JP2002528419A - Compounds for diabetic complications and their therapeutic use - Google Patents

Abstract

  (57) [Summary] A series of compounds are disclosed that inhibit the enzymatic conversion of fructose-lysine to fructose-lysine-3-phosphate in an ATP-dependent reaction in a newly discovered metabolic pathway. Through normal action on this pathway, fructose-lysine-3-phosphate (FL3P) is degraded to produce free lysine, inorganic phosphate and 3-deoxyglucosone (3DG). The latter are reactive protein modifiers. 3DG can be detoxified by reduction to 3-deoxyfructose (3DF), or react with endogenous proteins to produce advanced glycation end product modifying protein (AGE-protein) that is thought to be responsible for diabetic complications Can be generated. It also evaluates therapies that use such inhibitors to reduce, reduce and delay diabetic complications, thereby reducing diabetic complications, as well as the risk of developing diabetic complications. Also disclosed are methods for determining the effect of the disclosed inhibitor treatment by measuring the ratio of 3DG to 3DF in a biological sample after oral administration of a fructose-lysine containing food product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] 35U. S. C. Pursuant to §202 (c), this grants the U.S. Government certain rights in the invention described herein, which was partially funded by a fund from the National Institutes of Health (registration number DK44050, DK50317 and DK50364).

TECHNICAL FIELD [0002] The present invention relates to the treatment of diabetes, and in particular, to therapeutic agents for preventing, reducing or delaying the occurrence of diabetic complications or other disorders of related etiology and their use. More specifically, the present invention relates to fructose lysine (FL) fructose-
It relates to a series of enzyme inhibitors that inhibit the enzymatic conversion to lysine-3-phosphate (FL3P) and are considered an important step in the biochemical mechanism leading to diabetic complications. The invention also relates to a method of assessing the risk of a diabetic patient of suffering from diabetic complications, as well as a method of measuring the effect of a therapeutic intervention in preventing, reducing or delaying the onset of diabetic complications.

[0003] Diabetes has four particularly severe complications: diabetic nephropathy or kidney disease; diabetic retinopathy causing blindness due to retinal destruction; diabetic nerves including loss of peripheral nerve function. Disorders; and circulatory problems due to capillary damage. Both retinopathy and renal impairment are thought to be a subset of the common circulatory problems associated with the condition. The role of microvascular dysfunction in late stage diabetes has recently been summarized (Tooke, Diabetesm, 44: 721 (1995)). Throughout this disclosure, the terms and conditions associated with "diabetes associated with diabetes" are meant to encompass the various well-known retinopathies, neuropathies, renal disorders, macrovascular disorders, and other complications of diabetes. .

[0004] A number of similarities have been reported between conditions resulting from diabetes and those resulting from aging. Studies have shown that many diabetes-related conditions can be associated with normal aging,
It has been shown to be very similar clinically. For example, it has been shown that in diabetes, arteries and joints become prematurely stiff and lung elasticity and vital capacity prematurely decrease. In addition, atherosclerosis, myocardial infarction and stroke occur more frequently in diabetic patients than in non-diabetic individuals of the same age. Diabetic patients are also more susceptible to infection and are younger than non-diabetic patients, and appear to have high blood pressure, promoted bone loss, osteoarthritis and impaired T cell function.

[0005] The similarities between the pathology associated with diabetes and aging appear to suggest a common mechanism of action. Various mechanisms have been proposed as a common biochemical basis for both the pathology and aging associated with diabetes. The hypothesis most strongly supported by data from human subjects assumes a non-enzymatic glycosylation mechanism. The hypothesis states that the pathology associated with the aging process and diabetes as described above is caused, at least in part, by protein modification and cross-linking by glucose-derived metabolites via the Maillard reaction with glucose (Monnier et al., Proc. Natl. .Acad.Sci.USA, 81: 583 (1984)
) And Lee et al., Biochem. Biophys. Res. Comm., 123: 888 (1984)).
In the present specification, a modified protein obtained by such a sugar chain formation reaction is referred to as a modified protein ((advanced glycation end product modified prot
eins) (AGE protein). It is widely accepted that 3-deoxyglucosone (3DG) is a key intermediate in the multi-step reaction pathway leading to AGE protein formation. 3DG is a glucose-induced metabolite that can react with proteins to crosslink both intracellular and extracellular proteins such as collagen and basement membrane.

[0006] In the case of diabetic complications, the response to AGE protein is thought to be kinetically accelerated by the chronic hyperglycemia associated with the disease. Evidence supporting this mechanism includes data indicating that long-lived proteins such as collagen and lens from diabetic subjects contain significantly more AGE protein content than proteins from normal controls of the same age. Thus, the abnormal incidence of cataracts in diabetic patients at a relatively young age can be explained by the high modification and crosslinking rates of the lens. Similarly, the early onset of joint and arteriosclerosis and loss of vital capacity observed in diabetic patients is explained by the high modification and cross-linking rates of collagen, an important structural protein. Since these proteins are long-lived, the results of modification tend to be cumulative.

[0007] Another factor elucidating the causal link between diabetic complications and hyperglycemia is
Hyperglycemic memory. A particularly striking example of this phenomenon is that dogs who are initially diabetic and treated to return to normal blood glucose levels develop severe retinopathy. At the time of treatment, dog eyes were histologically normal, but over time these animals developed diabetic retinopathy despite normalization of glucose levels (Engerman et al., Diabetes 36 : 808 (1987)). Thus, during the initial period of hyperglycemia, before the onset of clinical signs, radical damage to the eyes occurred irreversibly. Diabetic humans and animals have higher than normal concentrations of early and late sugar-modified AGEs.
It was revealed to have protein. In fact, the increase in AGE protein is greater than the increase in blood glucose levels. The concentration of AGE protein can be measured by fluorometry, as some percent of the sugar molecules rearrange to form protein-bound fluorescent molecules.

[0008] The developmental pathological role of the AGE protein is not limited to diabetes. Protein glycation has been linked to Alzheimer's disease (Harrington et al., Nature 370:
247 (1994)). Increased protein fluorescence is also seen with aging. In addition, several theories have revealed that the aging process is a combination of oxidative damage and sugar-induced protein modification. Thus, treatments that reduce AGE protein production may also be useful in treating other etiologically similar conditions in humans, possibly delaying the aging process. It is generally assumed that AGE protein formation begins with the reaction of protein amino groups with sugars, mainly glucose. One exemplary reference is that the first addition product formed by glycation of the ε-amino group of a lysine residue is an Amadori compound,
Fructose lysine. . . It is. Glycation is the starting step in a complex series of reactions collectively known as the Maillard or browning reactions, which ultimately leads to the formation of cross-linked, precipitated, oxidized, brown fluorescent proteins. (KJ Knecht et al., Archives of Biochem. Biophys. 294: 130 (199).
2)).

[0009] The process of forming the AGE protein from the sugar is a multi-step process that involves first reacting reversibly with the sugar to produce a fructose-lysine containing protein. These modified proteins then continue to react, producing irreversibly modified AGE proteins. AG
Since antibodies obtained by antagonizing E protein do not react with fructose-lysine, AG
It is clear that the E protein is not the same as the protein containing glycated lysine residues. It is also clear that the AGE protein exists as a number of chemical species;
However, most of them have not been identified. The species (ε-amino- (carboxymethyl) lysine has been identified in one recent study as one important final AGE protein structure (Reddy et al., Biochem. 34: 19872 (1995) and Ik
eda et al., Biochemistry 35: 8075 (1996)). In this study, about 50%
Another AGE protein epitope occupying the modification site could not be identified chemically. A method for studying the rate of production of AGE protein from ribose has recently been developed (
Khalifah et al., Biochemistry 35: 4645 (1996)). However, this study suggests that ribose may play an important physiological role in AGE protein production, and supports the relatively broad definition of glycated lysine and fructose-lysine given below.

[0010] Other references point out the difference between proteins containing glycated lysine residues and AGE proteins, "Schiff base and Amadori products reach equilibrium levels in hours and weeks, respectively. Even for very long-lived proteins, the reversibly equilibrium nature of the product is important because the total amount of early glycosylation products reaches the stationary phase in a short period of time ... in chronic diabetes. Because these early glycosylation products do not continue to accumulate on collagen and other stable tissue proteins for many years, their concentrations correlate with either the presence or severity of diabetic retinopathy. Not doing nothing is surprising ... however, some of the early glycosylation products on vascular wall collagen and other long-lived proteins do not dissociate.
They perform a slow and complex series of chemical rearrangements to produce irreversible progressive glycation end products. "(M. Brownlee et al., New England
Journal of Medcine 318: 1315 (1988)). The only route to producing these modified proteins described in the scientific literature is the initiation reaction between the protein and the sugar molecule.

[0011] Numerous references indicate that the production of AGE proteins occurs via a multistep pathway, and that 3-deoxyglucosone (3-DG) is a key intermediate in the pathway (M.
Brownlee, Diabetes 43: 836 (1994); M. Brownlee, Diabetes Care.
15: 1835 (1992); T. Niwa et al., Nephron 69: 438 (1995);
WLDills, Jr., Am. J. Clin. Nutr. 58: S779 (1993); H. Yamadat et al., J.
Biol. Chem. 269: 20275 (1994); N. Igaki et al., Clin. Chem. 36:
631 (1990)). A generally accepted pathway for the formation of 3DG from a sugar and protein reaction is illustrated in FIG. As shown in FIG. 1, a sugar (glucose) molecule initially forms a Schiff base with a protein-lysine amino group (I). Next, the resulting Schiff base is rearranged to produce fructose-lysine-modified protein (II).
The reaction up to (II) is free and reversible. (II) is dislocated to 3D
G and free protein lysine can be produced. The subsequent reaction between 3DG and the protein is the first irreversible step in AGE protein formation.

[0012] To the best of our knowledge, it has not been reported that 3DG can be produced by alternative pathways, or indeed that the main source of 3DG is derived from the enzyme-catalyzed metabolic pathway rather than the non-catalytic reaction shown in FIG. Has not been reported. Diabetic patients have significantly more 3DG in their serum than non-diabetic patients (12.78 ± 2.49 μM vs. 1.94 ± 0.17 μM) (Toshimitsu N
Iwa et al., Nephron 69: 438 (1995)). Nevertheless, this toxic compound is found in normal healthy individuals. Therefore, it is not surprising that the body has developed a detoxification pathway for the molecule. One of these reactions is a reaction catalyzed by an aldehyde reductase, which reduces the 3DG to 3-deoxyfructose (3DF), which is efficiently excreted in urine, to reduce its activity.
It detoxifies DG (Takahashi et al., Biochem 34: 1433 (199).
5)). Another detoxification reaction involves the conversion of 3DG to 3-DG by oxoaldehyde dehydrogenase.
It oxidizes to deoxy-2-ketogluconic acid (DGA) (Fujii et al., Bio
Chem. Biophys. Res. Comm. 210: 852 (1995)).

[0013] The results of studies to date have revealed that the efficacy of at least one of these enzymes, aldehyde reductase, has a negative effect on diabetes. When isolated from normal rat liver, a fraction of the enzyme is partially glycated at lysines 67, 84 and 140 and has a lower catalytic efficiency compared to the normal unmodified enzyme (Takahashi et al., Biochem. 34). : 1433 (1995)).
Diabetic patients have higher levels of glycated protein than normal glycemic individuals and therefore have higher levels of 3DG and have a reduced ability to detoxify the reactive molecule by reduction to 3DF It seems. The mechanism of aldehyde reductase has been studied. These studies determined that this important detoxifying enzyme was inhibited by an aldose reductase inhibitor (ARI) (Barski et al., Biochem. 34: 11264 (1995)). ARI is currently in clinical trials for its potential to reduce diabetic complications. A series of these compounds has shown several effects on short-term diabetic complications. However, the compounds lack a clinical effect on long-term diabetic complications and impair kidney function in rats fed a high protein diet. As will be apparent from the following, this finding is consistent with the newly discovered metabolic pathway for lysine recovery underlying the present invention. A high protein diet can be used in the kidney lysine recovery pathway (lysi
The recovery of fructose-lysine to convert to 3DG will increase due to the ne recovery pathway. Detoxification by reducing generated 3DG to 3DF is AR
Inhibited by I therapy but results in increased kidney damage compared to rats not receiving ARI. This is because inhibition of aldose reductase by ARI reduced the effectiveness of aldose reductase to reduce 3DG and 3DF.

[0014] The role of 3DG in human disease has been studied to date, as evident from the patent bulletins summarized below. U.S. Pat. No. 5,447,849 (Ulrich et al.) Describes a method of inhibiting the production of AGE proteins using amino-benzoic acids and derivatives. These compounds are
Presumably, it acts by reacting with 3DG and removing it from the system before it reacts with the protein and initiates the irreversible step of AGE protein production. U.S. Pat. Nos. 4,798,583 and 5,128,360 (Cerami et al.)
It describes the use of aminoguanidine to prevent E protein production and diabetes induced arterial wall protein cross-linking. Aminoguanidine was shown to react with early glycosylation products. This initial product is 3DG as defined herein. These patents do not predict the possibility of inhibiting the production of 3DG. They concentrate exclusively on the complex formation of the toxic molecule. U.S. Pat. No. 5,468,777 (France et al.) Describes methods and agents for preventing tooth discoloration caused by non-enzymatic browning of proteins in the oral cavity.
Cysteine and cysteine derivatives are described as being particularly useful in such applications.

US Pat. No. 5,358,960 (Ulrich et al.) Describes a method of inhibiting AGE protein production using amino-substituted imidazoles. These compounds were shown to react with the initial glycosylation product (3DG). In that patent, 3
There is no mention that metabolic sources of DG may be present. The patent contemplates that 3DG is produced exclusively as an intermediate in non-enzymatic browning of proteins. US Patent No. 5,334,617 (Ulrich et al.) Describes amino acids useful as inhibitors of AGE protein production. Lysine and other bifunctional amino acids are described as being particularly useful in this regard. These amino acids are described as reacting with the initial glycosylation products from the reaction of glucose and protein. The initial glycosylation product described in the patent appears to be 3DG. U.S. Pat. No. 5,318,982 (Ulrich et al.) Describes the inhibition of AGE protein production using 1,2,4-triazoles as inhibitors. The inhibitors described in that patent contain a diamino substituent positioned to react with 3DG to form a complex. The patent describes these compounds as reacting with an initial glycosylation product (3DG as defined herein).

US Pat. No. 5,272,165 (Ulrich et al.) Describes the use of 2-alkylidene-aminoguanidines as inhibitors of AGE protein production. The inhibitors described in the patent appear to be highly reactive with 3DG. There is no mention in this patent of inhibiting the metabolic production of 3DG. U.S. Pat. No. 5,262,152 (Ulrich et al.) Describes the use of amidrazone and derivatives to inhibit AGE protein production. The compounds described in that patent are α-acting amines. WPJencks, Third Edition, McGraw Hill New York. Compounds in this category are known to react with dicarbonyl compounds, eg, 3DG. U.S. Pat. No. 5,258,381 (Ulrich et al.) Describes the use of 2-substituted-2-imidazolines to inhibit AGE protein production. The compounds described in this patent contain adjacent amino groups that can readily react with 3DG. U.S. Pat. No. 5,243,071 (Ulrich et al.) Describes the use of 2-alkylidene-aminoguanidine to inhibit AGE protein production. The compounds described in this patent are highly reactive with 3DG and function by forming a complex with this reactive toxic molecule.

US Pat. No. 5,221,683 (Ulrich et al.) Describes the use of diaminopyridine compounds to inhibit AGE protein production. Diaminopyridine compounds described as being particularly useful will react with 3DG to form a complex containing a stable 6-membered ring. U.S. Patent No. 5,130,337 (Ulrich et al.) Describes the use of amidrazone and derivatives to inhibit AGE protein production. The inhibitors described in this patent are α-acting amines well known in the art that will react rapidly with 3DG to form stable complexes. U.S. Pat. No. 5,130,324 (Ulrich et al.) Describes the use of 2-alkylidene-aminoguanidine to inhibit AGE protein production. The compound described in this patent is an initial glycosylation product (3D) resulting from the reaction of glucose and protein.
It functions by reacting with G). U.S. Pat. No. 5,114,943 (Ulrich et al.) Describes the use of amino-substituted pyrimidines to inhibit AGE protein production. The compounds described in this patent appear to react rapidly with 3DG and detoxify 3DG.

None of the aforementioned patents suggests inhibiting metabolic production of 3DG as a means of therapeutic intervention to prevent diabetic complications. Furthermore, none of these patents even suggests involvement of an enzymatic pathway in 3DG production. U.S. Pat. No. 5,108,930 (Ulrich et al.) Describes a method for detecting the level of aminoguanidine in a biological sample. The assay is described as having potential utility in determining renal function by measuring aminoguanidine elimination time. The main utility directed to the assay method described in the patent is to measure tissue levels of aminoguanidine so that dosages sufficient to inhibit AGE protein production can be maintained in animal and human studies. It is in. There is no statement in the patent that urine 3DG, 3DF or DGA ratio is used to determine the risk of complications in diabetic patients. U.S. Pat. No. 5,231,031 (Szwergold et al.) Describes a method for assessing the risk of medical conditions associated with diabetes and determining the efficacy of treatment for these complications. This patent describes the measurement of two phosphorylated compounds in red blood cells of diabetic patients. These two compounds were not chemically identified in the patent. However, neither compound was 3DG or 3DF whose levels were measured in urine in the diagnostic embodiments of the invention.

[0019] Methods of monitoring metabolic control in diabetic patients by measuring glycosylation end products are known. It is known that the concentration of glycosylated hemoglobin reflects the average blood glucose concentration in the last few weeks. US Patent No. 43713
No. 74 (A. Cerami et al.) Is a degradation product of glycosylated protein in urine, more specifically,
Methods for monitoring glucose levels by quantification of non-enzymatically glycosylated amino acids and peptides are described. The method utilizes the affinity of alkaline boric acid for forming a specific complex with the coplanar cis-diol groups found in the glycosylation end product to separate and quantify such end product. With the goal.

[0020] US Patent No. 4,761,368 (A. Cerami) describes the isolation and purification of chromophores present in browned polypeptides, such as bovine serum albumin and poly-L-lysine. Chromophore, 2- (2-furoil) -4 (5) -2 (furoil)
-1H-imidazole (FFI) is a complex heterocycle derived from the condensation of two molecules of glucose and two lysine-derived amino groups. The patent further provides a method for measuring "aging" (the degree of progressive glycosylation) in a protein sample,
Describes the use of FFI in a method in which sample "aging" is determined by measuring the amount of the chromophore in a sample and then comparing the measurement to a standard. With the amount of FFI correlated to "aging"). Existing treatment plans for diabetics include, in order to prevent, reduce or delay the onset of the condition associated with diabetes, and to determine the benefits of such treatment,
There is a long-standing unmet need for effective methods of identifying patients at risk for developing such conditions.

DISCLOSURE OF THE INVENTION The present invention involves an enzyme-mediated conversion of FL to FL3P and describes a metabolic pathway that produces relatively high concentrations of 3-deoxyglucosone (3DG) in diabetes-affected organs. It is obtained from the finding. Subsequent studies of the biological function of this newly discovered pathway are pointing in the way that the pathway plays an important role in the pathogenesis of diabetic kidney disease. It is also presumed that the pathway is involved in the pathogenesis of various known diabetes-associated pathologies. Based on this finding, in one embodiment, the present invention provides a series of compounds having enzyme inhibitory activity and effective in inhibiting the enzymatic conversion of fructose-lysine to fructose-lysine-3-phosphate. Practical applications have been found. The relevant enzyme inhibitory activity of the compounds of the invention can be readily determined in assays. The assay method comprises obtaining an aqueous solution of a compound of the present invention in an amount sufficient to demonstrate the source and inhibitory activity of fructose-lysine, adenosine triphosphate (ATP), fructose-lysine-3-phosphate kinase; The resulting solution is used as the product of the interaction between the kinase, fructose-lysine and adenosine triphosphate, as described above.
Subject to conditions that promote the formation of lysine-3-phosphate and adenosine diphosphate;
Measuring the production of at least one product of such products.
The same relative amounts of fructose-lysine, adenosine triphosphate and fructose-
The compounds of the present invention reduce the amount of such products as compared to an aqueous solution containing a source of lysine-3-phosphate kinase and no addition of the compounds of the present invention. The assay methods described herein are also within the scope of the present invention.

According to another aspect, the present invention relates to a formulation for preventing, reducing or delaying the onset of diabetic complications in diabetic patients, comprising as active agents a compound of the invention as defined above and A formulation comprising a pharmaceutically acceptable vehicle is provided. According to a further aspect of the present invention, there is provided a method of preventing, reducing or delaying the onset of diabetic complications in a patient at risk of developing diabetic complications, the method comprising providing the patient with fructose-lysine of fructose-lysine. There is provided a method comprising administering an amount of a compound of the invention effective to inhibit enzymatic conversion to 3-phosphate. The method may be used for the prevention or treatment of other etiologically similar conditions, as described further below.

According to yet another aspect, the present invention provides a method of assessing a diabetic patient's risk of having a complication associated with diabetes. The method comprises administering to a patient a source of glycated lysine residues in an amount that provides a predetermined amount of glycated lysine residues, and providing 3-deoxyglucosone in a biological sample obtained from the patient. 3-
The ratio to deoxyfructose consists of measuring the ratio of 3-deoxyglucosone to 3-deoxyfructose in normal subjects, ie non-diabetic subjects or subjects without clinical symptoms of diabetes. A higher ratio of 3-deoxyglucosone to 3-deoxyfructose in a sample of diabetic patients compared to the proportion of asymptomatic subjects indicates that the diabetic patient is more at risk of complications of diabetes-associated conditions. Is shown.

The present invention also provides a method for evaluating the effect of a therapeutic intervention in preventing diabetic complications. The method involves measuring the concentration of 3-deoxyglucosone, 3-deoxyfructose and fructose-lysine in a biological sample obtained from a diabetic patient both before and after initiation of the therapeutic intervention. The sum of the 3-deoxyglucosone and 3-deoxyfructose concentrations is then compared to the fructose-lysine concentration in the sample. Obtained after the start of the intervention, compared to the sum of the 3-deoxyglucosone and 3-deoxyfructose concentrations relative to the fructose-lysine concentration measured in the biological sample obtained prior to the start of the intervention. A decrease in the sum of the 3-deoxyglucosone and 3-deoxyfructose concentrations relative to the fructose-lysine concentration in the biological sample indicates an effect of the therapeutic intervention.

In yet another aspect of the present invention, there is provided a method of notifying a diabetic of a food product that may be involved in the development of a condition associated with diabetes. The method includes measuring the content of glycated lysine residues in the food and providing the information to the diabetic, for example, on a package of the food or in a publication for use by the diabetic.

According to the present invention, it has been found that the presence of high levels of 3DF in a biological sample, eg, urine, is associated with a significant risk of developing diabetic complications. Was issued. Thus, a further embodiment of the present invention provides for one or more 3D-like indicators as indicators of the likelihood that a diabetic will or will not develop diabetic complications.
With reference to the predetermined baseline level of F, 3
A method is provided for assessing the risk of a diabetic patient of developing a condition associated with diabetes based on measuring DF. Yet another aspect of the invention includes a method of reducing suspicion of carcinoma in a patient associated with glycated protein intake. The method comprises administering a pharmaceutical composition containing an active compound that has inhibitory activity with respect to the enzymatic conversion of fructose-lysine to fructose-lysine-3-phosphate. The present invention also embodies a method of preventing, reducing or delaying the onset of carcinoma caused by the formation of AGE-protein. The method comprises administering a therapeutic amount of an agent that inhibits the production of 3-deoxyglucosone.

Further, as a means for evaluating the molecular mechanism of transformation of a malignant tumor associated with ingestion of a diet containing a glycated protein, an animal can be fed a glycated protein diet with a 3-day diet.
A method is provided for inducing carcinoma in a susceptible test animal comprising providing deoxyglucosone for a sufficient period of time to be present in a biological fluid at a concentration at least three-fold. Such animals are evaluated against untreated control animals. A method of screening for a substance that affects the onset of carcinoma is also provided in the present invention. Feeding the glycated protein diet such that the 3DF concentration in the biological fluid is at least three times higher induces carcinoma in the test animals.
The animals are then divided into two groups, one of which is administered the compound to be evaluated,
The other group serves as a negative control. After an appropriate period of time, both groups of animals are sacrificed and the presence and / or absence of carcinoma in both groups is assessed.

Finally, in still another embodiment of the present invention, there is provided a method of screening for a substance that prevents, reduces or delays the occurrence of carcinoma. The method comprises the steps of providing a glycated protein to a susceptible test animal in the amounts and times indicated below:
This amount and time refers to the amount of 3-deoxyglucosone in biological fluid from a similarly susceptible test animal fed a diet substantially free of glycated protein.
3DG) amount and time sufficient to maintain the content. The test substance is administered to one part of the test animal but not to another animal. The animals are then sacrificed and the explants are compared to each of the susceptible test animals to evaluate the effect of the test substance.

DETAILED DESCRIPTION OF THE INVENTION The following definitions are provided to facilitate understanding of the invention, as described in more detail below:

[0030] 1. Glycated Lysine Residues As used herein, the expression "glycated lysine residue" refers to a modified lysine residue of a stable addition product formed by the reaction of a reducing sugar with a lysine-containing protein. Say. Most of the protein lysine residues are on the surface of the protein as expected for positively charged amino acids. Thus, lysine residues on proteins that come into contact with serum or other biological fluids are free to react with sugar molecules in solution. The reaction takes place in a number of stages. The initiation step involves the formation of a Schiff base between the lysine free amino group and the sugar keto group.
The starting product then undergoes Amadori rearrangement to produce a stable ketoamine compound. This series of reactions can take place with various sugars. If the sugar involved is glucose,
The first Schiff base product consists of an aldehyde group on glucose C-1 and a lysine ε-
An imine is formed between the amino groups. The Amadori rearrangement is referred to herein as fructose-lysine or FL, a lysine attached to the C-1 carbon of fructose, 1-
This leads to the formation of deoxy-1- (ε-aminolysine) -fructose.

A similar reaction will occur with other aldose sugars, such as galactose and ribose (Dills, Am. J. Clin. Nutr. 58: S779 (1993)). For the purposes of the present invention, the initial product of the reaction of any reducing sugar with the ε-amino residue of protein lysine is a significant glycated lysine residue, regardless of the exact structure of the modified sugar molecule. Is included. Also, the terms glycated lysine residue, glycated protein and glycosylated protein or lysine residue are used interchangeably herein, and such expressions are often used interchangeably in scientific journals. Consistent with recent practices.

[0032] 2. Fructose-Lysine The term “fructose-lysine” (FL) is used herein to mean any glycated lysine that is incorporated into a protein / peptide or released from the protein / peptide by proteolytic digestion. used. The term is not particularly limited to the chemical structure commonly referred to as fructose-lysine, which was reported to be formed from the reaction of protein lysine residues with glucose. As mentioned above, lysine amino groups can react with a wide variety of sugars. Furthermore, one report indicates that glucose is the least reactive sugar among the group of 16 different sugars tested (Bunn et al.,
Science 213: 222 (1981)). Thus, tagatose-lysine, formed from galactose and lysine as well as glucose, as well as condensates of all other sugars, natural or unnatural, include the term fructose-lysine in the description herein. Is always included. From the description herein, it will be appreciated that the reaction between a protein-lysine residue and a sugar involves a number of reaction steps. The final steps in this series of reactions involve the cross-linking of proteins and the generation of multimeric species known as AGE proteins, some of which are fluorescent. Proteolytic digestion of such modified proteins does not yield lysine covalently linked to the sugar molecule. Thus, these species are not encompassed by the meaning of "fructose-lysine" as used herein.

[0033] 3. Fructose-lysine-3-phosphate The compound is formed by the enzymatic transfer of a high energy phosphate group from ATP to FL. As used herein, fructose-lysine-3-phosphate (
The term FL3P) is meant to include all phosphorylated fructose-lysine moieties which can be free or bound to proteins and formed enzymatically.

[0034] 4. Fructose-lysine-3-phosphate kinase This term refers to one or more proteins capable of enzymatically converting FL to FL3P, as described above, when additionally providing a source of high energy phosphate.

5.3 3-Deoxyglucosone 3-Deoxyglucosone (3DG) is a 1,2-dicarbonyl-3-deoxysugar (also known as 3-deoxyhexulosone), which is free Produced in the degradation of FL3P to produce lysine and inorganic phosphate. In this specification, 3
The term deoxyglucosone is a FL3P having the broad definition of FL3P described above.
Is meant to include all possible dicarbonyl sugars produced in the decomposition of

[0036] 6. FL3P Lysine Recovery Pathway The lysine recovery pathway regenerates unmodified lysine present in human kidney or other tissues and either as free amino acids or incorporated into polypeptide chains. As further described below, the pathway is an important factor involved in diabetic complications.

[0037] 7. AGE Protein The term "AGE protein" (advanced glycation end product modified protein) is used in scientific journals to indicate the end product of the reaction between sugar and protein, and is used herein ( Brownlee, Diabetes Care 15: 1835 (1992) and Niwa et al., Nephron 69: 438 (1995)). For example, it is clear that the reaction between protein lysine residues and glucose does not end with the formation of fructose-lysine. FL can produce non-enzymatic 3DG via multiple dehydration and rearrangement reactions, which further reacts with free amino groups, leading to cross-linking and browning of related proteins. Indeed, as noted above, there is evidence that 3DG is a central intermediate in this modification reaction.

9. Glycated Diet As used herein, this expression refers to any diet in which a percentage of normal protein is replaced by glycated protein. "Glycated diet"
And the expressions "glycated protein diet" are used interchangeably herein. At least some or all of the diabetic complications result from covalent modification of the protein by glucose and other reactive sugars (M. Brownlee, Diabetes 4
3: 836 (1994)). As described above, it has been found that diabetic humans and animals have higher than normal concentrations of glycoproteins. In fact, the increase in AGE protein associated with diabetes is greater than the increase in blood glucose levels. Previously, it was generally recognized that the origin of 3DG in vivo came from the degradation of proteins containing glycated lysine residues. It was also generally believed that these glycated lysines could not be used as a source of amino acids. As evident from the following, this idea was wrong.

[0039] 10. Susceptibility Test Animal As used herein, this expression refers to a line of laboratory animals that are susceptible to malignant tumor transformation and tumor formation due to certain genetic variations. Eker rats with a mutated tuberous sclerosis gene (Tsc-2) were utilized for the studies described herein. Those skilled in the art will note that various other laboratory rat or mouse strains have increased properties of tumor formation. The term "similar susceptibility test animal" refers to an animal having a comparable genetic background used as a control, i.e., untreated animal.

As described above, the present invention has been developed based on the discovery of a previously unknown metabolic pathway for producing 3DG in an enzyme-catalyzed reaction. This enzymatic pathway allows for enzymatic inhibition, thereby reducing the production of toxic 3DG.

During the course of a series of studies in diabetic kidney, streptozotocin (streptozotocin)
An extract of kidney perchloric acid from otoxin-induced diabetic rats was subjected to ³¹ P NMR spectroscopy, which revealed an unusual new peak in the NMR spectrum. Previous studies by the present inventors have demonstrated the presence of fructose-3-phosphate in the rat lens and human erythrocytes (A. Petersen et al., Biochem. J. 2).
84: 363-366 (1992); Lal et al., Arch. Biochem. Biophys. 318: 1.
91 (1995); Szwergold et al., Science 247: 451 (1990) and L.
al et al., Investigative Opthalmology and Visual Science 36 (5): 969 (
1995)). Earlier studies identified other abnormal phosphorylated sugars in the rat lens (Szwergold et al., Diabetes 44: 810 (1995) and Kapple
r et al., Metabolism 44: 1527 (1995)). Therefore, it was reasonable to assume that this newly identified peak was another phosphorylated saccharide. Further laboratory studies have shown that this novel compound is not a simple sugar, but rather a fructose-lysine phosphorylated at position 3 of the fructose component.

This identification has been confirmed in two ways. Standard fructose-lysine-3-phosphate (FL3P) was synthesized according to the procedure disclosed in Example 2 below, and ³¹ P NMR
It was revealed that the spectrum resonated with the peak in diabetic rat kidney. In addition, synthetic fructose-lysine was injected into non-diabetic rats. These rats showed a substantial increase in FL3P levels in the kidney after injection.

To demonstrate that FL3P is derived directly from FL in an enzyme-catalyzed reaction, two experiments were performed. Fructose-lysine in which the C3 position of the fructose moiety was labeled with deuterium was synthesized and injected into rats. Three hours after injection, the kidneys of these rats were removed and extracted with perchloric acid. NMR spectroscopy showed that the FL3P material isolated from these rats contained a deuterium label at the C3 position of the fructose moiety. In addition, it demonstrates that rat kidney homogenate can produce FL3P in reactions requiring both ATP and fructose-lysine. This last described experiment confirms the presence of a specific FL3P kinase since FL3P is not formed when only fructose lysine and ATP are incubated together under physiological conditions. Further experiments involving fractions of the renal cortex indicate that the kinase activity is not evenly distributed in the kidney, but is one of the earliest anatomical sites showing damage in the diabetic kidney of humans and animals.
It was found to be concentrated in the proximal tubular region.

FL3P is unstable in aqueous solutions. It decomposes quickly and 3D
Produces G, lysine and inorganic phosphate. The reaction also occurs in vivo. Whether the in vivo degradation of FL3P is a spontaneous or enzyme-catalyzed reaction is currently unknown. However, 3DG from fructose-lysine
It is highly speculated that enzymatic catalysis is involved, since the production of PEG occurs very rapidly in intact kidneys.

FIG. 2 shows reaction steps in the FL3P lysine recovery pathway. In the first step,
Reacting fructose-lysine and ATP in a FL3P kinase-catalyzed reaction,
Produces fructose-lysine-3-phosphate (FL3P) and ADP. Phosphorylation occurs at the 3-position of the fructose moiety, and the fructose lysine molecule becomes unstable. Next, the obtained FL3P is decomposed to give 3-deoxyglucosone (3DG
), To produce unmodified free recyclable lysine which can be used in inorganic phosphate and protein synthesis. Aldehyde reductase is excreted in urine
3DG is detoxified by reduction to deoxyfructose (3DF).

Although FIG. 2 illustrates the pathway using the most commonly performed glycated lysine, fructose-lysine, it is readily apparent to one of skill that a wide variety of similar molecules can flow through the pathway. It will be obvious. Rather, as described in more detail below, the substrate selectivity of the FL3P lysine recovery pathway is very broad, ensuring a broad definition of the term.

[0047] Additional experiments have shown that the lysine recovery pathway can be found in sheep, pigs, dogs, rabbits, cattle,
It has been shown to be found in a wide variety of animal species, including mice and chickens. This pathway also exists in humans. The ubiquity of the FL3P lysine recovery pathway can be understood by the fact that lysine is an essential amino acid present in relatively low concentrations in most foods. In addition, a significant proportion of lysine residues in food is present in glycated form, and the proportion of modified lysine will increase when cooking food. Since these glycated lysine residues are not available for protein synthesis, the lysine recovery route is of great utility and offers selective advantages to organisms having it.

[0048] Diabetes has two effects on the lysine recovery pathway. Blood proteins contain higher concentrations of glycated lysine when isolated from diabetic patients than non-diabetic individuals. Thus, diabetic patients will have more flow through the lysine recovery pathway than non-diabetic patients. In addition, 3D in urine of diabetics and normal bodies
Preliminary observations on the ratio of G and 3DF appear that diabetics have a reduced ability to detoxify 3DG produced via the pathway. These two factors combine to produce higher urinary concentrations of 3DG in diabetic patients (see FIG. 7; also, Lal et al., Arch. Biochem. And Biophys. 342 (1): 254-60 (1).
997)).

[0049] Substances involved in the lysine recovery pathway have been identified in kidney as well as in other tissues, particularly erythrocytes, lens and peripheral nerve tissue. All of these tissues are affected by the complications of diabetes. Location in erythrocytes correlates with microvascular complications of diabetes, e.g., diabetic retinopathy, and location in the kidney correlates with diabetic nephropathy, while location in the peripheral nerves affects diabetic peripheral neuropathy. Correlate. These substances are also found in the pancreas. Experiments have been performed to determine the presence of these substances in the skin. If present, improve their harmful effects on the mind and body by typical treatment with inhibitors of the invention in a suitable vehicle to prevent collagen crosslinking, thereby improving skin elasticity It is considered possible.

Experiments were performed to demonstrate that humans produce both 3DG and 3DF from orally ingested proteins containing glycated lysine residues. These experiments, described in detail below, convincingly explain that the lysine recovery pathway exists in humans. These experiments also highlight the incomprehensible phenomenon: some diabetics develop diabetic complications, while others with poor glycemic control do not develop such complications. Clarified. The reason for this phenomenon is clear from the data provided herein. Diabetic patients have different abilities to detoxify 3DG. A subset of the diabetic population appears to have relatively higher aldehyde reductase activity than many diabetic patients. Therefore, these individuals can handle the increased flow through the lysine recovery pathway by efficiently detoxifying higher than normal levels of 3DG. Those with reduced abilities can hardly detoxify their increased 3DG levels and are therefore more at risk from developing diabetic complications.

As will be described in more detail below, it has been demonstrated that stimulation of the lysine recovery pathway can be effected by use of a glycated protein diet. When using the above FL, an increase in FL3P, 3DG and 3DF was observed in test animals fed a glycated protein diet. The enzyme inhibitor compounds of the present invention block the lysine recovery pathway and prevent the production of toxic 3DG from FL3P. The following describes a broad set of features that enzyme inhibitors suitable for use in the practice of the present invention should exhibit, as well as certain tests to determine whether any putative inhibitor combines these features. I do. Candidate kinase inhibitors for use in accordance with the present invention may be natural products isolated from plants or microorganisms. Alternatively, they may be synthetic molecules derived from enzymatic reactions and rational knowledge of the mechanism. Inhibitors may also be synthesized by combinatorial methods. Combinatorial libraries may be generated from random starting points. In addition, combinatorial methods can be used 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 combine all of the features listed below inhibit the lysine recovery pathway, thereby preventing the development of diabetic complications or disorders of the associated etiology, It is not considered a useful therapeutic agent that can be reduced or delayed. 1. Inhibitors are small molecules that should be easily absorbed by cells.
To combine these features, the inhibitor is less than 2,000, more typically about 1,
It must have a molecular weight of 000 daltons or less. 2. Inhibitors must exhibit antagonistic, non-antagonistic, irreversible or suicide inhibition of FL3P kinase. If the inhibitor is an antagonistic or non-antagonistic inhibitor, the inhibition constant Ki must be less than about 1 mM. Typically, it is
M, more typically less than about 40 μM. If the inhibitor exhibits suicide or other irreversible inhibition, the requirement for an inhibition constant is controversial.

[0053] 3. Inhibitors must be soluble in aqueous solution and stable in aqueous solution at physiological pH. The requirement for solubility is met only if the inhibitor or inhibitor salt can be dissolved in saline or serum at a concentration of 10 μM or more. The stability requirements are:
After incubation of the inhibitor solution in saline at 37 ° C. for 1 hour, 5
It is only satisfied if it retains 0% or more activity. Typically, inhibitors must retain greater than 50% activity in one or more days of incubation.

[0054] 4. Inhibitors must exhibit acceptable pharmacokinetics. That is, it must remain at a therapeutically effective concentration for at least one hour after administration of the substance. Ideally, it should maintain an effective concentration for at least 8 hours.
More ideally, once daily dosing is all that is required to maintain therapeutic concentrations of the inhibitor. This requirement does not imply that after the initial administration, the inhibitor must be able to establish a therapeutic concentration. Many examples of successful pharmaceuticals exist where those medical effects are only seen with prolonged administration. A feature means that as soon as an effective concentration is reached, the concentration should be able to be maintained for more than 1 hour after the last administration of the substance. Tests for therapeutic efficacy are described herein.

[0055] 5. Inhibitors must be non-toxic. This feature requires that the inhibitor be non-toxic to humans when administered at therapeutic doses. Typically, if the inhibitor is present at twice the blood and / or target tissue levels required for therapeutic effect, toxicity should not be overt. More typically, there should be no apparent toxicity at levels greater than six times the therapeutic range. Diabetic complications can only be prevented by long-term inhibitor treatment. Thus, the requirement for non-toxicity must include both acute and chronic toxicities that may become evident with prolonged long-term use. The toxicity of the candidate molecule can be easily assessed using well-established animal studies. Human toxicity is evaluated in a stage 1 clinical trial.

Among the compounds useful in the practice of the present invention are compounds of the formula:

Embedded imageWherein X is -NR'- or -O-, wherein R 'is H and a linear or
Branched alkyl group (C₁-C₄) And unsubstituted or substituted
Reel base (C₆-C₁₀) Or an aralkyl group (C₇-C₁₀Group)
R is H, amino acid residue, polyamino acid residue, peptide chain, substituted
Not substituted or substituted with at least one nitrogen- or oxygen-containing substituent
Linear or branched aliphatic group (C₁-C₈), Unsubstituted or low
At least one nitrogen- or oxygen-containing substituent and at least one
Straight or branched chain interrupted by -O-, -NH- or -NR "-groups
Aliphatic group (C₁-C₈Is a substituent selected from the group consisting of:
Linear or branched alkyl (C₁-C₆) And unsubstituted or substituted
Aryl group (C₆-C₁₀) Or an aralkyl group (C₇-C₁₀)
With the proviso that when X is -NR'-, R and R 'are the nitrogen to which they are attached
Together with the atom, at least one nitrogen and oxygen is the only
Substituted or unsubstituted heteroaryl having 5 to 7 ring atoms
And the aryl group (C₆-C₁₀) Or an aralkyl group (C ₇ -C₁₀) And the heterocyclic ring substituent is H, alkyl (C₁-C₆), Haloge
, CF₃, CN, NO₂And -O-alkyl (C₁-C₆From the group consisting of
Selected; R₁Is a linear polyol group having 1 to 4 carbon atoms; Y is carbonyl
Le group

Embedded image Or hydroxymethylene group

Embedded image Z is -H, -O-alkyl (C ₁ -C ₆ ), -halogen-C
F _3, is -CN, isomers and pharmaceutically acceptable salts of the compounds and the compound represented by] is selected from the group consisting of -COOH and _-SO 3 _{H 2.}

Representative examples of nitrogen or oxygen containing “R” substituents are γ-amino-α-hydroxybutyric acid (— (CH ₂ ) ₂ —CHOH—COOH), 1,2,4 triaminobutane (−
_{(CH 2) 2 -CHNH 2 -CH} 2 NH 3), 3,6- diamino-5-hydroxy-heptanoic acid _{(-CH 2 -CH (OH) -CH} 2 -CH (NH 2) -CH 2 -C
OOH) and the like. The structures of Formula I have asymmetric centers and occur as racemates, racemic mixtures and various stereoisomers and mixtures thereof, and all such isomeric forms are within the scope of the invention. Certain compounds having the structure of Formula I above were previously known, while others are believed to be novel and provide all compounds of Formula I for inhibiting the enzymatic catalyzed production of 3DG in vivo. Per se, as well as the use of compounds of the present invention are within the scope of the present invention.

The inhibitors of the above formula are prepared by reacting a suitable sugar, for example, glucose, galactose, mannose, ribose, xylose, or a derivative thereof, with an amino acid or other suitable primary or secondary amine. Thus, an inhibitor having a carbonyl moiety (ie, Y = -C (= O)-) may be obtained. Alternatively, sugar,
For example, glucose, galactose, mannose, ribose, type described such as the herein xylose amino - or hydroxyl - substituted reactant with NaBH ₃ C
N in the presence of a reagent that selectively reduces a Schiff base intermediate to an amine, such that an inhibitor having an alcohol moiety (ie, Y = -CH (-
OH)-). When an amino acid reactant is used, the reactive portion of the amino acid reactant may be an amine group on the α-carbon or an amine or hydroxyl group on the acid side chain. Suitable amino acids include essential amino acids. Particular examples include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, aspartic acid, phenylalanine, tyrosine, histidine and tryptophan. Other suitable reactants include a wider variety of aminocarboxylic acids, such as pyroglutamic acid, beta-
It is derived from alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid and the like. N-acyl derivatives of the above amino acids, such as formyl lysine, may also be used, if desired.

Other suitable reactants include, but are not limited to, unsubstituted or substituted aryl (C ₁ -C ₃ ), which may be an alkyl (C ₁ -C ₃ ), alkoxy, carboxy, nitro or halogen group. ₆ -C ₁₀₎ compound, an unsubstituted or substituted group at least one alkoxy group, which may be substituted alkane; or unsubstituted or substituted groups alkyl _(C 1 _-C 3), aryl _(C 6 _{-C 10} ), Substituted nitrogen-containing heterocyclic compounds which may be alkoxy, carboxy, nitro or halogen groups. Examples of the last-mentioned group of reactants are m-methyl-, p-methyl-, m-
Methoxy-, o-methoxy- and m-nitro-aminobenzene, o- and p
-Aminobenzoic acid; n-propylamine, n-butylamine, 3-methoxypropylamine; morpholine and piperidine.

[0060] Representative inhibitor compounds having the above formula are shown in the accompanying Table A. Known compounds that may be used as inhibitors in the practice of the present invention include, but are not limited to,
For example, 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, for example, alkali metal salts, alkaline earth metal salts, ammonium, substituted ammonium and other amine salts. Suitable acids include, for example, hydrochloric acid, hydrobromic acid and methanesulfonic acid. Pharmaceutically acceptable salts of the compounds of formula I can be prepared according to techniques well known to those skilled in the art.

The ability of a compound to inhibit FL3P kinase can be measured using a wide variety of kinase activity assays. One useful assay uses fructose-a possible inhibitor.
Incubating with lysine and ATP in the presence of kidney homogenate or other enzyme source. Typically, no more than 1 mmol of the inhibitor compound of the invention, fructose lysine (FL) in an amount ranging from 1-10 mmol, 0.1-1
Prepare a solution of the assay components containing an amount of enzyme source sufficient to convert ATP and FL to fructose lysine-3-phosphate in amounts ranging from 0 mmol. Incubations should be performed at a pH in the range of 4.5 to 9.5, typically at or near neutral. Incubations should be performed at a temperature compatible with the enzyme activity, 4 to 40 ° C. Typically, the incubation is performed at a physiological temperature. After incubation, the reaction is terminated by acidic precipitation of the protein and the production of FL3P is measured by ³¹ P-NMR spectroscopy. FL3P production will be reduced or eliminated in the sample containing the inhibitor compound as compared to a control sample containing no inhibitor.

Other assays have utility for rapid measurement of enzyme inhibition. One such assay involves the use of fructose-lysine and gamma-labeled ³² P or ³³ P-ATP. Although FL3P does not bind to Dow-1, ATP and most other phosphates do, so after a predetermined reaction time, typically 10 minutes, Dow-1.
The product, FL3P, can be separated from the remaining reaction mixture by passing the assay solution through a column of resin. The obtained solution is added to a scintillation solution, for example, a container of Ecoscint A, and the amount is counted to determine the amount of the generated radioactive substance.

Because of the difficulty in obtaining large amounts of human tissue, it is desirable to use a recombinant form of the cloned kinase in an expression system such as E. Coli. Cloned kinases can be readily obtained from "shotgun" cloning of a tissue-specific cDNA library. Such libraries are readily available from the market. For example, they can be purchased from Clontech, Palo Alto, California.
tech). This shotgun cloning may be performed using a lambda cloning system commercially available from Stratagen, San Diego, California. This cloning kit has detailed instructions for its use. The pharmaceutical preparations of the present invention contain one or more of the above compounds as active ingredients, in combination with a pharmaceutically acceptable carrier medium or adjuvant.

The components may be prepared in various forms for administration, including both liquid and solid. Thus, the formulations may be in the form of tablets, caplets, pills or dragees, or may be filled in suitable containers such as capsules, or in the case of suspensions, bottles. As used herein, the term "pharmaceutically acceptable carrier medium" refers to any and all solvents, diluents, or other liquid vehicles, dispersions, as appropriate for the particular desired dosage form. Or suspension aids,
Includes surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Representative examples of suitable carrier media include gelatin, lactose, starch, magnesium stearate, talc, vegetable and animal fats and oils, gums, polyalkylene glycols and the like. In Remington's Pharmaceutical Science, 15th Edition (Mack Publishing Co., Easton, PA 1975), EW Martin discloses various carriers used in the formulation of pharmaceutical compositions and known techniques for their preparation. Any conventional carrier medium may interact with the enzyme inhibitors of the invention, e.g., to produce any undesirable biological effect, or otherwise, to any other component of the formulation in a manner that is deleterious to mind and body. Except where otherwise incompatible, their use is considered to be within the scope of the present invention.

In the pharmaceutical preparations according to the invention, the active agent is present in at least 5% by weight, generally not more than 98% by weight, based on the total weight of the preparation, if any, including carrier medium and / or adjuvants. It may be present in an amount. The proportion of active agent may range from 65% to 9% by weight of the composition.
Preferably, it varies between 5% by weight. Preferred supplemental active agents are compounds that bind to 3DG in vivo. Such compounds include, but are not limited to, aminoguanidine, aminobenzoic acid and its derivatives, cysteine and its derivatives, amino-substituted imidazole, 1,2-disubstituted benzimidazole, substituted 1,2,4-triazole, Includes diaminopyridine and its derivatives, amino-substituted pyrimidines, amino alcohols, diamines and the like. Antihypertensives, including especially angiotensin-converting enzyme (ACE) inhibitors, can also be included in the formulations of the present invention as supplemental active agents.

Auxiliaries, such as compounds that protect the active compound from acidolysis in the stomach or promote absorption of the active compound into the bloodstream, can also be incorporated, if necessary or desired, into the pharmaceutical formulation. Such adjuvants can include, for example, complexing agents such as borates or other salts that partially offset acidic conditions in the stomach. Delivery of the active compound as a salt of a fatty acid (when the active compound has one or more basic functional groups) can increase absorption. A compound of the present invention may be administered with any supplemental active ingredient using any amount and any route of administration effective to inhibit the FL3P lysine recovery pathway. Thus, the expression "therapeutically effective amount" as used herein is a non-toxic, but non-toxic, desired to prevent diabetic complications or to inhibit the metabolic production of other pathogenic 3DG. Refers to an enzyme inhibitor in an amount sufficient to provide a treatment for, for example, reducing the effects of aging or other human conditions caused by AGE protein formation. The exact amount required may vary depending on the species, age and general conditions of the patient, the type of complication, the particular enzyme inhibitor and its mode of administration.

The compounds of the present invention are preferably formulated in dosage forms for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to a physically discrete unit of an enzyme inhibitor appropriate to the patient to be treated. Each dosage should contain the active substance, calculated by itself to produce the desired therapeutic effect, either on its own or together with a selected pharmaceutical carrier medium. Typically, the compounds of the present invention will contain from about 1 mg to about 2500 mg of compound, preferably from about 5 mg to about 2500 mg, by weight of the formulation.
It will be administered in dosage units containing a range of 50 mg. The compounds of the present invention may be administered orally, parenterally, for example, by intramuscular injection, intraperitoneal injection, intravenous infusion, etc., depending on the type of diabetic complication to be treated. The compounds of the present invention may be administered at a dosage level of from about 0.7 μg to about 20 mg, preferably from about 30 μg to about 3.5 mg / kg / day of the patient per day, to achieve the desired therapeutic effect. It may be administered one or more times, orally or parenterally.

[0068] Orally active enzyme inhibitors are particularly preferred, and the oral dosage provided can produce blood and / or target tissue levels of the therapeutically active inhibitor.
One of skill in the art can readily determine the levels of small molecule inhibitors in deproteinized samples of blood, kidney and other target tissues. The inhibitor concentration in these samples can be compared to a predetermined inhibition constant. Tissue levels much lower than the inhibition constant indicate a lack of therapeutic activity. In the case of irreversible inhibitors, this deficiency can be confirmed or challenged by assaying FL3P kinase levels in each tissue. In all cases, by feeding a 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 food intake, Therapeutic activity can be assessed. Subjects with a therapeutically active inhibitor in those systems have reduced secretion of both 3DG and 3DF as compared to secretion levels by these same subjects before inhibitor treatment, as described in more detail below. , The secretion of fructose-lysine into the urine will increase.

The compounds of the invention will typically be administered once daily or up to 4-5 times daily depending on the exact inhibitor chosen. A once-daily dosing regimen is preferred, however, as diabetic patients are accustomed to paying close attention to the condition,
More frequent dosing regimens will be readily accommodated, if necessary, to deliver a daily dose. However, the precise dosage regimen for the compounds and compositions described herein will necessarily depend on the needs of the individual patient to be treated, the type of treatment to be administered and the judgment of the attending physician. As used herein, "patient"
The term includes both humans and animals. The inhibitor compounds described herein are useful for the treatment of diabetic complications, especially in diabetic patients.
It is useful to prevent diabetic nephropathy, which is the leading cause of end-stage renal disease, which affects more than% and requires dialysis and transplantation. In addition, these inhibitors are AG
Other conditions that contribute to the production of E protein, such as hypertension, stroke, neurodegenerative disorders, such as Alzheimer's dementia, circulatory disease, atherosclerosis, osteoarthritis, cataract and general weakness of aging May be used for the prevention or treatment of adverse effects.

Preliminary experiments have shown that severe health detrimental effects result from stimulation of the lysine recovery pathway by prolonged consumption of glycated proteins. When using FL, an increase in FL3P, 3DG, and 3DF was observed in test animals fed a glycated protein diet. See Table B. Example 1 described later
As further described at 0, after eight months of such a diet, clear evidence of a renal pathology similar to that found in diabetic kidneys was found in animals on the glycated protein diet. Also, a transient increase in 3DG and 3DF levels was observed in the urine of human volunteers who consumed small amounts of glycated protein.

[0071]

[Table 1]

Since stimulation of the newly discovered lysine recovery pathway leads to a substantial increase in systemic 3DG levels, to determine whether a glycated diet causes a significant effect on pregnancy A study was conducted. The results obtained so far indicate that this route has a very strong effect, as is evident in the examples below.

Furthermore, in susceptible strains of rats and mice, diet given to animals at an early age (after weaning) has a significant effect on the incidence of type 1 diabetes with an incidence ranging from 10% to 90%. It is known that it is possible. Over the past decade, considerable effort has been made to study this phenomenon. For example, Diabetes 46 (4): 589-9
8 (1997) and Diabetes Metab. ReV. 12 (4): 341-59 (199).
6) and references cited therein. Studies on two extremes of diet to induce diabetes have been performed by some of the present inventors. AI
N-93 (Dyets, Inc.) causes the lowest incidence of diabetes, producing the lowest ratio of urine 3DF / creatinine (1.0) ever observed. Purina 500 induces the highest incidence of diabetes, producing a 2.5 fold increase in the 3DF / creatinine ratio. Since FL3P, 3DG and 3DF were observed in rat pancreas, fructose lysine kinase and metabolites of the metabolic pathway appear to be involved in the development of type I diabetes. Animals susceptible to this type of diabetes
A useful model of insulin dependence or type I diabetes in humans) sensitizes the animal to unknown antigens expressed on pancreatic β-cells, thus preventing autoimmune attacks on the β-cells by the animal's own immune system. Has an abnormal immune system that arises. This results in their destruction, thereby depriving the animal of its ability to produce insulin. The fact that 3DG reacting with protein can create a new antigenic site
well known. Thus, the origin of the antigenic nature of various diets appears to be 3DG, which is produced by the breakdown of fructoselysine-3-phosphate in the pancreas.

Also, since 3DG is generally known to interact with amines, it can interact with DNA and may exhibit mutagenesis and carcinogenic potential, as well as cross-linked proteins. Knowledge of the FL3P lysine recovery pathway makes it practical, for the first time, to distinguish between diabetic populations and determine that a subset of the population is likely to develop diabetic complications. This determination can conveniently be made on the biological fluid to be tested, for example, a blood fraction (especially plasma or serum), lymph, interstitial fluid, and the like.

After an overnight fast, human subjects are provided a food source containing relatively high concentrations of glycated lysine residues. By way of example, the food may be in the form of a casein / sugar "cookie" as described in Example 5 below, or some other suitable source of glycated lysine or synthetic fructose-lysine. it can. When utilizing a protein containing glycated lysine residues, the glycated lysine content is preferably 0.02 to 10% of the total protein amino acids, or more preferably about 0.2 to 0.4%. It is. The total amount of glycated lysine residues at an oral dose is about 0.3 grams. Preferably, urine samples are collected prior to consumption of the glycated lysine source, then at 1, 3 and 5 hours or at other appropriate times as may be warranted by the particular clinical situation.

The 3DG and 3DF levels in these urine samples are measured and their metabolite ratio is calculated. The particular methodology used for the measurements is not essential to the practice of the present invention.
The GC method described in Example 5 described below may be used if desired. As will be apparent to those skilled in the art, alternatively, a colorimetric or immunological assay method can be used. Recently completed Diabetes Control and Complication Test
It is clear that the major risk factor faced by diabetics is glycemic control, as evidenced by ons Traial). However, the incidence of diabetic complications cannot be explained solely by blood glucose levels; when comparing the incidence of diabetic complications to historical blood glucose levels, a truly widespread variance is seen. One method of determining a subset of the most at risk diabetic population developing diabetic complications is a particularly significant aspect of the present invention. The method involves measuring the levels of FL, 3DG and 3DF before and optimally after ingesting the source of glycated lysine.

For example, a normal subject has a fasting urine 3DG: 3DF ratio of about 0.025, but a diabetic patient has a ratio as high as 5 times or more. This is supported by the data in FIG. 7, where normal glycemic subjects have a 3DG / 3DF ratio of 0.025 (
1 / 39.77), which is a very narrow range around this value.
It has an average ratio greater than twice (mean 0.069) and is much more dispersed around its mean. As shown herein, diabetics have increased production of 3DG. Therefore, resistance to diabetic complications requires very effective removal of the toxic metabolite. The efficiency of the 3DG detoxification pathway can be evaluated by the ratio of 3DG to 3DF calculated by the method described herein. Individuals with a low ratio will generally be resistant to the development of diabetic complications. Individuals with higher ratios that include ratios falling within the normal range are at greater risk, while individuals with higher ratios above the normal range are at greater risk of developing these complications. In state.

Recently, fructoselysine (FL) in plasma and urine of four rat strains
) Revealed that there was a significant difference in the way each of their kidneys processed blood FL. Two of the four systems (Long Evans, Brown
Norway), based on the ratio of the compound and its metabolites to creatinine, virtually all FL filtered by the kidney appeared in the urine. In the other two lines (Sprague Dawley, Fischer), 10-20% of the plasma FL appeared in urine based on comparison with the creatinine fraction. These measurements strongly suggest a major difference in FL treatment in mammalian kidneys. Since rodent and human kidneys have been found to be nearly functionally equivalent, it makes sense to assume that similar changes in FL treatment will be present in humans.
Since FL is the first input into the fructose lysine recovery pathway, the entire pathway appears to be substantially stimulated in these humans, and large amounts of F
L is absorbed from the ultrafiltrate, and locally high levels of 3-deoxyglucosone (3DG) are found in the kidney as well as in the whole body. This observation may serve as a basis for a diagnostic test to determine the flow of FL to the kidney and the fraction of that flow that appears in the urine by comparison with a sample of plasma or serum obtained at the same time as the urine sample. Individuals whose ratio is substantially lower than 1 are then at risk for developing various renal conditions including, but not limited to, diabetic nephropathy, renal failure in old age and renal cancer. .

The therapeutic effect of the kinase inhibitors of the present invention can be readily and safely determined using tests for the lysine recovery pathway. The test protocol is the same as described immediately above, except that urinary fructoselysine levels are measured in addition to urinary 3DG and 3DF levels. It is useful to perform this test both before and after the start of FL3P kinase inhibitor treatment. Urinary 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 highest levels of 3DG and 3DF found in urine after ingesting foods rich in glycated lysine residues are induced by the activity of the lysine recovery pathway. Non-reactive fructose-lysine at these metabolite concentrations (a common component of human urine)
The ratio to reflects the activity of the pathway. Inhibition of the lysine recovery pathway will cause a decrease in the amount of 3DG and 3DF that is excreted and an increase in fructose-lysine excretion. Thus, the therapeutic effect of a kinase inhibitor can be quantified by measuring the decrease in the (3DG + 3DF) / fructose-lysine ratio after inhibition treatment. It should be noted that in interpreting the assay, only metabolite ratios are considered, not urine volume or metabolite concentration.

It will be appreciated from the foregoing disclosure that orally digested foods containing high concentrations of glycated lysine residues will lead to the production of kidney and serum 3DG.
It is reasonable to warn individuals at risk of kidney disease, for example, diabetics, to avoid foods that have high levels of them. The concentration of glycated lysine residues can be measured in a variety of different ways. One such measurement method is described in Example 4 below. However, any suitable assay that accurately measures the level of glycated lysine residues can be used in place of the assay methods exemplified below. Examples of particularly contemplated assay methods include, but are not limited to, colorimetric and immunological methods. Regardless of the assay used, measure the content of glycated lysine residues in cooked foods and refrain from ingesting foods high in glycated lysine in individuals at risk of developing renal failure Thus, notifying the individual of these measurements is within the scope of the present invention.

The following examples are provided to describe the invention in further detail. These examples are provided for illustrative purposes only and in any way,
It should not be construed as limiting the invention. Unless otherwise stated, all temperatures given in the examples are in degrees Celsius.

EXAMPLES Example 1 Isolation and Identification of FL3P: A ³¹ P NMR analysis of a perchloric acid extract from diabetic rat kidney showed 6.24 ppm.
Showed a novel sugar-monophosphate resonance, which is not observed in non-kidney tissues and is present at very low levels in non-diabetic kidneys. The compound responsible for the observed resonance was isolated by chromatography of the extraction on a microcrystalline cellulose column using 1-butanol-acetic acid-water (5: 2: 3) as eluent. Proton 2
The structure was determined by D COSY to be fructose-lysine triphosphate. This has been described previously (Finot and Mauson, Helv. Chim. Ac.
ta 52: 1488 (1969)) was confirmed later by injecting the prepared FL into animals and revealing the direct phosphorylation to FL3P. The position of the phosphate at carbon-3 was confirmed using FL specifically deuterated at the 3-position. This was done by analyzing both the coupled and decoupled ³¹ P NMR spectra. A typical POCH coupling is 10
. A double line is generated in FL3P with a J value of 3 Hz, but 3-deuterated FL
In the case of 3P, POCD has no coupling and yields a singlet, both coupled and decoupled. A unique property of FL3P is that when treated with sodium borohydride, it is converted to two new resonances at 5.85 and 5.95 ppm, corresponding to mannitol and sorbitol-lysine triphosphate.

Example 2 Synthesis of FL3P: 1 mmol of dibenzylglucose 3-phosphate and 0.25 mmol of α-
The carbobenzoxy-lysine was refluxed in 50 ml of MeOH for 3 hours. 1 solution
Diluted with 00 ml of water and in pyridinium form in a Dow-50 column (2.5 × 20
cm), first with water (200 ml) and then with 60 ml.
Elution was performed with 0 ml of buffer (0.1 M pyridine and 0.3 M acetic acid). The target compound eluted at the end of the water wash and at the beginning of the buffer wash. 20 psi hydrogen
Removal of the cbz and benzyl blocking groups using 5% Pd / C at gave FL3P in 6% yield.

Example 3 Enzymatic Production of FL and ATP to FL3P and Assay to Screen for Inhibitors: Initially, ³¹ P NMR was used to demonstrate kinase activity in the renal cortex.
A 3 g sample of fresh porcine kidney cortex was prepared with 9 ml of 150 mM KCl, 5 mM DTT.
Homogenized in 50 mM Tris HCl, pH 7.5, containing 15 mM MgCl ₂ . This was centrifuged at 10,000 g for 30 minutes, then the supernatant was
Centrifuged at 00000 g for 60 minutes. Ammonium sulfate was added to 60% saturation. After 1 hour at 4 ° C., the precipitate was collected by centrifugation and dissolved in 5 ml of the original buffer. Aliquot a 2 ml aliquot of the solution to 10 mM ATP and 10 mM
Incubated with M FL (prepared as in Example 1 above) at 37 ° C. for 2 hours. The reaction was quenched with 300 μl perchloric acid, the protein was removed by centrifugation and desalted on a Sephadex G10 column (5 × 10 cm). The formation of FL3P was detected by ³¹ P NMR analysis of the reaction mixture.

Based on the kinase activity tests obtained in this way, a radioactivity assay was developed. The assay was designed with the advantage that FL3P does not bind to Dow-1 anion exchange resin. This feature of FL3P was found during efforts to isolate it. Since most phosphates bind to the resin, it was believed that all compounds that reacted with ATP bound as well as any excess ATP, leaving FL3P in solution. The first step was to determine the amount of resin needed to remove ATP in the assay. This was done by mixing the mixture with Dow-1 (200 mg) in H ₂ O (0.1%).
9 ml) in a suspension, pipetted, stirred and centrifuged to solidify the resin. From now on, 0.8 ml of the supernatant is 200 m with a pipette.
g of fresh dry resin, agitated and centrifuged. A 0.5 ml volume of the supernatant was pipetted into 10 ml of Ecoscint A and counted. The residual count was 85 cpm. The technique was used for the assay. The precipitate from the 60% ammonium sulfate precipitation of the crude cortical homogenate was redissolved in homogenate buffer at 4 ° C. The assay consists of 10 mM γ ³³ P-ATP (40,000 cpm), 10 mM FL, 1 mM in 0.1 ml of 50 mM Tris HCl, pH 7.5.
Contains 50 mM KCl, 15 mM MgCl ₂ , 5 mM DTT. FL3P
The relationship between production and enzyme concentration was 30 ° C. at 37 ° C. with 1, 2 and 4 mg of protein.
The determination is made using a means of triple determination in minutes. Blanks run simultaneously without FL were subtracted and data recorded. The observed activity corresponded to a FL3P synthesis rate of approximately 20 nanomoles / hour / mg protein.

Example 4 Inhibition of 3-Deoxyglucosone Production by Meglumine and Various Polyol Lysines a. General Polyol Lysine Synthesis Sugar (11 mmol), α-carbobenzoxy-lysine (10 mmol) and NaBH ₃ CN (15 mmol) were dissolved in 50 ml of MeOH—H ₂ O (3: 2) and 25 Stirred at C for 18 hours. The solution was treated with excess Dow-50 (H) ion exchange resin to decompose excess NaBH ₃ CN. The mixture (liquid plus resin) was transferred onto a Dow-50 (H) column (2.5 x 15 cm) and washed well with water to remove excess sugar and boric acid. Carbobenzoxy-polyol lysine was eluted with 5% NH ₄ OH. The resin obtained by evaporation was dissolved in water-methanol (9: 1), and hydrogen gas (20 psi) was applied using 10% palladium catalyst on charcoal.
Reduced. Filtration and evaporation gives the polyol lysine.

B. Experimental protocol for reduction of urine and plasma 3-deoxyglucosone by sorbitol lysine, mannitol lysine and galactitol lysine Urine was collected from 6 rats for 3 hours. Plasma samples were also obtained. The animals were then given 10 micromolar of either sorbitollysine, mannitollysine or galactitollysine by intraperitoneal injection. Collect urine for another 3 hours
At the end of the time a plasma sample was obtained. As described in Example 5 below, 3-deoxyglucosone was measured in these samples and various volumes were normalized to creatinine. The mean reduction in urine 3-deoxyglucosone was 50% with sorbitol lysine, 35% with mannitol lysine and 35% with galactitol lysine. Plasma 3-deoxyglucosone was reduced by 40% by sorbitol lysine, 58% by mannitol lysine and 50% by galactitol lysine.

C. Use of meglumine to reduce urinary 3-deoxyglucosone Three rats were treated as in b) above, except that intraperitoneal injection of meglumine (100 micromolar) instead of the lysine derivative described above. . Three hours after injection, the average urinary 3-deoxyglucosone concentration decreased by 42%.

Example 5 Urinary FL, 3DG and 3DF in Humans After Injection of Glycated Protein
Increase a. Preparation of glycated protein-containing food: 260 g of casein, 120 g of glucose and 720 ml of water were mixed to obtain a homogeneous mixture. The mixture was transferred to a metal plate and cooked at 65 ° C. for 68 hours. The resulting cake was then crushed to a course powder. The powder contained 60% protein as determined by the Kjeldahl method.

B. Determination of glycated lysine content: 1 g of the powder prepared as in step a above was hydrolyzed by refluxing with 6N HCl for 20 hours. The resulting solution was adjusted to pH 1.8 with NaOH solution,
Dilute to 100 ml. Fructose lysine content was measured with an amino acid analyzer as furosine, a product obtained from acid hydrolysis of fructose lysine. In the method, it was determined that the cake contained 5.5% (w / w) fructose lysine.

C. Experimental Protocol: Volunteers spent 2 days on a diet without fructose lysine, then consumed 22.5 g of food prepared as described herein, thus effectively 2 g
Of fructose lysine. Urine was collected at 2 hour intervals for 14 hours, with a final collection at 24 hours.

D. Measurement of FL, 3DG and 3DF in urine: FL was measured at 46 ° C. on a Waters C18 Free Amino Acid column and acetonitrile-methyl alcohol-water (45:15:40 at 1 ml / min).
) Was measured by HPLC on a Waters 996 diode Array using a gradient elution system from acetonitrile-sodium acetate-water (6: 2: 92). For quantification, an internal standard of meglumine was used.

[0093] 3DF was measured by HPLC after deionization of the sample. Analysis was performed on PA1
Performed on a Dionex DX-500 HPLC system using a column (Dionex), eluting at 1 ml / min with 32 mM sodium hydroxide. Quantification is based on synthesis 3
DF was performed from the standard curve obtained on day 1. 3DG was measured by GC-MS after deionization of the sample. 3DG is P
Induced with a 10-fold excess of diaminonaphthalene in BS. Ethyl acetate extraction yielded a salt-free fraction, which was converted to trimethylsilyl ether with Tri-Sil (Pierce). Analysis was performed by Hewlett-Packard
The analysis was performed using a 5890 selective ion monitoring GC-MS system. GC is a fused silica capillary column (DB- 5,25mx.25mm). Quantification of 3DG used selected ion monitoring using an internal standard of U-13C-3DG.

The graph shown in FIG. 3 shows the production of FL, 3DF and 3DG in the urine of one volunteer after consumption of the glycated protein. The rapid appearance of all three metabolites is evident. Both 3DF and 3DG show a slight increase even after 24 hours. The graph shown in FIG. 4 shows the generation of 3DF in each of the seven test group members. A similar pattern is seen in all cases. As is evident in FIG. 4, 3DF excretion was highest at 4 hours after FL bolus and even at 24 hours after bolus, 3DF
It should be noted that there is a slight rise in.

Example 6 Feeding Experiment: N-Acetyl-β-glucosaminidase (NAGase) is an enzyme excreted in urine at high concentrations in diabetic patients. Although it is thought to be an early landmark of ductal injury, the pathology of urinary NAGase increase is not well understood.
It has been proposed that the increased urinary production of NAGase in diabetic patients is due to lysosomal activation in proximal tubules induced by diabetes with increased production in the urine rather than destruction of cells. The results obtained in this example show that, in all comparisons, 3DF and NAG
4 shows that ase levels are increased in the experimental group compared to the control. Therefore,
Animals fed glycated protein excrete excess NAGase in the urine, similar to the results obtained in diabetics. NGase excretion is increased by about 50% compared to control animals. These animals also have a 5-fold increase in urine 3DF compared to controls. As seen in FIGS. 5 and 6, urinary 3DF correlates very well with 3DG. Both compounds appear to be cleared from plasma at glomerular filtration values without reabsorption.

Example 7 SDS Gel of Kidney Protein: Two rats were injected daily with 5 micromolar of either FL or mannitol (used as control) for 5 days. Animals were killed, kidneys removed and dissected into cortex and bone marrow. Tissue was homogenized in 5 volumes of 50 mM Tris HCl, pH 7.5, containing 150 mM KCl, 15 mM MgCl ₂ and 5 mM DTT. Cell debris was removed by centrifugation at 10,000 g for 15 minutes, then the supernatant was centrifuged at 150,000 g for 70 minutes. SDS of soluble protein on 12% polyacrylamide gel and 4-15 and 10-20% gradient gel
Analyzed by PAGE. In all cases, lower molecular weight bands were absent or visually reduced from kidney extracts of animals injected with FL compared to animals injected with mannitol.

Example 8 Synthesis of 3-O-methylfructose lysine: A suspension of 19.4 g (0.1 mol) of 3-O-methylglucose anhydride and 1 g of sodium bisulfite in 30 ml of methanol and 15 ml of glycerol was prepared.
Reflux and then 0.035 mole of α-carbobenzoxy-lysine and 4
ml of acetic acid was added. The solution was refluxed for 3 hours. The solution was treated with one volume of water and chromatographed on a Dowex-50 column (4 × 50 cm) in pyridinium form, eluting first with water and then with pyridinium acetate. Fractions containing pure material were combined and evaporated. The resulting material was dissolved in 50 ml of water-methanol (9: 1) and reduced with hydrogen gas (20 psi) over 10% palladium on charcoal. Filtration and evaporation gave 3-O-methylfructose lysine. Other specific compounds having the structure of formula (I) above can be used, for example, to select selected nitrogen or oxygen containing starting materials, possibly amino acids, polyamino acids, peptides, etc., using glycating agents such as fructose. Manufactured by application, and if desired, it may be chemically modified by techniques well known to those skilled in the art.

Example 9 Additional Assays for FL3P Kinase Activity: a. Preparation of stock solution: 100 mM HEPES pH 8.0, 10 mM ATP, 2 mM MgCl ₂ ,
An assay buffer solution of 5 mM DTT, 0.5 mM PMSF was prepared. A fructosyl-spermine stock solution, 2 mM fructosyl-spermine HCl, was prepared. A spermine control solution that was 2 mM spermine HCl was prepared.

B. Synthesis of fructosyl-spermine: The synthesis of fructosyl-spermine is known in the art (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 50 ml methanol-water (1: 1) molar ratio of 8: 4: 1 (spermine: glucose: pyrosulfite). And refluxed for 12 hours. The product was diluted with water to 200 ml and a DOW-50 column (5 × 90
cm). Unreacted glucose was removed by water 2 column volumes of product and unreacted spermine were removed with 0.1 M NH 4 _OH. The products of the peak fractions were pooled and lyophilized and the concentration of fructosyl-spermine was determined by
Qualitative ¹³ C NMR spectrum of the product (NMR data is 45 ° pulse, 10
(Collected without second relaxation lag time and NOE decoupling) was determined by measuring the entire C-2 fructosyl peak.

C. Kinase Assay for Purification: An incubation mixture containing 10 μl enzyme preparation, 10 μl assay buffer, 1.0 μCi ³³ P ATP, 10 μl fructosyl-spermine stock solution and 70 μl water was prepared and incubated at 37 ° C. for 1 hour. Incubated for hours. At the end of the incubation, 90 μl (2 × 45 μl) of the sample was
. Spotted onto a 5 cm diameter cellulose phosphate disc (Whatman P-81) and dried. The disc was extensively washed with water. After drying, the disks were placed in scintillation vials and counted. Each enzyme fraction was assayed in duplicate using the appropriate spermine control.

Example 10 Kidney Pathology Observed in Test Animals on a Glycated Protein Diet Three rats were maintained on a glycated protein diet (20% total protein; 3% glycation) for 8 months and a control diet. Compared to 9 rats of the same age maintained.
The main finding was a substantial increase in damaged glomeruli in animals on a glycated diet. Typical lesions observed in these animals were segmental sclerosis of the glomerular sac (Bowman's capsule), tubular dysplasia of the parietal epithelium, and interstitial fibrosis. All three animals on the glycated protein diet and only one animal on the control diet showed greater than 13% injured glomeruli. The probability that this occurs is less than 2%. In addition to the pathology observed in the glomeruli, several hyaline casts were observed in the tubules. Although not quantified, more of these were found in animals on the glycated diet. Increased levels of NAGase were also observed in animals on the glycated diet. From the results of the experiment, the glycated diet appeared to cause the test animals to develop a series of histological lesions similar to those found in diabetic kidneys.

Example 11 Effect of Glycated Diet on Pregnancy In a preliminary experiment, 5 pairs of mice were given a glycated diet (18% total protein; 3
% Glycation) and gave birth to 6 offspring over a 7 month period. The six pregnancies resulted in 17, 23, 13, 0, 3, and 0 surviving offspring. Third
Taking into account this sharp drop in surviving offspring after birth, each of the two groups of 10 pairs had a glycated diet (13% total protein; 3% glycation) or a control diet (13% protein, 0%). % Glycation). So far, two groups of offspring were born four times, with similar results in both groups. First pregnancy yields 49/20 (glycation / control) offspring; second 18/41; third 3
On July 27, the fourth was 20/33. The fifth pregnancy is currently in progress. A set of mice was tested for hyperglycemia. Blood glucose levels are 120 and 112 mg / dl in the experimental and control groups, respectively. Preliminary measurements of 3DF levels in mouse urine show, as expected, a substantial increase (approximately 5-10 fold) in systemic 3DF for the glycated diet described herein.

Example 12 Carcinogenic effects of the fructose lysine pathway To investigate the potential carcinogenicity of metabolites generated in the fructose lysine pathway, experiments were performed in a rat strain highly susceptible to kidney cancer. Four rats received a glycated diet and three rats received a control diet. Feed 10
After a week, the animals were killed and their kidneys examined. In all four animals fed, 1
Kidney cancers of a size larger than mm were seen, while no such large lesions were found in control animals. The probability that this will happen is less than 2%. The data show that elevated 3DG levels caused by excess fructoselysine from glycated proteins in animal diets found in renal tubular cells (known to be cells of many renal cancer origins). Interacts with cellular DNA,
Demonstrates that various mutations can be induced, ultimately leading to an oncogenic event. The process can be important in the development of human cancer in the kidney and elsewhere.

Dietary effect of glycated protein diet on renal cell carcinoma in susceptible rats In an additional experiment evaluating the relationship between glycated diet and renal cell carcinoma, susceptibility to renal carcinoma development was demonstrated. Twenty-eight mutated rats were divided into two groups. One group was fed a glycated protein diet and the other group was fed a control diet. The glycated protein diet consists of a defined nutrient diet plus 3% glycated protein. The glycated protein is obtained by mixing casein and glucose (2: 1) together, adding water (twice the weight of dry matter),
The mixture was made by baking at 60 ° C. for 72 hours. A control was prepared in a similar manner, except that water was not used and casein and glucose were not mixed before baking. Rats were allowed to feed immediately after weaning at 3 weeks of age, and then remained free to eat for 16 weeks. The rats were then sacrificed, the kidneys fixed, and a hematoxylin and eosin incision performed. These were examined for the lesion by a trained pathologist. Four types of lesions were identified. These lesions included cysts; very small aggregates of typically less than 10 tumor-like cells; small tumors less than 0.5 mm; and tumors larger than 0.5 mm. For each type, as can be seen from the table below, more lesions were observed in the animals on the glycated diet than on the control diet.

[0105]

[Table 2]

To summarize the results, the average number of lesions per kidney section was computerized for each diet. These were 0.82 ± 0.74 and 2.43 ± 2.33 for the control and glycated diet, respectively. The chance of this happening by chance is about twice per 100,000. These results strongly support the premise that the findings underlie the present invention, and that the effects of the resin recovery pathway mutate and also cause carcinogenesis. These results provide the basis for developing therapeutics and therapeutics to inhibit this pathway and reduce cancer in the kidney, as well as in other organs where the resin retrieval pathway provides a similar effect. I do.

Urinary excretion of 3-deoxy-fructose, indicating progression to microalbuminate urine in patients with type 1 diabetes, as described above, glycation intermediate, 3deoxy-glucosone (3DG)
Serum levels of and its reduced detoxified product, 3deoxy-fructose (3DF), are high in diabetics. The relationship between baseline levels of these compounds and their subsequent progression to microalbuminuria (MA) was compared to insulin-dependent diabetes mellitus (IDDM) and diabetes in microalbuminuria at the Josulin Diabetes Center. ACE for a group of 39 people from a possible group
Not against inhibitors, but tested (based on multiple measurements of a 2-year baseline starting between 1990 and 1993).

The baseline levels of 3DF and 3DG in urine at random spots were measured by HPLC and GC-MS. Individuals who progressed to higher levels of MA or proteinuria in the next four years (n = 24) were compared to those who did not (n = 15),
The g3DF / urine creatinine ratio was at a significantly higher baseline level (p = 0.02)
. The baseline level measured in this study was about 0.24 micromol / mg creatinine in advanced individuals, while about 0.18 micromol / mg creatinine in non-advanced individuals. . Baseline 3DG / urine creatinine ratio did not differ between groups. Adjusting the baseline level of HgA _IC (the major fraction of glycosylated hemoglobin) did not substantially change these results. These results provide further evidence for a link between urinary 3DF and progression of renal complications in diabetes.

A. Determination of 3-deoxyfructose A 0.3 mL aliquot of the test sample was transferred to an ion exchange column (0.15 mL of AG).
The sample was processed by passing 1-X8 and 0.15 mL of AG 50W-X8 resin). The column was then washed twice with 0.3 mL of deionized water, aspirated to remove free liquid, and filtered through a 0.45 mm Millipole filter. Injections (50 μL) of the treated samples were analyzed using a Dionex DX500 chromatography system. Elution was performed using a Carbopak PA1 anion exchange column with an eluent consisting of 16% sodium hydroxide (200 mM) and 84% deionized water. 3DF was detected electrochemically using a pulsed current detector. A standard 3DF solution with a predetermined 3DF concentration was run before and after each unknown sample.

B. Measurement of urinary creatinine The urinary creatinine concentration was measured by a modified endpoint colorimetric method using a plate reader (Sigma Diagnostic kit 555-A). To determine the level of metabolites present in urine, creatinine concentrations were evaluated to normalize urine volume.

C. Measurement of albumin in urine In order to evaluate the albumin level in urine of a test subject, urine was collected in a spot, and immunoturbidimetry was performed using a BN100 apparatus equipped with an N-albumin kit (Behring). Anti-albumin antibodies are commercially available. Albumin levels in urine are E
It can be assessed by any suitable assay including, but not limited to, LISA assay, radioimmunoassay, western and dot blotting. Based on data obtained from a study of patients at the Josulin Diabetes Center, it is clear that high levels of urinary 3DF are associated with progression to microalbuminuria in diabetes. This observation provides a new diagnostic parameter for assessing the likelihood that a patient suffering from diabetes will progress to severe renal complications.

While certain embodiments of the present invention have been described and / or illustrated above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. Therefore, the present invention is not limited to the specific examples described and / or illustrated, but may take into account changes and modifications without departing from the scope of the appended claims.

[Brief description of the drawings]

FIG. 1 shows the starting steps involved in a multi-step reaction leading to an irreversibly modified AGE protein.

FIG. 2 shows the reactions involved in the lysine recovery pathway.

FIG. 3 is a graph showing the time course of 3DF, 3DG and FL from a single individual given 2 g of FL, showing the urine profile followed for 24 hours.

FIG. 4 is a graph showing 3DF versus time of urine excretion from seven volunteers who received 2 g of fructose lysine.

FIG. 5. 3DF and N-acetyl-β-glucosaminidase (NA) between a control animal group and an experimental group that maintained a diet containing 0.3% glycated protein.
7 is a graph comparing G).

FIG. 6 is a graph showing the linear relationship between urinary 3DF and 3DG levels in rats fed either a control diet or a diet rich in glycated protein.

FIG. 7 shows fasting urine 3DG levels of normal and diabetic patients on fasting 3
It is a graph plotted against the DF level.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07H 15/12 C07H 15/12 G01N 33/15 G01N 33/15 Z 33/50 33/50 Z (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 2G045 AA25 AA40 CA26 CB03 CB17 CB26 DA20 DA36 DA44 DA80 FB01 FB03 FB05 FB06 JA01 JA20 4C057 AA17 HH02 4C086 AA01 AA02 EA03 MA01 MA04 MA56 NA14 4C206 AA01 AA02 FA03 MA01 MA04 MA72 MA75 MA76 MA86 NA14 ZB26 ZC41 4H006 AA01 AB28 BN10 BU32

Claims (18)

[Claims] 1. A compound that reduces the susceptibility to carcinoma in a patient associated with the ingestion of the glycated protein by the patient. Wherein, X is -NR'- or -O-, where R 'is H and straight or branched chain alkyl group _(C 1 -C ₄₎ and unsubstituted or substituted aryl group R is selected from the group consisting of (C ₆ -C ₁₀ ) or an aralkyl group (C ₇ -C ₁₀ ); R is H, amino acid residue, polyamino acid residue, peptide chain, unsubstituted or at least one A linear or branched aliphatic group (C ₁ -C ₈ ) substituted with a nitrogen- or oxygen-containing substituent, unsubstituted or at least one
Substituted with at least one nitrogen- or oxygen-containing substituent and at least one -O-,-
A linear or branched aliphatic group interrupted by an NH- or -NR "-group (
C ₁ -C ₈ ), wherein R ″ is a linear or branched alkyl (C ₁ -C ₆ ) and an unsubstituted or substituted aryl group ( C ₆ -C ₁₀ ) or an aralkyl group (C ₇ -C ₁₀ ); provided that when X is —NR′—, R and R ′ together with the nitrogen atom to which they are attached, have at least one Substituted and unsubstituted heterocyclic rings having 5 to 7 ring atoms, wherein the nitrogen and oxygen are the only heteroatoms in the ring, and the aryl group (C ₆ -C ₁₀ ) or an aralkyl group (C ₇ -C ₁₀ ) and the heterocyclic ring substituent are H, alkyl (C ₁ -C ₆ ), halogen, CF ₃ , CN, NO ₂ and —O-alkyl (C ₁ -C ₁₀ ). It is selected from the group consisting of C ₆₎ ; R ₁ is linear polyols having 1 to 4 carbon atoms; Y is a carbonyl group ## STR2 ## Or a hydroxymethylene group Of either be; Z is -H, -O- alkyl _(C 1 _-C 6), - halogen _-CF 3, -CN, -
Isomers and pharmaceutically acceptable salts of the compounds and the compound represented by] is selected from the group consisting of COOH and -SO ₃ H _2. 2. A method for reducing the susceptibility to carcinoma in a patient associated with the ingestion of the glycated protein by the patient, comprising the step of converting fructose-lysine to fructose-lysine-3-phosphate. A method comprising administering a pharmaceutical composition comprising an active compound having an effective inhibitory activity in inhibiting conversion. 3. The method according to claim 2, wherein the carcinoma is renal cell carcinoma. 4. The method of claim 1, wherein the active compound exhibits competitive or non-competitive enzyme inhibition,
3. The method of claim 2, wherein the method has an inhibition constant (Ki) less than M. 5. An active compound comprising: (i) a molecular weight of less than about 2000 daltons; (ii) a solubility in saline or serum of 10 μM or more; and (iii) at least a solubility in saline or serum. 3. The method of claim 2, wherein the enzyme retains at least 50% of its enzyme inhibitory activity after 1 hour incubation. 6. An active compound having the structural formula: Wherein, X is -NR'- or -O-, where R 'is H and straight or branched chain alkyl group _(C 1 -C ₄₎ and unsubstituted or substituted aryl group R is selected from the group consisting of (C ₆ -C ₁₀ ) or an aralkyl group (C ₇ -C ₁₀ ); R is H, amino acid residue, polyamino acid residue, peptide chain, unsubstituted or at least one A linear or branched aliphatic group (C ₁ -C ₈ ) substituted with a nitrogen- or oxygen-containing substituent, unsubstituted or at least one
Substituted with at least one nitrogen- or oxygen-containing substituent and at least one -O-,-
A linear or branched aliphatic group interrupted by an NH- or -NR "-group (
C ₁ -C ₈ ), wherein R ″ is a linear or branched alkyl (C ₁ -C ₆ ) and an unsubstituted or substituted aryl group ( C ₆ -C ₁₀ ) or an aralkyl group (C ₇ -C ₁₀ ); provided that when X is —NR′—, R and R ′ together with the nitrogen atom to which they are attached, have at least one Substituted and unsubstituted heterocyclic rings having 5 to 7 ring atoms, wherein the nitrogen and oxygen are the only heteroatoms in the ring, and the aryl group (C ₆ -C ₁₀ ) or an aralkyl group (C ₇ -C ₁₀ ) and the heterocyclic ring substituent are H, alkyl (C ₁ -C ₆ ), halogen, CF ₃ , CN, NO ₂ and —O-alkyl (C ₁ -C ₁₀ ). It is selected from the group consisting of C ₆₎ ; R ₁ is linear polyols having 1 to 4 carbon atoms; Y is a carbonyl group embedded image Or a hydroxymethylene group Of either be; Z is -H, -O- alkyl _(C 1 _-C 6), - halogen _-CF 3, -CN, -
Isomers and pharmaceutically acceptable salts of the compounds and the compound represented by] is selected from the group consisting of COOH and -SO ₃ H _2,
The method of claim 2. 7. The method according to claim 2, wherein the pharmaceutical composition is administered orally. 8. The method of claim 1, wherein the patient is caused by AGE-protein formation.
A method for preventing, reducing or delaying the occurrence of carcinoma, comprising administering to the patient a therapeutically effective amount of a compound that inhibits the patient from producing 3-deoxyglucosone. 9. The method according to claim 8, wherein the carcinoma is renal cell carcinoma. 10. The compound of the formula: Wherein, X is -NR'- or -O-, where R 'is H and straight or branched chain alkyl group _(C 1 -C ₄₎ and unsubstituted or substituted aryl group R is selected from the group consisting of (C ₆ -C ₁₀ ) or an aralkyl group (C ₇ -C ₁₀ ); R is H, amino acid residue, polyamino acid residue, peptide chain, unsubstituted or at least one A linear or branched aliphatic group (C ₁ -C ₈ ) substituted with a nitrogen- or oxygen-containing substituent, unsubstituted or at least one
Substituted with at least one nitrogen- or oxygen-containing substituent and at least one -O-,-
A linear or branched aliphatic group interrupted by an NH- or -NR "-group (
C ₁ -C ₈ ), wherein R ″ is a linear or branched alkyl (C ₁ -C ₆ ) and an unsubstituted or substituted aryl group ( C ₆ -C ₁₀ ) or an aralkyl group (C ₇ -C ₁₀ ); provided that when X is —NR′—, R and R ′ together with the nitrogen atom to which they are attached, have at least one Substituted and unsubstituted heterocyclic rings having 5 to 7 ring atoms, wherein the nitrogen and oxygen are the only heteroatoms in the ring, and the aryl group (C ₆ -C ₁₀ ) or an aralkyl group (C ₇ -C ₁₀ ) and the heterocyclic ring substituent are H, alkyl (C ₁ -C ₆ ), halogen, CF ₃ , CN, NO ₂ and —O-alkyl (C ₁ -C ₁₀ ). It is selected from the group consisting of C ₆₎ ; R ₁ is linear polyols having 1 to 4 carbon atoms; Y is a carbonyl group embedded image Or a hydroxymethylene group Z is -H, -OH, -O-alkyl (C ₁ -C ₆ ), -halogen-CF ₃ ,-
CN, isomers and pharmaceutically acceptable salts of the compounds and the compound represented by] is selected from the group consisting of -COOH and _-SO 3 _{H 2,}
The method of claim 8. 11. A method for inducing carcinoma in a susceptible test animal, said method comprising the steps of inducing a biological fluid 3-deoxyglucose from a similar susceptible test animal fed a diet substantially free of glycated protein. Feeding the test animal a glycated protein diet in an amount and for a time sufficient to maintain the 3DG content of the biological fluid of the test animal at least three times higher compared to the Son (3DG) content. And how to. 12. The method according to claim 11, wherein the carcinoma is renal cell carcinoma. 13. A method of screening for a substance that affects carcinoma development, comprising: (i) the screening of a biological fluid from a similarly susceptible test animal fed a diet substantially free of glycated protein. The animal is provided with a glycated protein diet in an amount and for a time sufficient to maintain the 3DG content of the biological fluid of the test animal at least three times higher compared to the 3-deoxyglucosone (3DG) content. (Ii) administering the test substance to some of the test animals, but not to the other animals; (iii) killing the test animals; (ii) killing the test animals; iv) comparing nuclear carcinomas found in each of the diseased test animals. 14. The method of claim 13, wherein each is about half of the test animal. 15. The method according to claim 13, wherein the carcinoma is renal cell carcinoma. 16. A method of screening for a substance that prevents, reduces or delays the occurrence of carcinoma, comprising the same biological fluid from a similar susceptible test animal fed a diet substantially free of glycated protein. Glycated protein in the diseased test animal in an amount and for a time sufficient to maintain the 3DG content of the biological fluid of the test animal at least three times higher compared to the 3-deoxyglucosone (3DG) content of the test animal. Feeding; administering the test substance to some of the test animals but not to other animals; killing the test animals; comparing tissue portions from each of the diseased test animals;
A method comprising: 17. The method of claim 16, wherein each is about half of the test animal. 18. The method of claim 16, wherein the carcinoma is a renal cell carcinoma.