WO2000024405A9 - Compounds and their therapeutic use with diabetic complications - Google Patents
Compounds and their therapeutic use with diabetic complicationsInfo
- Publication number
- WO2000024405A9 WO2000024405A9 PCT/US1999/025358 US9925358W WO0024405A9 WO 2000024405 A9 WO2000024405 A9 WO 2000024405A9 US 9925358 W US9925358 W US 9925358W WO 0024405 A9 WO0024405 A9 WO 0024405A9
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- Prior art keywords
- group
- lysine
- carcinoma
- unsubstituted
- fructose
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates to therapeutic agents and their use for the treatment of diabetics and in particular for preventing, reducing or delaying the onset of diabetic complications and other disorders of related etiology. More particularly, the present invention relates to a class of enzyme inhibitors which inhibit the enzymatic conversion of fructose lysine (FL) to fructose-lysine-3-phosphate (FL3P) , which is believed to be an important step in the biochemical mechanism leading to diabetic complications. This invention also relates to a method of assessing a diabetic patient's risk of experiencing diabetic complications, as well as a method of determining the efficacy of therapeutic intervention in preventing, reducing or delaying the onset of diabetic complications.
- FL fructose lysine
- FL3P fructose-lysine-3-phosphate
- diabetes There are four particularly serious complications of diabetes, namely, diabetic nephropathy or kidney disease; diabetic retinopathy which causes blindness due to destruction of the retina; diabetic neuropathy involving the loss of peripheral nerve function; and circulatory problems due to capillary damage. Both retinopathy and nephropathy are thought to be subsets of the general circulatory problems associated with this disease state.
- the role of microvascular dysfunction in late stage diabetes has been recently summarized (Tooke, Diabetes, 4.: 721 (1995)).
- the terms "diabetes-associated pathologic conditions" and synonymous terms are meant to include the various well-known retinopathic, neuropathic, nephropathic, macroangiopathic, as well as other complications of diabetes.
- diabetes-associated pathologic conditions and aging would appear to suggest a common mechanistic rationale.
- a variety of mechanisms have been proposed as a common biochemical basis for both diabetes-associated pathologic conditions and aging.
- the hypothesis most strongly supported by data from human subjects is premised on a non-enzymatic glycosylation mechanism.
- This hypothesis states that the aging process and diabetes-associated pathologic conditions, such as those described above, are caused, at least in part, by protein modification and cross-linking by glucose and glucose-derived metabolites via the Maillard reaction (Monnier et al., Proc. Natl. Acad. Sci. USA, 81: 583 (1984) and Lee et al., Biochem. Biophys. Res. Comm.
- 3DG 3-deoxyglucosone
- 3DG is a glucose- derived metabolite that can react with proteins leading to the cross-linking of both intracellular and extracellular proteins, such as collagen and basement membranes.
- hyperglycemic memory Another factor demonstrating cause and effect relationship between diabetic complications and hyperglycemia is hyperglycemic memory.
- hyperglycemic memory Another factor demonstrating cause and effect relationship between diabetic complications and hyperglycemia is hyperglycemic memory.
- This phenomenon is the development of severe retinopathy in dogs that were initially diabetic, then treated to restore normal blood glucose levels. Although the dog eyes were histoic ically normal at the time of the treatment, over time diabetic retinopathy developed in these animals in spite of the normalized glucose concentrations (Engerman et al., Diabetes, 2__.z 808 (1987)). Thus, the underlying damage to the eyes irreversibly occurred during the period of early hyperglycemia, before clinical symptoms were evident. Diabetic humans and animals have been shown to have higher than normal concentrations of early and late sugar modified AGE-proteins. In fact, the increase in AGE-proteins is greater than the increase in blood glucose levels. The concentration of AGE- proteins can be estimated by fluorescence measurement, as
- AGE-proteins The pathogenic role of AGE-proteins is not limited to diabetes. Protein glycation has been- i plicated in Alzheimer's disease (Harrington et al., Nature, 370: 247 (1994)). Increased protein fluorescence is also seen with aging. Indeed, some theories trace the aging process to a combination of oxidative damage and sugar-induced protein modification. Thus, a therapy that reduces AGE-protein formation may also be useful in treating other etiologically-similar human disease states, and perhaps slow the aging process.
- AGE-proteins from sugars is a multi-step process, involving early, reversible reactions with sugars to produce fructose-lysine containing proteins. These modified proteins then continue to react to produce irreversibly modified AGE-proteins. It is clear that AGE-proteins are not identical to proteins containing glycated-lysine residues, as antibodies raised against AGE-proteins do not react with fructose-lysine. It is also clear that AGE-proteins exist as multiple chemical species; however few have been identified.
- FIG. 1 The generally accepted pathway for formation of 3DG from the reaction of sugars and proteins is illustrated in Figure 1.
- a sugar (glucose) molecule initially forms a Schiff base with a protein-lysine amino group (I) .
- the resulting Schiff base then rearranges to produce fructose-lysine modified proteins (II) .
- the reactions leading up to (II) are freely reversible.
- (II) can rearrange to produce 3DG and free protein lysine.
- Subsequent reaction between 3DG and protein is the first irreversible step in AGE-protein formation.
- 3-DG can be produced by alternative pathways, or indeed, that the major source of 3-DG is from an enzyme catalyzed metabolic pathway, rather than from the uncatalyzed reactions shown in Figure 1.
- Diabetic patients have significantly more 3DG in serum than do non-diabetic patients (12.78+2.49 ⁇ M versus 1.94+0.17 ⁇ M) .
- This toxic compound is found in normal healthy individuals.
- the body has developed a detoxification pathway for this molecule.
- One of these reactions is catalyzed by aldehyde reductase which detoxifies 3DG by reducing it to 3-deoxyfructose (3DF) which is efficiently excreted in urine (Takahashi et al. , Biochem, __£.: 1433 (1995)).
- Another detoxification reaction oxidizes 3DG to 3-deoxy-2- ketogluconic acid (DGA) by oxoaldehyde dehydrogenase (Fujii et al., Biochem. Biophys. Res. Comm. , 210.: 852 (1995)).
- aldehyde reductase inhibitors (Barski et al., Biochem., £ 11264 (1995)).
- ARIs aldose reductase inhibitors
- a high protein diet will increase the consumption of fructose-lysine, which undergoes conversion into 3DG by the kidney lysine recovery pathway.
- the detoxification of the resulting 3DG by reduction to 3DF will be inhibited by ARIs therapy, which consequently leads to an increase in kidney damage, as compared to rats not receiving ARIs. This is because inhibition of the aldose reductase by the ARIs would reduce availability of aldose reductase for reducing 3DG and 3DF.
- U.S. Patent 5,476,849 to Ulrich et al describes a method of inhibiting the formation of AGE-proteins using amino-benzoic acids and derivatives. These compounds presumably act by reacting with 3-DG and removing it from the system before it can react with proteins to begin the irreversible steps of AGE-protein formation.
- U.S. Patent 5,358,960 to Ulrich et al describe a method for inhibiting AGE-protein formation using aminosubstituted imidazoles. These compounds were shown to react with an early glycosylation product (3DG) . No mention is made in this patent that a metabolic source of 3DG may exist. This patent envisions that 3DG is made exclusively as an intermediate in the non-enzymatic browning of proteins.
- U.S. Patent 5,334,617 to Ulrich et al describes amino acids useful as inhibitors of AGE-protein formation. Lysine and other bifunctional amino acids are described as particularly useful in this regard. These amino acids are described as reacting with the early glycosylation product from the reaction of glucose and proteins. It appears that the early glycosylation product described in this patent is 3DG.
- U.S. Patent 5,318,982 to Ulrich et al describes the inhibition of AGE-protein formation - lo ⁇
- inhibitory agent 1,2,4-triazoles using as the inhibitory agent 1,2,4-triazoles.
- the inhibitors described in this patent contain diamino-substituents that are positioned to react with and complex 3DG.
- the patent describes these compounds as reacting with an early glycosylation product (3DG as defined herein) .
- U.S. Patent 5,272,165 to Ulrich et al describes the use of 2-alkylidene-aminoguanidines as inhibitors of AGE-protein formation.
- the inhibitors described in this patent are said to be highly reactive with 3DG. No mention is made of inhibiting the metabolic formation of 3DG in this patent.
- U.S. Patent 5,262,152 to Ulrich et al describes the use of amidrazones and derivatives to inhibit AGE-protein formation.
- the compounds described in this patent are ⁇ -effect amines. W.P. Jencks, 3rd ed. , McGraw Hill, New York. Compounds of this category are known to react with dicarbonyl compounds, e.g. 3DG.
- U.S. Patent 5,258,381 to Ulrich et al describes the use of 2-substituted-2-imidazolines to inhibit AGE-protein formation.
- the compounds described in this patent contain adjacent amino groups that can readily react with 3DG.
- U.S. Patent 5,243,071 to Ulrich et al describes the use of 2-alkylidene-aminoguanidies to inhibit AGE-protein formation.
- the compounds described in this patent are highly reactive with 3DG and function by complexing this reactive, toxic molecule.
- U.S. Patent 5,221,683 to Ulrich et al. describes the use of diaminopyridine compounds to inhibit AGE-protein formation.
- the diaminopyridine compounds described as particularly useful will react with 3DG to form a stable, six-member ring containing complex.
- U.S. Patent 5,130,337 to Ulrich et al describes the use of amidrazones and derivatives to inhibit AGE-protein formation.
- the inhibitors described in this patent are ⁇ -effect amines which, as is know in the art, will rapidly react with 3DG and form stable complexes.
- U.S. Patent 5,130,324 to Ulrich et al describes the use of 2-alkylidene-aminoguanidines to inhibit AGE-protein formation.
- the compounds described in this patent function by reacting with the early glycosylation product resulting from the reaction of glucose with proteins (3DG) .
- U.S. Patent 5,114,943 by Ulrich et al describes the use of amino-substituted pyrimidines to inhibit AGE-protein formation.
- the compounds described in this patent are said to rapidly react with and detoxify 3DG.
- U.S. Patent 5,108,930 to Ulrich et al describes a method for detecting the levels of aminoguanidine in biological samples. This assay is described as having potential utility in determining kidney function by measuring the aminoguanidine elimination time. The principal utility intended for the assay method described in this patent is in the measurement of tissue levels of aminoguanidine, so that doses sufficient to inhibit AGE-protein formation can be maintained in animal and human studies. No mention is made in this patent of using urine 3DG, 3DF or DGA ratios to determine diabetics at risk for complications.
- U.S. Patent 5,231,031 to Szwergold et al describes a method for assessing the risk of diabetic-associated pathologic conditions and determining the efficacy of therapies for these complications. This patent describes the measurement of two phosphorylated compounds in erythrocytes of diabetic patients. These two compounds were not chemically identified in this patent. However, neither compound is 3DG or 3DF, whose levels are measured in urine in the diagnostic embodiment of the present invention.
- U.S. Patent 4,761,368 issued to A. Cerami describes the isolation and purification of a chromophore present in browned polypeptides, e.g., bovine serum albumin and poly-L-lysine.
- the chromophore, 2-(2-furoyl) -4 (5) -2 (furoyl) -IH-imidazole (FFI) is a conjugated heterocycle derived from the condensation of two molecules of glucose with two lysine-derived amino groups.
- This patent further describes the use of FFI in a method for measuring "aging” (the degree of advanced glycosylation) in a protein sample wherein the sample “age” is determined by measuring the amount of the above-described chromophore in the sample and then comparing this measurement to a standard (a protein sample having an amount of FFI which has been correlated to the "age" of the sample) .
- Tnore is a lo g-standing, unfilled need in existing treatment regimens of diabetic patients for effective means to identify those at risk of developing diabetes-associated pathologic conditions, to prevent, reduce or delay the onset of such conditions by therapeutic intervention and to determine the benefit of such therapeutic intervention.
- the present invention arose from the discovery of a metabolic pathway that involves the enzyme-mediated conversion of FL to FL3P and produces relatively high concentrations of 3-deoxyglucosone (3DG) in organs affected by diabetes. Subsequent research into the biochemical function of this newly discovered pathway tends to indicate that it has an important role in the etiology of diabetic kidney disease. It is also suspected that this pathway contributes to the development of the various known diabetes-associated pathologic conditions.
- 3DG 3-deoxyglucosone
- the assay method comprises providing an aqueous solution of fructose-lysine, adenosine triphosphate (ATP) , a source of fructose-lysine-3-phosphate kinase and a compound of the present invention in an amount sufficient to demonstrate inhibitory activity, subjecting the resulting solution to conditions promoting the formation of fructose-lysine-3-phosphate and adenosii.f.
- ATP adenosine triphosphate
- the compounds of the present invention reducing the amount of such products, as compared to an aqueous solution of the same relative amounts of fructose-lysine, adenosine triphosphate and source of fructose-lysine- 3-phosphate kinase, without the addition of a compound of the present invention.
- the assay method just described is also within the scope of the present invention.
- the present invention provides a pharmaceutical preparation for preventing, reducing or delaying the onset of diabetic complications in a diabetic patient, comprising, as an active agent, a compound of the invention, as described above, and a pharmaceutically acceptable vehicle.
- a method for preventing, reducing or delaying the onset of diabetic complications in a patient at risk of developing same comprises administering to the patient a compound of the present invention in an amount effective to inhibit the enzymatic conversion of fructose-lysine to fructose-lysine-3-phosphate.
- This same method may be used for the prevention or treatment of other etiologically-similar disease states, as will be further described hereinbelow.
- the present invention provides a method for assessing a diabetic patient's risk of experiencing a diabetes- associated pathologic condition.
- This method comprises administering to the patient a source of glycated-lysine residues in an amount providing a predetermined dose of the glycated-lysine residues, and measuring the ratio of 3-deoxyglucosone to 3- deoxyfructose in a biological sample obtained from the patient, with reference to the ratio of 3- deoxyglucosone to 3-deoxyfructose in a normal subject, i.e., a non-diabetic subject or one having no clinical symptoms of diabetes.
- the higher ratio of 3- deoxyglucosone to 3-deoxyfructose in the diabetic patient sample, in comparison to that of the asymptomatic subject is indicative that the diabetic patient is at higher risk of experiencing a diabetes- associated pathologic condition.
- the present invention also provides a method for assessing the efficacy of therapeutic intervention in preventing diabetic complications.
- the method involves measuring the concentration of 3- deoxyglucosone, 3-deoxyfructose and fructose-lysine in biological samples obtained from a diabetic patient, both before and after initiation of the therapeutic intervention. The sum of the 3-deoxyglucosone and 3- deoxyfructose concentrations are then compared to the concentration of fructose-lysine in the samples.
- a method for apprising a diabetic person of the potential of a food product to contribute to the development of a diabetic-associated pathologic condition involves measuring the content of glycated-lysine residues in the food product an ⁇ providing this information to diabetic patients, e.g., on the package of the food product or in a publication intended for use by diabetics.
- an additional embodiment of the invention provides a method for assessing a diabetic patient's risk of experiencing a diabetes-associated pathologic condition based on measurement of the 3DF present in a biological sample of a diabetic patient with reference to one or more predetermined baseline level of 3DF as an indicator of the likelihood that the patient will develop diabetic complications, or not.
- Another aspect of the invention comprises a method of reducing suceptibility to carcinoma in a patient associated with the intake of glycated proteins.
- the method comprises the administration of a pharmaceutical composition which contains an active compound having inhibitory activity for the enzymatic conversion of fructose-lysine to fructose-lysine-3- phosphate.
- Also embodied in the present invention is a method of preventing, reducing, or delaying the onset of carcinoma caused by the formation of AGE- proteins.
- the method comprises administering a therapeutic amount of an agent that inhibits production of 3-deoxyglucosone.
- a method for inducing carcinoma in a susceptible test animal comprises feeding the animal with a glycated protein diet for a sufficient time period, such that 3-deoxyglucosone is elevated in biological fluids at least three fold. Such animals would be assessed relative to untreated control animals.
- a method of screening for substances which affect the development of carcinoma is also provided in accordance with the present invention.
- Carcinoma will be induced in test animals via feeding of a glycated protein diet such that 3DG levels are elevated at least 3 fold in biological fluids.
- the animals are then divided into two groups, one of which will receive the compound to be assessed, while the other group serves as a negative control. After a suitable time period, both groups of animals will be sacrificed and the presence and/or absence of carcinoma in both groups assessed.
- a method for screening for substances which prevent, reduce or delay the onset of carcinoma comprises the steps of feeding susceptible test animals a glycated protein diet in an amount and for a time sufficient to maintain 3-deoxyglucosone (3DG) content of a biological fluid from a similar susceptible test animal fed a diet substantially free of the glycated protein.
- a test substance will then be administered to one portion of the test animals but not to the other portion.
- the animals will then be sacrificed and tissue sections compared from each such portion of susceptible test animals to assess the effects of the test substance.
- FIGURE 1 shows the initial step involved in the multi-step reaction leading to irreversibly- modified AGE-proteins.
- FIGURE 2 illustrates the reactions involved in the lysine recovery pathway.
- FIGURE 3 is a graphical representation of a urinary profile showing the variation over time of
- 3DF, 3DG and FL from a single individual fed 2 g. of FL and followed for 24 hours.
- FIGURE 4 is a graphical representation of urinary excretion over time of 3DF from seven volunteers fed 2 g. of fructoselysine.
- FIGURE 5 shows a graphical comparison of 3DF and N-acetyl- ⁇ -glucosaminidase (NAG) between a group of control animals and an experimental group maintained on a feed containing 0.3% glycated protein.
- NAG N-acetyl- ⁇ -glucosaminidase
- FIGURE 6 is a graph showing the linear- relationship between 3DF and 3DG levels in urine of rats fed either a control diet or one enriched in glycated protein.
- FIGURES 7A and 7B are graphical representations of fasting levels of 3DG in the urine of normals and diabetic patients plotted against the fasting level of 3DF. DETAILED DESCRIPTION OF THE INVENTION
- Glycated-Lysine Residues refers to .he modified lysine residue of a stable adduct produced by the reaction of a reducing sugar and a lysine-containing protein. The majority of protein lysine residues are located on the surface of proteins as expected for a positively charged amino acid. Thus, lysine residues on proteins which come in contact with serum, or other biological fluids, can freely react with sugar molecules in solution. This reaction occurs in multiple stages. The initial stage involves the formation of a Schiff base between the lysine free amino group and the sugar keto-group. This initial product then undergoes the Amadori rearrangement, to produce a stable ketoamine compound.
- the early products of the reaction of any reducing sugar and the e-amino residue of protein lysine are included within the meaning of glycated-lysine residue, regardless of the exact structure of the modifying sugar molecule.
- glycated-lysine residue glycated protein and glycosylated protein or lysine residue are used interchangeably herein, which is consistent with current usage in scientific journals where such expressions are often used interchangeably.
- Fructose-lysine The term "fructose-lysine" (FL) is used herein to signify any glycated-lysine, whether incorporated in a protein/peptide or released from a protein/peptide by proteolytic digestion. This term is specifically not limited to the chemical structure commonly referred to as fructose-lysine, which is reported to form from the reaction of protein lysine residues and glucose. As noted above, lysine amino groups can react with a wide variety of sugars. Indeed, one report indicates that glucose is the least reactive sugar out of a group of sixteen (16) different sugars tested (Bunn et al., Science, 213: 222 (1981)).
- tagatose-lysine formed from galactose and lysine analogously to glucose is included wherever the term fructose-lysine is mentioned in this description, as is the condensation product of all other sugars, whether naturally-occurring or not.
- fructose-lysine residues and sugars involves multiple reaction steps. The final steps in this reaction sequence involve the crosslinking of proteins and the production of multimeriq species, known as AGE- proteins, some of which are fluorescent. Proteolytic digestion of such modified proteins does not yield lysine covalently linked to a sugar molecule. Thus, these species are not included within the meaning of "fructose-lysine", as that term is used herein.
- Fructose-lysine-3-phosphate This compound is formed by the enzymatic transfer of a high energy phosphate group from ATP to FL.
- FL3P fructose-lysine-3-phosphate
- FL3P is meant to include all phosphorylated fructose-lysine moieties that can be enzymatically formed whether free or protein-bound.
- Fructose-lysine-3-phosphate kinase - This term refers to one or more proteins which can enzymatically convert FL to FL3P, as defined above, when additionally supplied with a source of high energy phosphate.
- 3-Deoxyglucosone - 3-Deoxyglucosone is the 1, 2-dicarbonyl-3-deoxysugar (also known as 3-deoxyhexulosone) which is formed upon breakdown of FL3P to yield free lysine and inorganic phosphate.
- 3-deoxyglucosone is intended to include all possible dicarbonyl sugars which are formed upon breakdown of FL3P, having the broad definition of FL3P stated above.
- a lysine recovery pathway exists in human kidney, and possibly other tissues, which regenerates unmodified lysine as a free amino acid or incorporated in a polypeptide chain. As will be further explained below, this pathway is an important factor contributing to the complications of diabetes.
- AGE-Proteins The term “AGE-proteins” (Advanced Glycation End-product modified proteins) has been used in scientific journals, and is used herein, to refer to the final product of the reaction between sugars and proteins (Brownlee, Diabetes Care, 15.: 1835 (1992) and Niwa et al., Nephron, £__.: 438 (1995)). It is clear that the reaction, for example, between protein lysine residues and glucose does not stop with the formation of fructose-lysine. FL can undergo multiple dehydration and rearrangement reactions to produce non-enzymatic 3DG, which reacts again with free amino groups, leading to cross-linking and browning of the protein involved. Indeed, there is reasonable evidence that 3DG, as defined hereinabove, is a central intermediate in this modification reaction.
- Glycated Diet As used herein, this expression refers to any given diet in which a percentage of normal protein is replaced with glycated protein.
- the expression "glycated diet” and “glycated protein diet” are used interchangeably herein.
- Susceptible test animal As used herein this expression refers a strain of laboratory animals which, due to the presence of certain genetic mutations have a higher propensity towards malignant transformation and tumor formation.
- the Eker rat which has a mutation in the tuberous sclerous gene (T ⁇ c-2) was utilized in the studies described herein.
- T ⁇ c-2 tuberous sclerous gene
- One of ordinary skill in the art is aware of a variety of other laboratory rat or mouse strains with increased propensity for tumor formation.
- similar susceptible test animal refers to animals of a comparable genetic background which are used as control, untreated animals.
- the present invention evolved from the discovery of a previously unknown metabolic pathway which produces 3DG in an enzyme- catalyzed reaction.
- This enzymatic pathway is capable of enzymatic inhibition, thereby reducing the production of toxic 3DG.
- FL3P Authentic fructose-lysine-3-phosphate
- FL3P is derived directly from FL in an enzyme catalyzed reaction.
- Fructose-lysine labeled with deuterium at the C3 position of the fructose moiety was synthesized and injected into rats.
- the kidneys of these rats were removed and extracted with perchloric acid.
- NMR spectroscopy revealed that the FL3P material isolated from these rats contained the deuterium label at the C3 position of the fructose moiety.
- rat kidney homogenates demonstrate the ability to produce FL3P in a reaction requiring both ATP and fructose- lysine.
- FL3P is not stable in aqueous solution. It rapidly degrades to form 3DG, lysine and inorganic phosphate. This reaction also occurs in vivo. It is not currently know if the degradation of FL3P in vivo is a spontaneous or enzyme catalyzed reaction. It is strongly suspected, however, that enzymatic catalysis is involved, as the production of 3DG from fructose- lysine occurs very rapidly in intact kidney.
- fructose-lysine and ATP react to form fructose-lysine-3-phosphate (FL3P) and ADP in a reaction catalyzed by FL3P kinase.
- FL3P fructose-lysine-3-phosphate
- ADP in a reaction catalyzed by FL3P kinase.
- Phosphorylation occurs on the 3-position of the fructose moiety, leading to destabilization of the fructoselysine icclecule.
- the resulting FL3P then decomposes to form 3-deoxyglucosone (3DG) , inorganic phosphate, and unmodified, free, reusable lysine, which is available for utilization in protein synthesis.
- Aldehyde reductase detoxifies 3DG by reduction to 3- deoxyfructose (3DF) , which is excreted in urine.
- Figure 2 illustrates this pathway using the most prevalent glycated-lysine, fructose- lysine, it will be readily apparent to those skilled in the art that a wide variety of similar molecules can flux through this pathway. Indeed, as will be explained in further detail below, the substrate selectivity of the FL3P lysine recovery pathway is quite broad, warranting the broad definition of the terms given above.
- lysine recovery pathway is found in a wide variety of animal species, including sheep, pig, dog, rabbit, cow, mice and chicken. This pathway is also present in humans.
- the ubiquitous presence of the FL3P lysine recovery pathway can be understood, given that lysine is an essential amino acid which is present in relatively low concentrations in most foods.
- lysine residues in food will exist in the glycated form and the proportion of this modified lysine will increase when the food is cooked. Since these glycated lysine residues can not be utilized for protein synthesis, a recovery pathway for lysine is of great utility and affords a selective advantage to organisms which possess it.
- Diabetes has two effects on the lysine recovery pathway. Blood proteins contain higher concentrations of glycated-lysines when isolated from diabetics than from non-diabetic individuals. Thus, diabetics are subject to greater flux through the lysine recovery pathway than non-diabetics. Additionally, from preliminary observations on the ratios of 3DG and 3DF in the urine of diabetics and normals, diabetics appear to have a reduced ability to detoxify 3DG that is produced via this pathway. These two factors combine to produce higher urinary concentrations of 3DG in diabetics (See Figure 7; also Lai et al., Arch. Biochem. and Biophys. , 342 (1) : 254- 60 (1997) .
- the agents involved in the lysine recovery pathway have been identified in other tissues besides kidney, specifically red blood cells, lens, and peripheral nerve tissues. All of these tissues are affected by the complications of diabetes.
- the location in red blood cells correlates with the microvascular complications of diabetes, e.g., diabetic retinopathy, the kidney location correlates with diabetic nephropathy, while the location in peripheral nerve correlates with diabetic peripheral neuropathy.
- These agents are also found in pancreas. Experiments are in progress to determine the presence of these agents in skin. If found to be present, it is believed that their deleterious effects may be ameliorated by a topical treatment using the inhibitory compounds of the invention in a suitable vehicle to prevent collagen crosslinking, and thereby improve skin elasticity.
- the enzyme inhibitor compounds of the invention block the lysine recovery pathway, preventing formation of toxic 3DG from FL3P. Described below is a set of extensive criteria that a suitable enzyme inhibitor should display for use in the practice of this invention, as well as certain tests for determining if any putative inhibitor meets these criteria.
- Candidate kinase inhibitors for use in accordance with this invention may be natural products isolated from plants or microorganisms. Alternatively, they may be synthetic molecules derived from the rational knowledge of the enzymatic reaction and its mechanism. Inhibitors may also be synthesized by combinatorial methods. Combinatorial libraries may be generated from a random starting point.
- combinatorial methods can be utilized to generate a wide variety of compounds related to previously identified inhibitors of the target FL3P kinase. Regardless of the source of the putative inhibitor, compounds that do not meet all of the criteria listed below are not considered useful therapeutic agents capable of inhibiting the lysine recovery pathway and thereby preventing, reducing or delaying the onset of diabetic complications or disorders of related etiology.
- the inhibitor should be a small molecule and readily taken up by cells. In order to meet this criteria, the inhibitor must have a molecular weight of less than 2,000 and more ideally approximately 1,000 daltons or less.
- the inhibitor must show competitive, noncompetitive, irreversible or suicide inhibition of the FL3P kinase. If the inhibitor is a competitive or noncompetitive inhibitor, the inhibition constant, K i# must be less than about 1 mM. Ideally, it must be less than 100 ⁇ M and more ideally, about 40 ⁇ M or less. If the inhibitor shows suicide or other irreversible inhibition, this requirement for inhibition constant is rendered moot.
- the inhibitor must be both soluble in aqueous solution and stable in aqueous solution at physiological pH.
- the requirement for solubility is met only if the inhibitor, or a salt of the inhibitor, is soluble in physiological saline or serum at a concentration equal to or greater than 10 ⁇ M.
- This stability requirement is met only if a solution of inhibitor dissolved in physiological saline at 37°C retains greater than 50% of its activity after incubation for one hour. Ideally, the inhibitor must retain greater than 50% activity upon incubation for one day or more.
- the inhibitor must show acceptable pharmacokinetics. That is, it must remain at a therapeutically effective concentration for at least one hour following administration of the agent.
- the inhibitor should maintain effective concentration for at least eight hours. More ideally, once per day dosing should be all that is necessary in order to maintain a therapeutic concentration of the inhibitor. This requirement does not mean that the inhibitor must be able to establish a therapeutic concentration after the first dose. Numerous examples of successful pharmaceuticals exist where medical efficacy is seen only upon prolonged dosing. The criterion does mean that, once an efficacious concentration is reached, this concentration should be able to be maintained for greater than one hour following the last administration of medication. A test for therapeutic efficacy is described herein. 5. The inhibitor must be non-toxic. This criteria requires that the inhibitor not demonstrate human toxicity when administered at the therapeutic dose. Ideally, toxicity should not be evident when the inhibitor is present at blood and/or target tissue levels of twice that needed for therapeutic effect.
- X is -NR'- or -0-, R' being selected from the group consisting of H, and linear or branched chain alkyl group (C ⁇ -C 4 ) and an unsubstituted or substituted aryl group (C 6 -C 10 ) or aralkyl group (C 7 - C .o ) ;
- R is a substituent selected from the group consisting of H, an amino acid residue, a polyaminoacid residue, a peptide chain, a linear or branched chain aliphatic group (C_-C 8 ) , which is unsubstituted or substituted wit.h at least one nitrogen- or oxygen-containing substituent, a linear or branched chain aliphatic group (Cx-Cg) , which is unsubstituted or substituted with at least one nitrogen- or oxygen-containing substituent and interrupted by at least one -O-, -NH-, or -NR"- moiety, R" being linear or
- Z is selected from the group consisting of -H, -O-alkyl (C ⁇ C 6 ) , -halogen -CF 3 , -CN, -COOH, and -S0 3 H 2 , and optionally -OH; and the isomers and pharmaceutically acceptable salts of said compound.
- nitrogen- or oxygen-containing "R" substituents include those derived from ⁇ -amino- -hydroxy butyric acid (-(CH 2 ) 2 - CHOH-COOH) , 1,2,4 triaminobutane (-(CH 2 ) 2 -CHNH 2 -CH 2 NH 3 ) , 3 , 6-diamino-5-hydroxyheptanoic acid (-CH 2 -CH(OH) -CH 2 - CH(NH 2 )-CH 2 -COOH) , and the like.
- the structure of formula I has asymetric centers and may occur as racemates, racemic mixtures and various stereoisomers, all of such isomeric forms being within the scope of this invention, as well as mixtures thereof. Although certain of the compounds having the structure of formula I, above, were previously known, others are believed to be novel and as such are within the scope of the present invention, as is the use of all of the compounds of formula I for inhibiting the enzyme-catalyzed production of 3DG in vivo.
- appropriate sugar e.g. , glucose, galactose, mannonse, ribose, xylose, or derivative thereof.
- sugars such as glucose, galactose, mannose, ribose, xylose, or the like
- the reactive moiety of an amino acid reactant when used, may be the amine group on the alpha-carbon, or the amine group or hydroxyl group on the acid side chain.
- Suitable amino acids encompass the essential amino acids.
- glycine alanine, valine, leucine, isoleucine, serine, threonine, methionine, aspartic acid, phenylalanine, tyrosine, histidine and tryptophan.
- Other suitable reactants are from the broader class of aminocarboxylie acid, for example, pyroglutamic acid, beta-alanine, gamma-aminobutyric acid, epsilon-amino caproic acid and the like.
- N-acyl derivatives of the above-mentioned amino acids such as formyl lysine, may also be used if desired.
- aryl (C6-C10) compounds wherein the substituent may be alkyl -(Cl- C3) , alkoxy, carboxy, nitro or halogen groups, unsubstituted or substituted alkanes, wherein the substituent may be at least one alkoxy group; or unsubstituted or substituted nitrogen-containing heterocyclic compounds, wherein the substituents may be alkyl (C_-C 3 ) , aryl (C 6 -C 10 ) , alkoxy, carboxy, nitro or halogen groups.
- Illustrative examples of the last- mentioned group of reactants include m-methyl-, p- ethyl-, m-methoxy-, o-methoxy- and m-nitro- aminobenzenes, o- and p-aminobenzoic acids; n- propylamine, n-butylamine, 3-methoxypropylamine; morpholine and piperdine.
- inhibitor compounds having the above formula are set forth in the attached Table A.
- Examples of known compounds that may be used as inhibitors in practicing this invention include, without limitation, meglumine, sorbitol lysine and mannitol lysine.
- the inhibitor compounds described herein can form pharmaceutically acceptable salts with various inorganic or organic acids or bases.
- Suitable bases include, e.g., alkali metal salts, alkaline earth metal salts, ammonium, substituted ammonium and other amine salts.
- Suitable acids include, e.g., hydrochloric acid, hydrobromic acid and methanesulfonic acid.
- the ability of a compound to inhibit the FL3P kinase can be determined using a wide variety of kinase activity assays.
- One useful assay involves incubating the potential inhibitor with fructose-lysine and ATP in the presence of kidney homogenate or other enzyme source.
- a solution of the assay components is prepared, which typically contains 1 millimole or less of the inhibitor compound of this invention, an amount of fructose lysine (FL) in the range of 1-10 millimoles, an amount of ATP in the range of 0.1-10 millimoles and an amount of the enzyme source which is sufficient to convert FL to fructose lysine-3-phosphate.
- the incubation should be conducted within a pH range of 4.5 to 9.5 and ideally at neutral or near neutral pH.
- the incubation should be carried out at a temperature that is compatible with enzyme activity, between 4 and 40°C. Ideally, the incubation is carried out at physiological temperature.
- the reaction is stopped by acid precipitation of the protein and the production of FL3P measured by 31 P-NMR spectroscopy. FL3P production will be reduced or eliminated in samples containing an inhibitor compound when compared to control samples that are free of inhibitor.
- FL3P does not bind to Dow-1 but ATP and most other phosphates do, it is possible to separate the product FL3P from the remaining reaction mixture by passing the assay solution through a column of Dow-1 resin after a predetermined reaction time, typically 10 minutes. The resultant solution is added to a container of scintillation liquid, e.g., Ecoscint A, and counted to determine the amount of radioactivity produced.
- scintillation liquid e.g., Ecoscint A
- a recombinant version of the kinase that is cloned into an expression system such as E. Coli.
- the cloned kinase can be readily obtained from the "shotgun" cloning of tissue specific cDNA libraries. Such- libraries are readily available from commercial sources. For example they may be obtained from Clontech, Palo Alto, CA. The shotgun cloning envisioned may be performed using the lambda cloning system commercially available from Stratagen, located in San Diego, California. This cloning kit contains detailed instructions for its use.
- compositions of the present invention comprise one or more of the compounds described above, as the active ingredient, in combination with a pharmaceutically acceptable carrier medium or auxiliary agent.
- ingredients may be prepared in various forms for administration, including both liquids and solids.
- the preparation may be in the form of tablets, caplets, pills or dragees, or can be filled in suitable containers, such as capsules, or, in the case of suspensions, filled into bottles.
- pharmaceutically acceptable carrier medium includes- any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
- Suitable carrier media include gelatine, lactose, starch, magnesium stearate, talc, vegetable and animal fats and oils, gum, polyalkylene glycol, or the like.
- Remington's Pharmaceutical Sciences. Fifteenth Edition, E.W. Martin (Mack Publishing Co. , Easton, PA 19/5) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the enzyme inhibitors of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical preparation, its use is contemplated to be within the scope of this invention.
- the active agent(s) may be present in an amount of at least 5% and generally not more than 98% by weight, based on the total weight of the preparation, including carrier medium and/or auxiliary agent(s) , if any.
- the proportion of active agent varies between 65% - 95% by weight of the composition.
- Preferred supplementary active agents are compounds that bind to 3DG in vivo.
- This class of compounds includes, without limitation, aminoguanidine, amino benzoic acid and derivatives thereof, cysteine and derivatives thereof, amino- substituted imidazoles, 1,2-disubstituted benzimidazoles, substituted 1, 2, 4-triazoles, diaiui ⁇ opyridine and derivatives thereof, amino- substituted pyrimidines, aminoalcohols, diamines and the like.
- Anti-hypertensive drugs including particularly the angiotensin-converting enzyme (ACE) inhibitors, may also be included as supplementary active agents in the pharmaceutical preparations of this invention.
- ACE angiotensin-converting enzyme
- auxiliary agents such as compounds that will protect the active compound from acid destruction in the stomach or facilitate the absorption of the active compound into the bloodstream can also be incorporated into the pharmaceutical preparation, if necessary or desirable.
- auxiliary agents may include, for example, complexing agents such as borate or other salts which partially offset the acid conditions in the stomach, and the like. Absorption can be increased by delivering the active compound as the salt of a fatty acid (in those cases where the active compound contains one or more basic functional groups) .
- the compounds of the invention may be administered, using any amount and any route of administration effective for inhibiting the FL3P lysine recovery pathway.
- therapeutically effective amount refers to a nontoxic but sufficient amount of the enzyme inhibitor to provide the desired therapy to counteract diabetic complications or to inhibit the metabolic production of 3DG for other medical reasons, such as reducing the effects of aging or other human disease states where AGE-Protein formation has a causative role.
- the exact amount required may vary, depending on the species, age, and general condition of the patient, the nature of the complications, the particular enzyme inhibitor and its mode of administration, and the like.
- the compounds of the invention are preferably formulated in dosage form for ease of administration and uniformity of dosage.
- Dosage unit form as used herein refers to a physically discrete unit of enzyme inhibitor appropriate for the patient to be treated. Each dosage should contain the quantity of active material calculated to produce the desired therapeutic effect either as such, or in association with the selected pharmaceutical carrier medium.
- the compounds of the invention will be administered in dosage units containing from about 1 mg to about 2,500 mg of the compound, by weight of the preparation, with a range of about 5 mg to about 250 mg being preferred.
- the compounds of the invention may be administered orally, parenterally, such as by intramuscular injection, intraperitoneal injection, intravenous infusion or the like, depending on the nature of the diabetic complication being treated.
- the compounds of the invention may be administered orally or parenterally at dosage levels of about 0.7 ⁇ g to about 20 mg and preferably from about 30 ⁇ g to about 3.5 mg/kg, of patient body weight per day, one or more times a day, to obtain the desired therapeutic effect.
- Orally active enzyme inhibitors are particularly preferred, provided the oral dose is capable of generating blood and/or target tissue levels of the inhibitor that are therapeutically active.
- Those skilled in the art can readily measure the levels of a small molecule inhibitor in deproteinized samples of blood, kidney and other target tissues. The concentration of inhibitor in these samples can be compared with the predetermined inhibitory constant. Tissue levels that are far below the inhibitory constant suggest a lack of therapeutic activity. In the case of irreversible inhibitors, this lack can be confirmed or refuted by assay of the FL3P kinase levels in the respective tissue.
- therapeutic activity can be assessed by feeding the human or animal subject a food rich in glycated lysine residues or fructose-lysine and measuring the amount of 3DG and 3DF in their urine, both before and after feeding.
- Subjects that have therapeutically active inhibitor in their systems will experience decreased secretion of both 3DG and 3DF and increased urinary secretion of fructose-lysine when compared to levels secreted by these same subjects prior to inhibitor therapy as will be described in further detail hereinbelow.
- the compounds of the invention will typically be administered once per day or up to 4-5 times per day, depending upon the exact inhibitor chosen. While a dosing schedule of once-a-day is preferred, diabetic patients are accustomed to paying close attention to their disease state, and so will readily accept more frequent dosing schedules if required, so as to deliver the above-mentioned daily dosage. However, the exact regimen for administration of the compounds and compositions described herein will necessarily be dependent on the needs of the individual patient being treated, the type of treatment administered and the judgment of the attending physician. As used herein, the term "patient" includes both humans and animals.
- the inhibitor compounds described herein are useful in counteracting diabetic complications, especially diabetic nephropathy which affects greater than forty percent of diabetics and is the primary cause of end stage renal disease requiring dialysis and transplantation.
- these inhibitors may Le used for the prevention or treatment of other pathological conditions attributable to the formation of AGE-proteins, such as hypertension, stroke, neurodegenerative disorders, e.g., senile demential of the Alzheimers type, circulatory disease, atherosclerosis, osteoarthritis, cataracts and the general debilitating effects of aging.
- 3DG is known to interact with amines generally, it may be able to interact with DNA and show mutagenic and carcinogenic potential, as well as crosslink proteins.
- the discovery of the FL3P lysine recovery pathway makes it practical, for the first time, to differentiate the diabetic population and to determine which subset of the population is likely to develop to diabetic complications. This determination can be conveniently carried out on a biological fluid of the test subject, such as urine, blood fractions (particularly plasma or serum) , lymph fluid, interstitial fluid or the like.
- this food can be in the form of a casein/sugar "cookie", such as described in Example
- glycated- lysines or synthetic fructose-lysine.
- the content of glycated-lysine should be preferably between 0.02 and 10% of total protein amino acid, or more preferably between about 0.2 and 0.4%.
- the total amount of glycated-lysine residues in the oral dose should be about 0.3 grams.
- a urine sample is collected before consumption of the glycated-lysine source, then at one, three and five hours, or such other appropriate times as may be warranted by the individual clinical situation.
- the 3DG and 3DF levels in these urine samples are measured and the ratios of these metabolites calculated.
- the particular methodology utilized in this measurement is not essential to the practice of this invention.
- the GC method described in Example 5, below, may be utilized, if desired.
- colorimetric or immunological assay methods can be used, as will be apparent to those skilled in the art.
- Therapeutic efficacy of the kinase inhibitors of the invention can be easily and safely determined using a test of the lysine recovery pathway.
- the test protocol is identical to the one presented immediately above, with the exception that urinary fructoselysine levels are measured in addition to urinary 3DG and 3DF levels. It is useful to conduct this test both before and after initiating FL3P kinase inhibitor therapy.
- the urine levels of 3DG and 3DF are summed at each time point and compared to the levels of fructose-lysine measured in the same sample.
- the peak levels of 3DG and 3DF found in urine following ingestion of food rich in glycated-lysine residues are derived from the activity of the lysine recovery pathway.
- the ratio of the concentration of these metabolites to unreacted fructose-lysine (which is a normal component of human urine) reflects the activity of this pathway. Inhibition of the lysine recovery pathway will cause a decrease in the amount of 3DG and 3DF excreted, and an increase in the excreted levels of fructose-lysine.
- therapeutic efficacy of a kinase inhibitor can be quantitated by measuring the decrease of the (3DG + 3DF) /fructose-lysine ratio following initiation of therapy. It is noteworthy that urine volume or metabolite concentrations are not a factor in interpreting this assay, as only a ratio of metabolites is considered.
- assay methods specifically contemplated include but are not limited to colorimetric and immulogical methods.
- the structure was determined by proton 2D COSY to be fructose-lysine 3-phosphate. This was later confirmed by injecting animals with FL, prepared as previously described (Finot and Mauson, Helv. Chim. Acta, __2_: 1488 (1969)), and showing direct phosphorylation to
- FL3P Using FL specifically deuterated in position-3 confirmed the position of the phosphate at carbon-3. This was performed by analyzing the 31 P NMR spectra both coupled and decoupled. he normal P-O-C-H coupling produces a doublet in FL3P with a J value of 10.3 Hz, whereas P-O-C-D has no coupling and produces a singlet both coupled and decoupled, as was found for 3-deuterated FL3P.
- a unique property of FL3P is- that when treated with sodium borohydride it is converted into two new resonances at 5.85 and 5.95 ppm, which correspond to mannitol and sorbitol-lysine 3- phosphates.
- EXAMPLE 2 SYNTHESIS OF FL3P: 1 mmol of dibenzyl- glucose 3-phosphate and 0.25 mmol* of ⁇ -carbobenzoxy- lysine was refluxed in 50 ml of MeOH for 3 hours. The solution was diluted with 100 ml water and chromatographed on a Dow-50 column (2.5 x 20 cm) in the pyridinium form and eluted first with water (200 ml) and then with 600 ml buffer (0.1M pyridine and 0.3M acetic acid) . The target compound eluted at the end of the water wash and the beginning of the buffer wash. Removal of the cbz and benzyl blocking groups with 5% Pd/C at 20 psi of hydrogen gave FL3P in 6% yield.
- EXAMPLE 3 ENZYMATIC PRODUCTION OF FL3P FROM FL AND ATP AND ASSAY FOR SCREENING INHIBITORS: Initially 31 P NMR was used to demonstrate kinase activity in the kidney cortex. A 3 g. sample of fresh pig kidney cortex was homogenized in 9 ml. of 50 mM Tris-HCl containing 150 mM KC1, 5 mM DTT, 15 mM MgCl 2 , pH 7.5. This was centrifuged at 10,000 g for 30 minutes, and then the supernate centrifuged at 100,000 g for 60 minutes.
- a radioactive assay was developed. This assay was designed to take advantage of the lack of binding to Dow-1 anion exchange resin by FL3P. This characteristic of FL3P was discovered during efforts to isolate it. Since most phosphates bind to this resin, it was suspected that the bulk of all compounds that react with ATP as well as any excess ATP would be bound, leaving FL3P in solution.
- the first step was to determine the amount of resin required to remove the ATP in the assay. This was accomplished by pipetting the mixture into a suspension of 200 mg. of Dow-1 in 0.9 ml H 2 0, vortexing and centrifuging to pack the resin. From this 0.8 ml. of supernate was pipetted onto 200 mg.
- the assay contains 10 mM ⁇ 33 P-ATP (40,000 cpm), 10 mM FL, 150 mM KC1, 15 mM MgCl 2 , 5 mM DTT in 0.1 ml of 50 mM Tris-HCl, pH 7.5.
- Urine was collected from six rats for three hours. A plasma sample was also obtained. The animals were then given 10 ⁇ mols of either sorbitollysine, mannitollysine, or galactitollysine by intraperitoneal injection. Urine was collected for another three hours, and a plasma sample obtained at the end of the three hours.
- 3-deoxyglucosone was measured in these samples, as described in Example 5, below, and variable volumes were normalized to creatinine.
- the average reduction of urinary 3-deoxyglucosone was 50% by sorbitollysine, 35% by mannitollysine and 35% by galactitollysine.
- Plasma 3-deoxyglucosone was reduced 40% by sorbitollysine, 58% by mannitolysine and 50% by galactitollysine.
- EXAMPLE 5 ELEVATION OF URINARY FL, 3DG AND 3DF IN HUMANS FOLLOWING INGESTION OF GLYCATED PROTEIN: a. Preparation of glycated protein containing food product: 260 g. of casein, 120 g. of glucose and 720 ml. of water were mixed to give a homogeneous mixture. This mixture was transferred to a metal plate and cooked at 65° for 68 hours. The resulting cake was then pulverized to a course powder. This powder contained 60% protein as determined by the Kjeldahl procedure. b. Measurement of glycated lysine content: 1 g of the powder prepared as in step a .
- 3DG was measured by GC-MS after deionization of the sample. 3DG was derivatized with a 10-fold excess of diaminonaphthalene in PBS. Ethyl acetate extraction gave a salt free fraction which was converted to the trimethyl silyl ethers with Tri-Sil (Pierce) . Analysis was performed on a Hewlett-Packard 5890 selected ion monitoring GC-MS system. GC was performed on a fused silica capillary column (DB-5,25 mx.25 mm) using the following temperature program: injector port 250°, initial column temperature 150° which is held for 1 minute, then increased to 290° at 16°/minute and held for 15 minutes. Quantitation of 3DG employed selected ion monitoring using an internal standard of U-13C-3DG.
- the graph shown in Figure 3 represents production of FL, 3DF and 3DG in the urine of one volunteer after consuming the glycated protein. The rapid appearance of all three metabolites is clearly evident. Both 3DF and 3DG show a slight elevation even after twenty-four hours.
- the graph shown in Figure 4 represents the formation of 3DF in each of the members of a seven person test group. A similar pattern was seen in all cases. As appears in Figure 4, 3DF excretion peaks about 4 hours after the FL bolus and a slight elevation of 3DF is noticeable even 24 h after the bolus.
- N-acetyl- ⁇ - glucosaminidase is an enzyme excreted into the urine in elevated concentration in diabetics. It is thought to be an early marker of tubular damage, but the pathogenesis of increased NAGase in urine is not well understood. The increased urinary output of NAGase in diabetics has been proposed to be due to activation of lysosomes in proximal tubules induced by diabetes with an increased output into the urine rather than destruction of cells.
- EXAMPLE 7 SDS GEL OF KIDNEY PROTEINS: Two rats were injected daily with 5 ⁇ mols. of either FL or mannitol (used as a control) for 5 days. The animals were sacrificed and the kidneys removed and dissected into the cortex and medulla. Tissues were homogenized in 5 volumes of 50 mM Tris-HCl containing 150 mM KCl, 15 mM MgCl 2 and 5 mM DTT, pH 7.5. Cellular debris was removed by centrifugation at 10,000 g for 15 minutes, and the supernate was then centrifuged at 150,000 g for 70 minutes.
- the soluble proteins were analyzed by SDS PAGE on 12% polyacrylamide gels as well as on 4-15 and 10-20% gradient gels. In all cases, lower molecular weight bands were missing or visually reduced from the kidney extract of the animal injected with FL when compared with the animal injected with mannitol.
- EXAMPLE 8 SYNTHESIS OF 3-O-METHYLFRUCTOSE LYSINE: A suspension of 19.4 g (0.1 mol) of anhydrous 3-O-methyl glucose and 1 g of sodium bisulfite in 30 ml of methanol and 15 ml of glycerol was refluxed for 30 minutes, followed by the addition of 0.035 mol of ⁇ -carbobenzoxy-lysine and 4 ml of acetic acid. This solution was refluxed for 3 hours. The solution was treated with 1 volume of water and chromatographed on a Dowex-50 column (4x50 cm) in the pyridinium form, and eluted first with water and then with pyridinium acetate.
- An assay buffer solution was prepared which was 100 mM HEPES pH 8.0, 10 mM ATP, 2 mM MgCl 2 , 5 mM DTT, 0.5 mM PMSF.
- a fructosyl-spermine stock solution was prepared which was 2 mM fructosyl-spermine Hcl.
- a spermine control solution was prepared which was 2 mM spermine Hcl.
- Synthesis of Fructosyl-spermine Synthesis of fructosyl-spermine was performed by an adaptation of a known procedure (J. Hodge and B. Fisher, Methods Carbohydr. Chem., 2.: 99-107 (1963)).
- a mixture of spermine (500 mg) , glucose (500 mg) and sodium pyrosulfite (80 mg) was prepared in a molar ratio of 8:4:1 (spermine:glucose:pyrosulfite) in 50 ml of methanol-water (1:1) and refluxed for 12 hours.
- the product was diluted to 200 ml with water and loaded onto a DOW-50 column (5x90 cm) .
- the unreacted glucose was removed by 2 column volumes of water and the product and unreacted spermine were removed with 0.1 M NH 4 OH.
- EXAMPLE 11 EFFECTS OF GLYCATED DIETS ON PREGNANCY
- 5 mice pairs were placed on a glycated diet (18% total protein; 3% glycated) and bred six times over a period of 7 months.
- the resulting six pregnancies produced the following live pups; 17, 23, 13, 0, 3 and 0.
- two cohorts of ten pairs each were put on either a glycated diet (13% total protein; 3% glycated) or a control diet (13% total protein; 0% glycated) .
- the first pregnancy produced 49/20
- mice (glycated/control) pups; the second, 18/41; the third 37/27; and the fourth 20/33. The fifth pregnancy is currently underway. The mice pairs have been tested for hyperglycemia. The blood glucose levels are 120 and 112 mg/dl in the experimental and control groups, respectively.
- glycated protein diet was fed a glycated protein diet: the other cohort was on a control diet.
- the glycated protein diet consisted of a standard nutritious diet to which 3% glycated protein had been added.
- the glycated protein was made by mixing together casein and glucose (2:1) adding water (2X the weight of the dried material) and baking the mixture at 60°C for 72 hours.
- the control was prepared in the same way except that no water was used and the casein and glucose were not mixed prior to baking.
- Rats were placed on the diets immediately following weaning at three weeks of age and maintained on the diets ad libitum for the next 16 weeks. The animals were then sacrificed, the kidneys fixed and hemotoxylin and eosin sections were made. These were examined for lesions by a trained pathologist. Four types of lesions were identified. These included: cysts, very small collections of tumor-like cells, typically less than 10 cells; small tumors, 0.5 mm or less, and tumors greater than 0.5 mm. For every type, more lesions were observed in the animals on the glycated diet than on the control diet as shown in the following table.
- the average number of lesions per kidney section was computed for each diet. These were 0.82 ⁇ 0.74 and 2.43 ⁇ 2.33 in the control and glycated diet, respectively. The likelihood of this happening by chance is about 2 in 100,000.
- serum levels of the glycation intermediate, three deoxy-glucosone (3DG) and its reductive detoxification product, three deoxyfructose (3DF) are elevated in diabetes.
- the relationship between baseline levels of these compounds and subsequent progression of microalbuminuria (MA) has been examined in a group of 39 individuals from a prospective cohort of patients at the Jos1in Diabetes Center with insulin-dependent diabetes mellitus (IDDM) and microalbuminuria (based on multiple measurements during the two years of baseline starting between 1990-1993) and not on ACE inhibitors.
- IDDM insulin-dependent diabetes mellitus
- microalbuminuria based on multiple measurements during the two years of baseline starting between 1990-1993
- Injections (50 ⁇ L) of the treated samples were analyzed using a Dionex DX 500 chromatography system.
- a carbopac PAl anion-exchange column was employed with an eluant consisting of 16% sodium hydroxide (200 mM) and 84% deionized water.
- 3DF was detected electrochemically using a pulsed amperometric
- Urine creatinine concentrations were determined by the end-point colorimetrie method (Sigma Diagnostic kit 555-A) modified for use with a plate reader. Creatinine concentrations were assessed to normalize urine volumes for measuring metabolite levels present therein.
- albumin levels in the urine of the test subjects were assessed by any suitable assay including but not limited to ELISA assays, radioimmunoassays, Western and dot blotting.
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AU17094/00A AU773401C (en) | 1998-10-28 | 1999-10-28 | Compounds and their therapeutic use with diabetic complications |
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ES2689906T3 (en) * | 2012-08-09 | 2018-11-16 | Dynamis Therapeutics, Inc. | Meglumine to reduce high triglyceride levels |
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US5484735A (en) * | 1989-08-23 | 1996-01-16 | Northwestern University | Immunoassay of glycosylated proteins employing antibody directed to reductively glycosylated N-terminal amino acids |
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-
1999
- 1999-10-28 CA CA002348844A patent/CA2348844A1/en not_active Abandoned
- 1999-10-28 EP EP99960167A patent/EP1126856A4/en not_active Withdrawn
- 1999-10-28 CN CN99815023A patent/CN1331599A/en active Pending
- 1999-10-28 AU AU17094/00A patent/AU773401C/en not_active Ceased
- 1999-10-28 JP JP2000578014A patent/JP2002528419A/en active Pending
- 1999-10-28 WO PCT/US1999/025358 patent/WO2000024405A1/en active IP Right Grant
-
2002
- 2002-06-06 HK HK02104296.1A patent/HK1042253A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
AU773401C (en) | 2005-04-28 |
AU1709400A (en) | 2000-05-15 |
CN1331599A (en) | 2002-01-16 |
JP2002528419A (en) | 2002-09-03 |
CA2348844A1 (en) | 2000-05-04 |
EP1126856A4 (en) | 2008-05-14 |
EP1126856A1 (en) | 2001-08-29 |
WO2000024405A1 (en) | 2000-05-04 |
HK1042253A1 (en) | 2002-08-09 |
AU773401B2 (en) | 2004-05-27 |
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