Patent Application: US-25766703-A

Abstract:
the present invention relates to selective neuropeptide y y1 receptor antagonists . more closely , it relates to use of , and methods of using , selective npy y1 receptor antagonists for the treatment of inflammatory conditions . the invention also relates to use of a npy y1 receptor as a drug target in screening procedures to find anti - inflammatory compounds .

Description:
homologous recombination in embryonic stem cells was used to establish mice deficient in the npy y1 receptor . the disruption was generated by introducing an internal ribosomal entry site followed by a tau - lacz fusion minigene into the second exon of y1 ( fig1 a ). southern blot analysis confirmed that the y1 allele was disrupted and northern blot analysis showed that instead of the mrna transcripts encoding y1 , the mutant ( y1 −/− ) mice produced the expected mrna encoding β - galactosidase ( fig1 b - d ). as previously described , female y1 −/− mice display a late - onset overweight compared to their littennates 5 ( data not shown ). y1 receptors are abundant in the forebrain while little or nothing is present in the brainstei 6 . y1 receptors are also highly expressed in dorsal root ganglion neurons in preferentially small and medium size neurons 6 , 7 . however , the central termination of y1 nerve fibers in the dorsal horn , and whether y1 is expressed in both of the two major cytochemical subpopulations of pain neurons , the sp peptidergic and non - peptidergic pain neurons 8 , is unresolved . β - galactosidase histochemical and immunohistochemical staining of spinal cord sections from y1 −/− mice led to strong staining localised exclusively to the dorsal horn ( fig1 e and f ). immunohistochemical double staining for β - galactosidase ( staining y1 expressing neurons and fibers ) and the lectin ib4 ( staining somas and nerve fibers of unmyelinated non - peptidergic sensory nociception neurons 8 ) showed a strong staining for y1 nerve fibers in dorsal horn layer ii overlapping with ib4 terminals . a significant portion of the nerve fibers also terminated in layers i , iii and iv ( fig1 f ). y1 - positive dorsal horn interneurons were also found ( fig1 f , arrows ). many y1 expressing dorsal root ganglion neurons coexpressed sp ( fig1 h ; 38 % of y1 neurons contained sp ), however , a large number of y1 neurons also double stained for ib4 ( fig1 g ; 32 %). a presynaptic block of primary afferent sp release in the spinal cord may participate in central npy - induced analgesia 3 . we therefore examined if this primary afferent circuit was intact in the y1 −/− mice . no overt alteration of sp nor npy immunoreactivity was detected in the spinal cord of y1 −/− mice ( data not shown ). furthermore , total sp in spinal cord measured by eia was 2852 ± 328 . 9 pg / g tissue and 3919 ± 444 . 1 pg / g tissue in wild - type and y1 −/− mice , respectively ( p & gt ; 0 . 05 , st udent &# 39 ; s t - test ). immunohistochemical detection of the sp receptor revealed similar staining in the somatic and dendritic cell surface of neurons in the superficial spinal cord lamina ( i and ii ) of both wild - type and y1 −/− mice , as in previous results ( fig1 i and j ). sp release leads to sp receptor activation and internalisation . neurons containing internalised sp receptors were detected in y1 −/− mice following an intraplantar injection of capsaicin ( fig1 k and 1 ), indicating that there is no defect of receptor activation per se in the absence of the y1 receptor . thus , we conclude that the major neuronal pain circuit suggested to be modulated by npy is anatomically intact in the y1 −/− mice . behavioural acute nociceptive thresholds were markedly affected by an absence of y1 . sp / neurokinin a null mutant mice show a blunted response to painful stimulus only at moderate intensities , whereas response to mildly or intensely painful stimulus is intact 11 . we therefore tested whether also the npy y1 receptor act at specific thresholds in modulating nociception . withdrawal latency in the hot plate assay was examined at 48 , 50 , 52 , 55 and 58 ° c . at 48 ° c . no significant difference was observed ( data not shown ). the y1 −/− mice showed , however , a profound hyperalgesia and displayed a significantly reduced latency at all temperatures above 48 ° c . ( fig2 a ). the hot plate test involves supraspinal integration associated with the paw withdrawal . to test whether neuropeptide y1 receptor could be acting in a spinal circuitry , we characterised these mice in the tail flick assay . consistent with the hot plate assay , y1 −/− mice showed a markedly reduced latency at all temperatures between 46 - 54 ° c . ( fig2 b ). y1 −/− mice also displayed a significant decrease in mechanical threshold indicating mechanical hypersensitivity ( fig2 c ). we then examined a role for the npy y1 receptor in chemical nociception . during the first phase of the formalin assay , which provides a measure of the acute pain mediated by direct chemical activation of c - fibers , the nociceptive behaviour was augmented by 44 %, 60 % and 46 % at 1 . 2 , 2 and 5 % formalin injected , respectively , in y1 −/− mice ( fig2 d ). the second phase of nociception was not consistently altered by an absence of the npy y1 receptor . in two models of visceral pain , one that is secondary to an inflammatory response ( acetic acid ) and one that induces immediate pain independent of inflammation ( mgso 4 ) 11 , we also found significantly increased pain behaviour in y1 −/− mice ( fig2 e and f ). combined , the above results indicate that npy could be acting on y1 containing polymodal nociceptive afferents ( c - fibers ). consistent with our results , these neurons span different modalities , and mediate pain transduction from both visceral and cutaneous tissues . stress induces analgesia through endogenous opioid and non - opioid dependent mechanisms 12 . we tested whether these pathways interact with npy - dependent analgesia by letting the mice swim in 10 ° c . water producing a non - opioid , nada - dependent analgesia and 33 ° c . water resulting in opioid - dependent analgesia 13 , 14 . after a swim at 10 or 33 ° c ., the development of analgesia assayed in the hot plate assay was similar between wild - type and y1 −/− mice ( fig2 g ). these data suggest that the y1 receptor is not a critical component in stress - induced analgesia . the role of npy in neuropathic pain is incompletely defined and a number of conflicting results with regards to possible npy receptors involved have been reported 4 , 15 . we tested the physiological role of the y1 receptor in a model of neuropathic pain . a partial sciatic nerve ligation resulted in mechanical allodynia ( sensitisation to mechanical stimuli ) in wild - type mice ( 37 % and 52 % increase in sensitivity at 3 and 14 days after nerve injury , respectively , compared to day 0 ; fig2 h ). as indicated before , the basal threshold of mechanical sensitivity was significantly decreased in non - lesioned y1 −/− mice . despite this , the mechanical allodynia caused by nerve damage was significantly increased in y1 −/− mice compared to wild - type mice ( 55 % and 67 % increase in sensitivity at 3 and 14 days after nerve injury , respectively , compared to day 0 ; p & lt ; 0 . 01 for the slopes of the curves between day 0 - 14 after nerve injury ). pharmacological npy - induced analgesia to thermal stimuli following spinal delivery is well documented 2 , 16 . to identify the receptor involved in the pharmacological effects of intrathecally administered npy we injected npy ( 10 μg ) in the spinal cord of y1 −/− mice and measured heat sensitivity . the anti - nociceptive effect of npy on the spinal cord was completely abolished in y1 −/− mice ( fig2 i ). thus , the y1 receptor is exclusively responsible for the analgesic effects of centrally delivered npy . inflammation is caused by a neurogenic as well as a non - neurogenic component 17 . neurogenic inflammation does not occur in the denervated human skin , and can be prevented by a nerve block in rats 17 , 18 and is mediated by a peripheral release of sp / neurokinin a 11 . we tested whether the y1 receptor could participate in inflammation . a subcutaneous injection of capsaicin , which induces neurogenic inflammation , led to a marked inflammation seen by an increased paw diameter , plasma extravasation and hyperalgesia in wild - type mice . unexpectedly , y1 −/− mice displayed no overt or quantitative sign of plasma extravasation , increase in paw diameter and hyperalgesia ( fig3 a , b , c and d ). neither rectal and paw skin temperature nor heart rate differed between any of the groups before and after capsaicin administration ( data not shown ). both wild - type and y1 −/− mice exhibited an increased blood flow in the paw as a response to capsaicin ( 181 . 4 ± 31 . 3 % and 245 . 5 ± 71 . 4 %, respectively ) indicating that the y1 receptor is not influencing capsaicin - induced vasodilation . in contrast to capsaicin , mustard oil induces inflammation that is largely , but not exclusively , dependent on a neurogenic component including the release and proinflammatory effects of sp 19 y1 −/− mice showed markedly reduced plasma extravasation in response to mustard oil compared to wild - type ice . however , since the increase in plasma extravasation was significant ( although at a lower level ), some components of the effects of mustard oil , likely those of non - neurogenic origin , were intact in y1 −/− mice ( fig3 h ). in contrast to neurogenic inflammation , there was a similar increase of plasma extravasation , paw diameter and sensitisation following non - neurogenic inflammation induced by carrageenan 20 in y1 −/− nuce as in wild - type mice ( fig3 e , f and g ). since capsaicin and to a large extend mustard oil - induced inflammation depend on the integrity of primary c - fiber afferent release of sp 8 , we determined if y1 is required prior or after sp release in the sequence of events leading to inflammation by injecting sp in the paw . sp caused a similar inflammatory response in y1 −/− mice as it did in wild - type mice ( fig3 i ). sp receptor immunoreactivity was intact in the skin of wild - type and y1 −/− mice and was found occasionally in nerve endings in dermis and in scattered cells throughout dermis corresponding to mast cells , similar to wild - type mice ( data not shown ). our results are therefore consistent with that y1 could be required for capsaicin induced sp release . we measured by eia the quantity of total and released sp in the skin after capsaicin injection in wild - type and y1 −/− mice . capsaicin caused a marked increase of released sp in the skin of wild - type mice whereas it had no effect on sp release in y1 −/− mice ( fig4 a ). the lack of an increase of released sp was not caused by an overall reduction of sp peripherally because the quantity of total sp was similar in y1 −/− and wild - type mice ( 2269 ± 524 pg / g tissue and 2520 ± 860 pg / g tissue , respectively ). furthermore , the number of sp immunoreactive nerve fibers in the dermis and epidermis was similar in wild - type and y1 −/− mice ( 5 . 7 ± 0 . 5 and 6 . 2 ± 0 . 5 per cm , respectively ). in accordance with previous results , capsaicin led to a marked reduction of immunoreactive terminals in the epidermis of wild - type mice ( from 3 . 0 ± 0 . 3 to 1 . 8 ± 0 . 2 per cm ; p & lt ; 0 . 01 , student &# 39 ; s t - test ) possibly by a loss of immunoreactivity due to increased release . in contrast , y1 −/− mice displayed no reduction in sp immunoreactive terminals ( 3 . 1 ± 0 . 3 and 2 . 5 ± 0 . 6 per cm , respectively ). these results indicate that the absence of neurogenic inflammation in y1 −/− mice is caused by the requirement of y1 activation for sp release . the persistent vasodilation in these mice could be caused by a normal release of calcitonin gene related peptide , which induces vasodilation but not extravasation . furthermore , since close to one order of magnitude less sp is required for vasodilation than for plasma leakage 21 , a small residual sp release in these mice could also cause this phenotype . we challenged our results in wild - type mice by injecting the y1 agonist [ leu 31 - pro 34 ]- npy and by using a y1 receptor antagonist . [ leu 31 pro 34 ]- npy efficiently caused plasma extravasation in wild - type mice , and consistent with its specificity for the y1 receptor , no response was seen in y1 −/− mice ( fig4 b ). administration of the y1 antagonist bibp 3226 prior to intr - aplanltar injection of capsaicin markedly reduced plasma extravasation in wild - type mice ( fig4 c ), showing that the results oil the y1 −/− mice are directly caused by the absence of y1 signalling . we therefore conclude that the npy y1 receptor is both required and sufficient to induce neurogenic inflammation by controlling sp release , and that a y1 antagonist could provide an effective strategy for the treatment of neurogenic inflammatory diseases . we have shown that the y1 receptor play an essential anti - nociceptive role during pain transduction in many modalities including thermal , chemical and mechanical from both cutaneous and visceral tissues as well as during neuropathic pain . npy or another y1 receptor ligand 22 could mediate antinociception by reducing sp and excitatory neurotransmitter release from primary c - fiber afferents 3 , 23 , 24 and / or by inhibiting post synaptically the sp receptor expressing projection neurons of the spinal cord 25 , 26 . consistent with that npy does not modulate pain transmission only through a presynaptic regulation of sp release , the nociceptive phenotype of the y1 −/− mice does not fully correlate with sp and sp receptor null mutant mice . sp receptor null mutant mice show for instance a reduced stress - induced analgesia 27 . we also show that y1 receptor activation is both sufficient and required for neurogenic inflammation . because mustard oil - induced inflammation occur independent of the vanilloid receptor that is activated by capsaicin 19 , our results suggest that activation of the y1 receptor could be a shared and obligatory component in most , or all , neurogenic inflammatory conditions . y1 gene targeting . exon 2 of the y1 gene was partially deleted and replaced by a ires - tau - lacz cassette also containing a neomycin - resistance gene driven by the pgk promoter and polya ( etln ). a 0 . 7 kb dna fragment 3 ′ from the y1 targeting construct was used as external probe . homologous recombinant embryonic stem cells clones were injected to generate y1 mutant 129svxbalb / c hybrid mice . mice were analysed by southern blot and pcr using the primers 5 ′- atcaaattctgaccgacgag - 3 ′, 5 ′- catgatgttgattcgcttgg - 3 and 5 ′- gcagcctctgttccacataca - 3 ′. standard procedures were used for northern blot analysis and 60 μg / sample of total rna from adult brain was analysed . physiological studies . heart rate and body temperature was measured as previously described 28 . skin blood flow and temperature was monitored with a thermal probe ( physitemp bat - 12 ) subcutaneously inserted in the plantar region of the paw and a laser doppler flowmeter probe applied to the plantar surface ( penmided pf2b ) was used to measure basal and capsaicin - evoked changes . student t - test was used and differences were considered significant at p & lt ; 0 . 05 . behavioural studies . between 6 - 13 adult male f2 - 3 129sv × balb / c mice of each genotype were used for all studies . thermal 11 , mechanical 11 , chemical 11 . visceral 11 sensitivity , stress - induced analgesia 27 and neuropathic pain 29 , 30 was assessed as previously . tail - flick latency ( by directing a concentrated light beam to the tail of the mouse ) was monitored before and after intrathecal injection of 10 μg npy . for evans blue plasma extravasatioin the following were used : capsaicin , 3 μg in 10 μl ( sigma ; dissolved in 5 % ethanol , 5 % tween - 80 and 90 % saline ), 1 % carrageenan ( sigma ; dissolved in saline ), sp , 50 pmol / paw ( sigma dissolved in saline ), 5 % mustard oil ( fluka ; dissolved in mineral oil ), npy y1 receptor agonist [ leu 31 - pro 34 ]- npy , 10 μg / paw ( calbiochem ; dissolved in saline and 5 % acetic acid ) and npy y1 receptor selective antagonist bibp 3226 , 10 mg / kg in 10 ml / kg ( american peptide ; dissolved in saline and 5 % acetic acid ) briefly , mice were anaesthetised and injected intravenously with evan &# 39 ; s blue ( 50 mg / kg ) into the jugular vein . agents mentioned above were injected into one paw of the animal except for y1 receptor selective anatagonist , bibp 3226 , which was injected intravenously 10 min prior to injection into the paw . the other paw was injected with vehicle . after 30 min the plantar skin of the paw was removed , dried off excess liquid , weighed and incubated in formamide for 24 h at 56 ° c . extravasated evans blue was measured by spectrophotometer at 620 nm . mechanical sensitivity was determined before , 30 min and 3 h after capsaicin and carrageenan administration , respectively . carrageenan inflammation was induced similarly but extravasation was measured after 4 hours . the paw diameter was measured before and after capsaicin , carrageenan or vehicle administration using a spring - loaded calliper . immunohistochemistry . wild - type and y1 −/− mice were perfused with 4 % paraformaldehyde ( for sp receptor immunohistochemistry , mice were perfused with 4 % paraformaldehyde and 12 . 5 % picric acid 10 min after capsaicin injection into hindpaw ) and the spinal cord and dorsal root ganglia were sectioned coronally ( 15 μl in thickness ). capsaicin was injected intradermally into dorsal skin of mice . after 10 min the skin was removed , postfixed and sectioned as above . immunohistochemistry was performed as previously 28 using α - β - galactosidase ( 1 : 200 dilution , icn / cappel ) rabbit ctsp ( 1 : 5000 dilution , chemicon ), guinea pig α - sp ( 1 : 200 dilution , peninsula lab . ), rhodamine - conjugated bandeiraea simplicifolia lectin i ( isolectin b4 ; 1 : 100 dilution , vector ), α - npy ( 1 : 200 dilution , peninsula lab . ), and rhiodamine or fitc - conjugated secondary antisera ( jackson ). for sp receptor immunohistochemistry sections were incubated 30 min in pbs , 50 % methanol and 0 . 6 % h 2 o 2 prior incubation in 10 % goat sermum . the antiserum ( chemicon 1 : 2000 ) was used in the fluorescein tsa fluorescence system ( nen ). β - galactosidase histochemical staining was performed as previously 28 . eia . capsaicin or saline was injected into the paw of wt or y1 −/− mice . after 10 min , the paw was removed and the skin was cut open and washed in pbs and 0 . 1 % bsa for 10 min . the skin was then dried , weighed , transferred to a new container and frozen . the liquid was centrifuged at 4000 rpm for 15 min . supernatant was transferred to a new tube , weighed and frozen . the lumbar part of spinal cord was removed , weighed and frozen . the samples were then assayed for sp according to the manufacturer &# 39 ; s instructions using sp high sensitivity eia kit ( peninsula lab .). [ 0033 ] fig1 . targeted mutagenesis of the y1 receptor and expression analysis of y1 and sp receptors . a , y1 gene - targeting . top , targeting vector ( y1 coding exons = black boxes ). the disrupting cassette is indicated . bottom , restriction map of the resulting targeted allele ( b - bamhi sp - spei ; e - ecori ; p - paci ; pr , probe used in the southern blots ). b , southern blot analysis of es cells . c , pcr genotyping of wild - type , y1 +/− and y1 −/− mice . d , northern blot analysis of total brain rna of y1 ++ and y1 −/− mice losing a y1 probe ( y1 pr ) or lacz probe ( lacz pr ). probes used are underlined in red in ( a ). e , a transverse section from the spinal cord lumbar enlargement of y1 −/− mice histochemically stained for β - galactosidase . f , immunohistochemical staining of y1 −/− mice for β - galactosidase - positive nerve terminals and neurons ( arrows ) in the spinal cord dorsal horn ( green ) and the lectin ib4 ( red , layer iiinner ). g , double staining of l4 dorsal root ganglion for β - galactosidase ( green ) and ib4 ( red ). h , double staining of l4 dorsal root ganglion for β - galactosidase ( green , single stained neurons = arrows ) and sp ( red ). double stained neurons are shown by arrowheads . i , sp receptor distribution in the dorsal horn of wild - type mice . j , sp receptor distribution in dorsal horn of y1 −/− mice . k , sp receptor staining in lamina i of the contralateral vehicle injected side of y1 −/− mice . l , loss of cell surface and increase of intracellular sp receptor immunoreactivity in lamina i ten minutes after capsaicin injection into the hindpaw of y1 −/− mice . scale bar in ( e ) is 300 μm , in ( f ), ( i ) and ( j ) 80 μm , in ( g ) and ( h ) 30 μm , in ( k ) and ( l ) 20 μm . [ 0034 ] fig2 . cutaneous and visceral nociception of wild - type ( black bars ) and y1 −/− ( white bars ) mice in the hot - plate , tail - flick , formalin , acetic acid , mgso 4 , von frey hair and in neuropathic pain assays as well as in stress and npy produced analgesia . a , latency to shaking of hind - paw or jumping . b tail - flick latency . c , mechanical threshold assayed by von frey hairs . d , measurement of the number of events ( lifting , shaking , licking and biting of the injected paw ) in the formalin assay . the numbers on the x - axis indicate the concentration in percent of formalin administered subcutaneously . e and f , visceral pain response ( abdominal stretching ) produced by intraperitoneal injection of diluted acetic acid ( e ), or mgso 4 ( t ). g , stress - induced analgesia in the hot plate assay . l , development of mechanical allodynia of wild - type and y1 −/− mice in a chronic pain model . i , analgesic response to tail - flick following an intrathecal injection of npy . data are presented as % analgesia . all data are mean ± sem and statistical analysis was performed by unpaired student &# 39 ; s t - test ( a - g and i ) or two - tailed maru whitney u - test ( h ). *, p & lt ; 0 . 05 ; ** p & lt ; 0 . 01 ; ***, p & lt ; 0 . 001 . [ 0035 ] fig3 . neurogenic and non - neurogenic inflammation in wild - type and y1 −/− mice . a , paws of wild - type and y1 −/− mice 30 min after injection of capsaicin ( neurogenic inflammation ) or vehicle . b , quantification of evans blue extravasation after capsaicin or vehicle injection . c , percentage of paw diameter increase of vehicle and capsaicin injected paws . d , mechanical sensitisation before and after capsaicin - induced inflammation . e and f , evans blue extravasation ( e ) and paw diameter ( f ) 4 hours after carrageenan ( non - neurogenic ) induced inflammation in the wild - type and y1 −/− mice as indicated . g , mechanical sensitisation 3 h after carrageenan induced inflammation . in , quantification of evans blue extravasation of the paws 30 min after mustard oil administration . i , quantification of evans blue extravasation 30 min after vehicle or sp administration . in all experiments open bars are vehicle control side and black bars the experimental side . all data are mean ± sem . statistical analysis was performed by unpaired student &# 39 ; s t - test . *, p & lt ; 0 . 05 ; **, p & lt ; 0 . 01 ; ***, p & lt ; 0 . 001 . [ 0036 ] fig4 . measurement of sp release by capsaicin administration in the skin by eia and effects of y1 agonist and antagonist in inflammation - induced plasma extravasation . a , released sp in vehicle and capsaicin injected skin . b , evans blue extravasation 30 min after npy y1 receptor agonist [ leu 31 - pro 34 ]- npy or vehicle injection intraplantarly . c , capsaicin - induced evans blue extravasation in wild - type mice in the presence or absence of npy y1 receptor selective antagonist bibp 3226 . in all experiments open bars are the vehicle control side and black bars the experimental side . all data are mean ± sem and statistical analysis was performed by unpaired student &# 39 ; s t - test . *, p & lt ; 0 05 ; **, p & lt ; 0 . 01 ; ***, p & lt ; 0 . 001 . 1 . munglani , r ., hudspith , m . j . & amp ; hunt , s . p . the therapeutic potential of neuropeptide y . analgesic , anxiolytic and antihypertensive . drugs 52 , 371 - 389 ( 1996 ). 2 . hua , x y . et al . the antinociceptive effects of spinally administered neuropeptide y in the rat : systematic studies on structure - activity relationship . j pharmacol exp ther 258 , 243 - 248 ( 1991 ). 3 . duggan , a . w ., hope , p . j . & amp ; 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