Patent Application: US-201213613741-A

Abstract:
the present invention relates to the treatment and / or prevention of aortic valve stenosis and valve mineralization . particularly , the invention provides a target for intervention in the treatment or prevention of avs through the administration of the p2y 2 receptor agonists . also , the invention provides means to treat hypertension related to decreased arterial compliance and vascular calcification .

Description:
as : aortic stenosis ; cavd : calcific aortic valve disease ; vic : valve interstitial cells ; vsmc : vascular smooth muscle cells . the term “ prevent ” or “ prevention ” as used herein means the prevention of the disease as well as prevention of aggravation of symptoms . fig1 represents immunohistochemistry ( fig1 a ); q - pcr analyses ( fig1 b ); and transcript analyses ( fig1 c ) of the p2y 2 receptor in as / cavd valves . fig2 shows a functional assay with suramin , a purinergic receptor antagonist ( fig2 a ); treatment of isolated cells with 2 - thioutp ( a p2y 2 agonist ) ( fig2 b ); and expression levels of bone transcription factor , runx2 and osteocalcin ( fig2 c ) following treatment with 2 - thioutp . particularly , with respect to the different embodiments of the present invention such as methods and use , the mammal is particularly a human . particularly , with respect to the methods , use and compounds of the present invention , the test compound is particularly a competitive agonist of p2y 2 receptor activity , more particularly the agonist compound is an utp analog . with respect to the method of treatment and use of the invention , most particularly , the agonist is 2 - thioutp . particularly , with respect to the assay of the present invention , the 2 - thioutp may be labeled in such as way as to enable its detection by spectroscopic , photochemical , biochemical , immunochemical , chemical , or other physical means . for example , useful labels include fluorescent dyes , electron - dense reagents , enzyme substrates ( e . g ., as commonly used in an elisa ), biotin , digoxigenin , co - factors , ligands , chemiluminescent agents , radioisotope , fluorophores , colorimetric haptens , enzymatic label , and combinations thereof or other entities which can be made detectable , such that the compound bound to the label or marker can be determined by detecting the labeled marker compound in a complex . for example , compounds ( e . g ., label substrates ) can be labeled with 131 i , 125 i , 35 s , 14 c , or 3 h , either directly or indirectly , and the radioisotope detected by direct counting of radioemission or by scintillation counting . alternatively , assay components can be enzymatically labeled with , for example , horseradish peroxidase , alkaline phosphatase , beta - galactosidase or luciferase , and the enzymatic label detected by determination of conversion of an appropriate substrate to product . labels can also be proteins with luminescent properties , e . g ., green fluorescent protein and phycoerythrin . methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in sambrook et al . ( molecular cloning : a laboratory manual , cold spring harbor , n . y ., 1989 ) and ausubel et al . ( in current protocols in molecular biology , john wiley & amp ; sons , new york , 1998 ). alternatively , the assay may evaluate the activity of p2y 2 receptor agonists by way of decreased expression of runx2 or ostecalcin , particularly in a cell . the invention further provides an assay for identifying an agonist of p2y 2 receptor , comprising the steps of : a ) contacting a cell comprising a p2y 2 receptor with a purinergic receptor antagonist ; b ) measuring an amount of calcium in a cell culture from step a ) treated with a mineralizing medium ; c ) contacting the cell culture from step b ) with a test compound ; and d ) measuring calcium in cell culture treated in c ); whereby a decrease in cell culture calcium in d ) compared to b ) is an indication that said test compound is a potential agonist of p2y 2 receptor . cavd valves and control non - calcified valves were obtained . vics were isolated from control non calcified aortic valves . the protocol was approved by local ethical committee and informed consent has been obtained from the subjects . immunostaining analyses were performed with the following antibody : p2y 2 ( santa cruz biotechnology , usa ). slides were then incubated with envision dual link system - hrp , followed by aec substrate ( dako , carpinteria , calif ., usa ). rna was also extracted from as valves and cells during in vitro experiments . total rna was isolated with rneasy micro kit from qiagen ( qiagen , mississauga , ont , canada ). the rna extraction protocol was performed according to manufacturer &# 39 ; s instructions using 100 mg of tissue . the quality of total rna was monitored by capillary electrophoresis ( experion , biorad , mississauga , ont , canada ). four ( 4 ) μg of rna was reverse transcribed using the quantitec reverse transcription kit from qiagen . quantitative real - time pcr ( q - pcr ) was performed with quantitec sybr green pcr kit from qiagen in the rotor - gene 6000 system ( corbett robotics inc , san francisco , calif ., usa ). the following primers were obtained from ( invitrogen , burlington , ont , canada ): p2y 2 , runx2 , osteocalcin . the expression of hypoxanthine - guanine phosphoribosyltransferase ( hprt ) ( invitrogen , burlington , on , canada ) as a reference gene was chosen to normalize the results . in isolated cells , calcium was measured by the arsenazo iii method ( côte n , et al . j mol cell cardiol 2012 ; 52 : 1191 - 202 ) and results reported as percent changes . human valve interstitial cells ( vics ) were isolated by collagenase digestion . to provoke calcification , cells were incubated for 7 days with a pro - calcifying medium containing : dmem + 5 % fbs , 10 − 7 m insulin , 50 μg / ml ascorbic acid and nah 2 po 4 at 2 mm . p2y 2 receptor was documented in as / cavd valves by immunohistochemistry , where it was localized near mineralized areas , suggesting that it may have some connections with the calcifying process ( fig1 a ). the presence of p2y 2 receptor was then confirmed in as / cavd valves by q - pcr analyses ( fig1 b ). in isolated human vics we also found the presence transcripts for p2y 2 ( fig1 c ). in functional assay , suramin , a purinergic receptor antagonist , increased calcification of isolated vics suggesting that purinergic receptor ( s ) are involved in the control of the mineralizing process of the aortic valve ( fig2 a ). on the other hand , a treatment of isolated cells with a p2y 2 agonist , 2 - thioutp , reduced significantly calcification of isolated vics ( fig2 b ). of interest , the treatment with 2 - thioutp decreased the expression of the bone transcription factor , runx2 , as well as its downstream target , osteocalcin ( fig2 c ). when taken together , these facts indicate that p2y 2 may represent an important target in the treatment of as . calcific aortic valve disease ( cavd ) is the most frequent heart valve disorder . it is a progressive disorder related to the mineralization of the valvular tissue . 3 in this regard , progressive mineralization of the aortic valve causes in the initial stages no obstruction to the blood flow , and is often referred to as aortic sclerosis . 4 however , aortic sclerosis may progress to as , which is the resultant of an ongoing and constant mineralization of the valvular tissue . so far , there is no medical treatment that is available for the treatment of cavd / as . in the present work we demonstrate that p2y 2 receptor is a potential target in the treatment of cavd / as and / or cardiovascular calcification . to this effect , 2 - thioutp or other agonist molecules of the p2y 2 receptor may be used for the treatment of cavd / as , valve calcification and / or hypertension caused by vascular calcification . mineralization of the aortic valve is an active process . studies have shown that calcification of the aortic valve is associated with the expression of several osteoblastic genes . 3 it should be pointed out that runx2 is highly expressed during pathological mineralization and is considered as a key transcription factor that mediate osteoblastic differentiation of vics . 1 noteworthy , treatment of aortic vics with p2y 2 agonist prevented the rise of runx2 and its downstream target osteocalcin . it is thus likely that stimulation of p2y 2 receptor in the aortic valve prevents the phenotypic switch of resident cells toward bone - forming cells . in conclusion , we present herein evidence that 2 - thioutp and other related compounds that are agonists of the p2y2 receptor are potential pharmaceutical agents that could be used in the treatment and / or prevention of cavd / as , valve calcification and / or hypertension caused by vascular calcification . 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