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WO2000022094A2 - Procede permettant d'inhiber la modification oxydative des proteines - Google Patents

Procede permettant d'inhiber la modification oxydative des proteines Download PDF

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Publication number
WO2000022094A2
WO2000022094A2 PCT/US1999/023702 US9923702W WO0022094A2 WO 2000022094 A2 WO2000022094 A2 WO 2000022094A2 US 9923702 W US9923702 W US 9923702W WO 0022094 A2 WO0022094 A2 WO 0022094A2
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WIPO (PCT)
Prior art keywords
ribose
age
lysine
formation
amadori
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PCT/US1999/023702
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English (en)
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WO2000022094A3 (fr
Inventor
John Baynes
Joelle Onorato
Suzanne Thorpe
Raja Khalifah
Billy Hudson
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Kansas University Medical Center
University Of South Carolina
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Priority to AU14446/00A priority Critical patent/AU1444600A/en
Publication of WO2000022094A2 publication Critical patent/WO2000022094A2/fr
Publication of WO2000022094A3 publication Critical patent/WO2000022094A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4415—Pyridoxine, i.e. Vitamin B6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids

Definitions

  • the instant invention is in the field of Advanced Glycation End-products (AGEs), their formation, detection, identification, inhibition, and inhibitors thereof.
  • AGEs Advanced Glycation End-products
  • Nonenzymatic glycation by glucose and other reducing sugars is an important post-translational modification of proteins that has been increasingly implicated in diverse pathologies. Irreversible nonenzymatic glycation and crosslinking through a slow, glucose-induced process may mediate many of the complications associated with diabetes. Chronic hyperglycemia associated with diabetes can cause chronic tissue damage which can lead to complications such as retinopathy, nephropathy, and atherosclerotic disease. (Cohen and Ziyadeh, 1996, J. Amer. Soc. Nephrol. 7:183-190).
  • Glycated proteins have also been shown to be toxic, antigenic, and capable of triggering cellular injury responses after uptake by specific cellular receptors (see for example, Vlassara, Bucala & Striker, 1994, Lab. Invest.
  • the instant disclosure teaches previously unknown, and unpredicted mechanism of formation of post-Amadori advanced glycation end products (Maillard products; AGEs) and methods for identifying and characterizing effective inhibitors of post- Amadori AGE formation.
  • the instant disclosure demonstrates the unique isolation and kinetic characterization of a reactive protein intermediate competent in forming post- Amadori AGEs, and for the first time teaching methods which allow for the specific elucidation and rapid quantitative kinetic study of "late" stages of the protein glycation reaction.
  • Glycation reactions are known to be initiated by reversible Schiff-base (aldimine or ketimine) addition reactions with lysine side- chain ⁇ -amino and terminal ⁇ -amino groups, followed by essentially irreversible Amadori rearrangements to yield ketoamine products e.g. 1 -amino- 1 -deoxy-ketoses from the reaction of aldoses (Baynes et al., 1989, in The Maillard Reaction in Aging, Diabetes, and Nutrition, ed. Monnier and Baynes, (Alan R. Liss, New York, pp 43- 67).
  • sugars initially react in their open-chain (not the predominant pyranose and furanose structures) aldehydo or keto forms with lysine side chain ⁇ -amino and terminal ⁇ -amino groups through reversible Schiff base condensation (Scheme I).
  • the resulting aldimine or ketimine products then undergo Amadori rearrangements to give ketoamine Amadori products, i.e. 1 -amino- 1 -deoxy-ketoses from the reaction of aldoses (Means & Chang, 1982, Diabetes 31, Suppl. 3:1-4; Harding, 1985, Adv. Protein Chem. 37:248-334).
  • FFI 2-(2-furoyl)-4(5)-(2-furanyl)-lH- imidazole
  • Patents have issued in the area of inhibition of protein glycosylation and cross-linking of protein sugar amines based upon the premise that the mechanism of such glycosylation and cross-linking occurs via saturated glycosylation and subsequent cross-linking of protein sugar amines via a single basic, and repeating reaction. These patents include U.S.
  • Inhibition of AGE formation has utility in the areas of, for example, food spoilage, animal protein aging, and personal hygiene such as combating the browning of teeth.
  • Some notable, though quantitatively minor, advanced glycation end-products are pentosidine and N ⁇ -carboxymethyllysine (Sell and Monnier, 1989, J. Biol. Chem. 264:21597-21602; Ahmed et al., 1986, J. Biol. Chem. 261:4889-4894).
  • the Amadori intermediary product and subsequent post-Amadori AGE formation is not fully inhibited by reaction with aminoguanidine.
  • the formation of post-Amadori AGEs as taught by the instant disclosure occurs via an important and unique reaction pathway that has not been previously shown, or even previously been possible to demonstrate in isolation. It is a highly desirable goal to have an efficient and effective method for identifying and characterizing effective post-Amadori AGE inhibitors of this "late" reaction.
  • combinatorial chemistry can be employed to screen candidate compounds effectively, and thereby greatly reducing time, cost, and effort in the eventual validation of inhibitor compounds.
  • AGE post-Amadori advanced glycation end-product
  • This stable product is a presumed sugar saturated Amadori/Schiff base product produced by the further reaction of the early stage protein/sugar Amadori product with more sugar.
  • this post-Amadori/Schiff base intermediary has been generated by the reaction of target protein with ribose sugar.
  • the instant invention provides for a method of generating stable protein-sugar AGE formation intermediary precursors via a novel method of high sugar inhibition.
  • the sugar used is ribose.
  • the instant invention provides for a method for identifying an effective inhibitor of the formation of late Maillard products comprising: generating stable protein-sugar post-Amadori advanced glycation end-product intermediates by incubating a protein with sugar at a sufficient concentration and for sufficient length of time to generate stable post-Amadori AGE intermediates; contacting said stable protein-sugar post-Amadori advanced glycation end-product intermediates with an inhibitor candidate; identifying effective inhibition by monitoring the formation of post-Amadori AGEs after release of the stable protein-sugar post-Amadori advanced glycation end-product intermediates from sugar induced equilibrium.
  • Appropriate sugars include, and are not limited to ribose, lyxose, xylose, and arabinose. It is believed that certain conditions will also allow for use of glucose and other sugars. In a preferred embodiment the sugar used is ribose.
  • the instant invention teaches 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 endproducts in an assay comprising; generating stable protein-sugar post-Amadori advanced glycation end-product intermediates by incubating a protein with sugar at a sufficient concentration and for sufficient length of time to generate stable post-Amadori AGE intermediates; contacting said stable protein-sugar post-Amadori advanced glycation end-product intermediates with an inhibitor candidate; identifying effective inhibition by monitoring the formation of post-Amadori AGEs after release of the stable protein-sugar post- Amadori advanced glycation end-product intermediates from sugar induced equilibrium.
  • the assay uses ribose.
  • the methods of the instant invention allow for the rapid screening of candidate post-Amadori AGE formation inhibitors for effectiveness, greatly reducing the cost and amount of work required for the development of effective small molecule inhibitors of post-Amadori AGE formation.
  • effective inhibitors of post-Amadori "late" reactions of AGE formation include derivatives of vitamin B6 and vitamin B ⁇ , in the preferred embodiment the specific species being pyridoxamine and thiamine pyrophosphate.
  • the instant invention teaches new methods for rapidly inducing diabetes like pathologies in rats comprising administering ribose to the subject animal. Further provided for is the use of identified inhibitors pyridoxamine and thiamine pyrophosphate in vivo to inhibit post-Amadori AGE induced pathologies.
  • the present invention encompasses compounds for use in the inhibition of AGE formation and post-Amadori AGE pathologies, and pharmaceutical compositions
  • R is CH 2 NH 2 , CH 2 SH, COOH, CH 2 CH 2 NH 2 , CH 2 CH 2 SH, or CH 2 COOH;
  • R 2 is OH, SH or NH 2 ;
  • Y is N or C, such that when Y is N R is nothing, and when Y is C, R is NO 2 or another electron withdrawing group; and salts thereof.
  • the present invention also encompasses compounds of the general formula Formula II
  • R is CH 2 NH 2 , CH 2 SH, COOH, CH 2 CH 2 NH 2 , CH 2 CH 2 SH, or CH 2 COOH;
  • R 2 and R 6 is H, OH, SH, NH 2 , C 1-18 alkyl, alkoxy or alkene;
  • R and R 5 are H, C 1-18 alkyl, alkoxy or alkene
  • Y is N or C, such that when Y is N R 3 is nothing, and when Y is C, R 3 is NO 2 or another electron withdrawing group, and salts thereof.
  • R , R 5 and R are H.
  • the compounds of the present invention can embody one or more electron withdrawing groups, such as and not limited to -NH 2 , -NHR, -NR 2 , -OH, -OCH 3 , - OCR, and -NH-COCH 3 where R is C 1-6 alkyl.
  • the instant invention encompasses pharmaceutical compositions which comprise one or more of the compounds of the present invention, or salts thereof, in a suitable carrier.
  • the instant invention encompasses methods for administering pharmaceuticals of the present invention for therapeutic intervention of pathologies which are related to AGE formation in vivo.
  • the AGE related pathology to be treated is related to diabetic nephropathy.
  • the instant invention also teaches new methods to treat or prevent oxidative modification of proteins, including low density lipoproteins, to treat or prevent lipid peroxidation, and to treat or prevent atherosclerosis, comprising administering an amount effective of one of the compounds of the invention to treat or prevent the disorder.
  • Figure 1 is a series of graphs depicting the effect of vitamin B6 derivatives on AGE formation in bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • Figure 1A Pyridoxamine (PM);
  • FIG 2 is a series of graphs depicting the effect of vitamin Bi derivatives and aminoguanidine (AG) on AGE formation in bovine serum albumin.
  • Figure 2A Thiamine pyrophosphate (TPP);
  • Figure 2B thiamine monophosphate (TP);
  • Figure 3 is a series of graphs depicting the effect of vitamin B6 derivatives on AGE formation in human methemoglobin (Hb).
  • Figure 4 is a series of graphs depicting the effect of vitamin B i derivatives and aminoguanidine (AG) on AGE formation in human methemoglobin.
  • Figure 5 is a bar graph comparison of the inhibition of the glycation of ribonuclease A by thiamine pyrophosphate (TPP), pyridoxamine (PM) and aminoguanidine (AG).
  • Figure 6 A is a graph of the kinetics of glycation of RNase A (10 mg/mL) by ribose as monitored by ELISA.
  • Figure 6B is a graph showing the dependence of reciprocal half-times on ribose concentration at pH 7.5.
  • Figure 7 are two graphs showing a comparison of uninterrupted and interrupted glycation of RNase by glucose (7B) and ribose (7 A), as detected by ELISA.
  • Figure 8 are two graphs showing kinetics of pentosidine fluorescence (arbitrary units) increase during uninterrupted and interrupted ribose glycation of RNase.
  • Figure 9 is a graph which shows the kinetics of reactive intermediate buildup.
  • Figure 10 are graphs of Post-Amadori inhibition of AGE formation by ribose.
  • Figure 10A graphs data where aliquots were diluted into inhibitor containing buffers at time 0.
  • Figure 10B graphs data where samples were interrupted at 24h, and then diluted into inhibitor containing buffers.
  • Figure 11 is a graph showing dependence of the initial rate of formation of antigenic AGE on pH following interruption of glycation.
  • Figure 12 are two graphs showing the effect of pH jump on ELISA detected
  • Figure 13 is a series of graphs depicting the effect of vitamin B6 derivatives on
  • FIG 13A Pyridoxamine (PM); Figure 13B pyridoxal-5 '-phosphate (PLP); Figure 13C pyridoxal (PL); Figure 13D pyridoxine (PN).
  • PM Pyridoxamine
  • PLA pyridoxal-5 '-phosphate
  • PRP pyridoxal-5 '-phosphate
  • PL pyridoxal
  • PN pyridoxine
  • 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 Thiamine pyrophosphate (TPP);
  • Figure 14B thiamine monophosphate (TP); Figure 14C thiamine (T); Figure 14D 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 Pyridoxamine (PM);
  • Figure 15B pyridoxal-5 '-phosphate (PLP);
  • Figure 15C pyridoxal (PL);
  • Figure 15D pyridoxine
  • Figure 16 is a series of graphs depicting the effect of vitamin Bi derivatives and aminoguanidine (AG) on AGE formation during uninterrupted glycation of bovine serum albumin (BSA) by ribose.
  • Figure 16A Thiamine pyrophosphate (TPP);
  • TPP Thiamine pyrophosphate
  • Figure 17 is a series of graphs depicting the effect of vitamin B6 derivatives on AGE formation during uninterrupted glycation of human methemoglobin (Hb) by ribose.
  • Figure 17A Pyridoxamine (PM);
  • Figure 17B pyridoxal-5 '-phosphate (PLP);
  • Figure 17C pyridoxal (PL);
  • Figure 17D pyridoxine (PN).
  • Figure 18 is a series of graphs depicting the effect of vitamin B ⁇ derivatives on post-Amadori AGE formation after interrupted glycation by ribose.
  • Figure 18A BSA and Pyridoxamine (PM) Figure 18B BSA and pyridoxal-5 '-phosphate (PLP);
  • Figure 18D 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 RNase
  • Figure 19B BSA.
  • Figure 20 are graphs depicting the effect of aminoguanidine on post-Amadori AGE formation after interrupted glycation by ribose.
  • Figure 20A RNase
  • Figure 20B BSA
  • Figure 21 is a graph depicting the effect of N-a c etyl-L-lysine on post-
  • 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 22 A) and BSA ( Figure 22B) by ribose.
  • TPP thiamine pyrophosphate
  • PM pyridoxamine
  • AG aminoguanidine
  • Figure 23 is a bar graph showing the effects of Ribose treatment in vivo alone on rat tail-cuff blood pressure. Treatment was with 0.05 M, 0.30 M, and 1 M Ribose (R) injected for 1, 2 or 8 Days (D).
  • Figure 24 is a bar graph showing the effects of Ribose treatment in vivo alone on rat creatinine clearance (Clearance per 100 g Body Weight). Treatment was with
  • FIG. 25 is a bar graph showing the effects of Ribose treatment in vivo alone on rat Albuminuria (Albumin effusion rate). Treatment was with 0.30 M, and 1 M
  • Figure 26 is a bar graph showing the effects of inhibitor treatment in vivo, with or without ribose, on rat tail-cuff blood pressure.
  • Treatment groups were: 25 mg/kg body weight aminoguanidine (AG); 25 or 250 mg/kg body weight Pyridoxamine (P);
  • T 250 mg/kg body weight Thiamine pyrophosphate (T), or with 1 M Ribose (R).
  • Figure 27 is a bar graph showing the effects of inhibitor treatment in vivo, with or without ribose, on rat creatinine clearance (Clearance per 100 g body weight).
  • Treatment groups were: 25 mg/kg body weight aminoguanidine (AG); 25 or 250 mg/kg body weight Pyridoxamine (P); 250 mg/kg body weight Thiamine pyrophosphate (T), or with 1 M Ribose (R).
  • Figure 28 is a bar graph showing the effects of inhibitor treatment in vivo without ribose, and ribose alone on rat Albuminuria (Albumin effusion rate).
  • Treatment groups were: 25 mg/kg body weight aminoguanidine (AG); 250 mg/kg body weight Pyridoxamine (P); 250 mg/kg body weight Thiamine pyrophosphate (T), or treatment with 1 M Ribose (R) for 8 days (D). Control group had no treatment.
  • Figure 29 is a bar graph showing the effects of inhibitor treatment in vivo, with
  • Treatment groups were: 25 mg/kg body weight aminoguanidine (AG); 25 and 250 mg/kg body weight Pyridoxamine (P); 250 mg/kg body weight Thiamine pyrophosphate (T), or treatment with 1 M Ribose (R) for 8 days (D) alone. Control group had no treatment.
  • Figure 30A depicts Scheme 1 showing a diagram of AGE formation from protein.
  • Figure 30B depicts Scheme 2, a chemical structure of aminoguanidine.
  • Figure 30A depicts Scheme 1 showing a diagram of AGE formation from protein.
  • Figure 30B depicts Scheme 2, a chemical structure of aminoguanidine.
  • FIG. 30C depicts Scheme 3, chemical structures for thiamine, thiamine-5 '-phosphate, and thiamine pyrophosphate.
  • Figure 30D depicts Scheme 4, chemical structures of pyridoxine, pyridoxamine, pyridoxal-5 '-phosphate, and pyridoxal.
  • Figure 30E depicts
  • Scheme 5 kinetics representation of AGE formation.
  • Figure 30F depicts Scheme 6, kinetics representation of AGE formation and intermediate formation.
  • Figure 31 shows a 125 MHz C-13 NMR Resonance spectrum of Riobonuclease Amadori Intermediate prepared by 24 HR reaction with 99% [2-C13]Ribose.
  • Figure 32 are graphs which show AGE intermediary formation using the pentoses Xylose, Lyxose, Arabinose and Ribose.
  • Figure 33 is a graph showing the results of glomeruli staining at pH 2.5 with Alcian blue.
  • Figure 34 is a graph showing the results of glomeruli staining at pH 1.0 with Alcian blue.
  • Figure 35 is a graph showing the results of immunofluroescent glomeruli staining for RSA.
  • Figure 36 is a graph showing the results of immunofluroescent glomeruli staining for Heparan Sulfate Proteoglycan Core protein.
  • Figure 37 is a graph showing the results of immunofluroescent glomeruli staining for Heparan Sulfate Proteoglycan side-chain.
  • Figure 38 is a graph showing the results of analysis of glomeruli sections for average glomerular volume.
  • FIG 39 Structure and mass spectrum of the TFAME derivative of lysine. Lysine was derivatized to its methyl ester using methanolic-HCl then converted to its TFAME derivative with trifluoroacetic anhydride. The 180 ion (base peak) was used for quantification of lysine by GC/MS.
  • Figure 40 Structure of derivatized CML and CEL.
  • CML and CEL were derivatized to TFAMEs as described for lysine. Ions 392 and 379 were used for quantification of CML and CEL, respectively.
  • Figure 41 Mass Spectrum of derivatized CML and CEL. CML and CEL were derivatized to TFAMEs as described for lysine. Ions 392 and 379 were used for quantification of CML and CEL, respectively.
  • FIG 42 Structure of MDA-lysine (A) and HNE-lysine (B). MDA-lysine and HNE-lysine were derivatized to TFAMEs as described for lysine. Ions 474 and 323 were used for quantification of MDA-lysine and HNE-lysine, respectively.
  • Figure 43 Mass Spectrum of MDA-lysine (A) and HNE-lysine (B). MDA- lysine and HNE-lysine were derivatized to TFAMEs as described for lysine. Ions 474 and 323 were used for quantification of MDA-lysine and HNE-lysine, respectively.
  • FIG 44 Loss of free amino groups during reaction of PM with PUFAs.
  • PM (1 mM) was incubated alone ( ⁇ ) or in the presence of oleate ( ⁇ ), linoleate ( ⁇ ), or arachidonate (v) (5 mM) in 0.2 M sodium phosphate buffer pH 7.4 for 6 days at 37°C. Aliquots were withdrawn at various time intervals and analyzed for free amino groups using the TNBS assay. Data shown are the mean and standard deviation of 3 independent experiments.
  • Figure 45 LC-MS analysis of a PM/linoleate reaction at day 6. PM (1 mM) was reacted with linoleate (5 mM). The day 6 sample was analyzed by LC-MS as described in Materials and Methods. Panel A and panel B represent the mass spectra taken from the major products.
  • FIG. 46 Kinetics of formation of products 267, 339, and 305.
  • PM (1 mM) was reacted with linoleate (5 mM) as described previously.
  • the samples were analyzed by RP-HPLC using absorbance detection at 294 nm.
  • Panel A shows a chromatogram of a day 6 reaction and the formation of products 339, 267 and 305.
  • the products were quantified (Panel B) based on their area ratios to the internal standard PL (267 ( ⁇ ), 339 (v), and 305 ( ⁇ )). The data shown are the mean and standard deviation from 3 independent experiments.
  • Figure 47 Proposed structure of products 267 and 339. Products 267 and 339 were hypothesized to be hexamide and nonanedioic acid amide derivatives of PM.
  • Figure 48 TNBS reactivity of synthetic 339. An estimated 80 nmoles of synthetic 339 was analyzed by the TNBS assay. An equivalent amount of PM standard was analyzed for comparison.
  • Figure 49 Proposed mechanism to the formation of 267 and 339. Oxidation of linoleic acid proceeds through formation of linoleic acid 9- and 13- lipid hydroperoxides. These may then dehydrate to form ketoacids. Nucleophilic attack of
  • Figure 50 Preferential cleavage of the Schiff base to form the amide derivatives may be driven by radical stabilization within the conjugated system.
  • the carbinolamine, precursor of the Schiff base adduct, would then undergo hydrogen abstraction.
  • the resulting radical might be stabilized by the conjugated carbon system, directing the cleavage reaction to form the 6-carbon amide derivative.
  • the same mechanism would apply to formation of the nonanedioic acid amide derivative.
  • Figure 51 Proposed structure for product 305. Though complete characterization of 305 was not accomplished, a molecular formula obtained by high resolution FAB-MS suggests the product is either a pyrrole or furan derivative of PM.
  • FIG 52 Inhibition of CML and CEL formation by PM during reactions of RNase with arachidonate.
  • RNase (1 mM (10 mM lysine)
  • arachidonate 100 mM alone (Q) or in the presence of 1 mM PM ( ⁇ ) in 200 mM sodium phosphate buffer, pH 7.4 at 37°C for 6 days.
  • 1 mg of protein was removed and prepared for GC/MS analysis.
  • CML and CEL were analyzed by GC/MS as their trifluoroacetyl methyl esters. Data shown is the average and range of two independent experiments.
  • Figure 53 Inhibition of MDA-lysine and HNE-lysine by PM during reactions of RNase with arachidonate.
  • RNase (1 mM) was reacted with arachidonate (100 mM) alone ( ⁇ ) or in the presence of 1 mM PM ( ⁇ ) in 200 mM sodium phosphate buffer, pH 7.4 at 37°C for 6 days. At various time intervals 1 mg of protein was removed and prepared for GC/MS analysis. MDA-lysine and HNE-lysine were analyzed by GC/MS as their trifluoroacetyl methyl esters. Data shown is the average and range of two independent experiments.
  • FIG 54 Prolongation of conjugated diene formation by PM during copper catalyzed oxidation of LDL.
  • LDL 50 ⁇ g/ml
  • FIG. 55 PM inhibits formation of CML and CEL during copper-catalyzed oxidation of LDL.
  • LDL 50 ⁇ g/ml
  • the samples were dialyzed, delipidated and hydrolyzed in 6N HC1.
  • CML and CEL were analyzed as their trifluoroacetyl methyl esters. Results shown represent the average and range of two independent pools of LDL.
  • FIG 56 PM inhibits formation of MDA-lysine and HNE-lysine during copper catalyzed oxidation of LDL.
  • LDL 50 ⁇ g/ml
  • the samples were dialyzed, delipidated and hydrolyzed in 6N HC1.
  • MDA- lysine and HNE-lysine were analyzed as their trifluoroacetyl methyl esters. Results represent the average and range of two independent pools of LDL.
  • Figure 57 The average and range of two independent pools of LDL.
  • FIG 58 Effect of PM on TBARs formation during oxidation of linoleate.
  • Linoleate was oxidized alone (5 mM) (B) or in the presence of PM (1 mM) (• ) for 6 days at 37°C in sodium phosphate buffer, pH 7.4. Aliquots were removed at various time intervals and analyzed for TBARs as described above. Data shown represents the mean +/- standard deviation of 3 independent experiments.
  • FIG 59 Effect of PM on the rate of linoleate oxidation.
  • Linoleate (5 mM) was oxidized alone (B) or in the presence of PM (1 mM) (• ) for 6 days.
  • Linoleate was extracted using acidified chloroform:methanol (Folch, 1957). Following addition of the internal standard, palmitate, the samples were derivatized with borontrichloride- methanol and the fatty acids analyzed as their methyl esters by GC/MS. Quantification was based on standard curves generated from linoleate and palmitate standards. Data shown is the mean +/- standard deviation of 3 independent experiments.
  • Figure 60 Mechanism by which PM may act as an antioxidant.
  • PM (A) may donate a hydrogen to radicals produced either during the initiation or propagation phases of lipid peroxidation.
  • the resulting PM radical (B) is stabilized by the aromatic ring system (C).
  • Alloxan induced diabetic Lewis rats have been used as a model for protein aging to demonstrate the in vivo effectiveness of inhibitors of AGE formation.
  • the correlation being demonstrated is between inhibition of late diabetes related pathology and effective inhibition of AGE formation (Brownlee, Cerami, and Vlassara, 1988, New Eng. J. Med. 318(20): 1315-1321).
  • Streptozotocin induction of diabetes in Lewis rats, New Zealand White rabbits with induced diabetes, and genetically diabetic BB/Worcester rats have also been utilized, as described in, for example, U.S. Patent 5,334,617 (incorporated by reference).
  • a major problem with these model systems is the long time period required to demonstrate AGE related injury, and thus to test compounds for AGE inhibition.
  • An important tool for studying AGE formation is the use of polyclonal and monoclonal antibodies that are specific for AGEs elicited by the reaction of several sugars with a variety of target proteins.
  • the antibodies are screened for resultant specificity for AGEs that is independent of the nature of the protein component of the
  • thiamine is practically devoid of pharmacodynamic actions when given in usual therapeutic doses; and even large doses were not known to have any effects.
  • Thiamine pyrophosphate is the physiologically active form of thiamine, and it functions mainly in carbohydrate metabolism as a coenzyme in the decarboxylation of ⁇ -keto acids.
  • Tablets of thiamine hydrochloride are available in amounts ranging from 5 to 500 mg each.
  • Thiamine hydrochloride injection solutions are available which contain 100 to 200 mg/ml.
  • intravenous doses of as high as 100 mg/L of parenteral fluid are commonly used, with the typical dose of 50 to 100 mg being administered.
  • GI absorption of thiamine is believed to be limited to 8 to 15 mg per day, but may be exceed by oral administration in divided doses with food.
  • the instant invention has found, as shown by in vitro testing, that administration of thiamine pyrophosphate at levels above what is normally found in the human body or administered for dietary therapy, is an effective inhibitor of post- Amadori antigenic AGE formation, and that this inhibition is more complete than that possible by the administration of aminoguanidine.
  • Vitamin Bg is typically available in the form of pyridoxine hydrochloride in over-the-counter preparations available from many sources.
  • Beach pharmaceuticals Beelith Tablets contain 25 mg of pyridoxine hydrochloride that is equivalent to 20 mg of Bg, other preparations include Marlyn Heath Care Marlyn
  • Formula 50 which contain 1 mg of pyridoxine HCl and Marlyn Formula 50 Mega Forte which contains 6 mg of pyridoxine HCl, Wyeth-Ayerst Stuart Prenatal® tablets which contain 2.6 mg pyridoxine HCl, J&J-Merck Corp.
  • Stuart Formula® tablets contain 2 mg of pyridoxine HCl, and the CIBA Consumer Sunkist Children's chewable multivitamins which contain 1.05 mg of pyridoxine HCl, 150% of the U.S. RDA for children 2 to 4 years of age, and 53% of the U.S. RDA for children over 4 years of age and adults.
  • (Physician's Desk Reference for nonprescription drugs, 14th edition Medical Economics Data Production Co., Montvale, N.J., 1993).
  • pyridoxine is a primary alcohol
  • pyridoxal is the corresponding aldehyde
  • pyridoxamine contains an aminomethyl group at this position.
  • pyridoxine is a primary alcohol
  • pyridoxal is the corresponding aldehyde
  • pyridoxamine contains an aminomethyl group at this position.
  • Each of these three forms can be utilized by mammals after conversion by the liver into pyridoxal-5'-phosphate, the active form of the vitamin. It has long been believed that these three forms are equivalent in biological properties, and have been treated as equivalent forms of vitamin Bg by the art.
  • the most active antimetabolite to pyridoxine is 4-deoxypyridoxine, for which the antimetabolite activity has been attributed to the formation in vivo of 4- deoxypyridoxine-5-phosphate, a competitive inhibitor of several pyridoxal phosphate- dependent enzymes.
  • the pharmacological actions of pyridoxine are limited, as it elicits no outstanding pharmacodynamic actions after either oral or intravenous administration, and it has low acute toxicity, being water soluble. It has been suggested that neurotoxicity may develop after prolonged ingestion of as little as 200 mg of pyridoxine per day.
  • pyridoxine phosphate is involved in several metabolic transformations of amino acids including decarboxylation, transamination, and racemization, as well as in enzymatic steps in the metabolism of sulfur-containing and hydroxy-amino acids.
  • pyridoxal phosphate is aminated to pyridoxamine phosphate by the donor amino acid, and the bound pyridoxamine phosphate is then deaminated to pyridoxal phosphate by the acceptor ⁇ -keto acid.
  • vitamin B complex is known to be a necessary dietary supplement involved in specific breakdown of amino acids.
  • B vitamers especially pyridoxal phosphate (PLP) have been previously proposed to act as "competitive inhibitors" of early glycation, since as aldehydes they themselves can form Schiff bases adducts with protein amino groups (Khatami et al., 1988, Life Sciences 43: 1725-1731) and thus limit the amount of amines available for glucose attachment.
  • PBP pyridoxal phosphate
  • effectiveness in limiting initial sugar attachment is not a predictor of inhibition of the conversion of any Amadori products formed to AGEs.
  • the instant invention describes inhibitors of "late" glycation reactions as indicated by their effects on the in vitro formation of antigenic AGEs (Booth et al., 1996, Biochem. Biophys. Res. Com. 220: 113-119).
  • Bovine pancreatic ribonuclease A (RNase) was chromatographically pure, aggregate-free grade from Worthington Biochemicals.
  • Bovine Serum albumin Bovine Serum albumin
  • BSA fraction V, fatty-acid free
  • human methemoglobin (Hb) human methemoglobin (Hb)
  • D-glucose pyridoxine, pyridoxal, pyridoxal 5 'phosphate, pyridoxamine, thiamine, thiamine monophosphate, thiamine pyrophosphate, and goat alkaline phosphatase-conjugated anti-rabbit IgG were all from Sigma Chemicals.
  • Aminoguanidine hydrochloride was purchased from Aldrich Chemicals.
  • immunogen was prepared by glycation of BSA (R479 antibodies) or RNase (R618 antibodies) at 1.6 g protein in 15 ml for 60-90 days using 1.5 M glucose in 0.4 M sodium phosphate buffer of pH 7.5 containing 0.05% sodium azide at pH 7.4 and 37°C.
  • New Zealand white rabbit males of 8-12 weeks were immunized by subcutaneous administration of a 1 ml solution containing 1 mg/ml of glycated protein in Freund's adjuvant.
  • the primary injection used the complete adjuvant and three boosters were made at three week intervals with Freund's incomplete adjuvant. Rabbits were bled three weeks after the last booster.
  • the serum was collected by centrifugation of clotted whole blood.
  • the antibodies are AGE-specific, being unreactive with either native proteins (except for the carrier) or with Amadori intermediates.
  • the polyclonal anti- AGE antibodies have proven to be a sensitive and valuable analytical tool for the study of "late" AGE formation in vitro and in vivo.
  • CML carboxymethyl lysine
  • ELISA detection of AGE products The general method of Engvall (1981, Methods Enzymol. 70:419-439) was used to perform the ELISA.
  • glycated protein samples were diluted to approximately 1.5 ug/ml in 0.1 M sodium carbonate buffer of pH 9.5 to 9.7.
  • the protein was coated overnight at room temperature onto 96-well polystyrene plates by pippetting 200 ul of the protein solution in each well (0.3 ug/well). After coating, the protein was washed from the wells with a saline solution containing 0.05% Tween-20. The wells were then blocked with 200 ul of 1 % casein in carbonate buffer for 2 h at 37°C followed by washing.
  • Rabbit anti-AGE antibodies were diluted at a titer of about 1 :350 in incubation buffer, and incubated for 1 h at 37°C, followed by washing.
  • antibodies R479 used to detect glycated RNase were raised against glycated BSA
  • antibodies R618 used to detect glycated BSA and glycated Hb were raised against glycated RNase.
  • An alkaline phosphatase-conjugated antibody to rabbit IgG was then added as the secondary antibody at a titer of 1 :2000 or 1 :2500 (depending on lot) and incubated for 1 h at 37°C, followed by washing.
  • the p-nitrophenylphosphate substrate solution was then added (200 ul/well) to the plates, with the absorbance of the released p- nitrophenolate being monitored at 410 nm with a Dynatech MR 4000 microplate reader.
  • FIGS. 1 A-D are graphs which show the effect of vitamin B6 derivatives on post-Amadori AGE formation in bovine serum albumin glycated with glucose.
  • BSA (10 mg/ml) was incubated with 1.0 M glucose in the presence and absence of the various indicated derivative in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C for 6 weeks. Aliquots were assayed by ELISA using R618 anti-AGE antibodies. Concentrations of the inhibitors were 3, 15 and 50 mM.
  • Inhibitors used in Figures (1A) Pyridoxamine (PM); (IB) pyridoxal phosphate (PLP); (1C) pyridoxal (PL); (ID) pyridoxine (PN).
  • Figure 1 (control curves) demonstrates that reaction of BSA with 1.0 M glucose is slow and incomplete after 40 days, even at the high sugar concentration used to accelerate the reaction. The simultaneous inclusion of different concentrations of various B6 vitamers markedly affects the formation of antigenic AGEs.
  • Figure 1A-D Pyridoxamine and pyridoxal phosphate strongly suppressed antigenic AGE formation at even the lowest concentrations tested, while pyridoxal was effective above 15 mM. Pyridoxine was slightly effective at the highest concentrations tested.
  • FIGS. 1 A-D are graphs which show the effect of vitamin B i derivatives and aminoguanidine (AG) on 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 derivative in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C for 6 weeks. Aliquots were assayed by ELISA using R618 anti-AGE antibodies. Concentrations of the inhibitors were 3, 15 and 50 mM.
  • Inhibitors used in Figures (2A) Thiamine pyrophosphate (TPP); (2B) thiamine monophosphate (TP); (2C) thiamine (T); (2D) aminoguanidine (AG).
  • Figure 3 A-D are graphs which show the effect of vitamin B6 derivatives on AGE formation in human methemoglobin.
  • Hb (1 mg/ml) was incubated with 1.0 M glucose in the presence and absence of the various indicated derivative in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C for 3 weeks. Aliquots were assayed by ELISA using R618 anti-AGE antibodies. Concentrations of the inhibitors were 0.5, 3, 15 and 50 mM.
  • Inhibitors used in Figures (3A) Pyridoxamine (PM); (3B) pyridoxal phosphate (PLP); (3C) pyridoxal (PL); (3D) pyridoxine (PN).
  • Hb-AGE a component that binds to anti-AGE antibodies
  • HbAlc glycated Hb
  • FIGS. 4 A-D are graphs which show the effect of vitamin B i derivatives and aminoguanidine (AG) on AGE formation in human methemoglobin.
  • Hb (1 mg/ml) was incubated with 1.0 M glucose in the presence and absence of the various indicated derivative in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C for 3 weeks. Aliquots were assayed by ELISA using R618 anti-AGE antibodies. Concentrations of the inhibitors were 0.5, 3, 15 and 50 mM.
  • Inhibitors used in Figures (4A) Thiamine pyrophosphate (TPP); (4B) thiamine monophosphate (TP); (4C) thiamine (T); (4D) aminoguanidine (AG).
  • Figure 5 is a bar graph which shows a comparison of the inhibition of the glycation of ribonuclease A by thiamine pyrophosphate (TPP), pyridoxamine (PM) and aminoguanidine (AG).
  • TPP thiamine pyrophosphate
  • PM pyridoxamine
  • AG aminoguanidine
  • RNase (1 mg/ml) was incubated with 1.0 M glucose (glc) in the presence and absence of the various indicated derivative in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C for 6 weeks. Aliquots were assayed by ELISA using R479 anti-AGE antibodies. The indicated percent inhibition was computed from ELISA readings in the absence and presence of the inhibitors at the 6 week time point. Concentrations of the inhibitors were 0.5, 3, 15 and 50 mM.
  • pyridoxamine is a candidate amine potentially capable of forming a Schiff- base linkage with the carbonyls of open-chain sugars, with dicarbonyl fragments, with Amadori products, or with post-Amadori intermediates.
  • the mechanism of inhibition of B i compounds is not obvious. All the forms contain an amino functionality, so that the marked efficiency of only the pyrophosphate form suggests an important requirement for a strong negative charge.
  • Pentosidine Reverse-Phase HPLC Analysis Pentosidine production in RNase was quantitated by HPLC (Sell & Monnier, 1989, J. Biol. Chem. 264:21597-21602; Odetti et al., 1992, Diabetes 41:153-159). Ribose-modified protein samples were hydrolyzed in 6 N HCl for 18 h at 100°C and then dried in a Speed Vac. The samples were then redissolved, and aliquots were taken into 0.1% trifluoroacetic acid and analyzed by HPLC on a Shimadzu system using a Vydac C-18 column equilibrated with 0.1% TFA.
  • Glycation Modifications Modification with ribose or glucose was generally done at 37°C in 0.4 M phosphate buffer of pH 7.5 containing 0.02% sodium azide. The high buffer concentration was always used with 0.5 M ribose modifications. The solutions were kept in capped tubes and opened only to remove timed aliquots that were immediately frozen for later carrying out the various analyses. "Interrupted glycation" experiments were carried out by first incubating protein with the ribose at 37°C for 8 or 24 h, followed by immediate and extensive dialysis against frequent cold buffer changes at 4°C. The samples were then reincubated by quickly warming to 37°C in the absence of external ribose. Aliquots were taken and frozen at various intervals for later analysis.
  • the samples subjected to ELISA testing were also assayed for the production of pentosidine, an acid-stable AGE.
  • the content of pentosidine was measured for the same RNase samples analyzed for antibody reactivity by ELISA.
  • Glycation by ribose in 0.4 M phosphate buffer at pH 7.5 produced pentosidine in RNase A that was quantitated by fluroescence after acid hydrolysis.
  • Figure 8 A shows that under uninterrupted conditions, 0.05 M ribose produces a progressive increase in pentosidine.
  • the "interrupted glycation" experiments described above demonstrate that a precursor or precursors to both post-Amadori antigenic AGEs and pentosidine can be accumulated during glycation with ribose.
  • the kinetics of formation of this intermediate can be independently followed and quantitated by a variation of the experiments described above.
  • the amount of intermediate generated in RNase at different contact times with ribose can be assayed by the maximal extent to which it can produce antigenic AGE after interruption.
  • the free and reversibly-bound ribose is removed by dialysis in the cold or by rapid dilution (see below).
  • Figure 9 shows such an experiment where the kinetics of intermediate buildup 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 ribose removal at each interruption point is also shown (open symbols and dashed lines). As expected (cf. Figure 7A), little AGE is formed prior to removal (or dilution) of ribose, so that ELISA readings after the 5 day secondary incubation periods are mostly a measure of AGE formed after ribose removal. The results in Figure 9 show that the rate of buildup of intermediate in 0.5 M ribose is exponential and very fast, with a half-time of about 3.3 h. This is about 3-fold more rapid than the observed rate of conversion of the intermediate to antigenic AGEs after interruption (open symbols and dashed curve Figure 7A).
  • the interrupted glycation method was used to investigate the pH dependence of the post-Amadori kinetics of AGE formation from the reactive intermediate.
  • RNase A was first reacted for 24 h with 0.5 M ribose at pH 7.5 to generate the reactive intermediate.
  • the kinetics of the decay of the intermediate to AGEs were then measured by ELISA.
  • Figure 1 1 shows that an extremely wide pH range of 5.0-9.5 was achievable when the kinetics were measured by initial rates.
  • a remarkable bell- shaped dependence was observed, showing that the kinetics of antigenic AGEs formation are decreased at both acidic and alkaline pH ranges, with an optimum near pH 8.
  • the inhibition is unlikely to apply to the early step of formation of Amadori product due to the rapid rate of formation of the presumed 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 carried out (after sodium cyanoborohydrate reduction) before and after dialysis at the 24 h interruption point.
  • the unchanged residual lysine content indicates that any dischageable Schiff bases have already been irreversibly converted (presumably by Amadori rearrangement) by the 24 h time.
  • the interrupted glycation method allowed examination of aminoguanidine efficacy on only post-Amadori steps of AGE formation. Equally important, it allowed studies in the absence of free sugar or dicarbonyl-reactive fragments from free sugar (Wolff & Dean, 1987, Biochem. J. 245:243-250; Wells-Knecht et al., 1995, Biochemistry 34:3702-3709) that can combine with aminoguanidine.
  • the results of Figure 20 demonstrate that aminoguanidine has, at best, only a modest effect on post- Amadori AGE formation reactions, achieving 50% inhibition at concentrations above 100-250 mM.
  • aminoguanidine thus predominantly arises either from inhibiting early steps of glycation (Schiff base formation) or from scavenging highly reactive dicarbonyls generated during glycation. Contrary to the original claims, it does not appear to inhibit AGE fo ⁇ nation by complexing the Amadori intermediate.
  • interrupted glycation is not limited for kinetic studies. Interrupted glycation can simplify structural studies of glycated proteins and identifying unknown AGEs using techniques such as ⁇ C NMR that has been used to detect Amadori adducts of RNase (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 also be of special interest.
  • candidate AGEs such as the recently proposed (carboxymethyl)lysine (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 formation from the reactive intermediate that we have observed.
  • the availability of the interrupted kinetics approach will also help to determine the importance of the Amadori pathway to the formation of this AGE.
  • monitoring of the interrupted glycation reaction by techniques such as ⁇ C NMR should identify resonances of other candidate antigenic AGEs as being those displaying similar kinetics of appearance.
  • Table I lists the ⁇ C NMR peaks of the Amadori intermediate of RNase prepared by reaction with C-2 enriched ribose.
  • the downfield peak near 205 ppm is probably due to the carbonyl of the Amadori product.
  • the ability to remove excess free and Schiff base sugars through interrupted glycation will considerably simplify isolation, identification, and structural characterization.
  • Table I lists the peaks that were assigned to the Post-Amadori Intermediate due to their invariant or decreasing intensity with time. Peak positions are listed in ppm downfield from TMS.
  • Ribonuclease A was reacted for 24 hr with 0.5 M ribose 99% enriched at C-2, following which excess and Schiff base bound ribose was removed by extensive dialysis in the cold. The sample was then warmed back to 37°C immediately before taking a 2 hr NMR scan. The signals from RNase reacted with natural abundance ribose under identical conditions were then subtracted from the NMR spectrum. Thus all peaks shown are due to enriched C-13 that originated at the C-2 position. Some of the peaks arise from degradation products of the intermediate, and these can be identified by the increase in the peak intensity over time. Figure 31 shows the NMR spectrum obtained.
  • the interrupted glycation method for following post-Amadori kinetics of AGE formation allows for the rapid quantitative study of "late" stages of the glycation reaction. Importantly, this method allows for inhibition studies that are free of pathways of AGE formation which arise from glycoxidative products of free sugar or Schiff base (Namiki pathway) as illustrated in Scheme I. Thus the interrupted glycation method allows for the rapid and unique identification and characterization of inhibitors of "late" stages of glycation which lead to antigenic AGE formation.
  • pyridoxamine and thiamine pyrophosphate are unique inhibitors of the post-Amadori pathway of AGE formation. Importantly, it was found that efficacy of inhibition of overall glycation of protein, in the presence of high concentrations of sugar, is not predictive of the ability to inhibit the post-Amadori steps of AGE formation where free sugar is removed.
  • pyridoxamine, thiamine pyrophosphate and aminoguanidine are potent inhibitors of AGE formation in the overall glycation of protein by glucose, aminoguanidine differs from the other two in that it is not an effective inhibitor of post-Amadori AGE formation.
  • Aminoguanidine markedly slows the initial rate of AGE formation by ribose under uninterrupted conditions, but has no effect on the final levels of antigenic AGEs produced. Examination of different proteins (RNase, BSA and hemoglobin), confirmed that the inhibition results are generally non-specific as to the protein used, even though there are individual variations in the rates of AGE formation and inhibition.
  • Example 1 Preparation of polyclonal antibodies to AGEs As in Example 1 above.
  • Glycation was first carried out by incubating protein (10 mg/ml) with 0.5 M ribose at 37°C in 0.4 M phosphate buffer at pH 7.5 containing 0.2% sodium azide for 24 h in the absence of inhibitors. Glycation was then interrupted to remove excess and reversibly bound (Schiff base) sugar by extensive dialysis against frequent cold buffer changes at 4°C. The glycated intermediate samples containing maximal amount of Amadori product and little AGE (depending on protein) were then quickly warmed to 37°C without re-addition 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 prospective inhibitors. Aliquots were taken and frozen at various intervals for later analysis. The solutions were kept in capped tubes and opened only to remove timed aliquots that were immediately frozen for later carrying out the various analyses.
  • 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 did not produce any inhibition (data not shown). Similar experiments were carried out on RNase. Pyridoxamine caused nearly complete inhibition of AGE formation with RNase at 15 mM and 50 mM ( Figure 18D). Pyridoxal did not show any significant inhibition at 15 mM (the highest tested), but pyridoxal phosphate showed significant inhibition at 15 mM. Pyridoxal phosphate is known to be able to affinity label the active site of RNase (Raetz and Auld, 1972, Biochemistry 11:2229-2236).
  • Figure 20 shows the results of testing aminoguanidine for inhibition of post- Amadori AGE formation kinetics with both BSA and RNase. At 50 mM, inhibition was about 20% in the case of BSA ( Figure 20B), and less than 15% with RNase ( Figure 20A). The possibility of inhibition by simple amino-containing functionalities was also tested using N ⁇ -acetyl-L-lysine ( Figure 21), which contains only a free N ⁇ -amino group. N ⁇ -acetyl-L-lysine at up to 50 mM failed to exhibit any significant inhibition of AGE formation.
  • 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 on various phenomena that are induced by reducing sugars are widely considered as proof of the involvement of glycation in many such phenomena.
  • Aminoguanidine has recently entered into a second round of Phase III clinical trials for ameliorating the complications of diabetes thought to be caused by glycation of connective tissue proteins due to high levels of sugar.
  • Figure 22 are bar graphs which depict summarized 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 RNase AGE formation in ribose.
  • Figure 22B graphs the data for inhibition after interrupted glycation of BSA AGE formation by ribose.
  • Hyperglycemia can be rapidly induced (within one or two days) in rats by administration of streptozocin (aka. strep tozotocin, STZ) or alloxan. This has become a common model for diabetes melitus. However, these rats manifest nephropathy only after many months of hyperglycemia, and usually just prior to death from end-stage renal disease (ESRD). It is believed that this pathology is caused by the irreversible glucose chemical modification of long-lived proteins such as collagen of the basement membrane. STZ-diabetic rats show albuminuria very late after induction of hyperglycemia, at about 40 weeks usually only just prior to death.
  • Rats were then kept for 4 days with no further ribose administration, at which time they were sacrificed and the following physiological measurements were determined: (i) initial and final body weight; (ii) final stage kidney weight; (iii) initial and final tail-cuff blood pressure; (iv) creatinine clearance per 100 g body weight.
  • Albumin filtration rates were not measured, since no rapid changes were initially anticipated.
  • Past experience with STZ-diabetic rats shows that albuminuria develops very late (perhaps 40 weeks) after the induction of hyperglycemia and just before animals expire.
  • Renal Physiology Results a. Final body weight and final single kidney weight was same for low and high ribose treatment groups. b. Tail-cuff blood pressure increased from 66 ⁇ 4 to 83 ⁇ 3 to rats treated with low ribose (1 x 50 mM), and from 66 ⁇ 4 to 106 ⁇ 5 for rats treated with high ribose (2 x 300 mM). These results are shown in the bar graph of Figure 23.
  • Groups of rats (3-6) were intraperitoneally given 0.3 M “low ribose dose” (LR) or 1.0 M “high ribose dose” (HR) by twice-daily injections for either (i) 1 day, (ii) a “short-term” (S) of 4 days, or (iii) a "long-term” (L) of 8 days. Additionally, these concentrations of ribose were included in drinking water.
  • LR low ribose dose
  • HR high ribose dose
  • Renal Physiology Results a. Tail-cuff blood pressure increased in all groups of ribose-treated rats, confirming Phase I results. (Figure 23). b. Creatinine clearance decreased in all groups in a ribose dose-dependent and time-dependent manner ( Figure 24). c. Albumin Effusion Rate (AER) increased significantly in a ribose-dependent manner at 1-day and 4-day exposures. However, it showed some recovery at 8 day relative to 4 day in the high-dose group but not in the low-dose group. These results are shown in the bar graph of Figure 25. d. Creatinine clearance per 100 g body weight decreased for both low- and high- ribose groups to about the same extent in a time-dependent manner ( Figure 24).
  • ribose plus low dose of pyridoxamine (25 mg/kg body weight injected as 0.5 ml with 9 cc ribose);
  • ribose plus high dose of thiamine pyrophosphate (250 mg/kg body weight injected as 0.5 ml with 9 cc ribose); and (v) ribose plus low dose of amino guanidine (25 mg/kg body weight injected as 0.5 ml with 9 cc ribose).
  • Intervention compounds were pre-administered for one day prior to introducing them with ribose.
  • pyridoxamine and aminoguanidine both at 25 mg/kg, were apparently effective, and equally so, in preventing the ribose-induced decrease in creatinine clearance and ribose-induced mild increase in albuminuria.
  • Thiamine pyrophosphate was not tested at 25 mg/kg;
  • diabetic nephropathy Persistent hyperglycemia in diabetes mellitus leads to diabetic nephropathy in perhaps one third of human patients. Clinically, diabetic nephropathy is defined by the presence of: 1. decrease in renal function (impaired glomerular clearance)
  • Renal function depends on blood flow (not measured) and the glomerular clearance, which can be measured by either inulin clearance (not measured) or creatinine clearance. Glomerular permeability is measured by albumin filtration rate, but this parameter is quite variable. It is also a log-distribution function: a hundred-fold increase in albumin excretion represents only a two-fold decrease in filtration capacity.
  • Ribose Diabetic Rat Model By the above criteria, ribose appears to very rapidly induce manifestations of diabetic nephropathy, as reflected in hypertension, creatinine clearance and albuminuria, even though the latter is not large. In the established STZ diabetic rat, hyperglycemia is rapidly established in 1-2 days, but clinical manifestations of diabetic nephropathy arise very late, perhaps as much as 40 weeks for albuminuria. In general, albuminuria is highly variable from day to day and from animal to animal, although unlike humans, most STZ rats do eventually develop nephropathy.
  • pyridoxamine at 25 mg/kg body weight appears to effectively prevent two of the three manifestations usually attributed to diabetes, namely the impairment of creatinine clearance and albumin filtration. It did so as effectively as aminoguanidine.
  • the effectiveness of thiamine pyrophosphate was not manifest, but it should be emphasized that this may be due to its use at elevated concentrations of 250 mg/kg body weight. Pyridoxamine would have appeared much less effective if only the results at 250 mg/kg body weight are considered.
  • a typical adult human being of average size weighs between 66 - 77 Kg.
  • diabetic patients may tend to be overweight and can be over 1 12 Kg.
  • a range of doses for administration of pyridoxamine or thiamine pyrophosphate that is predicted to be effective for inhibiting post-Amadori AGE formation and thus inhibiting related pathologies would fall in the range of 1 mg/100 g body weight to 200 mg/100 g body weight.
  • the appropriate range when co-administered with aminoguanidine will be similar. Calculated for an average adult of 75 Kg, the range (at 10 mg/1 Kg body weight) can be approximately 750 mg to upwards of 150 g (at 2 g/1 Kg body weight). This will naturally vary according to the particular patient.
  • the interrupted glycation method allows for the rapid generation of stable well-defined protein Amadori intermediates from ribose and other pentose sugars for use in in vivo studies.
  • HSPG heparan sulfate proteoglycans
  • pyridoxamine can prevent both diabetic-like glomerular loss of heparan sulfate and glomerular deposition of glycated albumin in SD rats chronically treated with ribose-glycated albumin.
  • Rat serum albumin (RSA) fraction V, essentially fatty acid-free 0.005%; A2018
  • D-ribose, pyridoxamine, and goat alkaline phosphatase-conjugated anti -rabbit IgG were all from Sigma Chemicals.
  • Aminoguanidine hydrochloride was purchased from Aldrich Chemicals.
  • Rat serum albumin was passed down an Affi-Gel Blue column (Bio-Rad), a heparin- Sepharose CL-6B column (Pharmacia) and an endotoxin-binding affinity column (Detoxigel, Pierce Scientific) to remove any possible contaminants.
  • the purified rat serum albumin (RSA) was then dialyzed in 0.2 M phosphate buffer (pH 7.5). A portion of the RSA (20 mg/ml) was then incubated with 0.5 M ribose for 12 hours at 37°C in the dark.
  • the reaction mixture was dialyzed in cold 0.2 M sodium phosphate buffer over a 36 hour period at 4°C (this dialysis removes not only the free ribose, but also the Schiff-base intermediaries).
  • the ribated protein is classified as Amadori-RSA and is negative for antigenic AGEs, as determined by antibodies reactive with AGE protein (as described previously; R618, antigemglucose modified AGE-Rnase).
  • the ribated protein is then divided into portions that will be injected either as: a)Amadori-RSA, b)NaBH 4 -reduced Amadori- RSA, c)AGE-RSA.
  • the ribated 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 is reduced with NaBH to form NaBH 4 -reduced Amadori-RSA.
  • aliquots were reduced by adding 5 uL of NaBH stock 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 excess NaBH 4 , and then dialyzed extensively in cold PBS at 4°C for 36 hours.
  • the AGE-RSA was formed by reincubating the Amadori-RSA in the absence of sugar for 3 days.
  • Rats Male Sprague-Dawley rats (Sasco, lOOg) were used. After a 1 week adaptation period, rats were placed in metabolic cages to obtain a 24 hour urine specimen for 2 days before administration of injections. Rats were then divided into experimental and control groups and given tail vein injections with either saline, unmodified RSA (50 mg/kg), Amadori-RSA (50 mg/kg), NaBH 4 -reduced Amadori-RSA (50 mg/kg), or AGE-RSA (50 mg/kg).
  • Rats injected with Amadori-RSA and AGE-RSA were then either left untreated, or futher treated by the administration of either aminoguanidine (AG; 25 mg/kg), pyridoxamine (PM; 25 mg/kg), or a combination of AG and PM (10 mg/kg each) through the drinking water.
  • Body weight and water intake of the rats were monitored weekly in order to adjust dosages.
  • the rats were placed in metabolic cages to obtain 24 hour urine specimen for 2 days prior to sacrificing the animals.
  • Total protein in the urine samples was determined by Bio-Rad assay.
  • Albumin in urine was determined by competitive ELISA using rabbit anti-rat serum albumin (Cappell) as primary antibody (1/2000) and goat anti -rabbit IgG (Sigma Chemical) as a secondary antibody (1/2000).
  • Urine was tested with Multistix 8 SG (Miles Laboratories) for glucose, ketone, specific gravity, blook, pH, protein, nitrite, and leukocytes. None remarkable was detected other than some protein.
  • Creatinine measurements were performed with a Beckman creatinine analyzer II. Blood samples were collected by heart puncture before termination and were used in the determination of creatinine clearance, blood glucose (glucose oxidase, Sigma chemical), fructosamine (nitroblue tetrazolium, Sigma chemical), and glycated Hb (columns, Pierce chemicals). Aorta, heart, both kidneys and the rat tail were visually inspected and then quickley removed after perfusing with saline through the right ventricle of the heart. One kidney, aorta, rat tail, and the lower 2/3 of the heart were snap-frozen and then permanently stored at -70°C.
  • the other kidney was sectioned by removing both ends (cortex) to be snap-frozen, with the remaining portions of the kidney being sectioned into thirds with two portions being placed into neutral buffered formalin and the remaining third minced and placed in 2.5% glutaraldehyde/2% paraformaldehyde.
  • H&E Hams' alum hematoxylin and eosin
  • PAS perodic acid/Schiff reagent
  • alcian blue pH 1.0 and pH 2.5
  • Tissues were fixed in 2.5% glutaraldehyde/2% paraformaldehyde (0.1 M sodium cacodylate, pH 7.4), post-fixed for 1 hour in buffered osmium tetroxide (1.0%), prestained in 0.5% uranyl acetate for 1 hour and embedded in Effapoxy resin. Ultrathin sections were examined by electron microscopy.
  • Parrafin- embedded sections were deparaffmized and then blocked with 10% goat serum in PBS for 30 min at room temperature. The sections were then incubated for 2 hour at 37°C with primary antibody, either affinity purified polyclonal rabbit anti- AGE antibody, or a polyclonal sheep anti-rat serum albumin antibody (Cappell). The sections were then rinsed for 30 min with PBS and incubated with secondary antibody, either affinity purified FITC-goat anti-rabbit IgG (H+L) double stain grade (Zymed) or a Rhodamine-rabbit anti-sheep IgG (whole) (Cappell) for 1 hour at 37°C.
  • primary antibody either affinity purified polyclonal rabbit anti- AGE antibody, or a polyclonal sheep anti-rat serum albumin antibody (Cappell).
  • secondary antibody either affinity purified FITC-goat anti-rabbit IgG (H+L) double stain grade (Zymed) or a Rhodamine-rabbit anti-s
  • Kidney sections were then rinsed for 30 min with PBS in the dark, mounted in aqueous mounting media for immunocytochemistry (Biomeda), and cover slipped. Sections were scored in a blinded fashion. Kidney sections were evaluated by the number and intensity of glomerular staining in 5 regions around the periphery of the kidney. Scores were normalized for the immunofluorescent score per 100 glomeruli with a scoring system of 0-3.
  • Immunogen was prepared by glycation of BSA (R479 antibodies) or Rnase
  • Engvall (21) The general method of Engvall (21) was used to perform the ELISA. Glycated protein samples were diluted to approximately 1.5 ug/ml in 0.1 M sodium carbonate buffer of pH 9.5 to 9.7. The protein was coated overnight at room temperature onto a 96-well polystyrene plate by pippetting 200 ul of protein solution into each well (about .3 ug/well). After coating, the excess protein was washed from the wells with a saline solution containing 0.05% Tween-20. The wells were then blocked with 200 ul of 1% casein in carbonate buffer for 2 hours at 37°C followed by washing.
  • Rabbit anti-AGE antibodies were diluted at a titer of 1 :350 in incubation buffer and incubated for 1 hour at 37°C, followed by washing.
  • antibody R618 used to detect glycated RSA was generated by immunization against glycated Rnase.
  • An alkaline phosphatase-conjugated antibody to rabbit IgG was then added as the secondary antibody at a titer of 1 :2000 and incubated for 1 hour at 37°C, followed by washing.
  • Thej ⁇ -nitrophenolate being monitored at 410 nm with a Dynatech MR4000 microplate reader.
  • Immunofiuorescent glomerular staining for RSA showed elevated staining with Amadori-RSA and AGE-RSA injected animals (Figure 35). Significant reduction of this effect was seen in the rats treated with PM, and not with AG or combined AG & PM.
  • Immunofiuorescent glomerular staining for Heparan Sulfate Proteoglycan Core protein showed slightly reduced staining with Amadori-RSA and AGE-RSA injected animals but were not statistically significant(Figure 36). A reduction of this effect was seen in the rats treated with PM, and not with AG or combined AG & PM. However, immunofiuorescent glomerular staining for Heparan Sulfate Proteoglycan side-chain showed highly reduced staining with Amadori-RSA and AGE-RSA injected animals (Figure 37) A significant reduction of this effect was seen in the rats treated with PM, and not with AG or combined AG & PM. Analysis of average glomerular volume by blinded scoring showed that
  • Amadori-RSA and AGE-RSA caused significant increase in average glomeruli volume (Figure 38). A significant reduction of this effect was seen with treatment of the rats with PM. No effect was seen with treatment with AG or combined AG and PM at 10 mg/kg each.
  • LDL low density lipoprotein
  • the only characterized and chemically measurable lipoxidation products are N ⁇ -(carboxymethyl)lysine (CML), N ⁇ -(carboxyethyl)lysine (CEL), MDA- lysine and HNE-lysine. These products, however, account for less than 1% of the reducible lysine modifications formed during LDL oxidation. To better understand the changes that occur to LDL in vivo, other, more prevalent lipoxidation products need to be identified.
  • Heptafluorobutyric acid, trifluoracetic acid, acetyl chloride and trifluoroacetic anhydride were purchased from Acros.
  • Azelaic (nonanedioic) acid monomethyl ester was purchased from Aldrich.
  • Low-density lipoprotein was a gift from Dr. Alicia Jenkins of the Department of Medicine, Division of Endocrinology, Medical University of South Carolina.
  • RP-HPLC was performed on a Waters (Waters Associates, Milford, MA) 600S controller, 660 HPLC pump equipped with a Waters WISP model 723 autosampler, photodiode array detector (model 996) and Shimadzu (Kyoto, Japan) fluorescence detector (RF-535 fluorescence HPLC monitor) .
  • Amino acid analysis was performed using a SSI (Scientific Systems Inc., State
  • the buffers used were of the following composition: buffer A (Na315), pH 3.15, 98% water, 2% sodium citrate, 0.6% HCl; buffer B (Na740) pH 7.5, 93% water, 5% sodium chloride, 1.4% sodium acetate; buffer C (Na Reagent), pH 13, 94% water, 0.4% NaCl, 0.6% NaOH.
  • Gas chromatography/mass spectrometry was performed on a Hewlett-Packard (Palo Alto, CA) model 5890 gas chromatograph/5970 mass selective detector equipped with a Hewlett-Packard model 7673A autosampler.
  • ESI-MS and LC-ESI-MS were performed on a VG-70 triple quadropole mass spectrometer (Analytica ion source) interfaced to a Hewlett Packard series 1100 HPLC pump and degasser.
  • RNase (1 mM, 13.7 mg/ml) was reacted with arachidonate (100 mM) alone or in the presence of PM (1 mM) in 200 mM sodium phosphate buffer, pH 7.4.
  • the reactions were done in glass scintillation vials in a 37°C shaking water bath for 6 days, and were prepared using sterile technique to prevent bacterial growth. Aliquots were taken at 0, 1, 3 and 6 days and frozen at -20°C after addition of 1 mM DTP A/phytic acid to quench all metal catalyzed oxidation chemistry.
  • DTP A/phytic acid for GC/MS analysis of lipoxidation products, approximately 1 mg of protein was delipidated according to the method of Folch et al. (Folch, 1957).
  • the lower organic phase was discarded and the aqueous phase reduced with 500 mM NaBH 4 (in 0.1 N NaOH) in 0.1 M borate buffer, pH 9 for 4 hours at room temperature.
  • the samples were transferred to dialysis tubing (molecular weight cut-off 6,000-8,000) and dialyzed against deionized water for 24 hours at 4°C with 4 water changes.
  • the dialysates were dried in vacuo, internal standards (d 8 -lysine, d 4 -CML, d 4 - or d 8 -CEL, d 8 -MDA-lysine and d 4 -HNE-lysine) were added, then hydrolyzed in 6 N HCl at 1 10°C for 24 hours following.
  • the hydrolysates were reconstituted in 1 ml of 0.1% TFA and applied to a 1-ml Sep-Pak (Waters Corporation, Milford, MA), eluted with 3 ml of 0.1% TFA containing 20% methanol, then dried in vacuo.
  • Lysine (Figure 39) CML, CEL ( Figures 40 and 41), MDA-lysine and HNE-lysine ( Figures 42 and 4T) were derivatized to their trifluoroacetyl methyl esters for GC/MS analysis (Knecht, 1991) Methyl esters were prepared by addition of 1 ml of methanolic-HCl (18.7 ml of anhydrous MeOH - ⁇ - 1.3 ml acetyl chloride) lowed by heating at 65°C for 1 hour. The methyl esters were dried under nitrogen then converted to trifluoracetyl derivatives by addition of 1 ml ⁇ , L - trifluoroacetic acid anhydride and reaction at room temperature for 1 hour. The derivatized samples were dried under nitrogen and reconstituted in 150 ⁇ l methylene chloride for GC/MS analysis.
  • the injection port was maintained at 275°C and the temperature program was: 4 min at 140°C, 5°C/min ramp to 220°C, 25°C/min ramp to 300°C, then hold at 300°C for 5 min. Quantification was based on standard curves constructed from mixtures of deuterated and non-deuterated standards.
  • LDL was isolated by density centrifugation, then passed through PD-10 (Pharmacia, Sweden) columns pre-equilibrated with 30 ml of PBS (1.5 ml of LDL per column) to remove salts and EDTA, then passed through a Costar 0.4 ⁇ m syringe filter (Cambridge, MA) to remove aggregates.
  • the filtered LDL 50 ⁇ g/ml was oxidized in PBS with 5 ⁇ M Cu 2+ for 4-5 hours. Conjugated diene formation was measured at 294 nm at 10-15 minute intervals.
  • thiobarbituric reactive substances PM (1 mM) was reacted with linoleate (5 mM) in 200 mM sodium phosphate buffer for 6 days at 37°C. Aliquots were removed at 0, 1, 3 and 6 days, quenched with 1 mM DTPA and frozen at -20°C until analysis. TBARs were analyzed according to the method of Sawicki (1963).
  • Palmitate the internal standard, was added to all samples, followed by derivatization using boron trichloride-methanol in the presence of ImM butylated hydroxy toluene (BHT). Boron trichloride-methanol (0.5 ml) was added to the dried linoleate samples and heated at 80°C for 1 hour. Following derivatization, the samples were dried under nitrogen and the nonpolar methyl esters extracted twice with hexane: water (2:1 v/v).
  • BHT ImM butylated hydroxy toluene
  • the hexane layers were pooled and dried under nitrogen, The methyl esters were reconstituted in 150 ⁇ l of methylene chloride then analyzed by GC/MS using single ion monitoring (SIM) of m/z fragments 294 (linoleate) and 220 (palmitate).
  • the temperature program was as follows: 150°C, hold for 3 minutes; 5°C/minute ramp to 180°C; 8°C/minute ramp to 300°C, hold for 5 minutes. Linoleate was quantified based on standard curves generated from linoleate and palmitate standards.
  • N-hexanoyl-pyridoxamine was synthesized from pyridoxamine dihydrochloride and hexanoyl (caproyl) chloride. PM (20 mg) was dissolved in 50 ml of 2 ⁇ NaOH in a
  • Product 267 was hydrolyzed in 2 N HCl for 4 hours at 95°C. The hydrolysate was then dried in vacuo. The resulting free hexanoic acid was analyzed by GC/MS as its propyl ester. Briefly, 1 ml of HCl in dry propanol was added to hexanoic acid and allowed to react at 65°C for 1 hour. Following esterification, the hexanoic acid propyl esters were extracted with 2 ml hexane:water (2:1, v/v). After vortexing, the samples were centrifuged and the upper organic layer removed. Concentration of the hexane layer was performed under nitrogen and on ice to avoid loss of the volatile propyl esters. The temperature program for GC/MS analysis was as follows: initial temperature 75°C, 6°C/min ramp to 1 10°C, 10°C/min ramp to 180°C hold 5 minutes, 12°C/min ramp to 270°C/min hold 5 minutes.
  • the proposed structure for product 339 corresponds to a PM-nonanedioic acid- amide derivative.
  • synthetic 339 was made by reaction of PM with nonanedioic acid monomethyl ester. The reaction mixture was then analyzed by LC-MS, and the resulting extracted ion chromatogram and mass spectrum confirmed the formation of 339.
  • Synthetic 339 was isolated by RP-HPLC and analyzed by the TNBS assay to verify covalent attachment of the nonanedioic acid to the primary amino group of PM. The TNBS reactivity was compared to an equivalent amount of PM standard and the results are shown in Figure 48. No TNBS reactivity was present in 339, verifying that the amino group of PM participated in the amide bond.
  • synthetic 339 was hydrolyzed in 2N HCl at 95°C for 4 hours to release free PM and nonanedioic acid. The hydrolysates were derivatized and analyzed by GC/MS for acetylated PM and nonanedioic dimethyl ester. Hydrolysis of 339 yielded complete recovery of PM and of nonanedioic acid in a 1 :1 ratio.
  • products 267 and 339 As determined by RP-HPLC, of the 50-60% of PM that was consumed during reaction with linoleate, products 267 and 339 accounted for 10% and 5% of the consumed PM, respectively (product 305 accounts for about 2%). Considering the low levels of other lipoxidation markers, products 267 and 339 may constit ⁇ + major adducts on lipoxidized protein. Only four lipoxidation products have been characterized to date, and each of these has various limitations. For example, CML and CEL are both carbohydrate and iipid derived products, complicating conclusions regarding the origin of protein damage in vivo.
  • MDA-lysine and H ⁇ E-lysine while both purely lipid derived products, are subject to rearrangement to other products, including formation of crosslinks. Furthermore, all four markers represent less than 1% of the lysine modifications formed during lipoxidation reactions. Consequently, the need for additional markers of lipid-derived damage to proteins is evident, and the identification of amide derivatives of lysine may provide a convenient and sensitive tool for assessing protein oxidation. In fact, Kato and colleagues (1999) recently identified the hexamide derivative of N-benzoyl-glycyl-L-lysine following reaction with the 13-hydroperoxide of linoleic acid.
  • amide derivatives of lysine may constitute a significant fraction of the lysine modification formed in vivo.
  • these derivatives can serve as diagnostic markers for in vivo oxidative protein damage.
  • the presence and/or concentration of amide derivatives of lysine including but not limited to hexanoic acid and nonanedioic acid, are determined from urine, blood, or skin biopsy samples. In a most preferred embodiment, both blood plasma protein and skin biopsy samples are used.
  • amide derivatives in blood plasma and/or urine serves as an indicator of current exposure, since the half-life of plasma proteins is approximately 2-3 weeks. Conversely, the half life of skin proteins such as collagen and elastin is approximately 35 years, and thus determination of amide derivatives of lysine in skin collagen serves as a diagnostic m iLc; ⁇ :x ac. '.inul ⁇ tec 1 . chemical damage. Such determinations could utilize the chemical methods detailed herein, or could involve quantitative immunochemical techniques, which would require antibodies specific for the derivative form of the protein to be analyzed (for example, lipoprotein).
  • the immunochemical method could also utilize protein standards, (such as modified lipoprotein, where the extent of modification is known) and control samples (for example, the appropriate body fluid from someone with chronic atherosclerosis and/or from someone free of atherosclerosis.
  • protein standards such as modified lipoprotein, where the extent of modification is known
  • control samples for example, the appropriate body fluid from someone with chronic atherosclerosis and/or from someone free of atherosclerosis.
  • the trapping properties of PM suggest that it may be an effective inhibitor of the oxidative modification of lysine residues on protein during lipid peroxidation reactions.
  • Example 7 Pyridoxamine Inhibits Lipoxidation Reactions, In Vitro Introduction Clearance of native LDL from the circulation is dependent on recognition of
  • LDL by the LDL receptor in liver and peripheral tissues Interactions between this receptor and the apoB-100 component of LDL occur between a region of acidic amino acids on the receptor and a lysine-rich region on the protein (Goldstein, 1974). Covalent modification of the lysine residues (by acetylation, or by reaction with MDA, HNE and other lipid peroxidation products) in the receptor binding region of apoB-100 prevents recognition of LDL by the receptor (Steinbrecher, 1987). Consequently, the modified LDL is taken up the scavenger receptor of macrophages leading to foam cell formation, a hypothesized initiating event in the pathogenesis of atherosclerosis.
  • Oxidative modification of LDL in vitro interferes with its recognition by the LDL receptor and promotes its recognition by the macrophage scavenger receptor. Transition metal ions accelerate the oxidation of LDL, and for this reason, copper- catalyzed oxidation of LDL in vitro is a commonly used model for studying chemical modifications of LDL in vivo. Characteristic changes that occur to copper oxidized LDL include increased fluorescence (Ex. 350nm, Em. 433nm), increased conjugated diene formation (absorbance at 234 nm), increased anodic electrophoretic mobility, increased uptake by macrophages, and chemical modification of the lysine residues of apoB protein (Esterbauer, 1992).
  • CML, CEL, MDA-lysine and HNE-lysine were formed rapidly in RNase during incubation with arachidonate.
  • MDA-lysine and HNE-lysine are characteristic products of lipoxidation reactions.
  • Formation of CML was consistent with results of Fu et al. (1996) who first showed that CML was a product of lipoxidation as well as glycoxidation reactions.
  • the formation of CEL during reaction of RNase with arachidonate represents the first evidence that CEL, like CML, forms during both glycoxidation and lipoxidation reactions.
  • CML which is formed from oxidation of both arachidonate (20:4 ⁇ 5 ' 8 '"' 14 ) and linoleate (18:2 ⁇ 9 ' 12 )
  • CEL was formed in only trace amounts from oxidized linoleate compared to arachidonate, indicating that CML and CEL likely form from different sources in vivo.
  • Addition of 1 mM PM to the RNase/arachidonate reactions resulted in almost complete inhibition of formation of CML, CEL ( Figure 52), MDA-lysine and HNE- lysine ( Figure 53).
  • lysine by cation-exchange HPLC after hydrolysis of the protein.
  • Lipoxidation products such as MDA-lysine and HNE-lysine contain reactive Schiff base and aldehyde functional groups that are not stable to conditions of acid hydrolysis.
  • the protein was first reduced with NaBH , converting imines and aldehydes to amines and alcohols, respectively.
  • Amino acid analysis indicated that 58% of lysine residues were modified during reaction of RNase with arachidonate. Lysine loss decreased by approximately one-half in non-reduced samples (data not shown), confirming formation of acid-labile lysine modifications.
  • CEL was formed at concentrations approximately 10-fold lower than CML.
  • PM at 100 and 250 ⁇ M, inhibited CML (70% and 80%, respectively) CEL (43% and 71 %) (Figure 55), MDA-lysine (8% and 46%) and HNE- lysine (60% and 83%) formation (Figure 56).
  • total lysine modification was decreased by 62% by 100 ⁇ M PM and 87% by 250 ⁇ M PM in nonreduced and reduced samples ( Figure 57).
  • CML, CEL, MDA-lysine and HNE-lysine accounted for approximately 1% of the lysine modifications.
  • the TBARs assay recognizes primarily malondialdehyde, but also reacts with other compounds produced during PUFA oxidation (such as glyoxal). It is likely that some TBA-reactive compounds continue on to form other products, as shown in Figure 58. For example, aldehydes that may polymerize via aldol condensation reactions. This would explain the decrease in TBARs.
  • the production of TBARs during oxidation of linoleate increased during the first 3 days of reaction then decreased by day 6 ( Figure 58). PM slowed the rate of formation of TBARs by approximately 30%, but did not have a significant effect on the final yield of TBARs in the reaction mixture.
  • PM is not unlike many compounds that function as radical scavengers; most aromatic amines or phenols have antioxidant activity (Halliwell, 1989). It is possible that PM, like Vitamin E, reacts with lipid peroxy and alkoxy radicals, thus slowing the propagation phase of lipid peroxidation.
  • PM that PM retarded but did not prevent lipid peroxidation (as demonstrated by measurement of TBARs formation and rate of linoleate oxidation) suggests that the antioxidant activity of PM is a minor contributor to its mechanism of action.
  • the mild antioxidant effects of PM were not completely unexpected.
  • PM contains two functional groups common to many antioxidants; most antioxidants are aromatic amines or phenols. These types of antioxidants inhibit the propagation phase of lipid peroxidation by donation of a hydrogen atom to a peroxy or alkoxy radical. The resulting antioxidant radical delocalizes into the aromatic ring structure and is not reactive enough itself to perform hydrogen abstraction and further propagate the reaction.
  • Figure 60 represents a possible mechanism for the antioxidant activity of PM.
  • PM in vitro, appears to interrupt lipid peroxidation via two mechanisms (carbonyl trapping and antioxidant) is encouraging. PM may prove to be a powerful and multifunctional drug for the treatment of diabetes, atherosclerosis, and other chronic and inflammatory diseases in which chronic oxidative modifications of proteins is considered to be part of the pathogenic process.
  • N-hexanoyl-PM and N-nonanedioyl-PM from reactions of PM with linoleic acid suggests that amide derivatives may constitute a significant portion of the acid-labile lysine modifications formed during LDL oxidation. Formation of amide derivatives of lysine would also contribute to the increased electrophoretic mobility that occurs during LDL oxidation.
  • Kato and colleagues (1999) have i er ⁇ i ie the hexamide derivative of hippuryllysine as a product formed during incubation of hippuryllysine with the hydroperoxide of linoleic acid. They went on to show immunohistochemical evidence for the existence of ⁇ -hexanoyl-lysine in human atherosclerotic lesions.
  • Linoleate is the major PUFA in LDL; therefore, the formation of N-hexanoyl- and N-nonanedioyl-lysine would be expected during LDL oxidation based on our results from reactions of linoleate with PM.
  • LDL also contains arachidonate, albeit at a much lower concentration than linoleate (the ratio of arachidonate to linoleate in LDL is approximately 1 :8) (Esterbauer, 1992).
  • the oxidation of AA is more facile than that of linoleate because of the presence of 3 bis-aXlyXic carbons, but the formation of amide derivatives may be possible from this PUFA as well as from linoleate.
  • arachidonate is an ⁇ -6 PUFA, and produces a 15- hydroperoxide that, through the mechanism we proposed earlier for formation of 267 (nucleophilic addition of a primary amine on a ketoacid), could conceivably lead to formation of N-hexanoyl derivatives of lysine.
  • oxidation of arachidonate forms several other hydroperoxides that may lead to several amide derivatives of varying chain lengths, such as a pentadioic-amide derivative (formed from the 5- hydroperoxide) and longer chain unsaturated fatty acid derivatives.
  • amide derivatives are not reducible by sodium borohydride and are not stable to acid hydrolysis, their detection in lipoxidatively modified proteins would be indirect.
  • Modified protein, such as LDL could be hydrolyzed under relatively mild conditions, e.g. 2 ⁇ HCl at 95°C for 2 hours. This would release the amide derivatives as the free carboxylic acids, which in turn could be separated from the protein (by organic extraction) and analyzed as their ester derivatives by GC/MS.
  • Carboxylic acids containing less than 6 carbons may require either headspace GC analysis or conversion to heavy ester derivatives (such as butyl esters) because of their volatility.
  • Immunological methods for detection of protein modifications are an alternative approach.
  • antibodies could be raised against hexanoyl-modified proteins then used to detect related derivatives in tissues.
  • Production of both polyclonal and monoclonal antibodies is well known in the art. See, Antibodies: A Laboratory Manual, Harlow et al., eds., Cold Spring Harbor, N.Y. (1988).
  • a hexanoyl-modified protein(s) in the presence of an adjuvant, is injected into rabbits with a series of booster shots in a prescribed schedule optimal for high titers of antibody in serum.
  • mice are immunized with hexanoyl-modified protein(s). Following cell fusion, selection for hybrid cells' and subcloning, hybridomas are screened for a positive antibody against the hexanoyl-modified protein(s) using an indirect ELISA assay
  • N-hexanoyl and N- nonanedioyl derivatives of lysine in vivo should prove invaluable.
  • a significant marker of lipid-derived modification of protein has been identified that should be useful 1 ) to assess levels of oxidative damage to protein in vivo and 2) to better understand the processes that lead to the development of atherosclerosis.
  • Diabetes is associated with an increased risk of cardiovascular disease and is also accompanied by increased lipid peroxidation that may actually be catalyzed by hyperglycemia (Chisholm, 1992 and Tsai, 1994).
  • the modification of proteins b lipid peroxidation reactions is implicated in the pathology of various complications associated with diabetes and atherosclerosis.
  • Specific modifications of protein that have been identified on lysine residues that occur during lipid peroxidation include, CML, CEL, MDA-lysine and H ⁇ E-lysine. These products represent a very small fraction of the lysine modifications that occur during oxidation of LDL, but are the only protein modifications currently characterized. Inhibition of these 4 compounds, and inhibition of total lysine modification, by PM in vitro suggests that this B 6 vitamer may provide a useful therapeutic agent for the treatment of complications arising from the chemical modification of proteins in vivo.
  • PM will likely function through multiple mechanisms in vivo as well.
  • Our studies have shown that PM should prevent lipoxidative damage to protein, thereby impairing the development of atherosclerosis.
  • Previous examples above have demonstrated that PM inhibits both AGE formation from glucose and AGEs from post- Amadori reactions.
  • PM may also have inhibitory effects on damage to proteins derived from carbohydrates. By inhibiting protein damage occurring from both lipid and carbohydrate sources, PM would be expected to inhibit the development of complications in both atherosclerotic and diabetic animal models.
  • the present invention encompasses compounds, and pharmaceutical compositions containing compounds having the general formula:
  • ⁇ is CH 2 NH 2 , CH 2 SH, COOH, CH 2 CH 2 NH 2 , CH 2 CH 2 SH, or CH 2 COOH;
  • R 2 is OH, SH or NH 2 ;
  • Y is N or C, such that when Y is N R 3 is nothing, and when Y is C, R is NO 2 or another electron withdrawing group; and salts thereof.
  • the present invention also encompasses compounds of the general formula
  • R is CH 2 NH 2 , CH 2 SH, COOH, CH 2 CH 2 NH 2 , CH 2 CH 2 SH, or CH 2 COOH;
  • R 2 is OH, SH or NH 2 ;
  • Y is N or C, such that when Y is N R 3 is nothing, and when Y is C, R 3 is NO 2 or another electron withdrawing group;
  • R 4 is H, or C 1-18 alkyl
  • R 5 and R 6 are H, C 1-18 alkyl, alkoxy or alkane; and salts thereof.
  • the compounds of the present invention can embody one or more electron withdrawing groups, such as and not limited to -NH 2 , -NHR, -NR 2 , -OH, -OCH 3 , - OCR, and -NH-COCH 3 where R is C 1 -18 alkyl.
  • alkyl and “lower alkyl” in the present invention is meant straight or branched chain alkyl groups having from 1-18 carbon atoms, 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 3-methylpentyl.
  • the alkyl group substituents herein are optionally substituted with at least one group independently selected from hydroxy, mono- or dialkyl amino, phenyl or pyridyl.
  • alkoxy and “lower alkoxy” in the present invention is meant straight or branched chain alkoxy groups having 1-18 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.
  • alkene and “lower alkene” in the present invention is meant straight and branched chain alkene groups having 1-18 carbon atoms, such as, for example, ethlene, 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.
  • salts thereof in the present invention is meant compounds of the present invention as salts and metal complexes with said compounds, such as with, and not limited to, Al, Zn, Mg, Cu, and Fe.
  • salts and metal complexes with said compounds such as with, and not limited to, Al, Zn, Mg, Cu, and Fe.
  • One of ordinary skill in the art will be able to make compounds of the present invention using standard methods and techniques.
  • the instant invention encompasses pharmaceutical compositions which comprise one or more of the compounds of the present invention, or salts thereof, in a suitable carrier.
  • the instant invention encompasses methods for administering pharmaceuticals of the present invention for therapeutic intervention of pathologies which are related to AGE fon ⁇ ation in vivo.
  • the AGE related pathology to be treated is related to diabetic nephropathy.

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Abstract

L'invention concerne des compositions et des procédés, permettant de modéliser la formation de produits de glycosylation avancés (AGE) post-Amadori, d'identifier, et de caractériser des inhibiteurs efficaces de la formation d'AGE post-Amadori, et les compositions inhibitrices ainsi identifiées. L'invention concerne également de nouveaux procédés de traitement ou de prévention de la modification oxydative des protéines à savoir les lipoprotéines basse densité, de la peroxydation lipidique, et de l'athérosclérose, consistant à administrer une quantité efficace de l'un des composés.
PCT/US1999/023702 1998-10-09 1999-10-08 Procede permettant d'inhiber la modification oxydative des proteines WO2000022094A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU14446/00A AU1444600A (en) 1998-10-09 1999-10-08 Methods for inhibiting oxidative modification of proteins

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US10379598P 1998-10-09 1998-10-09
US60/103,795 1998-10-09

Publications (2)

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WO2000022094A2 true WO2000022094A2 (fr) 2000-04-20
WO2000022094A3 WO2000022094A3 (fr) 2001-02-22

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000021516A2 (fr) * 1998-10-14 2000-04-20 Kansas University Medical Center Research Institute, Inc. Procedes destines a empecher des complications diabetiques
US6521645B2 (en) 2000-11-20 2003-02-18 The University Of Kansas Medical Center Methods for the treatment and prevention of urinary stone disease
WO2014125376A3 (fr) * 2013-02-15 2014-12-04 Mediterranean Institute For Life Sciences Lésions protéiques dans le vieillissement et maladies liées à l'âge

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DE1915497A1 (de) * 1968-03-27 1970-11-12 Inst Rech Scient Irs Arzneimittel mit hypolipidaemischer und hypocholesterinaemischer Wirksamkeit
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CA2055990A1 (fr) * 1990-11-22 1992-05-23 David G. Schena Derives de vitamines phospholipidiques et methodes de preparation
WO1999025690A2 (fr) * 1997-11-17 1999-05-27 University Of Kansas Medical Center Intermediaires de produits terminaux de glycosylation avancee et inhibition post-amadori

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Publication number Priority date Publication date Assignee Title
GB1093546A (en) * 1965-05-19 1967-12-06 Laroche Navarron Lab Pyridoxamine salts
DE1915497A1 (de) * 1968-03-27 1970-11-12 Inst Rech Scient Irs Arzneimittel mit hypolipidaemischer und hypocholesterinaemischer Wirksamkeit
DE2461742A1 (de) * 1974-12-28 1976-07-08 Steigerwald Arzneimittelwerk Pyridoxin-derivate sowie deren herstellung und verwendung
DE3705549A1 (de) * 1987-02-18 1988-09-01 Ulrich Speck Verwendung von pyridoxin-derivaten bei der prophylaxe und therapie von hyperlipidaemien und atherosklerose
CA2055990A1 (fr) * 1990-11-22 1992-05-23 David G. Schena Derives de vitamines phospholipidiques et methodes de preparation
WO1999025690A2 (fr) * 1997-11-17 1999-05-27 University Of Kansas Medical Center Intermediaires de produits terminaux de glycosylation avancee et inhibition post-amadori

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BOOTH A A ET AL: "IN VITRO KINETIC STUDIES OF FORMATION OF ANTIGENIC ADVANCED GLYCATION END PRODUCTS (AGES)" JOURNAL OF BIOLOGICAL CHEMISTRY,US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 272, no. 9, 28 February 1997 (1997-02-28), pages 5430-5437, XP002068236 ISSN: 0021-9258 *
BOOTH A A ET AL: "THIAMINE PYROPHOSPHATE AND PYRIDOXAMINE INHIBIT THE FORMATION OF ANTIGENIC ADVANCED GLYCATION END-PRODUCTS: COMPARISON WITH AMINOGUANIDINE" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,US,ACADEMIC PRESS INC. ORLANDO, FL, vol. 220, no. 1, 1 January 1996 (1996-01-01), pages 113-119, XP002024845 ISSN: 0006-291X *
JACKSON R L: "ANTI-OXIDANTS FOR THE TREATMENT AND THE PREVENTION OF ATHEROSCLEROSIS" BIOCHEMICAL SOCIETY TRANSACTIONS,GB,COLCHESTER, ESSEX, vol. 21, no. 3, 1 August 1993 (1993-08-01), pages 650-651, XP000567472 ISSN: 0300-5127 *
RATH M. ET AL: "Nutritional supplement program halts progression of early coronary atherosclerosis documented by ultrafast computed tomography." JOURNAL OF APPLIED NUTRITION, (1996) 48/3 (68-78)., XP000914195 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000021516A2 (fr) * 1998-10-14 2000-04-20 Kansas University Medical Center Research Institute, Inc. Procedes destines a empecher des complications diabetiques
WO2000021516A3 (fr) * 1998-10-14 2001-01-04 Kansas University Medical Ct R Procedes destines a empecher des complications diabetiques
US6521645B2 (en) 2000-11-20 2003-02-18 The University Of Kansas Medical Center Methods for the treatment and prevention of urinary stone disease
WO2014125376A3 (fr) * 2013-02-15 2014-12-04 Mediterranean Institute For Life Sciences Lésions protéiques dans le vieillissement et maladies liées à l'âge

Also Published As

Publication number Publication date
AU1444600A (en) 2000-05-01
WO2000022094A3 (fr) 2001-02-22

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