Patent Application: US-201615288204-A

Abstract:
pharmaceutical compositions containing p2x purinergic agonists , e . g . p2x3 agonists , for increasing insulin secretion in a subject , methods of use , and methods of screening for related compounds and agents .

Description:
islet isolation . islets were isolated as previously described ( 57 ). monkey islets were isolated from cynomolgus monkeys ( macacca fascicularis ) & gt ; 4 years of age at the time of pancreas procurement , as previously described ( 58 ). pig pancreata were procured from the local slaughterhouse . mice ( c57bl / 6 ) and rat ( lewis rat ; harlan ) islets were isolated using a rodent - islet isolation technique ( 59 ). all animal protocols were approved by the university of miami care and use committee . human pancreatic islets were obtained from the human islet cell processing facility at the diabetes research institute , university of miami miller school of medicine or from the islet cell resource basic science islet distribution program , islet cell resource centers ( icrs ) consortium , division of clinical research , national center for research resources , national institutes of health . human islets were dissociated into single cells using enzyme - free cell dissociation buffer ( invitrogen ). islets and islets cells from q : 1 all species were cultured identically ( 37 ° c . and 5 % co 2 ) incmrl q : 2 medium - 1066 ( invitrogen ), niacinamide ( 10 mm ; sigma ), its ( bd biosciences ), zn 2 so 4 ( 15 μm , sigma ), glutamax ( 2 mm ; invitrogen ), hepes ( 25 mm ; sigma ), fbs ( 10 %; invitrogen ), and penicillinstreptomycin ( 100 iu / ml - 100 μg / ml ; invitrogen ). [ ca 2 + ] i ; imaging . [ ca 2 + ] i imaging was performed as previously described ( 8 , 36 ). dispersed islet cells were immersed in hepes - buffered solution ( 125 mmnacl , 5 . 9 mmkcl , 2 . 56 mmcacl 2 , 1 mmmgcl 2 , 25 mmhepes , and 0 . 1 % bsa , ph7 . 4 ). glucose was added to give a final concentration of 3 mm . islets or dispersed islet cells were incubated in fura - 2 am ( 2 μm ; 1 h ) and placed in a closed small volume imaging chamber ( warner instruments ). stimuli were applied with the bathing solution . islets loaded with fura - 2 were excited alternatively at 340 and 380 nm with a monochromator light source ( cairn research optoscan monochromator ; cairn research ltd ). images were acquired with a hamamatsu camera ( hamamatsu ) attached to a zeiss axiovert 200 microscope ( carl zeiss ). changes in the 340 / 380 fluorescence emission ratio over time were analyzed in individual islets and dispersed cells using kinetic imaging aqm advance software ( kinetic imaging ). peak changes in the fluorescence ratio constituted the response amplitude . beta cells were distinguished from other endocrine cells by their [ ca 2 + ] i , responses to high glucose concentrations , and alpha cells were identified by their [ ca 2 + ] i responses to kainate ( glutamate receptor agonist ) ( 8 , 36 ). insulin and glucagon secretion . insulin and glucagon secretion were measured as previously described ( 8 , 36 ). a high - capacity automated perifusion system was developed to dynamically measure hormone secretion from pancreatic islets . a low pulsatility peristaltic pump pushed hepes - buffered solution ( 125 mm nacl , 5 . 9 mm kcl , 2 . 56 mm cacl 2 , 1 mm mgcl 2 , 25 mm hepes , and 0 . 1 % bsa , ph 7 . 4 at a perifusion rate of 100 μl / min ) through a column containing 100 pancreatic islets immobilized in bio - gel p - 4 gel ( biorad ). except when otherwise stated , glucose concentration was adjusted to 3 mm for all experiments . stimuli were applied with the perifusion buffer . the perifusate was collected in an automatic fraction collector designed for a 96 - well plate format . the columns containing the islets and the perifusion solutions were kept at 37 ° c ., and the perifusate in the collecting plate was kept at & lt ; 4 ° c . perifusates were collected every 1 min . hormone release in the perifusate was determined with the human or mouse endocrine lincoplex kit following manufacturer &# 39 ; s instructions ( lincoresearch ). human islet preparations varied considerably in their quality . thus , the magnitudes of the responses to different stimuli were compared with the same recording or using recordings from the same preparation . immunohistochemistry . sections ( 14 μm ) were incubated overnight with anti - p2x receptor antibodies ( 1 - 7 ; alomone labs ), anti - insulin antibodies ( 1 : 500 ; accurate chemical & amp ; scientific ), antiglucagon antibodies ( 1 : 4 , 000 ; sigma ), and / or antisomatostatin antibodies ( 1 : 1 , 000 ; accurate chemical & amp ; scientific ). as a negative control , purified peptide ( 50 μg ) was preincubated with purinergic receptor primary antibodies ( 1 μg ) for 1 h ( room temperature ). pancreatic sections containing islets were examined using a zeiss lsm 510 scanning confocal microscope ( viewed at magnifications × 20 and × 40 ). in situ hybridization . in situ hybridization using dig - labeled rnaq : 3 probes for mrna detection of human p2xrs ( 1 - 7 ) was performed as described ( 60 ). a total of 30 ng of dig - labeled probe was diluted in 150 μl of hybridization buffer , applied to the slides , and allowed to hybridize at 70 ° c . overnight . slides were then washed for 1 h at 70 ° c . in 0 . 2 ssc solution ( ambionq : 4 applied biosystems ) and incubated with alkaline phosphatase - conjugated sheep anti - dig antibody ( roche ) overnight at 4 ° c . alkaline phosphatase reaction was carried out in pva with 200q : 5 μl of mgcl 2 1m and 140 μl of nbt / bcip stock ( roche ). senseq : 6 strand probes were used as a negative control for each p2xr . immunofluorescence localization of antigens , double - labeled immunofluorescence , and confocal microscopy were carried out as previously described ( 60 ). antibodies used were mouse antiinsulin ( 1 / 1 , 000 ; sigma ), guinea pig antiglucagon ( 1 / 50 ; dako ), alexa fluor 488 - conjugated goat anti - mouse ( 1 / 400 ; molecular probes ), and alexa fluor 568 - conjugated goat anti - guinea pig ( 1 / 400 ; molecular probes ). dapi was used as nuclear counterstaining . hybridization and immunofluorescence signals were merged by digitally converting the chromogen signal into a color signal in rgb scale . the hybridization signal was pseudocolored in red . q : 7 this signal was then merged with the insulin signal ( green ). both transformations were done using photoshop . western blotting . immunoblot analysis was carried out by standard methods using the antibodies used for p2x immunohistochemistry ( 1 : 1 , 000 ). in control experiments , primary antibodies were incubated with corresponding control peptide ( alomone labs ) at a ratio of 50 pg antigenic peptide / 1 μg antibody at room temperature for 5 h . statistical analyses . for statistical comparisons , we used a student t test or a one - way anova followed by multiple comparison procedures with the bonferroni t test . throughout the application , data are presented as average ± sem . to infer the role of atp as an autocrine / paracrine signal , we manipulated atp degradation and thus , the concentration of endogenously released atp in isolated human islets and recorded changes in hormone secretion by using a perifusion assay of dynamic secretory responses ( 36 ). released atp is rapidly cleared by membrane ecto - atpase , such as apyrase , that converts atp into adenosine ( 37 , 38 ). ecto - atpases are crucial in the duration and magnitude of purinergic signaling ( 39 ). a functional apyrase ( cd39 ) has been shown to be expressed in human β cells ( 40 ). application of the apyrase inhibitor arl67156 ( 50 μm ) ( 41 , 42 ) increased basal insulin secretion from islets incubated at low glucose concentration ( 3 mm ; fig1 a and b ), revealing that human islet cells released atp . under these conditions , the endogenous ecto - atpases are fully effective , explaining why exogenously added apyrase ( 5 u / ml ) did not reduce basal insulin secretion ( fig1 a and b ). because atp is already released at low glucose concentrations and has the potential to evoke insulin secretion , we hypothesized that atp potentiates glucose - induced insulin secretion at early stages . accordingly , exogenously added apyrase ( 5 u / ml ), during a step increase in glucose concentration from 3 mm to 11 mm , reduced insulin release by 15 % ( fig1 c and d ), indicating that endogenously released atp contributed to the β - cell response . adding the competitive apyrase inhibitor arl67156 during glucose stimulation , however , did not amplify the β - cell response ( fig1 d ), suggesting that endogenously released atp was high enough to saturate its potentiating effect . hence , stimulating with exogenous atp while the glucose concentration was increased did not add to the insulin response . apyrase may decrease glucose - induced insulin release either by reducing extracellular atp or by increasing adenosine ; this may act on p1 receptors to inhibit insulin release ( 43 ). degrading adenosine with adenosine deaminase did not change the effect of apyrase on glucose - stimulated insulin secretion ( fig1 d ), indicating that the presence of adenosine did not contribute to the inhibition of the insulin response . accordingly , neither the p1 receptor antagonist cgs15943 ( 10 μm ) nor adenosine ( 100 μm ) altered glucose - induced insulin secretion ( discussion ). because nerves are severed and neuronal remnants that could be additional sources or targets for atp do not survive under our experimental conditions ( 32 , 44 ), the most likely interpretation is that atp secreted by β cells provides a positive autocrine feedback loop to amplify insulin secretion . to examine the receptors involved in this autocrine feedback loop , we blocked purinergic receptors with specific receptor antagonists during stimulation with an increase in glucose concentration from 3 mm to 11 mm ( fig2 a ). insulin secretory responses to glucose stimulation were reduced in the presence of suramin ( 50 μm ; a broad antagonist of p2 receptors ), iso - ppadsq : 9 ( 50 μm ; an antagonist for p2x1 , p2x2 , p2x3 , and p2x5 receptors ), and oxidized atp ( oatp ; 500 μm ; an antagonist for p2x2 , p2x3 , and p2x7 receptors ) by 40 %, 30 %, and 65 %, respectively ( fig2 b ). insulin secretory responses to glucose stimulation in the presence of the specific p2x1 antagonist mrs2159 ( 10 μm ) and the two p2x7 receptor antagonists brilliant blue g ( 1 μm ) and kn - 62 ( 1 μm ) were not significantly reduced ( fig2 b ). antagonists for p2y receptors [ reactive blue 2 ( 50 μm ) and mrs2179 ( 10 μm ); specific for the p2y1 receptor ; fig2 b )] or the p1 receptor [ cgs15943 ( 10 μm )] did not inhibit glucose induced insulin release . to determine the direct effects of purinergic receptor activation on insulin secretion , we applied exogenous atp and other agonists . in human islets , application of atp , the universal agonist of p2 purinergic receptors , stimulated increases in insulin release concentration dependently at low ( 3 mm ) and high glucose concentrations ( 11 mm ) with similar thresholds ( fig2 c ). the concentration response relationship showed a high affinity component (˜ 0 . 5 μm ) that compared well with the reported ec50 for the human p2x3 receptor (˜ 0 . 39 μm ) and a second increase between 100 and 1000 μm that might correspond to activation of p2x7 receptors ( ec 50 100 μm ) ( 45 ). increasing extracellular atp & gt ; 1 mm did not further raise insulin release ( fig2 c ). the insulin responses to atp showed similar increases above basal as the responses stimulated by glucose . compared with the increase elicited by atp ( 1 mm ), the response to high glucose ( 11 mm ) was 101 %± 30 % or almost identical . similar results were obtained using monkey islets . by contrast , neither atp nor any of the other purinergic agonists tested stimulated insulin release in pig , mouse , or rat islets ( fig2 c and fig . s2 ). in rat islets , only high concentrations of atp ( 1 mm ) induced small increases in insulin release ( fig2 c ). atpγs ( 50 μm ; a nonhydrolysable atp analog ), the specific p2x receptor agonist bzatp ( 50 μm ), and the p2x 1 and p2x 3 agonist α , β - methylene atp ( 50 μm ) elicited strong insulin responses ( fig2 d ). p2y receptors were not involved in the response to endogenously released atp during glucose stimulation but could be directly activated by the selective agonists utp ( 100 μm ; an agonist of p2y 2 , p 2 y 4 , and p2y 6 ) and adp ( 100 μm ; an agonist of p2y 1 , p2y 12 , and p2y 13 ) to increase insulin release ( fig2 d ), suggesting the presence of multiple atp receptor subtypes in the human β cell . adenosine had a minor effect on insulin release , indicating that p1 receptors were only modestly involved ( fig2 d ). the magnitudes of the insulin responses to atp ( 100 μm ), atpγs ( 50 μm ), bzatp ( 50 μm ), utp ( 100 μm ), and adp ( 100 μm ) in islets kept at high glucose ( 11 mm ) were similar to the magnitudes of insulin responses to these agonists that were recorded in islets kept at low glucose concentrations , indicating that the effects of purinergic receptor activation are not altered at higher glucose levels . our results suggest that human islets express p2x receptors with activation that strongly stimulates insulin secretion . by using rtpcr , we found that all p2x receptor genes were expressed in human islets , confirming results from the beta cell biology consortium database ( website : betacell . org / resources / data / epcondb /). to localize p2x receptor expression in the islet , we performed in situ hybridization on human pancreatic sections . strong hybridization signals in human islets were detected for p2x 3 , p2x 5 , and p2x 7 ( fig3 a ). by combining in situ hybridization with immunofluorescence for islet hormones , we found that these receptors were expressed in β cells ( fig3 a ). no signals could be detected with p2x 1 , p2x 2 , p2x 4 , p2x 6 , or control sense riboprobes . immunofluorescence and western blots further showed that the p2x3 protein was present in β cells ( fig3 b and c ). although p2x5 and p2x7 immunoreactivities were seen in islets , they could not be blocked by control peptide preadsorption . therefore , it was not possible to determine if the staining could be considered a reliable indication of p2x5 and p2x7 receptor protein expression . isolated human islet cells were examined for the presence of functional p2x receptors using measurements of [ ca 2 + ] i . beta cells , identified by their response to high glucose ( 11 or 16 mm ) ( 8 ), responded to atpγs ( 50 μm ) and bzatp ( 50 μm ) with rapid [ ca 2 + ] i increases ( fig3 d ). a fraction of the cells ( 30 %) that responded to the alpha cell - specific stimulus kainate ( 100 μm ) ( 8 ) responded to atpγs ( 50 μm ) or bzatp ( 50 μm ) with rapid [ ca 2 + ] i increases ( fig3 d ). in line with these results , atp stimulated small increases in glucagon secretion in human , monkey , and mouse islets , and a subset of human alpha cells expressed p2x4 receptors ( fig . s3 ). what are the mechanisms by which atp induces insulin secretion in human β cells ? insulin responses to atp ( 10 μm ) were inhibited by the general p2 receptor antagonist suramin ( 100 μm ) and the specific p2x antagonist iso - ppads ( 50 μm ; ˜ 95 % inhibition ; fig4 a ). in the nominal absence of extracellular ca2 +, insulin responses to atp ( fig4 b ) and α , β meatp ( 100 μm ) were strongly diminished . by contrast , blocking the contribution of ca 2 + release from intracellular stores with thapsigargin ( 1 μm ) had no effect on insulin responses to atp ( fig4 b ). the ca 2 + needed for atp - induced insulin secretion could enter through the p2x receptor pore or voltage - dependent ca 2 + channels , which are activated as a consequence of p2x receptor - mediated membrane depolarization . the broad - spectrum voltage - gated ca2 + channel blocker cd 2 + ( 100 μm ; a concentration not affecting ca 2 + influx through p2x receptors ) ( 46 , 47 ) and the l - type ca 2 + channel blocker nifedipine ( 10 μm ) abolished insulin responses to atp ( fig4 b ) or α , β meatp . that atp failed to increase insulin secretion in the presence of cd 2 + or nifedipine indicates that p2x receptor activation caused sufficient depolarization to activate voltage - dependent ca 2 + channels ( 15 , 17 , 47 ), particularly l - type ca 2 + channels critical to the potential firing in human β cells ( 48 ). atp and the p2x receptor agonists bzatp and α , β meatp elicited repeatable [ ca 2 + ] i responses in β cells that were comparable with responses to glucose or kcl stimulation ( fig4 c ). [ ca 2 + ] i responses to atp were blocked by isoppads by 80 % in human β cells ( fig4 c ). thapsigargin ( 1 μm ) did not affect [ ca 2 + ] i responses to atp , indicating little contribution of ca 2 + released from intracellular stores ( fig4 d ). the nominal absence of extracellular ca 2 + or the addition of nifedipine ( 1004 ) reduced [ ca 2 + ] i responses to atp ( fig4 d ), indicating a major ca 2 + influx through the β - cell plasma membrane . the results detailed above demonstrate that human β cells express receptors for extracellular atp to mediate an essential positive autocrine feedback loop for insulin secretion . we have presented evidence that this autocrine feedback loop is present in human and nonhuman primate islets but not in the other species that we examined . these results support the conclusion that , in primates , p2x receptors predominate in the atp ( purinergic ) signaling pathways , amplifying the secretion of insulin in response to rapid increases in glucose concentration ( fig5 ). our findings have revealed a signaling pathway for atp in human β cells . we have found that atp is already released at low glucose concentrations , which is in agreement with recent studies in rodents showing that atp can be released from secretory granules while insulin is retained ( 12 , 34 ). therefore , atp signaling may precede secretion of insulin , sensitizing the β cell to respond appropriately to glucose stimulation . this notion is in line with studies showing that atp facilitates neurotransmitter release in presynaptic nerve terminals ( 49 , 50 ). our results further suggest that atp release seems to be strongest during sharp increases in glucose concentration . although exogenous atp promoted strong responses in islets kept at constant glucose concentrations ( 3 mm or 11 mm ), it was not effective during abrupt increases in glucose concentration , indicating that the receptors were fully activated by endogenously released atp under these conditions . thus , we have demonstrated that atp is a signal serving in an autocrine positive feedback loop for insulin release subsequent to glucose stimulation . our results showing substantial differences between human β cells and rodent β cells in terms of atp signaling reiterate that the structure and function of the human islets are distinctive ( 31 , 32 ). our studies revealed that atp is a potent stimulator of insulin release in islets of primate species but not in those of the other examined species . because we used the same technical approach for all species tested , the most likely explanation is that the differences in purinergic signaling suggest that β cells of various species express different subsets of purinergic receptors . our results show that both p2x and p2y receptors can be activated in human β cells , but the responses mediated by p2x receptors predominate . in mice , atp elicits [ ca2 +] i responses in β cells predominantly through p2y receptors , not p2 × receptors ( 26 , 51 ). there are only a few studies examining the expression of p2x receptors in the endocrine pancreas of any species . recently , p2x1 and p2x3 receptors were identified in isolated single mouse β cells ( 30 ), and p2x1 , p2x2 , p2x3 , p2x4 , and p2x6 have been detected in the mouse and rat pancreas ( 28 , 52 , 53 ). without being bound to any theory of the mechanism of the invention , p2x3 receptors most likely contribute to shape the electric activity of human β cells . direct application of atp at 3 mm glucose elicited large insulin and [ ca 2 + ] i , responses that were comparable with those elicited by high glucose or kcl depolarization . blocking atp receptors with p2x receptor antagonists reduced the insulin response to high glucose by up to 65 % ( fig2 ), revealing a strong contribution of atp receptor activation to the response . our results further indicated that most of the human β - cell response to atp was mediated by ionotropic p2x receptors ( fig4 ). this activation promotes considerably large inward currents in the na range and thereby , depolarizes the β - cell membrane , which results in increased electric activity ( 30 ). however , the exact magnitude of the currents will depend on the amount of atp released , the receptor density , and / or their localization . by using a combination of technical approaches , we have consistently identified p2x3 receptors in human β cells . p2x1 , p2x2 , p2x4 , and p2x6 receptors , reported to be expressed in rodent β cells ( 28 , 30 , 52 , 53 ), could not be detected in human β cells . in contrast , our studies revealed the presence of p2x5 and p2x7 . therefore , p2x receptors in human β cells may exist as monomers or heteromers of combinations of p2x3 , p2x5 , and p2x7 . the presence of a polymorphism at a critical position in the human p2x5 gene indicates that only a small subset of humans (˜ 14 %) will process and translate a functional protein ( 54 , 55 ), ruling out a contribution of p2x5 to atp signaling in β cells in most human beings . p2x7 receptors are unlikely to form heteromeric receptors with p2x3 ( 17 ) but may work as homomeric receptors . homomeric p2x7 receptors , however , likely do not participate in normal β - 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