Patent Application: US-74207103-A

Abstract:
the present invention is related to an inhibitor of the inositol polyphosphate 5 - phosphatase ship2 protein or its encoding nucleotide sequence identified by seq id no . 1 or of ship2 mrna expression . the present invention is also related to a pharmaceutical composition comprising said inhibitor and an adequate pharmaceutically acceptable carrier or diluent and to a non - human knock - out mammal comprising homozygously or heterozygously a partial or total deletion in its genome of the inositol polyphoshate 5 - phosphatase ship2 nucleotide sequence .

Description:
in order to characterize ship2 molecule functions in vivo , the inventors have generated and analysed a mouse strain deficient from the ship2 gene according to the following preferred method . the targeting vector was constructed by replacing a 7 . 3 kb genomic fragment containing exons 19 - 29 and the polyadenylation signal of the mouse ship2 gene by a neomycine resistance cassette ( schurmans et al . ( 1999 )). the cassette was flanked by a 4 . 0 kb and a 5 . 5 kb mouse genomic dna fragments at the 5 ′ and 3 ′ regions , respectively . r1 es cells were electroporated with the targeting plasmid linearized by sali . homologous recombination at the ship2 locus was confirmed by southern blotting , and ship2 +/− es cells were aggregated with morulae derived from cd1 mice . the resulting chimaeric mice transmitted the mutant allele to the progeny . for genotyping , southern analysis was performed with specific probes ( described in fig1 a ) or by polymerase chain reaction using specific primers to amplify the neo gene and a specific exon deleted in the mutant allele . messenger rna was extracted from mouse embryonic fibroblasts ( mef ) using a fasttrack kit ( invitrogen ), and total rna was purified from newborn liver and from adult skeletal and cardiac muscles using the rneasy mini kit ( qiagen ). 2 μg / lane of mrna or 20 μg / lane of total rna were loaded on a 1 % agarose gel and transferred to a nylon membrane . the membranes were hybridized with mouse cdna fragments coding for ship2 , tat - 5 ′, c / ebp □, c / ebp □, aldolase b , actin or g - 6 - pase as probes . antisense oligonucleotide probes were used for pepck mrna ( 5 ′- cagaccattatgcagctgaggaggcatt - 3 ′) and 18s rna ( 5 ′- gtgcgtacttagacatgcatg - 3 ′) detection ( lee et al . ( 1997 )). supernatants of mef homogenates isolated from ship2 +/+ , ship2 +/− and ship2 −/− day 13 . 5 embryos were analyzed by western blot . proteins ( 100 μg / lane ) were separated by sds - page and transferred to nitrocellulose sheets . saturation , incubation with rabbit anti - ship2 antiserum and ecl detection were performed as described ( bruyns et al . ( 1999 )). for in vivo glut4 expression , ship2 +/+ and ship2 +/− mice ( 6 - 10 week - old ) were overnight fasted , anaesthetized and intravenously injected or not with 1 mu / g ( body weight ) insulin . after 5 min , the skeletal muscles from the hind limbs were removed . a plasma membrane - rich fraction and a total lysate were prepared from myocytes as described ( simpson et al . ( 1983 ), higaki et al . ( 1999 )). aliquots of proteins ( 100 μg ) were separated by sds - page and blotted with anti - glut4 antibody . immune complexes were detected by using 125 i protein - a . ship2 +/+ and ship2 +/− mice ( 6 - 10 week - old ) were fasted for more than 12 h and intraperitoneally injected with either 1 . 5 mg / g ( body weight ) d - glucose or 1 mu / g ( body weight ) insulin ( actrapid ™, novo nordisc , denmark ). blood samples were drawn from the retro - orbital sinus at different time points . blood glucose concentration was determined enzymatically . plasma insulin levels were determined using a rat insulin elisa kit ™ ( mercodia ab , uppsala , sweden ). newborns received either repeated intraperitoneal injections of d - glucose ( 7 %, 100 μl per injection ) during the first 24 hours after birth , or a single intraperitoneal injection of a guinea pig anti - porcine insulin polyclonal ab ( 1 / 800 in 0 . 9 % nacl , 100 μl ; ref ab1295 ™, chemicon int inc , temecula , calif .) within the first postnatal hour . the neutralizing effect of this anti - insulin antibody was demonstrated after injection in adult mice loaded with glucose : significantly increased blood glucose levels were observed after 30 and 60 min , as compared to non - treated or normal guinea pig serum - treated mice . pancreatic islets ( malaisse et al . ( 1984 )), prepared by the collagenase procedure from the pancreas of 3 - 4 mice , were incubated in groups of 8 islets each for 90 min at 37 ° c . in 1 . 0 ml of a hepes - and bicarbonate - buffered medium containing 5 mg / ml bovine serum albumin and , as required , 11 . 1 mm d - glucose . the insulin released in the medium was measured by radioimmunoassay . the 2 soleus muscles were rapidly isolated from overnight fasted ship2 +/+ or ship2 +/− mice , and tied to stainless steel clips by the tendons ( stenbit et al . ( 1996 )). all incubations were carried out at 37 ° c . under an atmosphere of 95 % o 2 : 5 % co 2 in 1 ml of krebs - ringer bicarbonate buffer ( ph 7 . 35 ) supplemented with 1 % bovine serum albumin ( fraction v , ph 7 , intergen ) and 2 mm pyruvate . following a 15 min preincubation without insulin , muscles were incubated for 60 min without or with the indicated concentrations of insulin in the same medium without pyruvate but with 3 -[ 3 h ]- glucose ( 5 mm , 1 μci / ml ). upon completion , muscles were dissolved in 1n naoh , and aliquots of the alkaline solution were spotted onto whatmann papers . papers were dropped into ice - cold 60 % ethanol , and washed extensively ( three washes of 20 min each ) in 60 % ethanol before counting . all results were expressed per mg muscle protein ( determined by the well - known pierce assay ). after electroporation of embryonic stem ( es ) cells with the targeting vector , the inventors have used southern blotting and two different probes and restriction enzymes in order to identify recombinant clones with a 7 . 3 kb genomic dna deletion on one allele of the ship2 gene ( fig1 a , b ). crossing of chimeric males with c57bl / 6 females resulted in f1 heterozygous ( ship2 +/− ) mice that have no obvious abnormalities . the body weight at 8 weeks , life expectancy and spontaneous tumor incidence were not significantly different from wild - type ship2 +/+ mice . f1 ship2 +/− mice were then intercrossed and 182 viable offspring were genotyped at birth ; of these , 47 ( 26 %) were ship2 30 /+ , 94 ( 51 %) ship2 30 /− and 41 ( 22 %) ship2 −/− . these frequencies were within mendelian expectations for transmission of an autosomal gene , and suggest that disruption of both ship2 alleles does not cause embryonic lethality . day 13 . 5 embryo - derived mouse embryonic fibroblasts ( mef ) were analyzed to confirm the reduced levels and the complete absence of ship2 mrna ( fig1 c ) or protein ( fig1 d ) in ship2 +/− and ship2 −/− mice , respectively . at birth , ship2 −/− mice were phenotypically indistinguishable from their littermates ; most of them were able to feed , although progressively less efficiently than ship2 +/+ or ship2 +/− mice . close monitoring revealed that ship2 −/− mice became progressively cyanotic or pale and lethargic within the first 24 hours of life . some newborn ship2 −/− pups presented signs of respiratory distress and all failed to gain weight ( ship2 +/+ : 1 . 60 ± 0 . 02 g , ship2 +/− : 1 . 63 ± 0 . 04 g and ship2 −/− : 1 . 20 ± 0 . 01 g for a typical litter of 10 newborns , 18 hours after birth ; student t test , p & lt ; 0 . 005 ), and died within 3 days after birth . the cause of the respiratory distress observed in some of the ship2 −/− mice was not a lack of surfactant or an abnormal differentiation of surfactant - producing cells , since the expression of surfactant - associated proteins ( sp - a , sp - b and sp - c ) and of ttf - 1 or c / ebp transcription factors was normal in the lungs of ship2 −/− mice . moreover , hematoxylin and eosin staining of ship2 −/− lung , as well as of brain , heart , thymus , liver , stomach , pancreas , kidney , skin , muscle , spleen , bladder , and small and large intestines sections revealed no particular abnormalities . in newborns , severe hypoglycaemia is often associated with cyanotic episodes , apnea , respiratory distress , refusal to feed and somnolence . no difference in blood glucose concentrations among the different genotypes was observed 2 and 8 hours after delivery . however , when analyzed between 10 to 15 hours postpartum , blood glucose concentration in ship2 −/− mice was significantly lower than in ship2 +/− and ship2 +/+ mice ( fig2 a ; student t test , p & lt ; 10 − 6 ). hypoglycaemia was not due to glycosuria nor to an excessive secretion of insulin by the pancreatic beta cells : plasma insulin levels were significantly lower in ship2 −/− mice than in ship2 +/− and ship2 +/+ mice ( fig2 a ; student t test , p & lt ; 0 . 05 ). histology and immunohistochemistry of pancreatic islets using anti - insulin , - glucagon and - somatostatin antibodies did not reveal any abnormalities of antigen expression and distribution in ship2 −/− mice . to test whether the hypoglycaemia was the cause of postnatal death , ship2 −/− mice were injected with d - glucose . hypoglycaemic ship2 −/− mice were transiently rescued for up to 96 hours by repeated injections of d - glucose during the first 24 hours postpartum ( fig2 b ). about ten minutes after each injection , ship2 −/− newborns recovered a normal skin colour and were less lethargic than non - injected mice . prolonged survival was also observed when ship2 −/− mice were injected with a neutralizing antibody to insulin within the first hour after birth ( fig2 b ). in addition , anti - insulin ab injection of ship2 −/− mice significantly increased glucose concentrations at 10 - 15 hours postpartum as compared to non - injected mice ( fig2 a , student t test , p & lt ; 0 . 0002 ). these data indicate that hypoglycaemia in ship2 −/− mice is not caused by an increased production of insulin or decreased glucagon levels , but rather results from an increased sensitivity to insulin . the liver plays a major role in glucose homeostasis at birth : it provides glucose to the blood via gluconeogenesis . during that critical period , transcription of several hepatic enzymes involved in gluconeogenesis is activated in response to hormonal and dietary conditions . interfering with glucagon or insulin signalling cascades at or just before birth results in delayed or premature appearance of these enzymes , and in neonatal hypoglycaemia or diabetes . expression of phosphoenolpyruvate carboxykinase ( pepck ), a key enzyme of gluconeogenesis , was very low or absent in liver of ship2 −/− mice , as compared to ship2 +/+ mice ( fig2 c ). injection of ship2 −/− mice with a neutralizing anti - insulin ab restored a normal expression of pepck mrna ( fig2 c ). expression of hepatic tyrosine aminotransferase ( tat - 5 ′) and glucose - 6 - phosphatase ( g - 6 - pase ), two other gluconeogenic enzymes also induced after birth , were also decreased , albeit to a lesser extend than pepck ( fig2 c ). levels of c / ebp , c / ebp and aldolase b mrna were unaffected by the mutation . thus , the absence of ship2 leads to decreased expression of several key gluconeogenic enzymes , contributing to hypoglycaemia in newborn ship2 −/− mice . importantly , despite the low insulin levels found in mutant mice , the expression of pepck was induced when insulin is neutralized early after birth . taken together , these data indicate that ship2 −/− liver cells have enhanced sensitivity to insulin in vivo . since ship2 is a critical negative regulator of insulin sensitivity in vivo , and since loss of ship2 leads to lethal hypoglycaemia in newborn mice , it was investigated whether decreased amounts of ship2 expression in ship2 +/− mice ( fig1 c , 1 d , 3 c ) would alter insulin sensitivity . there was no significant difference in basal blood glucose or plasma insulin levels between adult ship2 +/+ and ship2 +/− mice , either after an overnight fasting ( fig3 a ), or when freely fed ( glucose levels in freely fed mice : 9 . 8 ± 0 . 5 mm versus 8 . 8 ± 0 . 2 mm in ship2 +/+ and ship2 +/− mice , respectively ; insulin levels in freely fed mice : 1 . 17 ± 0 . 29 μg / l versus 0 . 96 ± 0 . 16 μg / l in ship2 +/+ and ship2 +/− mice , respectively ). however , injection of d - glucose resulted in a more rapid glucose clearance in ship2 +/− than in ship2 +/+ mice : glycaemia was significantly lower at all time points in ship2 +/− mice ( fig3 a ). the increased glucose clearance in ship2 +/− mice was not a consequence of an increased release of insulin : thirty minutes after glucose administration , insulin levels were also significantly lower in ship2 +/− than in wild - type mice ( fig3 a ). moreover , the output of insulin from isolated pancreatic islets in response to glucose was not significantly different in ship2 +/+ and ship2 +/− mice . insulin hypersensitivity was demonstrated when mice were injected with insulin , that is , a significantly more profound hypoglycaemia was observed by 30 and 60 min after injection in ship2 +/− mice than in ship2 +/+ mice ( fig3 b ). insulin stimulates glucose transport and storage of glucose as glycogen into skeletal muscles through the translocation of the glut4 glucose transporter from intracellular stores to the cell surface ( czech et al . ( 1999 )), and glycogen synthase activation . loss of one ship2 allele resulted in reduced ship2 mrna and protein expression in skeletal muscle cells ( fig3 c ). after an overnight fasting , the amount of glut4 transporter in the myocyte plasma membrane fraction was low but similar in ship2 +/+ and ship2 +/− mice ( fig3 d ). however , when ship2 +/+ and ship2 +/− mice were loaded with insulin , plasma membrane glut4 levels were higher in ship2 +/− skeletal muscles , consistent with the increased glucose clearance found in these mice . glycogen synthesis in response to insulin stimulation , which reflects both glucose uptake and glycogen synthase activation , was analyzed in isolated soleus muscles from ship2 +/− and ship2 +/+ mice ( fig3 e ). in basal condition or in the presence of maximally stimulating insulin concentrations ( 2 and 50 nm ), a similar glycogen synthesis was observed in ship2 +/+ and ship 2 +/− muscles . however , stimulation with lower , physiologic insulin concentrations ( 0 . 1 or 0 . 3 nm ) resulted in significantly higher glycogen synthesis in ship2 +/− muscles than in ship2 +/+ muscles ( fig3 e ). when expressed in percent of maximal insulin effect , the insulin dose response curve was shifted towards the lower insulin concentrations in ship2 +/− mice , as compared to ship2 +/+ mice ( fig3 e ). together , data indicate that insulin sensitivity is significantly increased in skeletal muscles from heterozygous mice expressing only reduced amount of ship2 protein . the incidence of adult onset diabetes mellitus has dramatically increased and the disease is a major health care problem . resistance to the stimulatory effect of insulin on glucose utilization is a key pathogenic feature of most forms of adult onset ( type ii , or non - insulin dependent ) diabetes , and contributes to the morbidity associated with autoimmune ( type i , or insulin - dependent ) diabetes . it is crucial to better understand the molecular mechanisms that regulate insulin signaling . data in genetically modified mice identify ship2 as a critical and essential negative regulator of insulin signaling and insulin sensitivity in vivo . thus , ship2 is a novel therapeutic target for the treatment of type ii diabetes , and a candidate gene predisposing to the same disease . 1 . pesesse x . et al ., biochemical and biophysical research communications 239 , 697 - 700 ( 1997 ). 2 . habib , t . et al ., j . biol . chem . 273 , 18605 - 18609 ( 1998 ). 3 . pesesse x . et al ., febs letters 437 , 301 - 303 ( 1998 ). 5 . ishihara , h . et al ., biochem . biophys . res . commun . 260 , 265 - 272 ( 1999 ). 8 . czech , m . & amp ; corvera , s ., j . biol . chem . 274 , 1865 - 1868 ( 1999 ). 9 . lee , y .- h . et al ., mol . cell . biol . 17 , 6014 - 6022 ( 1997 ). 10 . bruyns , c . et al ., biol . chem . 380 , 969 - 974 ( 1999 ). 11 . simpson , i . a . et al . biochim . biophys . acta 763 , 393 - 407 ( 1983 ). 12 . higaki , y . et al ., j . biol . chem . 274 , 20791 - 20795 ( 1999 ). 13 . malaisse - lagae , f . & amp ; malaisse w . j ., in : lamer j ., pohl s . l . 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