gain elicited by aapds can be massive and associated with the “ metabolic syndrome ” leading to diabetes ( 5 , 7 , 27 , 28 ). thus , the orexigenic actions of aapds , especially olanzapine and clozapine , have precluded their use in large numbers of patients . ignorance of the mechanism of these orexigenic actions has hindered efforts to develop alternative agents . evaluation of candidate drugs for influences on histamine h1 receptors and hypothalamic ampk provides a straightforward approach to developing better drugs and may advance our understanding of the hypothalamic regulation of food intake . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . in hypothalamic slices , clozapine and olazapine markedly enhance levels of phospho - ampk , and quetiapine , which also is orexigenic , produces similar effects ( fig1 a ). however , risperidone , ziprasidone , haloperidol , and aripipirazole , which are much less orexigenic ( table 1 ), fail to stimulate ampk ( fig1 b ). increased ampk phosphorylation is observed as early as 5 min after treatment with clozapine or olanzapine ( fig1 c and d ). the drug actions are potent and substantial with ec50 values for both of about 10 nm and with six and 3 . 5 fold maximal increases respectively with clozapine and olanzapine ( fig1 e - h ). neuroleptic affinities for histamine h1 receptors correlate with orexigenic actions . receptor binding was assayed using rat brain membranes incubated with [ 3 h ] mepyramine and 12 concentrations of drugs ranging from 30 pm to 10 μm in triplicate . data are means of 3 independent determinations which varied less than 10 %. orexigenic action of drugs were obtained from published literatures indicating reproducible differences among aapds in eliciting weight gain , when administered at comparable therapeutic doses ( 5 , 8 , 14 ). hypothalami from 8 - 10 week old mice were cut at 0 . 4 mm intervals in sagittal and coronal planes using a mcllwain tissue chopper . the slices were dispersed in artificial cerebrospinal fluid buffer . male c57bl / 6 mice ( aged 6 weeks ) were purchased from charles river laboratories ( wilmington , mass . ), housed at 22 ° c ., maintained on a 12 - h / 12 - h light / dark cycle , and given access to standard rodent chow and water ad libitum . experiments were done at age 8 - 10 weeks . all drugs were administered by i . p . injection . for co - administration experiment , either leptin ( 3 mg / kg ) or insulin ( 3 mg / kg ) were first injected , and at 1 h , clozapine was administered . procedures were approved by guidelines of the johns hopkins university school of medicine institutional animal care and use committee . all drugs were purchased from toronto research chemicals inc . ( toronto , canada ). clozapine was dissolved in 0 . 1 n hcl ( 0 . 8 ml ) and neutralized by 0 . 1 n naoh ( 0 . 7 ml ). the drug was diluted with 8 . 5 ml saline solution , and the appropriate doses were administrated to mice . for control mice , the same solution was injected without a drug . for in vitro assays , drugs were dissolved in dmso . ampk enzyme activity was assayed as described previously ( 11 ). briefly , hypothalamic slices were maintained at 37 ° c . in artificial cerebrospinal fluid buffer and treated as indicated in the figure legends . subsequent to treatment , hypothalamic slices were lysed with ampk lysis buffer ( 50 mm tris . hcl ( ph 7 . 5 at 4 ° c . ), 50 mm naf , 5 mm na pyrophosphate , 1 mm edta , 1 mm egta , 1 mm dithiothreitol , 1 mm benzamidine , 1 mm phenylmethane sulfonyl fluoride , 1 % ( vol / vol ) triton x - 100 , and 10 % ( vol / vol ) glycerol by use of a motor - driven pestle . the crude homogenate was sonicated with 4 pulses of 3 s each before centrifugation at 18000 g for 3 min . protein concentrations were then determined using the bradford method , and each sample was diluted accordingly to an equivalent protein concentration . to 200 μl of the sample , 2 μl of α - ampk antibody ( cell signaling technologies , danvers , mass .) was added , and the immunoprecipitation was incubated overnight at 4 ° c . with gentle mixing . after 12 h immunoprecipitation , 30 μl of protein a beads ( 50 % slurry ) were added to each sample and incubated for 2 h at 4 ° c . with gentle mixing . the assay was begun with the addition of immunoprecipitated enzyme to assay buffer ( in mm : 80 hepes buffer , 160 nacl , 1 . 6 edta , 200 μm sams peptide ( alberta peptide institute , edmonton , ab , canada ), 200 μm amp , 200 μm atp , 16 % glycerol , 0 . 1 % triton x - 100 and 0 . 5 μci [ γ - 32 p ] atp per sample ). after the addition of enzyme to the reaction tube , samples were vortex - mixed for 5 s and incubated for 10 min at 30 ° c . after incubation , the reaction mixture was vortex - mixed and spotted on p81 whatman filter paper ( fisher scientific , pittsburgh , pa ., u . s . a . ), briefly allowed to dry , and washed three times in 1 % hcio 4 before a single wash in acetone . after sufficient time to allow the filter papers to air dry , samples were immersed in a scintillant - fluor cocktail , and radioactivity was measured in a beckman scintillation counter . unless otherwise listed , all reagents used in this assay were purchased from sigma . clozapine also potently and selectively augments hypothalamic ampk in intact animals . as little as 1 mg / kg of clozapine markedly stimulates levels of phospho - ampk ( fig2 a ) as well as ampk catalytic activity with 5 mg / kg producing a 3 . 5 folds , augmentation of activity ( fig2 b ). the increase of phospho - ampk and ampk catalytic activity is selective for the hypothalamus , as clozapine ( 1 mg / kg ) fails to increase phospho - ampk levels in the cerebellum and liver ( fig5 a and 5 b ) and ampk catalytic activity is not affected in the cerebral cortex or cerebellum by clozapine ( 1 mg / kg ) ( fig2 c ). at 5 mg / kg , clozapine elicits a 20 % increase in cortical ampk activity , much less than the quadrupling of hypothalamic ampk activity , while no increase is apparent in the cerebellum ( fig2 d ). the effect of clozapine is maximal 3 h after drug - administration and gradually decreases to basal levels in 24 h ( fig5 f ). kahn and colleagues ( 11 ) reported that the anorexigenic peptide leptin reduces hypothalamic ampk activity , which we confirm . clozapine reverses reductions in hypothalamic phospho - ampk elicited by leptin ( fig3 a ) and insulin ( 11 ) ( fig6 ). in intact mice , leptin ( 3 mg / kg ) reduces hypothalamic phospho - ampk ( fig3 b ) and catalytic activity ( fig3 c ), and clozapine reverses these actions . the arcuate and paraventricular hypothalamic nuclei display the greatest alterations of ampk activity in response to feeding stimuli ( 11 ). in immunohistochemical experiments phospho - ampk is selectively augmented in these two nuclei with clozapine ( 1 and 5 mg / kg ) while much lesser effects are evident in the cerebral cortex ( fig7 a and b ). by contrast , ziprasidone fails to alter phospho - ampk in the paraventricular nucleus ( fig8 ). phospho - ampkα immunohistochemistry was performed as previously described ( e . k . kim et al ., j . biol . chem . 279 , 19970 ( 2004 ); a . s . huang et al ., j . neurosci . 26 , 2814 ( 2006 )), and all solutions prior to and including the primary antibody incubation contained 2 mm sodium fluoride . c57bl / 6 mice or hr1 −/− mice ( 8 - 10 weeks ) were perfused with 4 % paraformaldehyde maintained at 37 ° c . organs were post - fixed 2 h at room temperature and cryoprotected overnight at 4 ° c . ( 30 % sucrose in pbs ). free - floating sections ( 45 μm ) were quenched with 3 % h 2 o 2 in water for 10 min at room temperature , washed in tbs - t ( 16 mm tris ph 7 . 4 , 140 mm sodium chloride , and 0 . 1 % tween - 20 ), and antigen retrieved for 30 min in a 70 ° c . water bath ( 10 mm sodium citrate in tbs - t ). sections were blocked ( 5 % ngs in tbs - t ) for 1 h at room temperature and incubated with a mouse anti - phospho - ampkα antibody ( cell signaling , danvers , mass .) diluted 1 : 200 into the blocking solution overnight at 4 ° c . subsequent washes were conducted in tbst , and labeling was visualized with the vectastain elite abc kit ( vector laboratories , burlingame , calif .). images were quantified with alphaeasefc program . we wondered whether the influence of aapds on hypothalamic ampk is secondary to actions of the drugs on specific neuropeptide receptors which have been implicated on appetite regulation . clozapine and olanzapine ( 10 , 100 nm ) fail to influence ligand binding to receptors for leptin , alpha - msh , and neuropeptide y ( data not shown ). relative potencies of aapds in blocking histamine h1 receptors have been reported to correlate with their orexigenic potencies ( 21 , 22 ), which we confirm ( table 1 ). moreover , in hypothalamic slices the h1 antihistamine triprolidine stimulates phospho - ampk to the same extent as clozapine both in hypothalamic slices ( fig4 a ) and in intact animals ( fig9 ). conversely , histamine decreases phospho - ampk with reversal of this effect by clozapine ( fig4 b ). to explore whether augmentation of phospho - ampk by drugs stems from h1 receptor blockade , we administered clozapine to h1 receptor deleted mice . whereas the drug elicits a quadrupling of phospho - ampk in wild - type mice , no effect is evident in the knockouts ( fig4 c and d ). the ic50 values of neuroleptics on histamine h1 receptors were determined as described previously ( r . s . chang , v . t . tran , s . h . snyder , eur . j . pharmacol . 48 , 463 ( 1978 )). briefly , rats were killed by decapitation and the forebrains removed . brains were homogenized in 30 vol of na - k phosphate buffer , ph 7 . 5 , and centrifuged at 48 , 000 × g for 10 min . the tissue was resuspended in buffer and re - centrifuged for an additional 3 times . tissue was resuspended in buffer at 15 mg / ml . tissue ( 0 . 2 ml ) was added to tubes containing 25 μl of drug and 25 μl of [ 3h ] mepyramine ( 30 nm ). non - specific binding was determined in the presence of 1 μm triprolidine . tubes were incubated for 1 h at 25 ° c ., and the samples were filtered over 0 . 5 % poly ( ethyleneimine )- coated filters washed with 2 × 5 ml of cold 50 mm nacl . 1 . j . kane , g . honigfeld , j . singer , h . meltzer , arch . gen . psychiatry 45 , 789 ( 1988 ). 2 . h . y . meltzer , hosp . community psychiatry 41 , 1356 ( 1990 ). 3 . r . w . buchanan , a . breier , b . kirkpatrick , p . ball , w . t . carpenter , jr ., am . j . psychiatry 155 , 751 ( 1998 ). 4 . a . tuunainen , k . wahlbeck , s . gilbody , schizophr . res . 56 , 1 ( 2002 ). 5 . j . a . lieberman et al ., n . engl . j . med . 353 , 1209 ( 2005 ). 6 . d . gothelf et al ., am . j . psychiatry 159 , 1055 ( 2002 ). 7 . j . w . newcomer , cns . drugs 19 suppl 1 , 1 ( 2005 ). 8 . d . b . allison et al ., am . j . psychiatry 156 , 1686 ( 1999 ). 9 . o . blin , j . micallef , j . clin . psychiatry 62 suppl 7 , 11 ( 2001 ). 10 . m . b . isaac , m . t . isaac , am . j . psychiatry 162 , 1764 ( 2005 ). 12 . j . p . lindenmayer et al ., am . j . psychiatry 160 , 290 ( 2003 ). 13 . v . l . albaugh et al ., obesity . 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