Patent Application: US-50841100-A

Abstract:
monohydroxamate vanadium complexes of the formula : r — co — nhoh . x , wherein r is a residue selected from : h 2 n — ch — n —, wherein n is 1 , 2 or 3 , and y is oh or nh 2 ; h 2 n — ch 2 — s — ch 2 —; and pyridyl , piperidyl or tetrahydroisoquinolinyl ; and x is a vanadium compound selected from a vanadyl , metavanadate or vanadate ) salt , are useful for inducing normoglycemia and / or reducing blood glucose levels in diabetic patients .

Description:
according to the present invention , a unique and specific potentiation of vanadium insulinomimetic potency is achieved by certain monohydroxamate complexants of vanadium . particularly , l - glutamic acid ( γ ) monohydroxamate ( glu ( γ ) hxm ) potentiates ≈ 7 - 10 fold vanadium (+ 5 ) in activating glucose metabolism in rat adipocytes , and elevates 5 - 7 - fold the efficacy of vanadate to lower blood glucose levels in stz - treated diabetic rats in vivo potentiation is maximal at a 2 : 1 molar ratio of l - glu ( γ ) hxm : vanadium . both nonmodified α - amino and α - carboxyl moieties of l - glu ( γ ) hxm are essential for the potentiation . moreover , the synergistic action of l - glu ( γ ) hxm is stereospecific and is not facilitated with d - glu ( γ ) hxm , although the latter also complexes with vanadium . interestingly enough , of all documented vanadium complexants that potentiate the insulinomimetic actions of vanadium , l - glu ( γ ) hxm is unique in the sense of activating lipogenesis in rat adipocytes in the absence of exogenously added vanadium as well . it is herein further established that this effect manifests through the vanadium pathway by experimental data indicating that l - glu ( γ ) hxm is capable of converting the minute physiological quantity of endogenously present vanadium in rat adipocytes into an insulinomimetic active species . chemophysical studies of this active complex , indicates a unique physicochemical features . vanadium is maintained at the + 5 oxidation state , at physiological ph value , at equilibrium also if prepared with the vanadyl + 4 cation . the in vitro screening assay used in the present invention indicates that , in addition to glu ( γ ) hxm , also l - asp ( β ) hxm and nicotinicacid - hxm ( at 1 : 1 molar ratio ) potentiate the insulinomimetic potency of vanadium (+ 4 ) as well . their synergizing effect is ˜ 85 % and ˜ 57 % of that exerted by glu ( γ ) hxm . in contrast , ( α - amino acid hydroxamates as well as the d - isomers of both glu ( γ ) hxm and asp ( β ) hxm , did not potentiate the insulinomimetic eficacy of vocl 2 . the invention will now be illustrated by the following non - limiting examples . ( a ) materials . d -[ u - 14 c ] glucose and 2deoxy - d -[ g - 3 h ] glucose were purchased from new england nuclear ( boston , mass .). collagenase type i ( 134 u / mg ) was obtained from worthington biochemicals ( freehold , n . j .). porcine insulin was purchased from eli lilly co . ( indianapolis , ind .). phloretin , 2 deoxyglucose , l - glutamic acid γ - monohydroxamate ( glu ( γ ) hxm ), glycine hydroxamate ( gly - hxm ), l - isoleucine hydroxamate ( isoleu - hxm ), l - tryptophan hydroxamate ( trp - hxm ), l - tyrosine hydroxamate ( tyr - hxm ) and l - cystine dihydroxamate ( cystine ( hxm 2 ) were purchased from sigma chemical co ., st . louis , mo . krebs - ringer bicarbonate ( krb ) buffer , ( ph 7 . 4 ), contained 110 mm nacl , 25 mm nahco 3 , 5 mm kcl , 1 . 2 mm kh 2 po 4 , 1 . 3 mm cacl 2 , 1 . 3 mm mgso 4 . all other chemicals and reagents used in his study were of analytical grade . ( b ) streptozocin ( stz )- treated rats : diabetes was induced by a single intravenous injection of a freshly prepared solution of streptozocin ( 55 mg / kg of body weight ) in 0 . 1 m citrate buffer ( ph 4 . 5 ). the effect of the tested compounds on blood glucose level was determined 14 days following induction of diabetes . ( c ) cell preparation and lipogenesis bioassay : rat adipocytes were prepared essentially by the method of rodbell , 1964 . the fat pads of male wistar rats were cut into small pieces with scissors and suspended in 3 ml of krb buffer . the digestion was performed with collagenase ( type 1 , 134 units / mg ; 1 mg / ml ) in a 25 - ml flexible plastic bottle under an atmosphere of carbogen ( 95 % o 2 , 5 % co 2 ) for 40 min at 37 ° c . with vigorous shaking . cell preparations showed more than 95 % viability by trypan blue exclusion , at least 3 hours after digestion . five ml of buffer was then added , and the cells were passed through a mesh screen . the cells were then allowed to stand for several minutes in a 15 - ml plastic test tube at room temperature , floating , and the buffer underneath was removed . this procedure ( suspension , floating , and removal of buffer underneath ) was repeated three times . in the lipogenic assay , for measurement of glucose uptake and its incorporation into lipids ( lipogenesis ), the adipocyte suspensions ( 3 × 10 5 cells / ml ) were divided into plastic vials ( 0 . 5 ml per vial ) and incubated for 60 min at 37 ° c . under an atmosphere of carbogen with 0 . 2 mm [ u - 14 c ] glucose ( 4 - 7 mci / mol ), in either the absence or presence of insulin ( 100 ng / ml ), and the complexes to be tested . lipogenesis was terminated by adding toluene - based scintillation fluid ( 1 . 0 ml per vial ) and the radioactivity in extracted lipids was counted ( moody et al ., 1974 ). in a typical experiment insulin - stimulated lipogenesis was 4 - 5 fold higher than basal . vbasal ≈ 2000 cpm per 3 × 10 5 cell / h ; vinsulin ≈ 8 , 000 - 10 , 000 cpm per 3 × 10 5 cell / h . in vitro potentiation of the lipogenic capacity of low concentrations of vocl 2 ( 10 mm ) by equimolar concentrations of monohydroxamates the following protocol was found to be a reliable assay in vitro , that is indicative for the potentiation effects of the amino acid monohydroxamates on vanadium (+ 4 ) in stz - rats in vivo . activation of lipogenesis was performed as described in experimental procedures , section ( c ), using 10 μm solutions of various free amino acid monohydroxamates ( hxm ), 10 μm solution of vocl 2 (+ 4 ) , or 10 μm solution of freshly prepared 1 : 1 complex of amino acid - hxm : vocl 2 . the following amino acid monohydroxamates were tested : l - glutamic acid γ - monohydroxamate [ glu ( γ ) hxm ], glycine hydroxamate ( gly - hxm ), l - isoleucine hydroxamate ( ile - hxm ), l - tryptophan hydroxamate ( trp - hxm ), l - tyrosine hydroxamate ( tyr - hxm ), l - cystine dihydroxamate [ cys ( hxm ) 2 ], l - lysine hydroxamate ( lys - hxm ), nicotinic acid hydroxamate ( nic - hxm ), l - arginine hydroxamate ( arg - hxm ), l - histidine hydroxamate ( his - hxm ), d - glutamic acid γ - monohydroxamate [ d - glu ( γ ) hxm ], n - acetyl - l - glutamic acid γ - monohydroxamate [ n - acetyl - glu ( γ ) hxm ], l - aspartic acid β - monohydroxamate [ asp ( β ) hxm ], aminoisobutyric acid monohydroxamate [ aib - hxm ]. the results are summarized in tables i and ii . as shown in table 1 , glu ( γ ) hxm ( 10 μm ), vocl 2 ( 10 mm ) or their 1 : 1 complex produced 22 %, 40 % and 117 %, respectively , of maximal insulin response . the net potentiating effect amounted therefore to 51 %. nic - hxm also potentiates the lipogenic capacity of vocl 2 ( 29 %, net potentiating effect table i ). other amino acid hydroxamates studied did not potentiate the effect of vanadium (+ 4 ). the same is valid for d - glu ( γ ) hxm and for n - acetyl - glu ( γ ) hxm , indicating that for glu ( γ ) hxm , the free α - amino group and the l - isomeric form are essential for the potentiation . insulinomimetic effect of various vocl 2 : hxm ( 1 : 1 ), in comparison to concentration - dependent in vitro activation of lipogenesis by a 1 : 1 to 1 : 5 complex of glu ( γ ) hxm : vocl 2 in comparison to free vocl 2 and free glu ( γ ) hxm in order to determine the most effective ratio of the glu ( γ ) hxm : vocl 2 complex to synergize the insulinominetic potency of vanadium , lipogenesis was performed as described in experimental procedures , section ( c ), using 1 : 1 to 5 : 1 complexes of glu ( γ ) hxm : vocl 2 , free vocl 2 , and free glu ( γ ) hxm . the results shown in fig1 demonstrate that , in comparison to the lipogenic potency of 5 mm vocl 2 alone and complexed with increasing concentrations of glu ( γ ) hxm ( 5 - 25 μm ), a 1 : 1 stoichiometric complex of both was found most effective in synergizing the insulinomimetic potency of vanadium (+ 4 ). effect of glu ( γ ) hxm : vocl 2 ( 1 : 1 complex ) on blood glucose levels ( bgl ) of stz - rats ; comparison to low vocl 2 alone in order to show that vanadium (+ 4 ) amino acid monohydroxamate chelators potentiate the normoglycemia effect of vanadium in vivo , stz - rats ( see experimental procedures , section ( b )), were given intraperitoneal ( i . p .) injections of either vocl 2 ( 0 . 02 mmol / kg / day ) or of vocl 2 : glu ( γ ) hxm 1 : 1 complex ( 0 . 02 mmol / kg rat / day ). blood glucose levels were measured over a period of 12 days . the results are depicted in fig2 showing that daily i . p . injections of a low dosage of vocl 2 by itself ( approximately 2 mg vanadium / kg / day ) had no significant effect on decreasing blood glucose levels , while the 1 : 1 complex of glu ( γ ) hxm : voci2 ( approximately 1 mg vanadium / kg / day ) was effective and produced a dramatic decrease in blood glucose levels of stz - rats toward normal values . stable normoglycemia has been achieved within 2 days after administrating the complex , and persisted for several days following administration ( fig2 dashed line represents blood glucose levels of control healthy rats ). the amount of free vanadium (+ 4 ) required to induce normoglycemia , is 9 . 3 mg / kg / day ( i . p . administration ). glu ( γ ) hxm : vocl 2 complex ( 1 : 1 ) reduced the daily dosage to about 1 mg / kg / day ( i . p . administration ). thus , in this in vivo stz - rat model , complexation of vanadium (+ 4 ) to glu ( γ ) hxm appears to potentiate vanadium (+ 4 ) about 9 - fold . monohydroxamates also synergize the insulinomimetic potency of vanadium (+ 5 ): l - glu ( γ ) hxm highly potentiates the vanadium (+ 5 ) to activate glucose metabolism in rat adipocytes in order to test the synergistic effect of glu ( γ ) hxm upon complexation with vanadate (+ 5 ), lipogenesis was performed as described in experimental procedures , section ( c ), with concentrations ranging from 10 μm to 50 μm of 1 : 1 complex of glu ( γ ) hxm : vo 4 + 3 (+ 5 ), free na 3 vo 4 and free glu ( γ ) hxm , respectively . unlike with the dihydroxamate chelator designated rl - 252 described in u . s . pat . no . 5 , 338 , 759 , that was found to potentiate the insulinomimetic capacity of vanadium (+ 4 ) but had no effect on , and even reduced , the potency of vanadium (+ 5 ) in vitro , glu ( γ ) hxm dramatically potentiated the insulin - like effect of vanadate (+ 5 ) ( fig3 ). it is estimated that glu ( γ ) hxm : vo 4 3 + (+ 5 ) is at least 7 - fold more potent in activating lipogenesis as compared to either free na 3 vo 4 or free glu ( γ ) hxm . in order to test the specific effect of free glu ( γ ) hxm and the glu ( γ ) hxm : navo 3 ( 2 : 1 ) complex on the entrance of glucose into cells , an in vitro assay was performed using 2 - deoxy - d -[ 6 - 3 h ] glucose ( 2 - dg ). 2 - dg is a non - metabolized analog of glucose and this assay thus represents the influence of a compound on glucose influx to cells , independent of glucose metabolism . freshly prepared adipocytes ( 3 × 10 5 cells / ml ) suspended in krb buffer ph 7 . 4 , containing 1 . 0 % bsa , were preincubated for 30 min , in the absence and the presence of insulin ( 17 nm ), and the indicated concentrations ( 20 and 40 μm ) of glu ( γ ) hxm , glu ( γ ) hxm : navo 3 ( 2 : 1 ) complex and navo 3 . aliquots ( 70 μl ) of the aforementioned samples were transferred into tubes that contained 2 - deoxy - d -[ 6 - 3 h ] glucose ( 0 . 1 mm final concentration ). after 3 minutes , phloretin ( 0 . 1 nm ) was added in order to terminate the penetration of 2 - dg into the cells . samples of the suspended cells were then transferred to tubes with silicone oil where , upon centrifugation , the cells separate from the krb medium and leftover 2 - dg . as shown in fig4 free glu ( γ ) hxm and glu ( γ ) hxm : navo 3 ( 2 : 1 ) complex was found to activate glucose entry into the cells independently of glucose metabolism . the magnitude of the effect amounted to about 60 % and 120 %, respectively , of maximal insulin response . glu ( γ ) hxm : navo 3 ( 2 : 1 ) complex lowers blood glucose levels in stz - treated rats in order to show that complexes of vanadium (+ 5 ) and monohydroxamate chelators potentiate the normoglycemic effect of vanadium in vivo and to test the normoglycemic effect of free glu ( γ ) hxm in vivo , stz - diabetic rats were divided into 4 groups of 4 - 5 rats each : diabetic control rats ; vanadate (+ 5 )- treated rats ; glu ( γ ) hxm : navo 3 ( 2 : 1 ) complex - treated rats ; and free glu ( γ ) hxm - treated rats . each group received daily i . p . injections of 0 . 05 mmol / kg ( at 11 . 00 a . m . ), of the corresponding compound . as shown in fig5 after the first day ( blood glucose level was measured at 8 . 00 a . m .) the blood glucose level of the complex - treated group reduced to normal levels . glu ( γ ) hxm activates lipogenesis in rat adipocytes in the absence of exogeneously added vanadium in order to investigate the normoglycemic potential of free glu ( γ ) hxm , lipogenesis was carried out as described above , using concentrations ranging between 10 - 100 μm of glu ( γ ) hxm . as shown in fig6 glu ( γ ) hxm is , among all vanadium binders tested herein , unique in the capacity to produce insulin effects in the absence of exogeneously added vanadium . it is assumed that l - glu ( γ ) hxm differs from all other amino acid - hxm in being capable of converting the minute quantity of intracellularly - located vanadium (˜ 20 nm ) into an insulinomimetically - active species . staurosporine , a potent inhibitor of rat - adipose cytptk ( ki ˜ 2 nm ) and a weak inhibitor of insrtk ( ki ˜ 1 μm ), preferentially inhibits the effect of vanadate in stimulating lipogenesis . in order to determine whether free glu ( γ ) hxm also works through the vanadium pathway , adipocytes were subjected to various concentrations of staurosporine ( as indicated in fig7 ) for 30 min at 37 ° c . lipogenesis was then carried out using adipocytes prepared as described above , in the presence of insulin ( 17 nm ), sodium metavanadate ( 0 . 8 mm ) or glu ( γ ) hxm ( 100 μm ). maximal activation ( 100 %) is that obtained with insulin , vanadate or glu ( γ ) hxm in the absence of staurosporine . fig7 shows the extent of lipogenesis evoked by free glu ( γ ) hxm , sodium metavanadate , or insulin at increasing concentrations of staurosporine . activation of lipogenesis by glu ( γ ) hxm was inhibited by staurosporine in a dose - dependent manner . the inhibition curve resembled that obtained for vanadate - ( rather than that for insulin -) evoked lipogenesis , indicating that free glu ( γ ) hxm works through vanadium ( insulin - independent ) pathway . lipogenesis : comparison between normal adipocytes and vanadiun - enriched adipocytes , treated with glu ( γ ) hxm free glu ( γ ) hxm is unique in the sense of activating lipogenesis in rat adipocytes in the absence of exogeneously added vanadium . it is herein established that this effect is manifest through the vanadium pathway by experimental data showing that free glu ( γ ) hxm is capable of converting the minute physiological quantity of endogeneously present vanadium in rat adipocytes into an insulinomimetically active species . to demonstrate this effect , male wistar rats received daily subcutaneous ( s . c .) injections of navo 3 ( 12 mg / kg / day ) for five days ( hereinafter designated “ enriched - vanadium rats ”). lipogenesis was performed as described above using free glu ( γ ) hxm to compare freshly prepared rat adipocytes ( 3 × 10 5 cells / ml ) from non - enriched vanadium rats to the enriched - vanadium rats . as shown in fig8 the free glu ( γ ) hxm was found to potentiate the effect of the intracellular vanadium in the enriched - vanadium rat cells to a much greater extent . to show that glu ( γ ) hxm forms stable complexes with navo 3 and vocl 2 that remain highly active during an extended period of time when kept as dry powder at room temperature , 2 : 1 complexes of glu ( γ ) hxm : navo 3 and glu ( γ ) hxm : vocl 2 were prepared by dissolving the monohydroxamate and the vanadium salt in 2 : 1 equimolar concentrations , respectively , in water . the water solutions were then mixed , frozen with liquid nitrogen , and lyophilized . the dry powder obtained was allowed to stand at room temperature for 4 weeks . lipogenesis was then performed as described above , using free glu ( γ ) hxm , navo 3 and vocl 2 , and the glu ( γ ) hnm : navo 3 and glu ( γ ) hxm : vocl 2 2 : 1 complexes as dry powders . as shown in fig9 both complexes maintained their level of insulinomimetic activity , indicating that they are stable . brichard , s . m ., and henquin , j . c . 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