MXPA00004785A - Advanced glycation end-product intermediaries and post-amadori inhibition - Google Patents

Abstract

The instant invention provides compositions and methods for modeling post-Amadori AGE formation and the identification and characterization of effective inhibitors of post-Amadori AGE formation, and such identified inhibitor compositions.

Description

INTERMEDIARIES OF THE FINAL PRODUCT OF THE ADVANCED GLICATION AND POST-AMADORI INHIBITION BACKGROUND OF THE INVENTION The present invention pertains to the area of the final products of the invention.

Advanced Glycation (AGEs), its information, detection, identification, inhibition and inhibitors thereof.

Final Products of Advanced Glycosylation and the Protein of Aging Non-enzymatic glycation by glucose and other sugar-reducing agents is an important post-translational modification of proteins that have been increasingly implicated in various pathologies. Non-enzymatic irreversible glycation and cross-linking through a slow induced glucose process can mediate many of the complications associated with diabetes. Chronic hyperglycemia associated with diabetes can cause chronic tissue damage that can lead to complications such as retinopathy, nephropathy and atherosclerosis disease. (Cohen and Ziyadeh, 1996, J. Amer. Soc. Nephrol., 7: 183-190). It has been shown that the result of chronic tissue damage associated with diabetes mellitus in the long term increases in part the formation of the immune complex in situ by accumulated immunoglobulins and / or antigens bound to long-lived structural proteins that have undergone the formation of final product of Advanced Glycosylation (AGE), via non-enzymatic glycosylation (Brownlee et al., 1983, J. Exp. Med. 158: 1739-1744). It is thought that the target of the primary protein is an extra-cellular matrix associated with collagen. The non-enzymatic glycation of proteins, lipids and nucleic acids can play an important role in the natural processes of aging. Recently, protein glycation has been associated with β-amyloid deposits and the formation of neurofibrillary tangles in Alzheimer's disease and possibly other neurodegenerative diseases that involve amyloidosis (Colaco and Harrington, 1994, NeuroReport 5: 859-861). . It has been shown that glycated proteins are also toxic, antigenic and capable of eliciting cellular damage responses after elevation by specific cellular receptors (see for example, Vlassara, Bucala &Striker, 1994, Lab. Invest. 70: 138-151; Vlassara et al., 1994, PNAS (USA) 91: 11704-11708; Daniels &Hauser, 1992, Diabetes 41: 1415-1421; Brownlee, 1994, Diabetes 43: 836-84; Cohen et al., 1994, Kidney Int. 45: 1673-1679, Brett et al., 1993, Am. J. Path. 143: 1699-1712 and Yan et al., 1994, PNAS (USA) 91: 7787-7791). The appearance of brown pigments during the cooking of food is a universally recognized phenomenon, the chemistry of which was first described by Maillard in 1912 and was subsequently investigated within the concept of protein aging. It is known that heat-treated and stored food suffers from nonenzymatic browning that is characterized by reticulated proteins that decrease their bioavailability. It was found that this Maillard reaction occurs in vivo as well as when it was found that a glucose was added via an Amadori rearrangement to the amino terminus of the -a chain of hemoglobin.

The present previously unknown description teaches and does not predict the mechanism of the formation of the final products of advanced post-Amadori glycation (Maillard products; AGEs) and the methods for their identification and the effective inhibitors characteristic of the post-Amadori AGE formation. The present description demonstrates the unique isolation and kinetic characterization of an intermediate reactive protein in the formation of post-Amadori AGEs and for the first time teaching methods that allow for specific elucidation and rapid quantitative kinetic study of "late" states. of the glycation reaction of the protein. In contrast to said "delayed" AGE formation, the "early" steps of the glycation reaction have been relatively well characterized and identified for several proteins (Harding, 1985, Adv. Protein Chem. 37: 248-334; Monnier & Baynes eds., 1989, The Maillard Reaction in Aging, Diabetes, and Nutrition (Alan R. Liss, New York); Finot et al., 1990, eds. The Maillard Reaction in Food Processing, Human Nutrition and Physiology (Birkhauser Verlag, Basel)). Glycation reactions are known to be initiated by the Schiff base of reversible addition reactions (aldimine or ketimine) with an e-amino side chain of lysine and terminal a-amino groups, followed by irreversible Amadori rearrangements essentially to produce ketoamine products by example, 1-amino-1-deoxy-ketoses from the reaction of aldoses (Baynes et al., 1989, in The Maillard Reaction in Aqinq, Diabetes, and Nutrition, ed. Monnier and Baynes, (Alan R. Liss, New York, pp 43-67) Typically, sugars initially react in their open chain (not the predominant structures of pyranose and furanose) keto or aldehyde forms with e-amino-side chain lysine and terminal amino groups through of the condensation of the reversible Schiff base (Scheme I) The resultant products of aldimine or ketimine subsequently undergo Amadori rearrangements to give Amadori ketoamine products, for example, 1-amino-1-deoxy-ketose s from the reaction of aldoses (Mean & Chang, 1982, Diabetes 31, Suppl. 3: 1-4; Harding, 1985, Adv. Protein Chem. 37: 248-334). These Amadori products subsequently suffer for a period of weeks or months, slow and irreversible "darkening" Maillard reactions, forming fluorescent products and other products via rearrangement, dehydration, oxidative fragmentation and cross-linking reactions. These post-Amadori reactions (slow "darkening" Maillard reactions) lead to poorly characterized Advanced Glycation (AGEs) end products. As with the Amadori intermediates and other glycation intermediates, free glucose by itself can undergo oxidative reactions that lead to the production of peroxide and highly reactive fragments such as glyoxal dicarbonyl and glycoaldehyde. Therefore, the elucidation of the mechanism of formation of a variety of AGEs has been extremely complex since most of the in vitro studies have been performed in extremely high concentrations of sugar. In contrast to the relatively well-characterized formation of these "early" products, there was a clear lack of understanding of the mechanisms of the formation of "late" Maillard products produced in post-Amadori reactions, due to their heterogeneity, to their prolonged reaction times and their complexity. The absence of detailed information about the chemistry of the "delayed" Millard reaction stimulated search to identify AGE fluorescent chromophores derived from the reaction of glucose with amino groups of polypeptides. One such chromophore, 2- (2-furoyl) -4 (5) - (2-furanyl) -1H-imidazole (FFI) was identified after non-enzymatic dimming of serum albumin of bovine and polylysine with glucose and would be representative of the chromophore present in the intact polypeptides. (Pongor et al., 1984, PNAS (USA) 81: 2684-2688). Later studies established that the FFI would be an artifact formed during acid hydrolysis by analysis. A series of US Patents have been issued. in the area of inhibition of protein glycosylation and the cross-linking of sugar amines in proteins based on the premise that the mechanism of glycosylation and cross-linking occurs via saturated glycosylation and the subsequent cross-linking of sugar amines in proteins via a simple and repeated reaction. These patents include the Patents of E.U.A. 4,665,192; 5,017,696; 4,758,853; 4,908,446; 4,983,604; 5,140,048; 5,130,337; 5,262,152; 5,130,324; 5,272,165; 5,221, 683; 5,258,381; 5,106,877; 5,128,360; 5,100,919; 5,254,593; 5,137,916; 5,272,176; 5,175,192; 5,218,001; 5,238,963; 5,358,960; 5,318,982 and 5,334,617. (All of the cited U.S. Patents are incorporated herein by reference in their entirety). The approach of these U.S. Patents, is a method for the inhibition of the AGE formation focused on the carbonyl moiety of the Amadori product of initial glycosylation and in particular the most effective inhibition shown shows the use of the ammoniguanidine administered exogenously. The effectiveness of aminoguanidine as an inhibitor of AGE formation is currently being tested in clinical trials. The inhibition of AGE formation has utility in the areas of, for example, food waste, aging of animal protein and personal hygiene such as combating tooth darkening. Some notable end products of advanced glycation, although quantitatively lower are pentosidine and Ne-carboxymethyllysine (Sell and Monnier, 1989, J. Biol. Chem. 264: 21597-21602; Ahmed et al., 1986, J. Biol. Chem. 261: 4889-4894). The Amadori intermediate product and its subsequent post-Amadori AGE formation, as demonstrated by the present invention, is not completely inhibited by the reaction with aminoguanidine. Therefore, the formation of post-Amadori AGEs as shown in this description occurs via a unique and important reaction route that had not been previously demonstrated or even previously it had not been possible to demonstrate it in isolation. It is a highly desirable objective to have an effective and efficient method for the identification and effective characterization of post-Amadori AGE inhibitors of this "late" reaction. By providing model systems and efficient research methods, combinatorial chemistry can be used to effectively screen candidate compounds and thereby greatly reduce the time, cost and effort in the eventual validation of inhibitor compounds. It would be very useful to have in vivo methods for the modeling and study of the effects of the post-Amadori AGE formation that would later allow the efficient characterization of effective inhibitors.

Inhibitor compounds that are biodegradable and / or naturally metabolized are more desirable for use as therapeutics than highly reactive compounds that can have toxic side effects, such as aminoguanidine.

SUMMARY OF THE INVENTION According to the present invention, a final product precursor (AGE) of the post-Amadori stable advanced glycation has been identified that can subsequently be used to rapidly complete the post-Amadori conversion within post-Amadori AGEs. This stable product is presumed to be a base product of an Amadori / Schiff saturated sugar produced by an additional reaction of the Amadori protein / sugar product from the initial stage with more sugar. In a preferred embodiment, this post-Amadori / Schiff intermediate base has been generated by the reaction of the white protein with ribose sugar. The present invention provides a method for generating the formation of intermediate AGE precursors of stable protein-sugar via a novel method of high sugar inhibition. In a preferred embodiment the sugar used is ribose. The present invention provides a method for the identification of an effective inhibitor of the formation of late Maillard products comprising: the generation of intermediates of the final product of advanced post-Amadori glycation of the stable protein-sugar by incubation of a protein with sugar in sufficient concentration and for a sufficient period of time to generate stable post-Amadori AGE intermediates; the contact of said end product intermediates of the post-Amadori advanced glycation of the stable protein-sugar with an inhibitory candidate; the identification of effective inhibition by monitoring the formation of post-Amadori AGEs after the release of intermediates from the final product of advanced post-Amadori glycation of protein-sugar stable sugar induced in equilibrium. Suitable sugars include but are not limited to ribose, lixose, xylose and arabinose. It is believed that certain conditions also allow the use of glucose and other sugars. In a preferred embodiment the sugar used is ribose. The present invention demonstrates that an effective inhibitor of post-Amadori AGE formation via "late" reactions can be identified and characterized by the ability to inhibit the formation of post-Amadori AGE end products in a test comprising: the generation of the intermediates of the final product of the post-Amadori advanced glycation by incubating a protein with sugar in a sufficient concentration and for a period of time sufficient to generate stable post-Amadori AGE intermediates; contacting said intermediates of the final product of the post-Amadori advanced glycation of the protein-stable sugar with an inhibitory candidate; the identification of effective inhibition by monitoring the formation of post-Amadori AGEs after the release of intermediates from the final product of advanced post-Amadori glycation of protein-sugar stable sugar induced in equilibrium. Ribose is used in a preferred mode of testing.

Therefore, the methods of the present invention allow the rapid investigation of candidate inhibitors for post-Amadori AGE formation because of their effectiveness, their great cost reduction and the amount of work required for the development of small molecule inhibitors effective in the post-Amadori AGE training. The present invention demonstrates that effective inhibitors of the post-Amadori "late" reactions of the AGE formation include vitamin & and vitamin B- ?, in the preferred embodiment the specific species being pyridoxamine, pyridoxamine-5'-phosphate and thiamine pyrophosphate. The present invention shows new methods for rapidly inducing diabetes similar to the pathologies in rats comprising the administration of ribose to the subject animal. In addition, the use of inhibitors identified as pyridoxamine, pyridoxamine-5'-phosphate and thiamine pyrophosphate in vivo is provided to inhibit post-Amadori AGE-induced pathologies. The present invention comprises compounds for use in the inhibition of AGE formation and post-Amadori AGE pathologies and pharmaceutical compositions containing said compounds of the general formula: R, Formula I wherein Ri is CH2NH2, CH2SH, COOH, CH2CH2NH2, CH2CH2SH or CH2COOH; R2 is OH, SH or NH2; And it is N or C, so that when Y is N, R3 is nothing and when Y is C, R3 is NO2 or another electron of the elimination group and salts thereof. The present invention also comprises compounds of the general formula Formula II wherein R-, is CH2NH2) CH2SH, COOH, CH2CH2NH2, CH2CH2SH or CH2COOH; R2 is OH, SH or NH2; And it is N or C, so that when Y is N, R3 is nothing and when Y is C, R3 is NO2 or another electron of the elimination group; R is H, or C 1-6 alkyl; Rs and RT are H, C 1-6 alkyl and salts thereof. In a preferred embodiment at least one of R 4, R 5 and Re are H. The present invention also comprises compounds wherein R and R 5 are H, C 1-6 alkyl, alkoxy or alkene. In connection with the present invention, it is also understood that R 2 and RT can be H, OH, SH, NH 2, C 1-6 alkyl, alkoxy or alkene. It is also envisaged that R4, R5 and Re may be larger functional groups, such as and not limited to aryl, hetero-pyl and alkoxy-cycloalkyl groups. In addition, the present invention also provides compounds of the formulas The compounds of the present invention can incorporate one or more electrons from the elimination groups, such as and not limited to -NH2, -NHR, -NR2, -OH, -OCH3, -OCR and -NH-COCH3 wherein R is C 1-6 alkyl. The present invention comprises pharmaceutical compositions comprising one or more of the compounds of the present invention or salts thereof, in an appropriate carrier. The present invention comprises methods for administering pharmaceuticals of the present invention for the therapeutic intervention of pathologies that are related to AGE formation in vivo. In a preferred embodiment of the present invention, the related AGE pathology is related to diabetic neuropathy.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a series of graphs depicting the effect of vitamin B derivatives on AGE formation in bovine serum albumin (BSA). Figure 1A Pyridoxamine (PM); Figure 1 B pyridoxal phosphate (PLP); Figure 1C pyridoxal (PL); Figure 1 D pyridoxine (PN), where in all the graphs: a) Elisa reading (410 nm), b) Time in Days Figure 2 is a series of graphs representing the effect of vitamin derivatives Bi and aminoguanidine (AG) in the AGE formation in bovine serum albumin. Figure 2A Thiamine pyrophosphate (TPP); The figure 2B thiamine monophosphate (TP); Figure 2C thiamine (T); Figure 2D aminoguanidine (AG). Figure 3 is a series of graphs depicting the effect of vitamin B derivatives on AGE formation in human methemoglobin (Hb). Figure 3A pyridoxamine (PM); Figure 3B pyridoxal phosphate (PLP); Figure 3C pyridoxal (PL); Figure 3D pyridoxine (PN). Figure 4 is a series of graphs depicting the effect of the derivatives of vitamin Bi and aminoguanidine (AG) on AGE formation in human methemoglobin (TP); Figure 2C thiamine (T); Figure 2D aminoguanidine (AG). Figure 5 is a bar chart comparing the inhibition of glycation of ribonuclease A by pyrophosphate thiamine (TPP), pyridoxamine (PM) and aminoguanidine (AG), where: c is the percent inhibition.

Figure 6A is a graph of the glycation kinetics of RNase A (10 mg / ml) by ribose as monitored by ELISA. Figure 6B is a graph showing the dependence of reciprocal half-times (d) on a concentration of ribose (e) at pH 7.5. Figure 7 are two graphs showing a comparison of an uninterrupted (II) and interrupted (I) glycation of the RNase by glucose (f) (7B) and ribose (7A) as detected by ELISA. Figure 8 are two graphs showing the kinetics of the increase in fluorescence of pentosidine (g) (arbitrary units) during the glycation of interrupted (I) and uninterrupted (II) ribose of the RNasa. Figure 8A shows the uninterrupted glycation in the presence of 0.05 M ribose. Figure 8B shows the glycation interrupted after 8 and 24 hours of incubation. Figure 9 is a graph showing the kinetics of reactive intermediate accumulation. Figure 10 are graphs of Post-Amadori (PA) inhibition of AGE formation by ribose. Figure 10A are data plots in which the aliquots were diluted within the inhibitor containing stabilizers at time 0. Figure 10B shows plots of data where the samples were stopped at 24 hours and then diluted into the inhibitor containing stabilizers. Figure 11 is a graph showing the dependence of the initial velocity (vi) of the AGE formation of antigens at pH followed by the interruption of glycation.

Figure 12 shows two graphs showing the effect of the pH jump (pHs) on the AGE formation detected in ELISA after the interrupted glycation. The interrupted samples were left for 12 days at 37 ° C in a stabilizer with pH 5.0 producing substantial EFAs (33%, Figure 12 B) when the pH changed to 7.5, as compared to the normal control sample that was not exposed to a Low pH (Figure 12A). Figure 13 is a series of graphs showing the effect of depots of vitamin B6 on the AGE formation during the uninterrupted glycation of ribonuclease A (RNase A) by ribose. Figure 13A shows Pyridoxamine (PM); Figure 13B shows pyridoxal-5'-phosphate (PLP); Figure 13C shows pyridoxal (PL); Figure 13D shows pyridoxine (PN). Figure 14 is a series of graphs depicting the effect of vitamin Bi derivatives and aminoguanidine (AG) on AGE formation during uninterrupted glycation of ribonuclease A (RNase A) by ribose. Figure 14A shows Thiamine pyrophosphate (TPP); Figure 14B shows thiamine monophosphate (TP); Figure 14C shows thiamine (T); Figure 14D shows aminoguanidine (AG). Figure 15 is a series of graphs depicting the effect of vitamin B6 derivatives on AGE formation during uninterrupted glycation of bovine serum albumin (BSA) by ribose. Figure 15A shows pyridoxamine (PM); Figure 15B shows pyridoxal-5'-phosphate (PLP); the Figure 15C shows pyridoxal (PL); Figure 15D shows pyridoxine (PN). Figure 16 is a series of graphs depicting the effect of vitamin B-i and aminoguanidine (AG) derivatives on AGE formation during uninterrupted glycation of bovine serum albumin (BSA) by ribose. Figure 16A shows thiamine pyrophosphate (TPP); Figure 16B shows thiamine monophosphate (TP); Figure 16C shows thiamine (T); Figure 16D shows aminoguanidine (AG). Figure 17 is a series of graphs depicting the effect of vitamin B derivatives on AGE formation during the uninterrupted glycation of human metemoglobin (Hb) by ribose. Figure 17A shows Pyridoxamine (PM); Figure 17B shows pyridoxal-5'-phosphate (PLP); Figure 17C pyridoxal (PL); Figure 17D shows pyridoxine (PN). Figure 18 is a series of graphs representing the effect of vitamin B6 derivatives on post-Amadori AGE formation after glycation interrupted by ribose. Figure 18A shows BSA and Pyridoxamine (PM); Figure 18B shows BSA and pyridoxal-5'-phosphate (PLP); Figure 18C shows BSA and pyridoxal (PL); Figure 18D shows RNase and pyridoxamine (PM). Figure 19 are graphs depicting the effect of thiamine pyrophosphate on post-Amadori AGE formation after interrupted glycation by ribose. Figure 19A shows RNase, Figure 19B shows BSA. Figure 20 are graphs showing the effect of aminoguanidine on post-Amadori AGE formation after glycation interrupted by ribose. Figure 20A shows RNase, Figure 20B shows BSA. Figure 21 is a graph depicting the effect of Na-acetyl-L-lysine on post-Amadori AGE formation after interrupted glycation by ribose.

Figure 22 are bar graphs showing a comparison of post-Amadori inhibition of AGE formation by thiamine pyrophosphate (TPP), pyridoxamine (PM) and aminoguanidine (AG) after interrupted glycation of RNase (Figure 22A) and BSA (Figure 22B) by ribose. Figure 23 is a bar graph showing the effects of in vivo treatment of Ribose alone on blood pressure in the tail-leg of the rat (cp) in the treatment groups (gt) in phase I, phase II and Phase III (f1, f2, f3 control, respectively). The treatment was with Ribosa (R) 0.05 M, 0.30 M and 1 M injected for 1, 2 or 8 Days (D). Figure 24 is a bar graph showing the effects of in vivo treatment with Ribose alone on the separation (s) of creatinine in the rat (Separation per 100 g of body weight). The treatment was with Ribosa (R) 0.05 M, 0.30 M and 1 M injected for 1, 2 or 8 Days (D). Figure 25 is a bar graph showing the effects of in vivo treatment with Ribose alone in rat albuminuria (Albumin effusion rate, ve). The treatment was with Ribarosa (R) 0.30 M and 1 M injected for 1, 2 or 8 Days (D). Figure 26 is a bar graph showing the effects of in vivo inhibitory treatment with or without ribose, of rat tail-leg blood pressure (cp). The groups were treated with: 25 mg / 1000 g body weight of aminoguanidine (AG); 25 or 250 mg / 1000 g of body weight of pyridoxamine (P); 250 mg / 1000 g of body weight of thiamine pyrophosphate (T) or with Ribose (R) 1 M.

Figure 27 is a bar graph showing the effects of inhibitory treatment in vivo, with or without ribose, on the separation of creatinine (Separation per 100 g of body weight). The groups were treated with: 25 mg / 1000 g body weight of aminoguanidine (GA); 25 or 250 mg / 1000 g of body weight of pyridoxamine (P); 250 mg / 1000 g of body weight of thiamine pyrophosphate (T) or with Ribose (R) 1 M. Figure 28 is a bar graph showing the effects of inhibitor treatment in vivo without ribose, and ribose alone in the Rat albuminuria (albumin effusion rate). The groups were treated with: 25 mg / 1000 g body weight of aminoguanidine (AG); 250 mg / 1000 g of body weight of Pyridoxamine (P); 250 mg / 1000 g body weight of Thiamin pyrophosphate (T), or treated with 1 M Ribose (R) for 8 days (D). The control group had no treatment. Figure 29 is a bar graph showing the effects of inhibitory treatment in vivo, with 1 M ribose, in rat albuminuria (Albumin effusion rate). The groups were treated with: 25 mg / 1000 g body weight of aminoguanidine (AG); 25 and 250 mg / 1000 g of body weight of pyridoxamine (P); 250 mg / 1000 g of body weight of thiamine pyrophosphate (T), or treated with only 1 M Ribose (R) for 8 days (D). The control group had no treatment. Figure 30A represents Scheme 1 (E1) showing a diagram of the AGE formation from proteins. Figure 30B depicts Scheme 2 (E2), a chemical structure of aminoguanidine. Figure 30C depicts Scheme 3 (E3), chemical structures for thiamine, thiamine-5'-phosphate and thiamine pyrophosphate. Figure 30D depicts Scheme 4 (E4), chemical structures of pyridoxine, pyridoxamine, pyridoxal-5'-phosphate and pyridoxal. Figure 30E represents Scheme 5 (E5), the kinetic representation of the AGE formation. Figure 30F represents Scheme 6 (E6), kinetic representation of the AGE formation and intermediate formation, where: P = amino protein group Q = Base Schiff R = Product Amadori T = Rearrangement Amadori DC = Dicarbonylose PE = Transverse links Protein O = Other AGEs AN = Antigens AGEs PT = Pentosidine Cl = Inhibited Complex Figure 31 A and Figure 31 B show a Resonance spectrum of 125 MHz C-13 NMR of the Amadori Ribonuclease Intermediate prepared by a 24 hour reaction with 99% [2-Cl13] of Ribose. Figure 32A is a set of graphs showing the formation of the AGE intermediate using the pentoses: Xylose (X), Lixose (L), Arabinose (A) and Ribose (R). The graphs illustrate the dependence of the post-Amadori AGE formation on pre-incubation time with 0.5 M of pentose sugar.

The RNase was mixed with 0.5 M pentose during the indicated time, later it was tested 7 days after the removal of the pentose by dilution.

Figure 32B is a graph showing the inhibition of AGE formation by pyridoxamine (PM) and pyridaxamine-5'-phosphate (PMP). The graph illustrates the effect of PM and PMP on post-Amadori AGE formation in Bovine Serum Albumin (BSA) modified by interrupted glycation with 0.5 M ribose. Figure 33 is a graph showing the results of the glomerulus remaining in a pH of 2.5 with Alciano blue. Figure 34 is a graph showing the results of the glomerulus remaining at a pH of 1.0 with Alcian blue. Figure 35 is a graph showing the results of the immunofluorescent glomerulus labeled by RSA. Figure 36 is a graph showing the results of the immunofluorescent glomerulus marked by the Nuclear Proteoglycan protein of Heparan Sulfate.

Figure 37 is a graph showing the results of the immunofluorescent glomerulus marked by the side chain of Heparan Sulfate Proteoglycan.

Figure 38 is a graph showing the results of the analysis of the sections of glomeruli by the percentage of the average glomerular volume (vgp).

DETAILED DESCRIPTION I Animal Models for Protein Aging Lewis diabetic rats induced by Alloxan have been used as a model for protein aging to demonstrate in vivo the effectiveness of inhibitors of AGE formation. The correlation demonstrated is between the inhibition of late diabetes related to pathology and the effective inhibition of AGE formation (Brownlee, Cerami and Vlassara, 1988, New Eng. J. Med. 318 (20): 1315-1321). Streptozotocin induction of diabetes has also been used in Lewis rats, New Zealand White rabbits with induced diabetes and diabetic BB / Worcester rats genetically, as described in, for example, U.S. Pat. 5,334, 617 (incorporated herein by reference). A major problem with these model systems is the long period of time required to demonstrate the damage related to the AGE and thus test the compounds for AGE inhibition. For example, 16 weeks of treatment were required for the studies with rats described in the U.S. Patent 5,334,617 and 12 weeks for studies with rabbits. Therefore it would be highly desirable and useful to have a model system for the AGE related to the pathology of diabetes that manifests in a short period of time, a greater efficiency and expeditious determination of the damages related to the AGE and the effectiveness of the inhibitors of the post-Amadori AGE training.

Antibodies to AGEs An important tool for the study of AGE formation is the use of monoclonal and polyclonal antibodies that are specific for AGEs achieved by the reaction of several sugars with a variety of white proteins. Antibodies are investigated for the resulting specificity for AGEs that is independent of the nature of the AGE protein component (Nakayama et al., Biochem. Biophys., Res. Comm. 162: 740-745; Nakayama et al., 1991, J. Immunol Methods 140: 119-125; Horiuchi et al., J. Biol. Chem. 266: 7329-7332; Araki et al., J. Biol. Chem. 267: 10211-10214; Makita et al., 1992, J. Biol. Chem. 267: 5133-5138). Such antibodies have been used to monitor AGE formation in vivo and in vitro.

Thiamine - Vitamin Bi The first member of the Vitamin B complex to identify itself, is thiamine practically devoid of pharmaceutical actions when it is provided in usual therapeutic doses and includes at higher doses it is not known to have any effect. Thiamine pyrophosphate is the physiologically active form of thiamine and functions mainly in the metabolism of carbohydrates as a coenzyme in the decarboxylation of a-keto acids. The thiamine hydrochloride tablets are available in amounts ranging from about 5 to 500 mg each. Injectable solutions of thiamine hydrochloride are available, which contain 100 to 200 mg / ml. To treat thiamine deficiency, intravenous doses as high as 100 mg / l of parenteral fluid are commonly used, administered at the typical dose of 50 to 100 mg. It is believed that the Gl absorption of thiamine is limited to 8 to 15 mg per day, but may be exceeded by oral administration in divided doses with food. Repeated administration of glucose can precipitate thiamine deficiency in poorly nourished patients and this has been noted during the correction of hyperglycemia. Surprisingly, the present invention has found, as shown by an in vitro test, that the administration of thiamine pyrophosphate at levels above those normally found in the human body or administered by diet therapy is an effective inhibitor of the post-Amadori antigenic AGE formation and that this inhibition is more complete than that possible by the administration of aminoguanidine.

Pyridoxine - Vitamin B6 Vitamin B is typically available in the form of pyridoxine hydrochloride in excessive preparations available from many sources. For example, Beelith Tablets from Beach Pharmaceutical Laboratories containing 25 mg of pyridoxine hydrochloride which is equivalent to 20 mg of Be, other preparations include the Marlyn 50 Formula of Marlyn Health Care containing 1 mg of pyridoxine HCl and the Marlyn Formula 50 Mega Forte containing 6 mg of pyridoxine HCl, Wyeth-Ayerst Stuart Prenatal® tablets containing 2.6 mg of pyridoxine HCl, J & amp;J-Merck Corp. Stuart Formula® containing 2 mg of pyridoxine HCl and chewable multivitamins for Children of CIBA Consumer Sunkist containing 1.05 mg of pyridoxine HCl, 150% of the RDA of E.U.A. for children 2 to 4 years old and 53% of RDA of E.U.A. for children above 4 years of age and adults. (Physician's Desk Reference for nonprescription drugs, 14th Edition (Medical Economics Data Production Co., Montvale, N.J., 1993)). There are three related forms of pyridoxine, which differ in the nature of the substitution at the carbon atom in the 4-position of the pyridine nucleus: pyridoxine is a primary alcohol, pyridoxal is the corresponding aldehyde and pyridoxamine contains an aminomethyl group in this position. Each of these three forms can be used by mammals after conversion in the liver to pyridoxal-5'-phosphate, the active form of the vitamin. It has been believed for a long time that these three forms are equivalent in their biological properties and have been treated as equivalent forms of vitamin B in the art. The Council in Pharmacy and Chemistry has assigned the name of pyridoxine to the vitamin. The most active antimetabolite to pyridoxine is 4-deoxypyridoxine, for which the antimetabolic activity has been attributed to the in vivo formation of 4-deoxypyridoxine-5-phosphate, a competent inhibitor of several pyrixodal phosphate dependent enzymes. The pharmacological actions of pyridoxine are limited, but it does not show any pharmacodynamic action after oral or intravenous administration and has low acute toxicity, being soluble in water. It has been suggested that neurotoxicity may develop after a prolonged intake as small as 200 mg pyridoxine per day. Physiologically, as a coenzyme, pyridoxine phosphate is involved in several metabolic transformations of amino acids including decarboxylation, transamination and racemization, as well as enzymatic steps in the metabolism of amino acids containing sulfur and hydroxy. In the case of transamination, the pyridoxal phosphate is aminated to the pyridoxamine phosphate by the donor amino acid and the pyridoxamine phosphate linkage is subsequently deaminated to pyridoxal phosphate by the α-keto acid acceptor. Therefore the vitamin B complex is known to be a necessary dietary supplement involved in the specific breakdown of amino acids. For a general review of the vitamin B complex see The Pharmacological Basis of Therapeutics, 8th. edition, ed. Gilman, Rail Nies and Taylor (Pergamon Press, New York, 1990, pp. 1293-4, pp. 1523-1540). Surprisingly, the present invention has discovered that the effective doses of the pyridoxal amine form transiently metabolizes vitamin B6 (pyridoxamine) to levels above those normally found in the human body, is an effective inhibitor of post-antigen AGE formation. Amadori and that this inhibition may be more complete than that possible by the administration of aminoguanidine.

Formation of stable base Amadori / Schiff Intermediary The typical study of the reaction of a protein with glucose to form AGEs has been by ELISA using antibodies directed through AGEs antigens and detection of the production of a stable acid fluorescent AGE, pentosidine, by HPLC followed by hydrolysis of the acid. Glycation of white proteins (e.g., BSA or RNase A) with glucose and ribose were compared by monitoring the ELISA reactivity of the anti-Glucose-AGE-RNase from polyclonal rabbits and the anti-Glucose-AGE-BSA antibodies. The antigen was generated by the reaction of 1 M glucose with RNase for 60 days and the BSA for 90 days. The antibodies (R618 and R479) were investigated and reactivity was demonstrated with only the AGEs and not with the protein, except for the carrier nmunogen BSA.

Example 1 Thiamine Pyrophosphate and Pyridoxamine Inhibit Antigen Formation in the Final Products of Advanced Glucose Glycation: Comparison with Aminoguanidine Some B6 vitamins, especially pyridoxal phosphate (PLP), have previously been proposed to act as " competitive inhibitors "of the initial glycation, since the aldehydes themselves can form Schiff base adductions with amino protein groups (Khatami et al., 1988, Life Sciences 43: 1725-1731) and therefore limit the amount of amines available for glucose adhesion. However, the effectiveness in the initial limitation of glucose adhesion is not a prognosis of the inhibition of the conversion of any Amadori product formed to AGEs. The present invention describes inhibitors of "late" glycation reactions as indicated by their effects on the in vitro formation of AGEs antigens (Booth et al., 1996, Biochem. Biophys. Res. Com. 220: 113-119). .

The chemicals of pancreatic bovine ribonuclease A (RNase) were chromatographically pure, of aggregate-free degree from Worthington Biochemicals. Bovine serum albumin (BSA, fraction V, free of fatty acids), human metemoglobin (Hb), D-glucose, pyridoxine, pyridoxal, pyridoxal-5'-phosphate, pyridoxamine, thiamine, thiamine monophosphate, thiamine pyrophosphate and goat alkaline phosphatase - conjugated anti-rabbit IgG all were from Sigma Chemicals. Aminoguanidine hydrochloride was purchased from Aldrich Chemicals.

Uninterrupted glycation with Glucose Bovine serum albumin, ribonuclease A and human hemoglobin were incubated with glucose at 37 ° C 0.4 M sodium phosphate stabilizer at pH 7.5 containing 0.02% sodium azide. The protein, glucose (at 1.0 M) and the anticipated inhibitors (at 0.5, 3, 15 and 50 mM) were introduced into the incubation mixture simultaneously. The solutions were stored in the dark in encapsulated tubes. The aliquots were taken and immediately frozen until they were analyzed by ELISA until the conclusion of the reaction. Incubations were for 3 weeks (Hb) or 6 weeks (RNase, BSA).

Preparation of polyclonal antibodies to AGE proteins The immunogen preparation followed by initial protocols (Nakayama et al., 1989, Biochem Biophys. Res. Comm. 162: 740-745; Houriuchi et al., 1991, J. Biol. Chem. 266: 7329-7332; Makita et al., 1992, J. Biol. Chem. 267: 5133-5138). Briefly, the immunogen was prepared by glycation of BSA (antibodies R479) or RNase (antibodies R618) in 1.6 g of protein in 15 ml for 60-90 days using 1.5 M glucose in a 0.4 M sodium phosphate stabilizer of pH 7.5 containing 0.05% sodium azide at a pH of 7.4 and 37 ° C. The New Zealand male white rabbits of 8-12 weeks were immunized by subcutaneous administration of a 1 ml solution containing 1 mg / ml glycated protein in a adjuvant of Freund. The primary injection used to complete the adjuvant and all three boosters was performed at three week intervals with incomplete Freund's adjuvant. The rabbits bled three weeks after the last pressure increase. The serum was collected by centrifugation of the coagulated whole blood. The antibodies were AGE-specific, being non-reactive with any native protein (except for the carrier) or with Amadori intermediates. It has been shown that polyclonal anti-AGE antibodies are a sensitive and valuable analytical tool for the study of "late" AGE formation in vitro and "in vivo". The nature of the dominant antigen AGE epitope or hapten remains unclear, although it has recently been proposed that the carboxymethyl lysine of the protein glycoxidation product (CmL) may be a dominant antigen of several antibodies (Reddy et al., 1995, Biochem. 34: 10872-10878). Previous studies have failed to reveal the reactivity by ELISA with the compounds of the CmL models (Makita et al., 1992, J. Biol. Chem. 267: 5133-5138).

ELISA detection of AGE products The general method of Engvall (1981, Methods Enzymol, 70: 419-439) was used to perform the ELISA. Typically, the glycated protein samples were diluted to approximately 1.5 ug / ml in a 0.1 M sodium carbonate stabilizer at a pH of 9.5 to 9.7. The protein was covered overnight at room temperature in 96-well polystyrene plates by pipetting 200 ul of the protein solution into each dish (0.3 ug / dish). After coating, the protein was washed from the dishes with a saline solution containing 0.05% Tween-20. The dishes were subsequently blocked with 200 ul of 1% casein in a carbonate stabilizer for 2 hours at 37 ° C followed by washing. Anti-AGE antibodies from rabbits were diluted to a solution concentration of approximately 1: 350 in an incubation stabilizer and incubated for 1 h at 37 ° C, followed by washing.

To minimize the background readings, the R479 antibodies used to detect the glycated RNase were raised against the glycated BSA and the R618 antibodies used to detect the glycated BSA and the glycated Hb were raised against the glycinated RNase. A conjugated alkaline phosphatase antibody for rabbit IgG was subsequently added as the secondary antibody at a concentration of a solution of 1: 2000 or 1: 2500 (depending on the batch) and incubated for 1 hour at 37 ° C, followed by washing. The p-nitrophenyl phosphate substrate solution was subsequently added to the plates (200 ul / dishes), with the absorbance of the released p-nitrophenolate being monitored at 410 nm with a 4000 Dynatech MR microplate reader. The controls containing the unmodified protein were routinely included and their readings were subtracted, the corrections are usually insignificant. The validity of the use of the ELISA method in the quantitative study of the kinetics of the AGE formation depends on the linearity of the test (Kemeny &Challacombe, 1988, ELISA and Other Solid Phase Immunoassays, John Wiley &Sons, Chichester, UK) . The control experiments were carried out, for example to demonstrate that the linear velocity for the RNase is below a coating concentration of approximately 0.2-0.3 ug / dish.

Results Figures 1A-D are graphs showing the effect of the vitamin Be derivatives in the post-Amadori AGE formation in bovine serum albumin glycated with glucose. BSA (10 ml / ml) was incubated with 1.0 M glucose in the presence and absence of several indicated derivatives in a 0.4 M sodium phosphate stabilizer at a pH of 7.5 at 37 ° C for six weeks. The aliquots were tested by ELISA using anti-AGE R618 antibodies. The concentrations of the inhibitors were 3, 15 and 50 mM. The inhibitors used in Figures (1A) were Pyridoxamine (PM); (1 B) pyridoxal phosphate (PLP); (1C) pyridoxal (PL); (1 D) pyridoxine (PN). Figure 1 (control curves) shows that the reaction of BSA with 1.0 M glucose is slow and incomplete after 40 days, even at a high concentration of sugar used to accelerate the reaction. The simultaneous inclusion of the different concentrations of several B vitamins markedly affects the formation of AGEs antigens. (Figure 1A-D) pyridoxamine and pyridoxal phosphate strongly suppressed antigen AGE formation in even the lowest concentrations tested, while pyridoxal was effective at approximately 15 mM. Pyridoxine was slightly effective at high concentrations tested. Figures 2 A-D are graphs showing the effect of the derivatives of vitamin Bi and aminoguanidine (AG) in AGE formation in bovine serum albumin. BSA (10 mg / ml) was incubated with 1.0 M glucose in the presence and absence of the various indicated derivatives in a 0.4 M sodium phosphate stabilizer at a pH of 7.5 at 37 ° C for 6 weeks. The aliquots were tested by ELISA using the anti-AGE antibodies R618. The concentrations of the inhibitors were 3, 15 and 50 mM. The inhibitors used in Figures (2A) were thiamine pyrophosphate (TPP); (2B) thiamine monophosphate (TP); (2C) thiamine (T); (2D) aminoguanidine (AG).

Of the several vitamin Bi similarly tested (Figure 2A-D), thiamine pyrophosphate was effective at all concentrations tested (Figure 2C), while thiamin and thiamine monophosphate were not inhibitory. More significantly, it is remarkable to take into account the decrease in the final levels of formed AGEs observed with thiamine pyrophosphate, pyridoxal phosphate and pyridoxamine. The aminoguanidine (Figure 2D) produced some inhibition of the AGE formation in the BSA, but not less than the above compounds. Similar studies were carried out with human metemoglobin and bovine ribonuclease A. Figures 3 A-D are graphs showing the effect of vitamin B6 derivatives on the AGE formation in human metemoglobin. Hb (1 mg / ml) was incubated with 1.0 M glucose in the presence and absence of the various indicated derivatives in a 0.4 M sodium phosphate stabilizer at a pH of 7.5 at 37 ° C for 3 weeks. The aliquots were tested by ELISA using anti-AGE R618 antibodies. The concentrations of the inhibitors were 0.5, 3, 15 and 50 mM. The inhibitors used in Figures (3A) were pyridoxamine (PM); (3B) pyridoxal phosphate (PLP); (3C) pyridoxal (PL); (3D) pyridoxine (PN). Previously it has been reported that the Hb of a diabetic patient contains a component that binds to anti-AGE antibodies and it was proposed that this glycated Hb (Hb-AGE termed, not to be confused with HbAic) could be useful in the measurement of glucose at Prolonged exposure time. The in vitro incubation of Hb with glucose produces AGEs antigens in a range apparently faster than that observed with BSA. However, the different vitamin Be (Figure 3A-D) and B1 (Figure 4 AC) showed that they tend to the same inhibition in Hb, with pyridoxamine and thiamine pyrophosphate being the most effective inhibitors in each of their respective families . Significantly, in Hb, aminoguanidine only inhibited the speed of the AGE formation and not the final levels of the AGE formed (Figure 4D). With RNase the rate of antigenic AGE formation by glucose was intermediate between Hb and BSA, but the extent of inhibition within each series of vitamins remained. Again, pyridoxamine and thiamine pyrophosphate were more effective than aminoguanidine (Figure 5). Figures 4 A-D are graphs showing the effect of the derivatives of vitamin Bi and aminoguanidine (AG) in the AGE formation in human metemoglobin. Hb (1 mg / ml) was incubated with 1.0 M glucose in the presence and absence of the various derivatives indicated in a 0.4 M sodium phosphate stabilizer at a pH of 7.5 at 37 ° C for 3 weeks. The aliquots were tested by ELISA using the anti-AGE antibodies R618. The concentrations of the inhibitors were 0.5, 3, 15 and 50 mM. The inhibitors used in Figures (4A) were thiamine pyrophosphate (TPP); (4B) thiamine monophosphate (TP); (4C) thiamine (T); (4D) aminoguanidine (AG). Figure 5 is a bar graph showing a comparison of the inhibition of ribonuclease A glycation by thiamine pyrophosphate (TPP), pyridoxamine (PM) and aminoguanidine (AG). RNase (1 mg / ml) was incubated with 1.0 m glucose (glc) in the presence and in the absence of the various indicated derivatives in a 0.4 M sodium phosphate stabilizer at a pH of 7.5 at 37 ° C for 6 weeks . The aliquots were tested by ELISA using anti-GE R479 antibodies. The percentage inhibition indicated was calculated from the ELISA readings in the absence and in the presence of the inhibitors in a time of 6 weeks. The concentrations of the inhibitors were 0.5, 3, 15 and 50 mM.

Discussion These results show that certain derivatives of vitamins Bi and B are able to inhibit "late" AGE formation. Some of these vitamins successfully inhibited the final levels of AGE produced, in contrast to aminoguanidine, it is suggested that they have greater interactions with the post-Amadori or Amadori precursors to the AGEs antigens. The Amadori and post-Amadori intermediaries represent a crucial junction in which the "classical" route of nonenzymatic glycation begins to be essentially irreversible (Scheme I). In preliminary studies of "glycation" inhibition it was usually measured as the formed Schiff base (after reduction with labeled cyanoborohydride) or as the Amadori product formed (after acid precipitation labeled with sugar). Said experiments did not produce information regarding the inhibition of the post-Amadori conversion stages to "late" AGE products, since said stages continue without change in the amount of labeled sugar that is added to the proteins. Other "glycation" experiments have relied on increased non-specific protein fluorescence induced sugar, but this can also be induced by free sugar dicarbonyl oxidative fragments, such as glycoaldehyde or glyoxal (Hunt et al., 1988 , Biochem. 256: 205-212), independently of the Amadori product formation. In the case of pyridoxal (PL) and pyridoxal phosphate (PLP), the data carrier of the simple inhibition mechanism involves the condensation of the competitive Schiff base of these aldehydes with the amino protein groups at the glycation sites. Due to internal hemiacetal formation in pyridoxal but not pyridoxal phosphate, the strongest inhibition of AGE formation by PLP was expected by this competitive mechanism. This is observed in the data (Figure 1 B, 1C, Figure 3B, 3C). Inhibition by pyridoxamine is necessarily different, since pyridoxamine lacks an aldehyde group. However, pyridoxamine is an amine candidate potentially capable of forming a Schiff base bond with the carbonyls of the open chain sugars with dicarbonyl fragments with Amadori products or with post-Amadori intermediates. The mechanism of inhibition of the Bi compounds is not obvious. All forms contain an amino functionality, so that the marked efficiency of only the pyrophosphate form suggests an important requirement for a strong negative charge. A significant unexpected observation is that the extent of inhibition by aminoguanidine and some other compounds is considerably less in the "late" stages of the reaction than during the initial phase. This suggests a different mechanism of action than that of pyridoxamine and thiamine pyrophosphate, suggesting that the therapeutic potential of these compounds will be improved by co-administration with aminoguanidine.

Example 2 Kinetics of Nonenzymatic Glycation: Paradoxical Inhibition by Ribose and Easy Isolation of Protein Intermediate for Rapid Post-Amadori AGE Formation While glucose concentrations are used to cause non-enzymatic protein glycation, paradoxically, it was found that ribose in high concentrations is inhibitory of the post-Amadori AGE formation in the ribonuclease by the action of the post-Amadori "late" stages of the glycation reaction. This inhibitory effect unexpectedly suggests that the "initial" reactive intermediates, presumably Amadori products, can accumulate with a small formation of "late" post-Amadori products (Maillard products; AGEs). Research within this phenomenon has demonstrated: (1) ability to define the conditions for the kinetic isolation of the Amadori (or post-Amadori) glycosylated intermediary (s); (2) the study of the ability of rapid kinetics of the accumulation of such intermediary; (3) the ability to study the surprisingly rapid kinetics of the conversion of said intermediates to AGE products in the absence of free or reversibly bound sugar; (4) the ability to use these intermediaries to study and characterize the post-Amadori inhibition stages of the AGE formation so that a novel system is provided to investigate the mechanism of reaction and the efficacy of potential agents that can block formation AGE and (5) with this knowledge it is also possible to use 13C NMR to study the reactive intermediates and monitor their conversion to several AGEs candidates (Khalifah et al., 1996, Biochemistry 35 (15): 4645-4654).

Chemicals and Materials As in Example 1 above.

Preparation of polyclonal antibodies to AGEs As in Example 1 above.

ELISA detection of AGE products As in Example 1 above.

Amino Acid Analysis The amino acid analyzes were carried out in the Biotechnology Support Laboratory of the Medical Center of the University of Kansas. The analyzes were carried out after hydrolysis of the glycated protein (reduced with sodium cyanoborohydride) with 6 N HCl at 110 ° C for 18-24 hours. The phenyl isothiocyanate was used for derivatization and the PTH derivatives were analyzed by the reverse-phase HPLC in an amino acid analyzer of Applied Biosystems (derivative 420A, separation system 130A, data analysis system 920A).

Phase-Inverse HPLC Analysis of Pentosidine The production of pentosidine in the RNase was quantified by HPLC (Sell &Monnier, 1989, J. Biol .. Chem. 264: 21597-21602; Odetti et al., 1992, Diabetes 41 : 153-159). The modified ribose protein samples were hydrolysed in 6N HCl for 18 hours at 100 ° C and subsequently dried in a Speed Vac. The samples were subsequently redissolved and the aliquots were placed in 0.1% trifluoroacetic acid and analyzed by HPLC in a Shimadzu system using a Vydac C-18 column equilibrated with 0.1% TFA. A gradient of 0-6% acetonitrile (0.1% in TFA) was run in 30 minutes at a flow rate of about 1 ml / min. Pentosidine was detected by fluorescence emission 385 nm / excitation 335 nm and its elution time was determined by running a synthesized standard. Due to the extremely small levels of pentosidine expected (Grandhee &Monnier, 1991, J. Biol .. Chem. 266: 11649-11653; Dyer et al., 1991, J. Biol. Chem. 266: 11654-11660) , no attempt was made to quantify the absolute concentrations. Only the relative concentrations were determined from the peak areas.

Modifications of Glycation Modification with ribose or glucose is generally carried out at 37 ° C in a 0.4 M phosphate stabilizer at a pH of 7.5 containing 0.02% sodium azide. The high concentration of stabilizer was always used with modifications of 0.5 M ribose. The solutions were preserved in encapsulated tubes and only opened to remove the synchronized aliquots that were immediately refrigerated to subsequently perform several analyzes. The experiments of "interrupted glycation" were carried out first with the incubation of the protein with ribose at 37 ° C for 8 or 24 hours, followed by extensive and immediate dialysis against frequent changes of the color stabilizer at 4 ° C. The samples were subsequently re-incubated by rapid heating at 37 ° C in the absence of external ribose. The aliquots were taken and frozen at various intervals for further analysis. Due to the low molecular weight of the RNase, protein concentrations were re-routed after dialysis even when the low molecular weight of the dialysis tube isolate was used. An alternate procedure was also devised (see below) in which the interruption was achieved by a simple 100-dot dilution from the reaction mixtures containing 0.5 M ribose. The protein concentrations were estimated from a UV spectrum. The difference in molar extinction between peak (278 nm) and channel (250 nm) was used for RNase concentration determinations to compensate for the general increase in UV absorbance that accompanies glycation. Studies of the difference between UV spectrum and time dependence were carried out to characterize the contributions of the UV spectrum glycation.

Data Analysis and Numerical Simulations of Kinetics Kinetic data were routinely adjusted to bioexponential or monoexponential functions using nonlinear least squares methods. The kinetic mechanism of schemes 5-6 has been examined by numerical simulations of the differential equations of the reaction. Both simulations and adjustments of the observed kinetic data were carried out with the SCIENTIST 2.0 software package (Micromath, Inc.). The determination of the apparent mean times (Figure 6B) of the adjustment of the kinetic data to the two-exponential functions (Figure 6A) was carried out with the "solve" function of the MathCAD 4.0 program (MathSoft, Inc.).

RESULTS Comparison of Glucose and Ribose Glucose The reaction of RNase A with ribose and glucose was first followed with ELISA tests, using rabbit-specific R 479 AGE antibodies developed against modified BSA-glucose. To a lesser degree, the production of pentosidine, the only known stable acid fluorescent AGE, was quantified by HPLC followed by acid hydrolysis. Preliminary studies using 0.05 M ribose at 37 ° C demonstrate that the antigenic AGE formation rate increases modestly (measured at 2-3 points by the apparent mean time) as the pH increases from 5.0 to 7.5, with a small apparent period of induction at the beginning of the kinetics in all cases. The glycation of RNase with 0.05 M ribose at a pH of 7.5 (half-time about 6.5 days) is almost an order of magnitude faster than that of glycation with 1.0 M glucose (half-time exceeding 30 days; see Figure 7B, solid line). Subsequent kinetics also displayed a small period of induction but incomplete even after 60 days, making it difficult to estimate a real mean time. When the dependence of the kinetics on the ribose concentration was examined at pH 7.5, a more unexpected result was obtained. The reaction rate initially increased with the increase in ribose concentration, but at concentrations above 0.15 M the rate of the reaction was leveled and subsequently significantly decreased (Figure 6A). A plot of the dependence of the reciprocal mean time on the ribose concentration (Figure 6B) shows that the high concentrations of ribose are paradoxically inhibitory to the post-Amadori antigen AGE formation. This unusual but consistent effect is independent of changes in concentration and in the stabilizer (2-point) or RNase (10-point) and was not changed by its purification affinity of the R479 antibody in an immobilized AGE-RNase column. . Similarly it is not due to the effects of ribose in the ELISA test itself. The inhibitory effect measured by ribose in the post-Amadori AGE formation is not similar due to the interference of ribose with the recognition of the antibody from the AGE antigen sites in the protein in the ELISA test. Prior to the first contact with the primary anti-AGE antibody in the ELISA plates, the glycated protein had been diluted over 1000-dot, washed extensively with Tween-20 after absorption and blocked with a 1% casein coating followed by a Additional washing with Tween-20.

Kinetics of AGE Formation Post-Amadori Antigens by "Interrupted Glication" In view of the small period of induction observed, an attempt was made to determine if there was any accumulation during the reaction, of an initial precursor such as an Amadori intermediate, capable of produce the AGEs post-Amadori antigens detectable by the ELISA test. The RNase was glycated at a pH of 7.5 and at 37 ° C with a high concentration of 0.5 M ribose and the reaction was stopped after 24 hours by immediate cooling at 4 ° C and dialysis against several changes of cold stabilizer over a period 24 h to remove freely and reversibly the limit of ribose (Schiff base). Since a sample of free ribose subsequently heated rapidly to 37 ° C without re-adding ribose and sampled for post-Amadori AGE formation for several days. The AGE antigen production of this "interrupted glycation" sample of 24 h is shown by the dotted line and the open triangles in Figure 7A, the time elapsed in cold dialysis is not included. An uninterrupted control (solid line and full circles) is also shown for comparison. In both samples, the kinetics of post-Amadori antigen AGE formation are evident and dramatically different. The kinetics of the antigenic AGE production of the interrupted free ribose sample shows: (1) monoexponential kinetics without induction period, (2) a greatly improved rate of antigen AGE formation with remarkable average times of the order of 10 hours and (3) ) production of antigen levels comparable to those observed in long incubations in the continuous presence of ribose (see Figure 6A). Equally significant, the data also demonstrate that the insignificant AGE antigen was formed during the cold dialysis period, as shown by the small difference between the points of the open triangle and the full circle in 1 day time in Figure 7A. Very little AGE was formed, if any, by "interrupting" the procedure itself. These observations show that a fully competent isolatable intermediate or precursor of the AGE antigen was generated during 24 h of contact with the ribose before free and reversibly removing the sugar limit. The samples interrupted after only 8 hours produced a final amount of AGE antigen that was approximately 72% of the sample interrupted for 24 h. The samples of RNase glycated with only 0.05 M ribose and interrupted at 8 hours by cold dialysis and reincubated at 37 ° C revealed less than 5% of ELISA reagent antigen production after 9 days. Disruption at 24 hours, however, produced a rapid increase in the ELISA antigen (similar to Figure 7A) to a level approximately 50% of that produced in the uninterrupted presence of 0.05 M ribose. The same effects of the general interruption they were also observed with other proteins (BSA and Hemoglobin). Except for an absolute value different from the rate constants and the amount of AGEs antigens formed during the 24 hours of the 0.5 M ribose incubation, the same dramatic increase in the speed of the AGE antigen formation was observed after the removal of ribose 0.5 M. The glycation is much slower with glucose than with ribose (note the difference in time scales between Figure 7A and Figure 7B). However, unlike the case with ribose, the interruption after 3 days of glycation with 1.0 M glucose produced an insignificant accumulation of the AGE antigen precursor ELISA reagent (dotted curve, Figure 7B).

Kinetics of Pentosidine Formation Samples subjected to the ELISA test were also examined for the production of pentosidine, a stable acid AGE. The content of pentosidine was measured for the same RNase samples analyzed for antibody reactivity by ELISA. Glycation by ribose in a 0.4 M phosphate stabilizer at a pH of 7.5 produced pentosidine in RNase A which was quantified by fluorescence after acid hydrolysis. Figure 8A shows that under uninterrupted conditions, 0.05 M ribose produces a progressive increase in pentosidine. However, when glycation is carried out under "interrupted" conditions using 0.5 M ribose, a dramatic increase in the rate of pentosidine formation is observed immediately after removal of excess ribose (Figure 8B), which is similar to, but slightly faster than the kinetics of the AGEs antigens (Figure 7A). A greater amount of pentosidine was also produced with a 24 hr interruption compared with that of 8 hr. The reproducible differences between the kinetics of pentosidine formation and the AGEs antigens can also be noticed. A significant amount of pentosidine was formed during the 24-h incubation and also during cold dialysis, resulting in an omission of the dotted vertical line in Figure 8B. Therefore, observations demonstrate that a pentosidine precursor accumulates in ribose during glycation that can rapidly produce pentosidine after ribose removal (see Odetti et al., 1992, Diabetes 41: 153-159).

Rate of Accumulation of the Intermediary (s) Reagent (s) The "interrupted glycation" experiments described above demonstrate that a precursor or precursor of AGEs post-Amadori antigens and pentosidine can accumulate during glycation with ribose. The kinetics of formation of this intermediate can be followed independently and quantified by a variation of the experiments described above. The amount of the intermediate generated in RNase in different contact spaces with ribose can be tested by the maximum extension at which the AGE antigen can be produced after the interruption. In variable periods after initial glycation, free and reversibly limited ribose is removed by cold dialysis or by rapid dilution (see below). Sufficient time is then followed (5 days, representing several half-lives according to Figure 7A) after heating to 37 ° C for maximum development of AGEs post-Amadori antigens. The 5-day ELISA readings after each interruption point represent the maximum AGE development, which could later be proportional to the intermediate concentration present at the time of interruption. Figure 9 represents said experiment wherein the kinetics of the intermediate accumulation are measured for RNase A in the presence of 0.5 M ribose (solid symbols and curve). For comparison, the amount of AGE present before the removal of ribose at each breakpoint is also shown (open symbols and dotted lines). As expected (Figure 7A), a small AGE is formed before the removal (or dilution) of the ribose, so that the ELISA readings after 5 days of the secondary incubation periods are mainly a measure of the AGE formed after the removal of ribose. The results in Figure 9 show that the buffering speed of the intermediate in ribose 0.5 M is exponential and very fast, with an average time of approximately 3.3 h. This is approximately 3-points faster than the observed rate of conversion of the intermediate to AGEs antigens after interruption (Figure 7A open symbols and dotted curve) In these experiments the removal of ribose at each interruption time was achieved by a 100-point dilution and not by dialysis. The simple dilution reduced the ribose concentration from 0.05 M to 0.005 M.

Independently it was determined (Figure 6A) that the small AGE was produced at the time of the scale with 5 mM of residual ribose. The dilution approach was dictated primarily by the need for quantitative point-to-point accuracy. This accuracy would not have been achieved by the dialysis process that would be carried out independently for each sample at each point of interruption. The results show that the dilution was equivalent to dialysis. A separate control experiment (see Figure 10 below) showed that 100-dot instantaneous dilution gave almost identical results to the dialysis procedure. These control experiments demonstrated that the binding equilibrium of reversible ribose protein (Schiff base) is very rapid in this time scale. This is consistent with the data of Bunn and Higgins (1981, Science 213: 222-224) which indicate that the average time of formation of the Schiff base with ribose 0.5 M should be of the order of a few minutes. The 100-point rapid dilution method to reduce ribosomes is a valid method where quantitative accuracy is essential and can not be achieved by multiple dialysis of many samples.

Direct Inhibition of AGE Post-Amadori Intermediary Formation by Ribose and Glucose The increase in the speed of AGE formation after interruption and dilution of sugar suggests, but does not prove that high ribose concentrations are inhibitory to the reaction in or in addition to the first "stable" intermediary, presumably the Amadori derivative (indicated in Scheme I). A test of this was carried out to study the effect of the direct addition of ribose in the post-Amadori reaction. The RNase was first incubated for 24 hours in 0.5 M ribose to prepare the intermediate. Two protocols were carried out to measure the possible inhibition of post-Amadori formation of AGEs antigens by different concentrations of ribose. In the first experiment, the lowered sample of 24 h was simply diluted 100-points in solutions containing several final concentrations of ribose in the range of 0.005 M to 0.505 M (Figure 10A). It is clearly observed that the speed and extension of AGE formation decreases by increasing ribose concentrations. Significantly, above the highest concentration of 0.5 M ribose, the kinetics is exponential and does not show the induction period that occurs with uninterrupted glycation (Figures 6A and 7A) at high concentrations of ribose. A second experiment (Figure 10B) was also carried out in which the interrupted sample of 24 h was dialyzed extensively in the cold to liberate freely and reversibly the limit of the ribose as well as any inhibitor product that had been formed during the inhibition of 24 h with ribose. Following this, the aliquots were diluted 100-points in fresh varying concentrations of ribose and the formation of AGE antigen products was monitored as explained above. These results were clearly identical to the experiment in Figure 10A where the dialysis step was omitted. In both cases, the speed and extension of AGE formation were decreased by increasing ribose concentrations and the kinetics was exponential with the period of non-induction.

The question of whether glucose or other sugars can also inhibit AGE formation of the reactive intermediate obtained by interrupted glycation in 0.5 M ribose was also investigated. The effects of glucose at concentrations of 1.02-2.0 M were tested (data not shown). Glucose was clearly not as inhibitory as ribose. When the interrupted 24-hour sample of ribose was diluted 100-points in these glucose solutions, the amount of AGE antigen formed was decreased by approximately 30% but there was a small decrease in the apparent rate constant. Again, the kinetics was exponential.

Effect of pH on Post-Amadori Kinetics of AGE Formation The interrupted glycation method was used to investigate the pH dependence of the post-Amadori kinetics of AGE formation from the reactive intermediate. In these experiments, RNase A was first reacted for 24 h with 0.5 M ribose at a pH of 7.5 to generate the reactive intermediate. The kinetics of the deterioration of the intermediate to AGEs was subsequently measured by ELISA. Figure 11 shows that an extremely wide pH range of 5.0-9.5 was achieved when the kinetics were measured at the initial rates. A well-configured remarkable dependence was observed, showing that the kinetics of the antigenic AGEs formation decreased in both ranges of alkaline and acid pH, with an optimum close to pH 8. An individual experiment of "pH jump" was also carried out in the pH 5.0 sample studied previously that had the slowest rate of AGE antigen formation. After 12 days at 37 ° C in a stabilizer with a pH of 5.0, the pH was quickly adjusted to 7.5, and the antigen AGE formation was monitored by ELISA. Within the experimental error, the sample showed identical kinetics (same first order rate constant) of AGE formation for the interrupted glycation samples that had been studied directly at a pH of 7.5 (Figure 12). In this experiment, the relative amounts of antigen AGE could not be directly compared in the same ELISA plate, but the pH jump sample was formed substantially through at some diminished levels of the AGEs antigens. These results demonstrate that the intermediate can be prepared free of AGE and stored at a pH of 5 for further studies of the conversion to AGEs.

Inhibition of Post-Amadori AGE formation by Aminoguanidine The efficacy of aminoguanidine was tested by this method of interrupted glycation, for example, by testing its effect on the post-Amadori formation of AGEs antigens after removing the excess limit of ribose. Figure 20A demonstrates that aminoguanidine has modest effects on the block of AGE formation antigens in RNase under these conditions, with little inhibition below 50 mM. Approximately 50% of the inhibition was achieved only at or above 100-250 mM. Note again that in these experiments, the protein was exposed to aminoguanidine only after the interruption and the free and reversible removal of the ribose limit. Comparable results were also obtained with the interrupted glycation of BSA (Figure 20B).

Amino acid analysis of the Interrupted Glycation Samples The amino acid analysis was carried out in RNase after a 24 h contact with 0.5 M ribose (without dialysis) after extensive dialysis of the 24 hr glycated sample after 5 days incubation of the late sample at 37 ° C. These determinations were made after the reduction of sodium cyanoborohydride which reduces the Schiff base present in the usinas or the terminal amino group. All three samples normalized to alanine (12 residues), showed the same residual lysine content (4.0 ± 0.5 out of the original 10 in RNase). This indicates that after 24 h of contact with 0.5 M ribose, most of the adducts of the Schiff base formed had been converted to Amadori or subsequent products. No arginine or histidine residue was lost by modification.

Discussion The use of the rapid reaction of ribose and the discovery of its reversible inhibition of post-Amadori stages has enabled the dissection and determination of the kinetics of different stages of protein glycation in RNase. Preliminary studies of protein "glycation" kinetics studies have now been restricted to the "initial" stages of Schiff base formation and the subsequent Amadori arrangement. Some kinetic studies have been carried out starting with synthesized fructosilamines, for example, small models of Amadori glucose compounds (Smith and Thornalley, 1992, Eur. J. Biochem. 210: 729-739, and the references cited in this document), but such studies, with few exceptions, have not so far they have been possible with proteins. A notable exception is the demonstration by Monnier (Odetti et al., 1992, supra) that BSA partially glycated with ribose can rapidly produce pentosidine after the removal of ribose. The increased reactivity of ribose has also proven a distinct advantage in the quantitative definition of the time course of AGE training. It is noted that glucose and ribose are both capable of producing similar AGE products, such as pentosidine (Grandhee & amp;; Monnier, 1991, supra; Dyer et al. 1991, supra), and the work of the model compound 13C NMR was done with ADP-ribose (Cervantes-Laurean et al., 1993, Biochemistry 32: 1528-1534). The present work shows that the AGE products of ribose antigens completely cross-react with anti-AGE antibodies directed against modified glucose proteins, suggesting that ribose and glucose produce AGEs similar antigens. The primary kinetic differences observed between these two sugars are probably due to their relative differences in the rate constants of the stages that guide the post-Amadori AGE formation, rather than in the mechanism. The results presented reveal a paradoxical and marked inhibition of the whole AGE formation by high concentrations of ribose (Figure 6) that have not been anticipated by preliminary studies. This inhibition is rapidly reversible in the sense that it is removed by dialysis of the initially modified protein (Figure 7A) or by a simple 100-dot dilution (as used in Figure 11). The experiments in Figure 10 demonstrate that it is not due to the accumulation of the dialyzable inhibitor intermediates during the initial glycation, since the 24 h dialysis of modified protein followed by the addition of different concentrations of fresh ribose induces the same inhibition. The data in Figure 10A, B show that the inhibition occurs by the rapid and reversible interaction of ribose with the protein intermediate that contains the reactive Amadori products. Inhibition can not be applied to the initial stage of Amadori product formation due to the rapid formation rate of the Amadori intermediate that was determined in the experiment of Figure 9. The identification of the reactive intermediate as an Amadori product is well supported by the amino acid analysis that was carried out (after reduction with sodium cyanoborohydride) before and after dialysis at the 24 h breakpoint. The residual lysine content without change indicates that no ZIF base has previously been irreversibly converted (presumably by the Amadori rearrangement) for 24 h. The suppression of secondary ribose from the "late" but not "initial" glycation stages significantly enhanced the accumulation of a fully competent reactive Amadori intermediate containing a small AGE. Its isolation by the process of interruption is of importance for the structural and kinetic studies, since it allows to make studies in the absence of the sugar limit of Schiff base or free and its reactions and accompanying complications. For example, Post-Amadori conversion rates to AGE antigen products and pentosidine AGE have been measured (Figure 7A, open symbols and Figure 8B) and proved to be much faster (t ~ 10 h) than those reflected in all the kinetics under non-interrupting conditions (Figure 6A and Figure 8A). The rapid formation of pentosidine that was measured is consistent with an initial interrupted ribose experiment in BSA by Odetti et al. (1992, supra). Since ribose and its derivatives such as ADP-ribose are normal metabolites, the very high rates of AGE formation observed suggest that they should be considered more seriously as sources of potential glycation in several cellular compartments (Cervantes-Lauren et al., 1993, supra), it was even thought that their concentrations were below those of the less reactive glucose. Another new application of intermediary isolation is in the study of the pH dependence of this complex reaction. The unusually bell-shaped pH profile observed for the post-Amadori AGE formation (Figure 11) stands in marked contrast to the moderate pH dependence of the entire reaction. Late kinetics reflect a compound effect of pH at all stages of the reaction, including the Schiff base and the Amadori product formation, each of which may have a unique pH dependence. This complexity is especially well illustrated by hemoglobin glycation studies (Lowery et al., 1985, J. Biol. Chem. 260: 11611-11618). The profile of the pH configured in the shape of a bell suggests, but does not prove, the participation of two ionizing groups. If true, the data may involve the participation of a second amino group, such as that of a nearby lysine, in the formation of AGEs dominant antigens. The pH profile observed and the pH jump observations described suggest that a useful route for the isolation and maintenance of the reactive intermediate would be by rapidly decreasing the pH to about 5.0 after the 24 hr. Kinetic studies provide new insights into the mechanisms of action of aminoguanidine (guanilhidrazine), an AGE inhibitor proposed by Cerami and its collaborators to combine with Amadori intermediates (Brownlee et al., 1986, supra). This proposed pharmacological agent is now in Phase III of clinical trials for possible therapeutic effects in the treatment of diabetes (Vlassara et al., 1994, supra). However, its AGE inhibition mechanism is equally complex, since it is multifunctional. As a nucelophilic hydrazine, it can be reversibly added to activate carbonyls, including the open chain sugar and ribose aldehyde carbonyls (Khatami et al., 1988, Life Sci. 43: 1725-1731; Hirsch et al., 1995, Carbohyd. Res. 267: 17-25), as well as keto carbonyls of Amadori compounds. Also a guanidinin compound that can produce highly reactive dicarbonyl glycation intermediates such as glyoxal and glucosones (Chen &Cerami, 1993, J. Carbohyd.Chem.12: 731-742; Hirsh et al., 1992, Carbohyd Res. 232: 125-130; Ou &Wolff, 1993, Biochem. Pharmacol. 46: 1139-1144). The interrupted glycation method allowed the examination of the efficacy of aminoguanidine in only the post-Amadori stages of AGE formation. Equally important, this allowed studies in the absence of free sugar or reactive dicarbonyl fragments of free sugar (Wolf &Dean, 1987, Biochem J. 245: 243-250, Wells-Knecht et al., 1995, Bichemistry 34: 3702-3709) that can be combined with aminoguanidine. The results of Figure 20 demonstrate that aminoguanidine has, at most, only a modest effect on post-Amadori AGE formation reactions, achieving 50% inhibition at concentrations above 100-250 mM. The efficacy of aminoguanidine is therefore predominantly increased in the initial stages of inhibition (Schiff base formation) or the production of highly reactive dicarbonyls generated during glycation. Contrary to the original claims, it does not inhibit AGE formation through the Amadori intermediate complex. The use of interrupted glycation is not limited to kinetic studies. The interrupted glycation can simplify structural studies of the glycated proteins and identify unknown AGEs using techniques such as 13C NMR that have been used to detect the RNase Amadori adducts (Neglia et al., 1983, J. Biol. Chem. 258 : 14279-14283; 1985, J. Biol .. Chem. 260: 5406-5410). The combined use of structural and kinetic approaches should be of special interest. For example, although the identity of the AGEs dominant antigens that react with the polyclonal antibodies remains uncertain, the candidate AGEs, such as the recently proposed lysine (carboxymethyl) (Reddy et al., 1995, Biochemistry 34: 10872-10878; cf. Makita et al., 1992, J. Biol. Chem. 267: 5133-5138) should display the same kinetics of the reactive intermediate that have been observed. The availability of the interrupted kinetic approach will also help to determine the importance of the Amadori route to the formation of this AGE. Similarly, the monitoring of the glycation reaction interrupted by techniques such as 13C NMR should identify the resonances of other candidate antigen AGEs as those that display similar appearance kinetics. Table I lists the 13C NMR peaks of the Amadori intermediate of the RNase prepared by the reaction with ribose enriched C-2. The lower peak near 205 ppm is probably due to the carbonyl of the Amadori product. In all cases, the ability to remove excess Schiff base and free sugars through interrupted glycation will considerably simplify isolation, identification and structural characterization.

Table I lists the peaks that were assigned to the Post-Amadori Intermediary due to its intensity of decrease or invariable with time. Peak positions are listed in ppm in the lower fields of the TMS.

Table I Resonances 125MHz C-13 NMR of the Amadori Intermediary Ribonuclease Prepared by Reaction 24 HR with 99% Ribose [2- C13] 216.5 ppm 108.5 ppm 211.7 105.9 208 103.9 103 172 95.8 165 163.9 73.65 162.1 70.2 69.7 Ribonuclease A was reacted for 24 hr with 0.5 M ribose enriched 99% with C-2, followed by an excess and the Schiff base boundary ribose were removed by extensive cold dialysis. The sample was subsequently heated again to 37 ° C immediately before taking a 2 hr NMR scan. The RNase signals reacted with ribose of natural abundance under identical conditions were subsequently subtracted from the NMR spectrum. For the all the peaks shown are due to the enriched C-13 that originated at position C-2. Some of the peaks increase from the intermediary degradation products and these can be identified by increasing the intensity of the peak by time. Figure 31 shows the NMR spectrum obtained.

Example 3 In Vitro Inhibition of the Formation of the Final Products of Advanced Antigen Glycation (AGEs) by Derivatives of Vitamins Bi and B6 and Aminoguanidine. Inhibition of Post-Amadori Kinetic Differences from All Glycation The interrupted glycation method for the following post-Amadori kinetics of AGE formation allows rapid quantitative study of the "late" stages of the glycation reaction. Importantly, this method allows for inhibition studies that are free of the routes of the AGE formation that increases from the glycoxidative products of the Schiff base or free sugar (Namiki route) as illustrated in Scheme I. therefore, the interrupted glycation method allows the identification and unique and rapid characterization of inhibitors of "late" stages of glycation that leads to AGE antigen formation. Among the derivatives of vitamin B- \ and B6 examined, pyridoxamine and thiamine pyrophosphate are unique inhibitors of the post-Amadori route of AGE formation. Importantly, it was found that the efficacy of the inhibition of total glycation of the protein, in the presence of high concentrations of sugar, does not predict the ability to inhibit the post-Amadori stages of AGE formation where the free sugar was removed . Therefore while pyridoxamine, thiamine pyrophosphate and aminoguanidine are potent inhibitors of AGE formation in the total glycation of the protein by glucose, aminoguanidine differs from the other two in that it is not an effective inhibitor of AGE formation after -Amadori. Aminoguanidine markedly retards the initial rate of AGE formation by ribose under uninterrupted conditions, but has no effect on the final levels of AGEs produced antigens. The examination of different proteins (RNase, BSA and hemoglobin) confirmed that the inhibition results are generally non-specific according to the protein used, although there are individual variations in the rates of inhibition and AGE formation.

Chemicals and Materials As in Example 1 above.

Preparation of polyclonal antibodies to AGEs As in Example 1 above.

ELISA detection of AGE products As in Example 1 above.

Non-interrupted ribose glycation assays Bovine serum albumin, ribonuclease A and human hemoglobin were incubated with ribose at 37 ° C in a 0.4 M sodium phosphate stabilizer at a pH of 7.5 containing 0.02% sodium azide. . The protein (10 mg / ml or 1 mg / ml), ribose 0.05 M and the anticipated inhibitors (at 0.5, 3, 15 and 50 mM) were introduced into the incubation mixture simultaneously. The solutions were stored in the dark in encapsulated tubes. The aliquots were taken and immediately frozen until they were analyzed by ELISA at the end of the reaction. Incubations were for 3 weeks (Hb) or 6 weeks (RNase, BSA). The glycation reactions were monitored for constant pH throughout the duration of the experiments.

Tests for interrupted glycation of ribose (post-Amadori) The glycation was first carried out by incubation of the protein (10 mg / ml) with 0.5 M ribose at 37 ° C in a 0.4 M phosphate stabilizer at a pH of 7.5 containing 0.2% sodium azide for 24 h in the absence of inhibitors. The glycation was subsequently interrupted to reversibly remove excess and limit sugar (Schiff base) by extensive dialysis against the frequent cold stabilization changes at 4 ° C. Samples of glycated intermediates containing a maximum amount of Amadori product and little AGE ( depending on the protein) they were heated rapidly to 37 ° C without replenishment of ribose. This initiated conversion of Amadori intermediates to AGE products in the absence or presence of various concentrations (typically 3, 15 and 50 mM) of anticipated inhibitors. The aliquots were taken and frozen at various intervals for further analysis. The solutions were stored in encapsulated tubes and opened only to remove the synchronized aliquots that were immediately frozen to subsequently carry out several analyzes.

Numerical analysis of kinetic data Kinetic data (time progression curves) were routinely adapted to mono or bi- exponential functions using nonlinear least-squares methods using the SCIENTIST 2.0 program (MicroMath, Inc.) or the ORIGIN program (Microcal , Inc.) that allows the user to define functions and control the parameters to iterate them. The standard deviations of the parameters of the adjusted functions (final and initial ordinary values and velocity constants) were returned as measures of the accuracy of the adjustment. The apparent mean times for the bi-exponential kinetic adjustments were determined with the "solve" function of the MathCad program (MathSoft, Inc.).

RESULTS Inhibition by the vitamin B6 derivatives of the total kinetics of the AGE formation from Ribose. The inhibitory effects of vitamin Bi and B6 derivatives on the kinetics of AGE antigen formation were evaluated by polyclonal antibodies specific for AGEs. The initial inhibition studies were carried out on the glycation of bovine ribonuclease A (RNase) in the continuous presence of 0.05 M ribose, which is the concentration of ribose when the speed of the AGE formation is close to the maximum. Figure 13 (control curves, full rectangles) shows that the formation of antigen AGEs in RNase when incubated with 0.05 M ribose is relatively rapid, with an average time of about 6 days under these conditions. Pyridoxal-5'-phosphate (Figure 13B) and pyridoxal (Figure 13C) significantly inhibited the rate of AGE formation in the RNase at concentrations of 50 mM and 15 mM. Surprisingly, pyridoxine, the alcoholic form of vitamin B6, also modestly inhibited AGE formation in RNase (Figure 13D). Of the vitamin Be derivatives examined, pyridoxamine at 50 mM was the best inhibitor of the "final" levels of the AGE formed in RNase in the 6-week monitored period of time (Figure 13A).

Inhibition by the derivatives of vitamin B, of the total kinetics of the AGE formation from Ribose. All Bi vitamin inhibited the antigenic AGE formation in RNase at high concentrations, but the inhibition was more complex than for the B derivatives (Figure 14A-C). in the case of thiamine pyrophosphate as the inhibitor (Figure 14A), both the rate of AGE formation and the final levels of the EFA produced in the dish were decreased. In the case of thiamine phosphate as the inhibitor (Figure 14B) and thiamin (Figure 14C), they had less effect on the rate of AGE formation, but a substantial decrease in the final level of the AGE formed in the presence of high concentration of the inhibitor. In general, thiamine pyrophosphate showed greater inhibition than the other two compounds at the lowest concentrations tested.

Inhibition by aminoguanidine of the total kinetics of AGE formation from Ribose The inhibition of AGE formation by aminoguanidine (Figure 14D) was distinctly different than that observed in the experiments with vitamin Bi and B6. The increase in aminoguanidine concentration decreased the rate of AGE formation in RNase, but did not reduce the final level of the AGE formed. The final AGE level formed after the 6-week was almost identical to that of the control for all the examined concentrations of aminoguanidine.

Inhibition of the total kinetics of AGE formation in serum albumin and hemoglobin from Ribose Comparative studies were carried out with BSA and human methemoglobin (Hb) to determine if the observed inhibition was specific protein. The different derivatives of vitamin Be (Figure 15 AD) and vitamin Bi (Figure 16 AC) exhibited similar tendencies of inhibition when incubated with BSA as with RNase, pyridoxamine and thiamine pyrophosphate being the most effective inhibitors of each family . Pyridoxine did not inhibit AGE formation in BSA (Figure 15D). Pyridoxal phosphate and pyridoxal (Figure 15B-C) mainly inhibited the rate of AGE formation, but not the final levels of the AGE formed. Pyridoxamine (Figure 15A) exhibited some inhibition at low concentrations and at high concentrations tested inhibited the final levels of the AGE formed more effectively than with any other Be derivative. In the case of the Bi derivatives, the total extension of the inhibition of AGE formation with BSA (Figure 16 A-C) was lower than that observed with RNase (Figure 14 A-C). The high concentrations of thiamine and thiamine pyrophosphate inhibited the final levels of AGEs formed without greatly affecting the speed of the AGE formation (Figure 16C). The aminoguanidine also displayed the same effects of inhibition with BSA as those observed with RNase (Figure 16D), decreasing the rate of AGE formation without significantly affecting the final levels of the AGE formed.

The kinetics of AGE formation were also examined using Hb in the presence of the derivatives of vitamin Be and Bi and aminoguanidine. The apparent absolute rates of AGE formation were significantly higher with Hb than with RNase or BSA. However, the inhibitors tested showed essentially similar behavior. The effects of vitamin B derivatives are shown in Figure 17. Pyridoxamine showed the highest inhibition at concentrations of 3 mM and higher (Figure 17A) and was more effective when compared to pyridoxal phosphate (Figure 17B), pyridoxal (Figure 17C) and pyridoxine (Figure 17D). In the case of the vitamin Bi derivatives (data not shown), the inhibitory effects were more similar to the BSA inhibition tendencies than to RNase. Inhibition was only modest at high concentrations tested (50 mM), being approximately 30-50% for all three Bi derivatives. The main manifestation of the inhibition was in the reduction of the final levels of AGE formed.

Inhibition by the vitamin B6 derivatives of the post-Amadori AGE ribose formation kinetics Using the interrupted glycation model to test the inhibition of the "late" post-Amadori formation, the kinetics were examined by incubation of Amadori intermediates isolated from Rnasa or BSA at 37 ° C in the absence of the free ribose limit. The ribose sugar that was initially used to prepare the intermediates was removed by cold dialysis after an initial glycation reaction period of 24 h. After the AGE training was summarized, AGE formation is very fast (average times of approximately 10 h) in the absence of any inhibitor. Figure 18 shows the effects of pyridoxamine (Figure 18A), pyridoxal phosphate (Figure 18B) and pyridoxal (Figure 18C) on the post-Amadori kinetics of BSA. Pyridoxine produced no inhibition (data not shown). Similar experiments were carried out in RNase. Pyridoxamine caused a close complete inhibition of AGE formation with RNase at 15 mM and 50 mM (Figure 18D). Pyridoxal showed no significant inhibition at 15 mM (the highest tested), but pyridoxal phosphate showed significant inhibition at 15 mM. Pyridoxal pyrophosphate is known for its affinity for labeling the active site of RNase (Raetz and Auld, 1972, Biochemistry, 11: 2229-2236). With BSA, other than RNase, a significant amount of AGE antigen formed during the initial 24 h incubation with BSA (25-30%) as evidenced by the high ELISA readings after the removal of ribose at time zero for the Figures 18 AC. For BSA and RNase, the inhibition, when observed, manifests as a decrease in the final levels of the AGE formed rather than as a decrease in the rate of AGE formation.

Inhibition by the Vitamin Bi derivatives of the post-Amadori AGE ribose formation kinetics Thiamine pyrophosphate inhibited AGE formation more effectively than other vitamin Bi derivatives with RNase and BSA (Figure 19). Thiamine showed no effects, while thiamine phosphate showed some intermediate effect.

As with the B6 tests, the post-Amadori inhibition was more apparently manifested as a decrease in the final levels of the AGE formed.

Effects of aminoguanidine and? / O '-acetyl-L-lysine on the post-Amadori AGE ribose formation kinetics Figure 20 shows the results of the aminoguanidine test for the inhibition of post-Amadori AGE formation kinetics with BSA and RNase. At 50 mM the inhibition was about 20% in the case of BSA (Figure 20B) and less than 15% with RNase (Figure 20A). The possibility of inhibition was also tested by simple amino-containing functionalities groups using Na-acetyl-L-lysine (Figure 21), which contains only one free Ne-amino group. Na-acetyl-L-lysine at above 50 mM did not exhibit any significant inhibition of AGE formation.

Discussion Numerous studies have shown that aminoguanidine is an apparently potent inhibitor of many manifestations of nonenzymatic glycation (Brownlee et al., 1986, Brownlee, 1992, 1994, 1995). The inhibitory effects of aminoguanidine in several phenomena that are induced by the reduction of sugars are widely considered as proof of the participation of glycation in many of these phenomena. Aminoguanidine has recently introduced a second cycle of Phase III clinical trials (such as pimagedine) to improve the complications of diabetes seen as the cause of glycation of connective tissue proteins due to high sugar levels. The data from the kinetic study of "slow" AGE formation uninterrupted with glucose-induced RNase (Example 1) confirmed that aminoguanidine is an effective inhibitor and also identified a number of derivatives of vitamins Bi and B6 as equal or slightly more effective inhibitors . However, inhibition by aminoguanidine unexpectedly decreased the effect in late stages of the AGE formation reaction. Due to the slow glycation of the protein with glucose, this surprising observation could not be fully examined. In addition, it has been suggested that there are questions about the long-term stability of aminoguanidine (Ou and Wolff, 1993, supra). The analysis using the much faster glycation by ribose allowed the total time course of AGE formation to be observed in a total and quantified manner during the uninterrupted glycation of the protein. The use of interrupted glycation only allowed the isolation and examination of the post-Amadori antigenic AGE formation in the absence of the free ribose (base Schiff) limit. The comparison of the data from these two approaches with the initial glucose glycation kinetics has provided novel insights into the mechanisms and effectiveness of several inhibitors. Figure 22 are bar graphs representing the synthesized comparative data of percent inhibition at defined time points using various concentrations of inhibitor. Figure 22A graphs the data for inhibition after interrupted glycation of the AGE RNase formation. Figure 22B graphs the data for inhibition after interrupted glycation of AGE BSA formation by ribose. The total results unambiguously demonstrate that aminoguanidine delays the rate of AGE antigen formation in the presence of sugar but that it has little effect on the final amount of the post-Amadori AGE formed. Therefore, observations limited to only the "initial" stages of AGE formation that indicate efficacy as an inhibitor can, in fact, lead to misinterpretation of the actual efficacy of the inhibition of post-Amadori AGE formation. Therefore, the ability to observe a complete reaction course using ribose and interrupted glycation provides a more complete picture of the efficacy of post-Amadori AGE formation inhibition.

Example 4 Animal model & In vivo effects test of the AGE / inhibitors training Hyperglycemia can be rapidly induced (in one or two days) in rats by the administration of streptozocin (aka streptozotocin, STZ) or alloxan. This has become a common model for diabetes mellitus. However, these rats manifest nephropathy only after many months of hyperglycaemia and usually just before dying from end-stage renal disease (ESRD). It is believed that this pathology is caused by the irreversible chemical modification of glucose from long-lived proteins such as collagen from the base membrane. STZ diabetic rats show very late albuminuria after the induction of hyperglycemia at approximately 40 weeks usually only just before death. Due to the dramatic fast effects of the ribose demonstrated in vitro in the previous examples, the effects of the administration of ribose to rats and the possible induction of AGEs by rapid glycation of ribose were examined. From this study, a rat model for the induced pathology of accelerated ribose has been developed.

Effects of short-term administration of ribose in vivo Phase I Protocol Two groups of six rats each were given, in one day: a. 300 mM ribose (two intraperitoneal infusions 6-8 separate hours, each 5% of body weight as ml) or b. 50 mM ribose (an infusion) The rats were kept for 4 days without additional administration of ribose, at which time they were sacrificed and the following physiological measures were determined: (i) initial and final body weight; (ii) weight of the kidney in the final state; (iii) final and initial tail-leg blood pressure; (V) separation of creatinine per 100 g of body weight. The albumin filtration ranges were not measured, since rapid changes were not initially anticipated. Past experiences with STZ diabetic rats show that albuminuria develops very late (perhaps 40 weeks) after the induction of hyperglycemia and just before the animals expire.

Kidney Physiological Results a. The final body weight and final kidney weight was the same for the treatment groups with high and low concentration of ribose. b. The tail-leg blood pressure was increased from 66 ± 4 to 83 ± 3 for rats treated with low-concentration ribose (1 × 50 mM) and from 66 ± 4 to 106 ± 5 for rats treated with high-concentration ribose (2). x 300 mM). These results are shown in the bar graph of Figure 23. c. Separation of creatinine, as cc per 100 g of body weight, was decreased (expected normal range approximately 1.0-1.2) in a dose-dependent manner of 0.87 ± 0.15 for the low ribose group and further decreased 30% to 0.62 ± 0.13 for the high concentration ribose group. These results are shown in the bar chart of Figure 24.

Conclusion Phase I A single-day treatment caused a dose-dependent hypertension and a dose-dependent decrease in a glomerular separation function was manifested 4 days later. These are significant metabolic changes of diabetes observed only much later in STZ diabetic rats. This phenomenon may be hypothetical due to the irreversible chemical modification of ribose (glycation) of the protein in vivo.

Effect of exposure to high concentrations of ribose for a long time Phase II Protocol Groups of rats (3-6) were given intraperitoneally "low concentration ribose dose" 0.3M (LR) or "high concentration ribose dose" 1.0 M (HR) by two daily injections per (i) 1 day, (ii) a "short term" (S) of four days or (iii) a "long term" (L) of 8 days. Additionally, these ribose concentrations were included in the drinking water.

Kidney Physiological Results a. The blood pressure of the tail-leg was increased in all groups of rats treated with ribose, confirming the results of Phase I (Figure 23). b. The separation of creatinine decreased in all groups in a dose-dependent and time-dependent manner (Figure 24). c. The rate of Albumin Effusion (AER) was significantly increased in a ribose-dependent manner at the 1-day and 4-day exposures. However, he showed some recovery at day 8 relative to day 4 in the high-dose group but not in the low-dose group. These results are shown in the bar graph of Figure 25. d. The separation of creatinine per 100 g of body weight decreased in both groups of high and low concentration of ribose to approximately the same extent in a time-dependent manner (Figure 24).

Phase II Conclusion Exposure to ribose in as little as 4 days; leads to renal dysfunction and hypertension, manifested by decreased creatinine clearance and increased albumin filtration. These changes are typical of diabetes and are observed much later in diabetic rats STZ.

Intervention through two new therapeutic compounds and aminoguanidine Phase III Protocol Six rats were randomly placed in 9 different groups, including those exposed to 1 M ribose for 8 days in the presence and absence of aminoguanidine, pyridoxamine and thiamine pyrophosphate as follows: Control Groups: (i) no treatment; (ii) high dose (250 mg / kg body weight) of pyridoxamine ("compound -P"); (iii) high dose (250 mg / kg body weight of thiamine pyrophosphate) ("T-compound" or "TPP") and (iv) low dose (25 mg / kg body weight) of aminoguanidine ("AG") ). Test Groups: (i) only 1 M ribose saline solution (2 x 9 cc daily IP for 8 days); (ii) ribose plus low dose ("LP") of pyridoxamine (25 mg / kg of body weight injected as 0.5 ml with 9 cc of ribose); (iii) ribose plus high dose ("HP") of pyridoxamine (250 mg / kg of body weight injected as 0.5 ml with 9 cc of ribose); (iv) ribose plus high dose ("HT") of thiamin pyrophosphate (250 mg / kg of body weight injected as 0.5 ml with 9 cc of ribose) and (v) ribose plus low dose of aminoguanidine (25 mg / kg of body weight injected as 0.5 ml with 9 cc of ribose). Unlike phase II, ribose was not administered in drinking water. The intervention compounds were pre-administered for one day before introducing them with ribose.

Kidney Physiological Results a. Blood pressure was increased very slightly by the three compounds alone (control groups); The elevated ribose BP was not enhanced by the co-administration of compounds. These results are shown in the bar graph of Figure 26. b. The creatinine separation in the controls was unchanged, except for the TPP that decreased it. c. The separation of creatinine was normalized when the ribose was coadministered with low dose (25 mg / kg) of aminoguanidine or pyridoxamine. These results are shown in the bar graph of Figure 27. d. High concentrations (250 mg / kg) of pyridoxamine and TPP showed only partial protection against the decrease in ribose induced in the separation of creatinine (Figure 27). and. The rate of albumin effusion (AER) was elevated by ribose, as well as by the high dose of pyridoxamine and TPP and the low dose of aminoguanidine in the absence of ribose. These results are shown in the bar graph of Figure 28. f. The rate of albumin effusion was restored to the normal level by the co-administration of low doses of aminoguanidine and pyridoxamine. These results are shown in the bar graph of Figure 29.

Phase III Conclusion As measured by two indices of renal function, pyridoxamine and aminoguanidine at 25 mg / kg were apparently effective in the same way in the prevention of the decrease in ribose induced in the separation of creatinine and the moderate increase in Ribose induced in albuminuria. (i) Thiamine pyrophosphate was not tested at 25 mg / kg; (ii) thiamine pyrophosphate and pyridoxamine at 250 mh / kg were partially effective in preventing decreases in creatinine clearance but possibly not in the prevention of moderate proteinuria; (iii) at these high concentrations and in the absence of ribose, thiamine pyrophosphate alone caused a decrease in creatinine separation and both produced moderate increases in albuminuria.

Abstract Renal Function and Diabetes Persistent hyperglycemia in diabetes mellitus leads to diabetic nephropathy in at least one third of human patients. Clinically, diabetic nephropathy is defined by the presence of: 1. decrease in renal function (odd glomerular separation) 2. an increase in urinary protein (odd filtration) 3. The simultaneous presence of hypertension Renal function depends on blood flow (not measured) and glomerular separation, which can be measured by separation of inulin (not measured) or separation of creatinine. Glomerular permeability is measured by the filtration rate of albumin but this parameter is very variable. It is also a logarithmic distribution function: a hundred-point increase in albumin excretion represents a two-point decrease in filtration capacity.

Diabetic Rat Model by Ribose, By the above criteria, ribose very quickly induces manifestations of diabetic nephropathy as reflected in hypertension, separation of creatinine and albuminuria, although the latter is not representative. In the established STZ diabetic rat, hyperglycemia is rapidly established in 1-2 days but the clinical manifestations of diabetic nephropathy increase very slowly, perhaps as much as 40 weeks for albuminuria. In general, albuminuria is highly variable from day to day and from animal to animal, although different in humans, most STZ rats eventually develop nephropathy.

Compound Intervention Using animals treated with ribose, pyridoxamine at 25 mg / kg body weight effectively prevents two of the three manifestations usually attributed to diabetes, namely the impairment of creatinine separation and albumin filtration. This was as effective as aminoguanidine. The effectiveness of thiamine pyrophosphate was not manifested, but it should be emphasized that this may be due to its use at high concentrations of 250 mg / kg body weight. Pyridoxamine is much less effective if only results at 250 mg / kg of body weight are considered.

Effect of Single Compounds In the end, the rats seemed to tolerate the compounds well. The kidney weights were not remarkable and they developed little hypertension. The physiological effects of the compounds were tested only at 250 mg / kg. Thiamine pyrophosphate, but not pyridoxamine, decreased the creatinine separation at this concentration. Both appeared to slightly increase albuminuria, but these measurements were perhaps the least reliable.

Administration in Humans A typical adult human being of average weight of 66-77 kg.

Typically, diabetic patients tend to be overweight and may be up to 112 kg. The recommended diet allows for a male adult between 66-77 kg revised in 1989, 1.5 mg per day of thiamine and 2.0 mg per day of Vitamin (Merck Manual of Diagnosis and Therapy, 16th, edition (Merck &Co., Rathaway , NJ, 1992) pp 938-939). Based on the rat model approach, a dose range of administration of pyridoxamine or thiamine pyrophosphate that is predicted to be effective for the inhibition of post-Amadori AGE formation and thus inhibit related pathologies would fall within the range of 1 mg / 100 g of body weight at 200 mg / 100 g of body weight. The appropriate range when co-administered with aminoguanidine would be similar. Calculated for an average adult of 75 Kg, the range (in for example 1 mg / 1 kg of body weight) can be approximately 75 mg to above 150 g (in for example 2 g / 1 kg of body weight). This will naturally vary in accordance with the particular patient.

Example 5 Inhibition of the Formation (AGE) of the Final Glycation Product Advanced by Pyridoxamine-5'-Phosphate (PMP) Recent data (Figure 32B) using the interrupted glycation test as described above have shown that the AGE formation is inhibited by the administration of Pyridoxamine-5'-Phosphate (PMP) compared to PM.

The present invention demonstrates pharmaceutical compositions comprising PMP or salts thereof in pharmaceutical carriers suitable for the treatment of disorders related to AGE. Therefore the present invention further demonstrates a method for the inhibition of post-Amadori AGE formation comprising the administration of an effective post-Amadori AGE inhibiting amount of pyridoxamine-5'-Phosphate. It also comprises a method of cross-linking protein inhibition by administering an effective post-Amadori AGE inhibiting amount of pyridoxamine-5'-Phosphate.

EXAMPLE 6 In Vivo Inhibition of End-product Formation (AGE) of Advanced Glycation by Derivatives of Vitamin B6 and Aminoguanidine. Inhibition of diabetic nephropathy. The interrupted glycation method as described in the previous examples, allows the rapid generation of Amadori well-defined protein intermediates stable from ribose and other pentose sugars for use in in vivo studies. The effects of 25 mg / kg / day of pyridoxamine (PM) and aminoguanidine (AG) in the renal pathology induced by the injection of 50 mg / kg / day of ribose glycated in rat serum albumin-Amadori in Sprague-Dawley rats daily and RSA not modified for 6 weeks. Transient hyperfiltration (increased creatinine separation) was observed transiently with rats receiving Amadori-RSA and AGE-RSA, despite the presence of PM and AG. Individuals from each group receiving Amadori-RSA and AGE-RSA exhibited microalbuminuria, but none were observed if PM was co-administered. Immunoblotting with anti-RSA revealed glomerular staining in rats treated with AGE-RSA and with Amadori-RSA and this staining was decreased by treatment with PM but not by AG treatment. A decrease in glomerular sulfated glycosaminoglycans (Alcian blue stain with pH 1.0) was also found in rats treated with RSA (Amadori and AGE) glycoside. This appears to be due to reduced heparan sulfate proteoglycans (HSPG) as evidenced by decreased staining with mAb JM-403 which is specific for side chain HSPG. These HSPG changes were improved by treatment with PM, but not by AG treatment. It is therefore concluded that pyridoxamine can prevent both glomerular and diabetic loss of heparan sulfate and glomerular deposition of glycated albumin in SD rats chronically treated with glycated-ribose albumin.

Materials and Chemical Methods Rat serum albumin (RSA) (fraction V, essentially free of fatty acids 0.005% A2018), ribose-D, pyridoxamine and goat alkaline phosphatase-IgG conjugated anti-rabbit were all from Sigma Chemicals. Aminoguanidine hydrochloride was purchased from Aldrich Chemicals.

Preparation of lowered RSA Rat serum albumin was passed on an Affi-Gel Blue column (BioRad), a CL-6B heparin-Sepharose column (Pharmacy) and an endotoxin binding-affinity column (Detoxigel, Pierce Scientific) to remove any possible contaminant. Purified rat serum albumin (RSA) was subsequently dialyzed in a 0.2 m phosphate stabilizer (pH 7.5). A portion of the RSA (20 mg / ml) was subsequently incubated with 0.5 M ribose for 12 hours at 37 ° C in the dark. After the 12 h incubation, the reaction mixture was dialysed in a 0.2 M cold sodium phosphate stabilizer for a period of 36 hours at 4 ° C (this dialysis removes not only the free ribose, but also the Schiff base intermediates ). At this stage of the glycation process, the lowered protein was classified as Amadori-RSA and is negative for AGEs antigens, as determined by the reaction of antibodies with AGE protein (as previously described; R618, antigen: modified glucose AGE- RNase). The lowered protein is then divided into portions that will be injected as: a) Amadori-RSA, b) NaBH4-reduced Amadori-RSA, c) AGE-RSA. The lowered protein to be injected as Amadori-RSA is simply dialyzed against cold PBS at 4 ° C for 24 hours. A portion of the Amadori-RSA in 0.2 M sodium phosphate was reduced with NaBH4 to form NaBH4-reduced Amadori-RSA. Briefly, the aliquots were reduced by the addition of 5 uL of NaBH4 of stored solution (100 mg / ml in 0.1 M NaOH) per mg of protein, incubated for 1 hour at 37 ° C, treated with HCl to discharge the excess of NaBH and subsequently extensively dialyzed in cold PBS at 4 ° C for 36 hours. AGE-RSA was formed by re-incubation of Amadori-RSA in the absence of sugar for 3 days. The mixture was then dialyzed against cold PBS at 4 ° C for 24 hours. All solutions were filtered (22 um filter), sterilized and monitored by endotoxins by a test of limulus amoebocyte lysate (E-Toxato, Sigma Chemicals) and contained < 0.2 ng / ml before being frozen (-70 ° C) below the individual aliquots until the time of injection.

Animal Studies Male Sprangue-Dawley rats (Sasco, 100 g) were used. After an adaptation period of 1 week, the rats were placed in metabolic cages to obtain a urine specimen for 24 hours for 2 days before the administration of the injections. The rats were subsequently divided into the control and experimental groups and injected into the tail veins with either saline, unmodified RSA (50 mg / kg), Amadori-RSA (50 mg / kg), NaBH4-reduced Amadori-RSA (50 mg / kg), or AGE-RSA (50 mg / kg). Rats injected with Amadori-RSA and AGE-RSA were subsequently left untreated or were further treated by administration of either aminoguanidine (AG, 25 mg / kg), pyridoxamine (MW, 25 mg / kg) or a combination of AG and PM (10 mg / kg each) through drinking water. The body weight and ingested water of the rats were monitored weekly to adjust the doses. At the end of the experimental study, the rats were placed in metabolic cages to obtain a urine specimen for 24 hours for 2 days before slaughter of the animals. The total protein in the urine samples was determined by the Bio-Rad test. Albumin in the urine was determined by competitive ELISA using rabbit anti-rat serum albumin (Cappell) as the main antibody (1/2000) and goat anti-rabbit IgG (Sigma Chemical) as a secondary antibody (1/2000) . The urine was tested with Multistix 8 SG (Miles Laboratories) for glucose, ketone, specific density, per block, pH, protein, nitrite and leukocytes. Nothing remarkable was detected except for a little protein.

Creatinine measurements were made with a Beckman II creatinine analyzer. Blood samples were collected by puncture of the heart before termination and used in determining the separation of creatinine, blood glucose (glucose oxidase, Sigma Chemical), fructosamine (nitro blue tetrazolium, Sigma Chemical) and Hb glycated (columns, Pierce Chemicals). The aorta, heart, both kidneys and tail of the rat were visually inspected and subsequently removed quickly after perfusion with saline through the right ventricle of the heart. One kidney, the aorta, the tail of the rat and the lower 2/3 of the heart were hermetically frozen and subsequently stored permanently at -70 ° C. The other kidney was sectioned removing both ends (cortex) to freeze tightly, with the remaining portions of the kidney being sectioned in three with two portions placed in stabilized formalin and the remaining third chopped and placed in 2.5% glutaraldehyde / paraformaldehyde. to 2%.

Light microscope After perfusion with saline, sections of the kidney were fixed on ice with 10% neutral stabilized formalin. Paraffin-embedded tissue sections from all groups of rats (n = 4 per group) were processed to stain with Harris haematoxylin alum and eosin (H &E), acid peroxide / Schiff reagent (PAS) and Alcian blue spots (pH 1.0 and pH 2.5) for histological examination. The alciano blue sections were recorded by two researchers confidentially.

Electron microscope Tissues were fixed in 2.5% glutaraldehyde / 2% paraformaldehyde (0.1 M sodium cacodylate, pH 7.4), post-fixed for 1 hour in stabilized osmium tetroxide (1.0%), pre-stained in uranyl acetate 0.5% for 1 hour and mounted on Effapoxi resin. The ultrathin sections were examined with an electron microscope. Immunofluorescence Paraffin-embedded sections were removed with paraffin and placed in blocks with 10% goat serum in PBS for 30 min at room temperature. The sections were subsequently incubated for 2 hours at 37 ° C with primary antibodies, either polyclonal anti-AGE antibody purified from rabbit by affinity or a polyclonal sheep anti-rat serum albumin antibody (Cappell). Subsequently, the sections were rinsed for 30 minutes with PBS and incubated with secondary antibodies, either FITC-goat purified by double-stained affinity (H + L) affinity (Zymed) or a sheep anti-sheep IgG. rabbit (complete) (Cappell) for 1 hour at 37 ° C. Sections were subsequently rinsed for 30 min with PBS in the dark, mounted on aqueous mounting media for immunocytochemistry (Biomeda) and slide cover. Sections were recorded confidentially. Kidney sections were evaluated by the number and intensity of glomerular spotting in 5 regions around the periphery of the kidney. The annotations were normalized by the immunofluorescence labeling per 100 glomeruli with a marking system of 0-3.

Preparation of Polyclonal Antibodies to AGE Proteins The immunogen was prepared by glycation of BSA (R479 antibodies) or RNase (antibodies R618) to 1.6 g of protein in 15 ml for 60-90 days using 1.5 M glucose in 0.4 M phosphate containing 0.05% sodium at a pH of 7.4 and at 37 ° C. The New Zealand male white rabbits of 8-12 weeks were immunized by subcutaneous administration of 1 ml of solution containing 1 mg / ml glycated protein in Freund's adjuvant. The main injection used the complete adjuvant and three pressure increases were made at three-week intervals with incomplete Freund's adjuvant. The rabbits bled for three weeks after the last pressure increase. The serum was collected by centrifugation of the blood completely clotted. The antibodies are AGE-specific, being non-reactive with any natural protein or with Amadori intermediates.

ELISA Detection of Products A GE The general method of Engvall (21) was used to perform the ELISA. Samples of glycated proteins were diluted at approximately 1.5 ug / ml in a 0.1 M sodium carbonate stabilizer at pH 9.5 to 9.7. The protein was coated overnight at room temperature in 96-well polystyrene plates by pipetting 200 ul of protein solution into each dish (approximately .3 ug / dish). After coating, the excess protein was washed from the dishes with a saline solution containing 0.05% Tween-20. The plates were subsequently blocked with 200 ul of 1% casein in a carbonate stabilizer for 2 hours at 37 ° C followed by washing. The rabbit anti-AGE antibodies were diluted to a concentration of 1: 350 solution in an incubation stabilizer and incubated for 1 hour at 37 ° C, followed by washing. To minimize the above readings, the R618 antibodies used to detect the glycated RSA were generated by immunization against the glycated RNase. A conjugated antibody-alkaline phosphatase to rabbit IgG was subsequently added as the secondary antibody at a solution concentration of 1: 2000 and incubated for 1 hour at 37 ° C, followed by washing. The p-nitrophenolate was monitored at 410 nm with a Dynatech MR4000 microplate reader.

Results The rats in this study survived the treatments and showed no outward signs of any significant pathology. Some of the rats showed some small change in weight and scabs on the tail. Initial investigation of the kidney sections with PAS and H &E spots did not reveal any obvious changes and some EM sections did not reveal any major changes in the glomerular base membrane (GBM). However, in the spotting with Alcian Blue, differences in the pulse were discovered. Spotting with Alcian blue was directed towards negatively charged groups in tissues and can be made selective via changes in staining pH. At a pH of 1.0, Alcian blue is selective for mucopolysaccharides and at pH 2.5 detects glucuronic groups. Therefore, the negative changes are detected depending on the pH of the stain.

At a pH of 2.5 the Alcian blue stain showed that the Amadori-RSA (p <0.05) and the AGE-RSA (p <0.01) induced the increased staining for the acid glycosaminoglycans (GAG) at the control levels ( Figure 33). For both, AGE-RSA and Amadori-RSA, treatment with pyridoxamine (PM) prevented the increase in spotting (p <0.05 compared to controls). In contrast, treatment with aminoguanidine (GA) or combined PM and AG at 10 mg / kg each did not prevent the increase. At a pH of 1.0 the Alcian blue spotting was significantly decreased by the AGE-RSA (p <0.001) (Figure 34). However, no significant difference was observed with the Amadori-RSA. Due to the light staining, the treatment with PM, AG and combined can not be quantified. Immunofluorescent glomerular staining for RSA showed high staining with Amadori-RSA and AGE-RSA in injected animals (Figure 35). The significant reduction of this effect was observed in the rats treated with PM and not with AG or AG & PM combined. Immunofluorescent glomerular staining for the Nuclear Proteoglycan protein of Heparan Sulfate showed a slightly reduced staining with Amadori-RSA and AGE-RSA in injected animals but was not statistically significant (Figure 36). A reduction of this effect was observed in the rats treated with PM and not with AG or AG & PM combined. However, immunofluorescent glomerular staining for the chain of Heparan Sulfate Proteoglycan showed slightly reduced staining with Amadori-RSA and AGE-RSA in injected animals (Figure 37). A significant reduction of this effect was observed in the rats treated with PM and not with AG or AG & PM combined. The analysis of the average glomerular volume by means of the confidential record showed that the Amadori-RSA and the AGE-RSA cause a significant increase in the average volume of the glomerulus (Figure 38). A significant reduction of this effect is observed with the treatment of rats with PM. No effect was observed in the treatment with AG or PM and AG combined at 10 mg / kg of each.

Example 7 AGE Inhibitory Compounds The present invention comprises compounds and pharmaceutical compositions containing compounds having the general formula: Formula I wherein Ri is CH2NH2, CH2SH, COOH, CH2CH2CH2, CH2CH2SH or CH2COOH; R2 is OH, SH or NH2; And it is N or C, so that when Y is N, R3 is nothing and when Y is C, R3 is NO2 or another electron of the elimination group and salts thereof.

The present invention also comprises compounds of the general formula Formula II wherein R ^ is CH2NH2, CH2SH, COOH, CH2CH2NH2, CH2CH2SH or CH2COOH; R2 is OH, SH or NH2; And it is N or C, so that when Y is N, R3 is nothing and when Y is C, R3 is NO2 or another electron of the elimination group; R is H or C 1-6 alkyl; R 5 and Re are H, C 1-6 alkyl, alkoxy or alkane and salts thereof . u-fa «...

In addition, the present invention also provides compounds of the formulas The compounds of the present invention can incorporate one or more of the electrons of the elimination group, so that they are not limited to -NH2, -NH ', -NR'2l -ON, -OCH3, -OCR' and -NH- COH3 wherein R 'is C 1-6 alkyl. In a preferred embodiment at least one of R 4, R 5 and Re are H. The present invention also comprises compounds wherein R and R 5 are H, C 1-6 alkyl, alkoxy or alkene. In connection with the present invention it is also understood that R2 and Re can be H, OH, SH, NH2, C1-6 alkyl, alkoxy or alkene. It is also envisioned that R, R 5 and R 6 may be major functional groups such that they are not limited to phosphate, aryl, heteroaryl and cycloalkyl alkoxy groups. As used herein, the term "aryl" refers to aromatic carbocyclic groups having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl) or multiple fused rings in which at least one is aromatic , (eg, 1, 2, 3, 4-tetrahydronaphthyl, naphthyl, anthryl or phenanthryl), which may be optionally substituted with, for example, halogen, lower alkyl, lower alkylthio, trifluoromethyl, lower acyloxy, aryl and heteroaryl.

A preferred aryl group is phenyl optionally substituted with up to five groups independently selected from halogen, cyano, hydroxy, long or branched chain lower alkyl having from 1-6 carbon atoms or cycloalkyl having from 3-7 carbon atoms, mono or dialkylamino , wherein each alkyl is independently long or branched chain lower alkyl having 1-6 carbon atoms or cycloalkyl having 3-7 carbon atoms, long or branched chain lower alkoxy having 1-6 carbon atoms, alkoxy cycloalkyl having 3-7 carbon atoms or NR1 COR2, COR2, CONR1R2 or CO2R2 wherein R1 and R2 are the same or different and represent hydrogen or long or branched chain lower alkyl having 1-6 carbon atoms or cycloalkyl having of 3-7 carbon atoms. By "heteroaryl" is meant that the aromatic ring systems have at least one or more than four hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur. Examples of the heteroaryl groups are pyridyl, pyrimidinyl, pyrrolyl, pyrazolyl, pyrazinyl, pyridazinyl, oxazolyl, naphthyridinyl, isoxazolyl, phthalazinyl, furanyl, quinolinyl, isoquinolinyl, thiazolyl and thienyl which can be optionally substituted with, for example, halogen, lower alkyl, alkoxy lower, lower alkylthio, trifluoromethyl, lower acyloxy, aryl, heteroaryl and hydroxy. The aryl and heteroaryl groups in this document are systems characterized by the electrons 4n + 2, where n is an integer.

In addition to those mentioned above, the present invention comprises other examples of the aryl and heteroaryl groups and are the following: As noted above, each of these groups may be optionally mono or polysubstituted with selected groups independently of, for example, halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, lower acycloxy, aryl, heteroaryl and hydroxy. Other examples of various aryl and heteroaryl groups are shown in Table D of published International Application WO 93/17025 (incorporated herein by reference). As used herein, the term "alkoxy cycloalkyl" refers to the groups of the formula where a is an integer from 2 to 6; R 'and R "independently represent hydrogen or alkyl and b is an integer from 1 to 6. By" alkyl "and" lower alkyl "in the present invention refer to long or branched chain alkyl groups having 1-12 carbon atoms. carbon, such as, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and -methylpentyl Unless otherwise indicated herein, the substituents of the alkyl group may optionally be substituted by at least one group independently selected from hydroxy, mono or dialkyl amino, phenyl or pyridyl, by "alkyl" and "lower alkyl" "in the present invention refer to long or branched chain alkyl groups having 1-12 carbon atoms, such as, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl and 3-methylpentyl, unless otherwise indicated In this manner, the substituents herein are optionally substituted with at least one group independently selected from hydroxy, mono or dialkyl amino, phenyl or pyridyl. By "alkoxy" and "lower alkoxy" in the present invention refer to long or branched chain alkoxy groups having 1-6 carbon atoms, such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentyl, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy and 3-methylpentoxy. "Alkylene" and "lower alkylene" in the present invention refer to long or branched chain alkene groups having 1-6 carbon atoms, such as, for example, ethylene, propylene, 1-butene, 1-pentene, 1-hexene, cis and trans-2-butene or 2-pentene, isobutylene, 3-methyl-1-butene, 2-methyl-2-butene and 2,3-dimethyl-2-butene. By "salts thereof" in the present invention refers to compounds of the present invention as salts and metal complexes with said compounds, such as with and not limited to Al, Zn, Mn, Cu and Fe. One skilled in the art ordinarily he will be able to make compounds of the present invention using standard methods and techniques. The present invention comprises pharmaceutical compositions comprising one or more of the compounds of the present invention, or salts thereof in an appropriate carrier. The present invention comprises methods for the administration of the pharmaceuticals of the present invention for the therapeutic intervention of pathologies that are related to the AGE formation in vivo. In a preferred embodiment of the present invention, the pathology related to AGE to be treated is related to diabetic nephropathy.

The compounds of the present invention can be formulated as a solution of lyophilized powders for parenteral administration. Powders can be reconstituted by the addition of an appropriate diluent or other pharmaceutically acceptable carrier before use. The liquid formulation is generally an aqueous, isotonic stabilized solution. Examples of suitable diluents are normal isotonic saline, standard 5% dextrose in water or sodium stabilized from an ammonium acetate solution. Said formulation is especially suitable for parenteral administration, but can also be used for oral administration. It may be desirable to add excipients such as polyvinyl pyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate. Alternatively, the compounds of the present invention can be encapsulated, placed in tablets or prepared in an emulsion syrup (oil in water or water in oil) for oral administration. The pharmaceutically acceptable solid or liquid carriers that are generally known in the art of pharmaceutical formulations can be added to improve or stabilize the composition or facilitate the preparation of the composition. Solid carriers include starch (corn or potato), lactose, calcium sulfate dihydrate, alba earth, croscarmellose sodium, magnesium stearate or stearic acid, talc, pectin, acacia, agar, gelatin or colloidal silica dioxide. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier can also include a sustained release material such as glyceryl mono-stearate or glyceryl distearate alone or with wax. The amount of the solid carrier varies but will preferably be between about 1 mg to about 1 g per unit dose. The present invention can be included in other forms or carried out in other routes without departing from the spirit or essential characteristics thereof. The present description and the enumerated examples thereof will be considered in all aspects as illustrative and not restrictive, the scope of the invention being indicated in the accompanying claims. An expert in the art will be able to recognize the equivalent embodiments of the present invention and will be able to practice such modalities using the teachings of the present disclosure and only routine experimentation.

Claims (18)

1. A compound of the general formula: wherein R1 is CH2NH2, CH2SH, COOH, CH2CH2NH2, CH2CH2SH, or CH2COOH; R2 and Re is H, OH, SH, NH2, C1-6 alkyl, alkoxy or alkene; R 4 and R 5 are H, C 1-6 alkyl, alkoxy or alkene; And it is N or C, so that when Y is N, R3 is nothing and when Y is C, R3 is NO2 or another electron of the elimination group and salts thereof, wherein said compound is not pyridoxamine.

2. A pharmaceutical composition comprising a compound according to claim 1, or a salt thereof in a suitable carrier.

3. A method for the inhibition of post-Amadori formation comprising the administration of an effective post-Amadori AGE inhibitory amount of a compound according to claim 1.

4. A method of inhibiting cross-linking protein by administering an effective post-Amadori AGE inhibitory amount of a compound according to claim 1.

5. A method for the treatment of a patient with AGE-related pathology comprising the administration of an effective therapeutic amount of a compound according to claim 1.

6. A compound that has the formula CHjNH,

7. A compound that has the formula

8. A pharmaceutical comprising the compound according to claim 6 or a salt thereof in a suitable carrier.

9. A pharmaceutical comprising the compound according to claim 7 or a salt thereof in a suitable carrier.

10. A method for the inhibition of AGE formation comprising the administration of an effective AGE inhibitory amount of a compound according to claim 6.

11. A method of inhibiting the cross-linking protein by administering an effective AGE inhibitory amount of a compound according to claim 6.

12. A method for the treatment of a patient with AGE-related pathology comprising the administration of an effective therapeutic amount of a compound according to claim 6.

13. A method for the inhibition of AGE formation comprising the administration of an effective AGE inhibitory amount of a compound according to claim 7.

14. A method of inhibiting the cross-linking protein by administering an effective AGE inhibitory amount of a compound according to claim 7.

15. A method for the treatment of a patient with AGE related pathology comprising the administration of an effective therapeutic amount of a compound according to claim 7.

16. A pharmaceutical composition comprising an effective AGE inhibitory amount of pyridoxamine-5'-phosphate or a salt thereof in an appropriate carrier.

17. A method for the inhibition of AGE formation comprising the administration of an effective AGE inhibitory amount of pyridoxamine-5'-Phosphate.

18. A method of inhibiting the cross-linked protein by administering an effective post-Amadori AGE inhibitory amount of pyridoxamine-5'-Phosphate.