Patent Abstract:
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 .

Detailed Description:
we have previously reported new classes of compounds which are aryl ( and heterocyclic ) ureido and aryl ( and heterocyclic ) carboxamido phenoxyisobutyric acids and also benzoic acid derivatives and related compounds as inhibitors of glycation and age formation ( rahbar et al ., 1999 ; rahbar et al ., 2000 ). in the course of screening different classes of organic compounds for investigation of their possible inhibitory effects on advanced glycation endproducts ( ages ), 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 aim of the present study was to develop classes of novel inhibitors of glycation , age formation and age - crosslinking and to investigate their effects through in vitro chemical and immunochemical assays . a total of 102 compounds were designed and synthesized . the first 92 compounds have been reported elsewhere . the ten novel compounds reported here were designed and developed based upon the previously reported lr23 ( 4 -( 3 , 5 - dichlorophenylureido )- phenoxyisobutyryl - 1 - amidocyclohexane - 1 - carboxylic acid )) which was one of the most powerful inhibitors reported previously ( rahbar et al ., 1999 ; u . s . patent application ser . no . 09 / 543 , 703 which is incorporated herein by reference ). these compounds are based upon lr3 ( see fig1 ), the synthesis of which is reported in lalezari and lalezari ( 1989 ) which is incorporated herein . considerable increase in the inhibitory potencies , particularly on inhibition of age - protein - crosslinking , were found in the three compounds lr96 , lr99 and lr102 ( see below ) as compared to the prototype lr23 and are 2 to 3 times more effective than pyridoxamine ( khalifah etal ., 1999 ). the mechanism ( s ) by which this class of compounds inhibits glycation , age - formation , and crosslinking is yet to be known . the present study indicates that these compounds are powerful inhibitors that act at multiple steps of glycation and age - formation , i . e ., early stage , as evidenced by lowering hbalc levels in the δ - glu assay , a specific assay for the early stage of glycation ( type a or b inhibitor ). most of these compounds strongly inhibit the post - amadori glycation as demonstrated by the bsa - glucose and g . k .- ribose assays ( type d inhibitors ), and a good number of them are powerful inhibitors of age protein crosslinking , as evidenced by a specific elisa assay ( type e inhibitors as described by baynes classification ( khalifah et al ., 1999 )). the mechanism of the inhibitory activities of guanidino compound inhibitors such as two known inhibitors of glycation ( aminoguanidine and metformin ) is that they are postulated to trap mg and other α - dicarbonyl intermediates of glycation . a most recent study has documented the reaction of metformin with mg and glyoxal ( go ), forming guanidino - dicarbonyl adducts further supporting this idea ( ruggiero - lopez et al ., 1999 ). however , the structures of our novel compounds suggest that they are unlikely to trap α - dicarbonyls . they may be working by a different mechanism distinct from that of aminoguanidine . using new assay methods specific for the early ( amadori ) and late ( post - amadori ) stages of glycation revealed some inhibitors to have greater effects in the early stage and some in the late stage of glycation . however , most of the inhibitor compounds we have investigated are multistage inhibitors . the reaction of reducing sugars with α - and ε - amino groups of proteins is not a random process but rather a site specific reaction which depends on the nature and the vicinity of these chemical groups . the future task is to specifically define the site ( s ) of interaction of an inhibitor compound in the complex series of reactions and intermediate substrates , leading to age formation and cross - linking . the development of the novel inhibitors of glycation , age formation , and age - protein crosslinking not only expands the existing arsenals of inhibitors of glycation reaction that can find therapeutic applications for the prevention of diabetic complications , as well as the prevention of other diseases associated with increased glycation of proteins or lipids . furthermore , the availability of these compounds may prove useful as tools to study the cascade of reactions and intermediate substrate in the process of age - formation and age - protein cross - linking . 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 ( u . s . pat . no . 5 , 661 , 139 ). the present agents are also useful in the area of oral hygiene as they prevent discoloration of teeth . compounds of the present invention are shown in fig6 - 15 as lr93 - lr102 , and were screened for inhibitory effects on protein glycation and age - formation . 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 . the δ - glu assay is a specific method for investigation of inhibitors of the early stage of glycation . 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 16 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 fig1 . the results , calculated as percent inhibition of hba 1c formation , are shown in table 1 . levels of hba 1c in the δ - glu treated blood is twice as high as the baseline control . various inhibitors ( compounds lr93 - lr102 ) show different levels of hba 1c depending on their inhibitory potencies . half of the new compounds exhibited better inhibition than the prototype lr23 . 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 ). triplicate samples of bsa ( fraction v , essentially fatty acid free , low endotoxin ) 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 , 330 nm ; emission , 410 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 . the results are shown in table 2 . fig2 shows the inhibitory effects of 1 millimole / l of the new inhibitor versus 50 millimoles / l of aminoguanidine . this assay method is mostly for the inhibitors of late glycation and age formation ( post - amadori ). the results obtained by this assay show all ten compounds investigated here have strong inhibitory effects on post - amadori glycation , age formation and age crosslinking . evaluation of the late glycation products ( ages ), 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 triplicate samples of equal volumes ( 0 . 1 ml ) of the 3 stock solutions were mixed together , filtered through a 0 . 2 micron filter ( corning ) 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 , 330 nm ; emission , 415 nm ). the % inhibition by different concentrations of inhibitor was calculated as described above . aminoguanidine was used at 50 mm as a positive control . fig3 shows the inhibitory effects of the compounds to block specific fluorescence of protein - age in these separate determinations , using g . k . peptide - ribose assay . results are shown in table 3 . the results of this assay indicate that all ten compounds investigated here have strong inhibitory effects and block specific fluorescence of proteins age in these separate determinations . 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 cell environment , becton dickinson ). 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 in our laboratory . 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 compounds lr96 , lr99 and lr102 are shown in fig4 a - c . these compounds are among a number of strong inhibitors of age - protein crosslinking . the above examples indicate 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 up to 250 times more potent inhibitors of age - fornation in vitro as compared to aminoguanidine which is in phase 2 / 3 clinical trial to prevent diabetic complications . these compounds can be administered orally at variable dosages depending on the activity of each agent in a single or individual amounts . in addition , the compounds can 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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