Patent Application: US-54370300-A

Abstract:
derivatives of aryl and heterocyclic ureido and aryl and heterocyclic carboxamido phenoxy isobutyric acids have been found to inhibit the nonenzymatic glycation of proteins which often results in formation of advanced glycation endproducts and crosslinks . many other phenoxyisobutyric acid derivatives as well as certain other compounds as set out in this disclosure also have been found to inhibit the nonenzymatic glycation of proteins . the nonenzymatic glycation and crosslinking of proteins is a part of the aging process with the glycation endproducts and crosslinking of long - lived proteins increasing with age . this process is increased at elevated concentrations of reducing sugars in the blood and in the intracellular environment such as occurs with diabetes . the structural and functional integrity of the affected molecules become perturbed by these modifications and can result in severe consequences . the compounds of the present invention can be used to inhibit this process of nonenzymatic glycation and therefore to inhibit some of the ill effects caused by diabetes or by aging . the compounds are also useful for preventing premature aging , spoilage of proteins in food and can prevent discoloration of teeth .

Description:
in the course of screening different classes of organic compounds for investigation of their possible inhibitory effects on advanced glycation endproducts ( age &# 39 ; s ), we found that most of the phenylureido substituted phenoxy propionic acid derivatives tested have inhibitory effects and several of these compounds were potent inhibitors of age - formation at concentrations much lower than an equally inhibiting concentration of aminoguanidine . the compounds and their useful compositions utilized in the present invention contain agents capable of reacting with the highly active carbonyl intermediate of an early glycation product thereby preventing those early products from later forming the advanced glycation endproducts which lead to protein crosslinking and to protein aging . other utilities envisioned for the present invention are : prevention of premature aging and of spoilage of the proteins in foodstuffs . the present agents are also useful in the area of oral hygiene as they prevent discoloration of teeth . the compounds of the present invention collectively are defined as derivatives of aryl and heterocyclic ureido and aryl and heterocyclic carboxamido phenoxy isobutyric acids ( rahbar et al ., 1999 ). a general formula encompassing several compounds of the invention is demonstrated in fig1 . these compounds are part of a series of compounds originally developed for their effects on the modification of oxygen affinity of hemoglobin by lowering the affinity of hemoglobin for oxygen and shifting the hemoglobin - oxygen - dissociation curve to the right ( rahbar et al ., 1987 ; lalezari et al ., 1988 ; lalezari and lalezari , 1989 ; u . s . pat . no . 5 , 268 , 500 ; u . s . pat . no . 5 , 292 , 935 ; u . s . pat . no . 5 , 093 , 367 ; u . s . pat . no . 4 , 921 , 997 ). representative compounds of the present invention are shown in fig5 - 10 as lr1 to lr92 , and were screened for possible inhibitory effects on protein glycation and age - formation . for purposes of this disclosure , the names assigned to these structures are : lr1 4 -[ 3 -( 6 - chloro - 2 , 4 -( 1h , 3h ) quinazolinedione )] phenoxyisobutyric acid , mw = 374 . 5 * the symbols in parentheses are taken from lalezari and lalezari ( 1989 ). although there are 92 compounds in the above list , many of the compounds are quite similar to each other and can be grouped together . for example , compounds lr1 and lr16 are both cyclic ureidophenoxyisobutyric acids ; compounds lr2 , lr9 , lr17 , lr20 , lr28 , lr29 , lr31 , lr34 , lr37 , lr38 , lr42 , lr47 , lr50 , lr51 , lr52 , lr61 and lr62 are amidophenoxyisobutyric acids ; compounds lr3 - lr8 , lr10 - lr15 , lr19 , lr21 - lr27 , lr30 , lr32 , lr33 , lr40 , lr41 , lr43 , lr44 , lr46 , lr48 , lr49 , lr53 , lr55 , lr58 - lr60 , lr63 , lr89 and lr90 are arylureidophenoxyisobutyric acids ; compounds lr35 , lr39 , lr54 , lr56 , lr57 , lr66 , lr74 , lr76 , lr77 , lr82 , lr86 and lr87 are clofibric acid derivatives ; compounds lr91 and lr92 are formimide derivatives ; and compounds lr64 , lr65 , lr67 - lr75 , lr78 and lr83 - lr85 are arylcarboxylic acid derivatives . the above compounds are capable of inhibiting the formation of advanced glycation end products on target proteins and the resulting protein crosslinking . the rationale of the present invention is to use agents which block the post - glycation step , i . e ., the formation of fluorescent chromophores , the presence of which chromophore is associated with and leads to adverse sequelae of diabetes and aging . an ideal agent would prevent the formation of the chromophore and its associated crosslinks of proteins and trapping of proteins on the other proteins , such as occurs in arteries and in the kidneys . the compounds of the invention may be administered to mammals including humans to prevent or reduce protein glycation and crosslinking ( protein aging ). the compounds may be administered orally at variable dosage depending on the activity of each agent in a single or individual amounts . in addition the compounds may be administered parenterally or rectally . the compounds of the invention , the rationale behind the different assay methods of the present invention , and their use are illustrated by the following examples . evaluation of early glycation products ( amadori ) formation on hemoglobin ( hba 1c ) is performed by incubating red blood cells with an oxidized form of glucose in the presence and the absence of the inhibitor compound followed by determination of ( hba 1c ) in the test versus the control ( rahbar and nadler ., 1999 ). this test is based on a recent report by lindsay et al . ( 1997 ). δ - glu , an oxidized analogue of glucose , can react rapidly with hemoglobin within the red cells and significantly increases the hba 1c levels within hours after incubation . by contrast , glucose requires weeks for an equivalent reaction to occur . we have used this finding to devise an assay method to measure early stage glycation of hemoglobin ( amadori product ) and an assay to evaluate the ability of an inhibitor to inhibit hba 1c formation . briefly , fresh blood was drawn in potassium - edta and prepared for incubation within 30 minutes of collection by mixing 200 μl of blood with 40 μl of either phosphate buffered - saline ( pbs ), ph 7 . 4 , alone , pbs containing 50 millimoles / l δ - glu ( sigma ), or pbs containing 50 millimoles / l δ - glu plus 1 millimole / l inhibitor . after incubation for 5 hours at 37 ° c ., the percentage of glycated hemoglobin present was determined . the percentage of glycated hb ( hba 1c ) was determined using a dedicated ion - exchange hplc system ( biorad diamat ). blood samples were analyzed in triplicate . the % inhibition of hba 1c formation by the compound was calculated according to the following formula : where a is hba 1c concentration in the baseline control tube not treated with δ - glu , b is the hba 1c concentration in blood incubated with δ - glu , c is the hba 1c content of the test tube treated both with δ - glu and the inhibitor compound . the amount of ( hba 1c ) formation using δ - glu treated whole blood from normal volunteers using 1 millimole / l of the compounds is shown in fig2 a - b for selected compounds . the results , calculated as percent inhibition of hba 1c formation , for all 92 lr compounds are shown in table 1 . fig2 a - b show results from a single assay whereas the results shown in table 1 are an average of 3 determinations . the above experiment suggests that this type of drug therapy has benefits in reducing the pathology associated with the formation of early glycation products , a preliminary step in the advanced glycation end product formation . this test is used to evaluate the ability of the inhibitors to inhibit glucose - mediated development of fluorescence of bsa ( ikeda et al ., 1996 ). bsa ( fraction v ) from sigma 50 mg / ml and 800 mm glucose ( 144 mg / ml ) in 1 . 5 m phosphate buffer ph 7 . 4 containing nan 3 0 . 2 g / l was incubated under aseptic conditions at 37 ° c . for 7 days in the presence or absence of various concentrations of the compounds . after 7 days of incubation each sample was examined for the development of specific fluorescence ( excitation , 370 nm ; emission , 440 nm ). the % inhibition of age formation in the test sample versus control was calculated for each inhibitor compound . aminoguanidine ( 50 mm ) was used as a positive control . fig3 a - b show for a selection of the tested compounds the inhibitory effects of 1 millimole / l of the new inhibitor versus 50 millimoles / l of aminoguanidine . the data presented in fig3 a - b are from 1 determination . results which are averaged for 3 determinations for each of the 92 compounds are tabulated in table 1 . evaluation of the late glycation products ( age &# 39 ; s ), and age - inhibition by the new inhibitor compounds was tested by incubation of g . k . peptide in ribose in the presence or the absence of the agent , followed by determination of chromophores generated in the course of glycation and age formation through determination of their specific fluorescence . the nagaraj et al . ( 1996 ) method used to evaluate the ability of the compounds of the present invention to inhibit the crosslinking of n - acetylglycyl - lysine methyl ester in the presence of ribose was as follows : 0 . 5 m sodium phosphate buffer ph 7 . 4 containing nan 3 0 . 2 g / l gk peptide ( sigma ) 80 mg / ml in 0 . 5 m sodium phosphate buffer ph 7 . 4 equal volumes ( 0 . 1 ml ) of the 3 stock solutions were mixed together , filtered through a 0 . 2 micron filter ( coming ) and incubated under aseptic conditions for 24 hours at 37 ° c . the inhibitor compounds were added to a final concentration of 1 millimole / l . at the end of the incubation period , samples were analyzed for their specific fluorescence ( excitation , 340 nm ; emission , 420 nm ). the % inhibition by different concentrations of inhibitor was calculated as described above . aminoguanidine was used at 50 mm as a positive control . fig4 a - b show the inhibitory effects of a selection of the compounds to block specific fluorescence of protein - age in these separate determinations , using g . k . peptide - ribose assay . results for all 92 compounds are shown in table 1 . fig4 a - b show the results of a single assay whereas the data in table 1 are the averaged results for 3 assays of each compound . the results obtained from the above two experiments ( examples 2 and 3 ) suggest this type of drug therapy has benefits in reducing the pathology associated with the formation of late glycation products and protein crosslinking . lysozyme - glucose or fructose crosslinking assays according to taneda and monnier ( 1994 ) are in vitro assays which were performed to evaluate the inhibitory effect of compounds on age - derived crosslinking and age - protein formation . egg white lysozyme ( sigma ) and glucose or fructose in 0 . 2 m sodium phosphate ph 7 . 4 containing 0 . 2 g / l nan 3 were mixed with various test compounds to give a final concentration of 1 millimole / l of test compound , 100 mg / ml egg white lysozyme , and 200 mm glucose or 100 mm fructose . all samples were incubated under aseptic conditions at 37 ° c . for 7 days . after 7 days , each sample was analyzed for the determination of age - derived crosslinking and age - formation . aliquots were applied to 20 % sds - page gels under reducing conditions and stained with coomassie blue . a special elisa technique ( al - abed et al ., 1999 ) was used to evaluate the ability of the compounds being studied to inhibit the crosslinking of glycated - bsa ( age - bsa ) to a rat tail - tendon - collagen coated 96 well plate ( biocoat microtiter plates from collaborative research . crosslinking of age - bsa to a rat tail - tendon - collagen coated plate was performed with and without the testing compound at the desired concentrations . the uncross - linked age - bsa was then removed by washing the wells . the age - bsa crosslinked to the tail - tendon - collagen coated plate was then quantified by a polyclonal antibody raised against age - rnase . positive results in the assay indicate that the inhibitor is capable of reducing the amount of age - bsa which crosslinks with collagen . aminoguanidine was used as positive control . the results using five representative compounds are shown in fig1 a - b . the five compounds ( lr33 , lr41 , lr20 , lr23 and lr62 ) are among a number of strong inhibitors of age - protein crosslinking . percent inhibitions of the control were calculated to be 61 % for lr33 and 27 . 4 % for lr41 at 1 mmole / l and 45 . 5 % for aminoguanidine at 50 mm . bovine pancreatic ribonuclease a ( rnase a ) has been extensively used as a model protein to study protein glycation and age formation , as well as the kinetics of age - formation ( khalifah et al ., 1996 ). rnase a has the advantage of not precipitating during glycation reaction whereas lysozyme tends to precipitate during glycation with ribose and glucose . the rnase - ribose assay measures the fluorescence generated as a result of age - formation in the presence or absence of the inhibitor as compared to aminoguanidine . this assay is also used to detect the inhibitory effects of a compound on protein - crosslinking and the formation of dimers and trimers as shown by sds - page . both uninterrupted and interrupted versions of this assay ( nagaraj et al ., 1996 ) have been performed successfully . however , the interrupted technique worked better in our hands . bovine rnase a type i - a ( sigma ) was used throughout our assays . for the uninterrupted method , rnase , 1 mg / ml , was incubated with ribose ( 0 . 2 m ) at 37 ° c . in 0 . 4 m sodium phosphate buffer ph 7 . 5 containing 0 . 02 % sodium azide for 7 days in the dark . prospective inhibitor was added to the reaction at the beginning of the incubation period . all solutions were prepared in sterile condition by filtering through a 0 . 2 micron filter ( corning ). at the end of the incubation , samples were analyzed for their fluorescence ( excitation , 330 nm ; emission , 400 nm ). the percent of inhibition of age - formation by different concentrations of the inhibitor were calculated as described before . interrupted rnase - ribose assays were carried out as follows : rnase , 10 mg / ml , was incubated with ribose ( 0 . 5 m ) at 37 ° c . in 0 . 4 m sodium phosphate buffer ph 7 . 5 containing 0 . 02 % sodium azide for 24 hours in the absence of inhibitor compound . glycation was then interrupted by diluting the reaction 1 : 100 in 0 . 4 m phosphate buffer and adding the inhibitor under study to the reaction at the desired concentrations . the reaction was filtered through a 0 . 2 micron filter and incubated at 37 ° c . in the dark for four additional days . at the end of the incubation period , samples were analyzed for their fluorescence as described before , and the percent inhibition was calculated . for detection of the inhibitory effects of the compound under the study , the inhibitory effect being the inhibition of rnase crosslinking and dimer - trimer formation , at the end of the incubation period the samples were analyzed by sds - page technique as described before . in the case of the interrupted glycation assays , the samples were concentrated by using centricon 10 ( amicon ) and then applied to the gels . the above examples suggest that this type of drug therapy will be beneficial in reducing the pathology associated with the formation of nonenzymatic glycation products ( early and late products ) and protein - protein crosslinking . compounds of the present invention are found to be 10 to 40 times more potent inhibitors of age - formation in vitro as compared to aminoguanidine which is in phase 2 / 3 clinical trial to prevent diabetic complications . previous studies have shown these compounds to be non - toxic . they may be administered orally at variable dosages depending on the activity of each agent in a single or individual amounts . in addition , the compounds may be administered parenterally or rectally . while the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention , it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense , as it is contemplated that modifications will readily occur to those skilled in the art , within the spirit of the invention and the scope of the appended claims . airaksinen k e j , et al . 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