CN1331599A - Compounds and their therapeutic use with diabetic complications - Google Patents

Abstract

The invention discloses a class of compounds which inhibit the enzymatic conversion of fructose-lysine into fructose-lysine-3-phosphate in an ATP dependent reaction in a recently discovered metabolic pathway. According to the normal functioning of this pathway, fructose-lysine-3-phosphate (FL3P) is broken down to form free lysine, inorganic phosphate and 3-deoxyglucosone (3DG), the latter being a reactive protein modifying agent. 3DG can be detoxified by reduction to 3-deoxyfructose (3DF), or it can react with endogenous proteins to form advanced glycation end-product modified proteins (AGE-proteins), which are believed to be a contributing cause of diabetic complications. Also disclosed are therapeutic methods of using such inhibitors to reduce formation of AGE-proteins and thereby lessen, reduce and delay diabetic complications, as well as methods for assessing a diabetic's risk of developing complications and for determining the efficacy of the disclosed inhibitor therapy by measuring the ratio of 3DG to 3DF in a biological sample following an oral dose of a fructose-lysine-containing food product.

Description

Chemical compound and be used for the treatment of the purposes of diabetic complication

According to 35U.S.C. § 202 (c), state that hereby U.S. government has certain rights and interests in invention disclosed herein, the present invention obtains state-run commune hospital fund and partly supports (number of supporting DK44050, DK50317 and DK50364).

Background of invention

The present invention relates to medicine and be used for diabetes especially prevent, alleviate or postpone to take place diabetic complication and relevant etiologic etiological other treatment of conditions purposes.More particularly, the present invention relates to the class of enzymes inhibitor, their suppress fructose lysine (FL) enzyme and are converted into fructose-lysine-3-phosphate (FL3P), and this conversion is considered to cause important step in the biochemical mechanism of diabetic complication.The invention still further relates to method and the prevention of definite therapeutic intervention of estimating diabetics generation diabetic complication risk, the method that alleviates or postpone to take place the usefulness of diabetic complication.

The diabetic complication that has 4 kinds of especially severes, that is: diabetic nephropathy, diabetic renal papillary necrosis, the diabetic neuropathy that relates to the peripheral nervous afunction and because the circulatory disturbance that the blood capillary damage causes because of the destroying retinal blinding.Retinopathy and nephropathy all are considered to the ingredient of the systemic circulation obstacle relevant with this disease state of an illness.Summarized the blood capillary disorder effect (Tooke, Diatetes, 44:721 (1995)) in the diabetes late recently.In the whole disclosure of the present invention, term " diabetes dependency disease (diabetes-associatedpathologic conditions) " and synonymous term mean and comprise various known retinopathys, neuropathy, nephropathy, macroangiopathy and other diabetic complication.

The existing report very widely of the similarity of pathology that causes about diabetes and the old and feeble pathology that causes.Studies show that many diabetes dependency diseases are very similar to the old and feeble pathological change that causes usually clinically.For example confirmed that diabetics tremulous pulse and joint are stiff too early, elastance of lung and vital capacity reduce too early.In addition, the non-diabetic individuality suitable with the age compared, and the frequency of diabetics generation atherosclerosis, myocardial infarction and apoplexy is higher.The also easier infection of diabetes, and compare with the non-diabetic individuality, hypertension, bone loss acceleration, osteoarthritis and the damage of T cell function just may take place at the younger age.

As if diabetes dependency disease and old and feeble similarity prompting, there is common mechanism (mechanistic rationale) in they.As diabetes dependency disease and old and feeble common biochemical basis, various mechanism have been proposed.The human subject data hypothesis of strong support is a prerequisite with non-enzymatic glycosylation mechanism.This hypothesis proposes, aging course and diabetes dependency disease such as above-mentioned disease are at least in part because protein modification and the crosslinked (Monnier etc. that cause that the deutero-metabolite of glucose and glucose is undertaken by the Maillard reaction, Proc.Natl.Acad.Sci.USA, 81:583 (1984) and Lee etc., Biochem.Biophys.Res.Comm., 123:888 (1984)).Because modified protein this paper that such glycosylation produces is called advanced glycation dead end product-modified protein (AGE-albumen).What be widely known by the people is that 3-deoxyglucosone (3DG) is the important intermediate that forms in the rapid reaction sequence of the proteic multistep of AGE-.3DG be can with the deutero-metabolite of the glucose of albumino reaction, cause in the cell and extracellular protein (for example collagen protein and basement membrane) crosslinked.

During diabetic complication, it is believed that the chronic hyperglycemia with this disease association dynamically quickens to produce the proteic reaction of AGE-.Support the data that this machine-processed evidence comprises to show, the long-life albumen of diabetes study subject (long-lived protein) is as collagen protein and the lenticular AGE-protein content normal control apparently higher than the suitable age.Therefore, lenticular modification and cross-linked speed are accelerated soluble diabetics why at the cataract incidence height at younger age.Equally, important structural protein are what the modification of collagen protein and cross-linked speed accelerated may be interpreted as and can be observed early stage generation joint and arteriosclerosis and lung capacity at diabetics and reduce.Because these albumen are long-standing, so its modification effect is cumulative often.

Causal another factor that confirms diabetic complication and hyperglycemia is the hyperglycemia memory.A typical especially example of this phenomenon is that the initial stage diabetes through the Canis familiaris L. that treatment recovers the euglycemia level serious nephropathy take place.Though the histology of the eyes of described Canis familiaris L. when treatment is normal, although blood sugar concentration is normal, As time goes on diabetic nephropathy (Engerman etc., Diabetes, 36:808 (1987)) still takes place in these Canis familiaris L.s.Therefore, in hyperglycemia the potential eye infringement of irreversibility takes place in early days, clinical symptoms is just obvious afterwards.

Confirmed that the early stage of diabetic and animal and the proteic concentration of AGE-that late period is sugar-modified are normal high.In fact, the proteic increase of AGE-is higher than the increase of blood sugar level.Fluoremetry can be estimated the AGE-protein concentration, produces protein bound fluorescence molecule because a certain proportion of glycan molecule is reset.

The proteic pathological effect of AGE-is not limited to diabetes.The albumen saccharifying relates to presenile dementia (Harrington etc., Nature, 370:247 (1994)).The increase of albumen fluorescence is also shown in aging.In fact, some theory with aging course owing to oxidative damage and the protein modified synergy that causes of sugar.Therefore, the therapy that reduces the formation of AGE-albumen also can be used for treating the similar human disease states of other cause of disease, and may delaying senility course.

Generally believe that AGE-albumen forms and starts from proteinic amino and sugar (mainly being glucose) reaction." it is the Amadori chemical compound that the amino saccharifying of the ε of lysine residue forms initial adduct; fructose lysine ... saccharifying is the initial step in the complicated serial reaction; described reaction is generically and collectively referred to as Maillard or browning reaction, and it finally causes forming the fluorescin of crosslinked, precipitation, oxidation, brown " described and be to a kind of typical reference citation.K.J.Knecht etc., Archivesof Biochem.Biophys., 294:130 (1992).

Forming AGE-albumen by sugar is a rapid process of multistep, comprises the albumen that contains fructose-lysine with early stage, the reversibility reaction generation of sugar.The albumen of these modifications continues the irreversible modification AGE-albumen of reaction generation then.Obviously AGE-albumen is different with the albumen that contains the saccharifying lysine residue, because do not react with fructose-lysine at the proteic antibody of AGE-.It is evident that in addition AGE-albumen exists with the number of chemical form; Yet the AGE-albumen that does not almost have evaluation.In nearest research, (epsilon-amino-(carboxymethyl) lysine is accredited as a kind of important final AGE-protein structure (Reddy etc., Biochem., 34:10872 (1995) and Ikeda etc. with chemical type, Biochemistry ,-35:8075 (1996)).Can not the chemical identification another kind of AGE-albumen epi-position of this research, it constitutes about 50% and modifies the position.Recently developed a kind of research and formed the proteic dynamics methods of AGE-(Khalifah etc., Biochemistry, 35:4645 (1996)) by ribose.Yet this researchs and proposes ribose may play important physiological action in AGE-albumen forms, support the more generalized definition of saccharifying-lysine proposed below and fructose-lysine.

It is proteic different with AGE that other list of references has pointed out to contain the albumen of saccharifying lysine residue, " reaches the equilibrium level of Schiff alkali and Amadori product respectively after a few hours and several weeks.The reversible equilibrium response of early stage glycation product is important, because even it means unusual long-life albumen, the total amount of this product also reaches the stable state platform in the short period of time ... .. be because these early stage glycation products continue to accumulate on chronic diabetes patient's the collagen protein and other stabilizing tissue albumen in can not several years, thus the existence of its concentration and diabetic nephropathy or the order of severity uncorrelated be not wonderful ... yet collagen protein can not dissociate with some early stage glycation product on other blood vessel wall long-life albumen.On the contrary, they form irreversible advanced glycosylation end product through the chemical rearrangement of a slow complex series ".M.Brownlee etc., New England Journal of Medicine, 318:1315 (1988).The unique channel of these modified proteins of generation that scientific literature is described comprises the primary response of albumen and glycan molecule.

Many lists of references point out that taking off glucosone (3DG) by rapid approach formation AGE-albumen of multistep and 3-is the important intermediate of this approach.M.Brownlee, Diabetes, 43:836 (1994); M.Brownlee, Diabetes Care, 15:1835 (1992); T.Niwa etc., Nephron, 69:438 (1995); W.L.Dills, Jr., Am.J.Clin.Nutr., 58:S779 (1993); H.Yamadat etc., J.Biol.Chem., 269:20275 (1994); N.Igaki etc., Clin.Chem., 36:631 (1990).What Fig. 1 had illustrated common acceptance forms the approach of 3GD by sugar and albumino reaction.As seen from Figure 1, sugar (glucose) molecule begins to form Schiff alkali with protein-lysine amino (I).The Schiff alkali that produces is reset generation fructose-polylysine modification albumen (II) then.The reaction that produces (II) is free reversible.(II) rearrangeable generation 3DG and free albumen lysine.3DG subsequently and albumino reaction are first irreversible steps during AGE-albumen forms.

Known so far, never can produce the report of 3DG about other approach, report in fact never that perhaps the enzyme catalysis metabolic pathway is the main source of 3-DG, rather than uncatalyzed reaction shown in Figure 1.

The serum 3DG of diabetics is significantly higher than ND (12.78 ± 2.49 μ M and 1.94 ± 0.17 μ M).(Toshimitsu Niwa etc., Nephron, 69:438 (1995)).However, this toxic chemical still sees the normal health individuality.So it is not wonderful that body produces the detoxification approach to this molecule.A reaction in these reactions of aldehyde reductase catalysis, this enzyme makes the 3DG detoxification by 30G being reduced to 3-deoxidation fructose (3DF), and 3DF can effectively be secreted into (Takahashi etc., Biochem, 34:1433 (1995)) in the urine.Another detoxification reaction is by keto-aldehyde (oxoaldehyde) dehydrogenase 3DG to be oxidized to 3-deoxidation-2-ketone group gluconic acid (DGA) (Fujii etc., Biochem.Biophys.Res.Comm., 210:852 (1995)).

Result of study so far shows, the efficient of one of them kind enzyme aldehyde reductase of negative effect during diabetes.When the normal rat liver separates,, compare the catalytic efficiency of this enzyme low (Takahaski etc., Biochem., 34:1433 (1995)) in the part of lysine 67,84 and 140 these enzymes of top saccharifying with normal non-modification enzyme.Because the ratio of the glycated protein of diabetics is than blood glucose amount normal individual height,, and removes the toxic ability of this bioactive molecule and may reduce by 3DG being reduced to 3DF so the 3DG level of diabetics may be high.

After deliberation the mechanism of aldehyde reductase.These studies confirm that aldose reductase inhibitor (ARI) suppresses this important detoxification enzyme (Barski etc., Biochem., 34:11264 (1995)).At present the potentiality that ARI are being used to reduce diabetic complication are carried out clinical research.Verified these chemical compounds that are classified as a class have certain effect to the short-term diabetic complication.Yet they lack clinical effectiveness to long-term diabetic complication, and the renal function of the rat of feed high protein feed is worsened.Find out that from following this finds consistent with newfound lysine recovery metabolic pathway as basis of the present invention.High protein diet will increase fructose-lysine consumption, and fructose-lysine is converted into 3DG by kidney lysine recovery approach.The 3DG that the ARI treatment suppresses to be produced compares with the rat of not accepting ARI by being reduced to the 3DF detoxification, and the result causes kidney damage to increase the weight of.This is because ARI has reduced the effectiveness of the aldose reductase of reduction 3DG and 3DF to the inhibitory action of aldose reductase.

Patent by following general introduction is summarized as can be known, has studied the effect of 3-DG in human diseases in the past.

The United States Patent (USP) 5,476,849 of Ulrich etc. has been described with amino-benzoic acid and derivant and has been suppressed to form the proteic method of AGE-.Infer that these compound effects are: can it be removed in by system before forming the proteic irreversible step of AGE-to start with albumino reaction with 3-DG reaction and at it.

The United States Patent (USP) 4,798,583 and 5,128,360 of Cerami etc. has been described and has been used aminoguanidine to prevent to form the arterial wall proteins that AGE-albumen and diabetes cause crosslinked.Confirm that aminoguanidine is with early stage glycation product reaction.This early stage product of this paper definition is 3DG.These patents are the probability of imagination inhibition formation 3-DG not.They only are absorbed in this toxicity molecule of complexation.

The United States Patent (USP) 5,468,777 of France etc. has been described method and the medicine that prevents the tooth staining that caused in the oral cavity by non-enzymatic brown albumen.This application thinks that cysteine and cysteine derivative are particularly useful.

The United States Patent (USP) 5,358,960 of Ulrich etc. has been described with the amino imidazoles that replaces and has been suppressed the method that AGE-albumen forms.These chemical compounds and early stage glycation product (3DG) reaction have been confirmed.In this patent, do not address the 3DG metabolism source that may exist.This patent imagination 3DG only produces as the intermediate in the non-enzymatic brown albumen.

The United States Patent (USP) 5,334,617 of Ulrich etc. has been described the aminoacid that forms inhibitor as AGE-albumen.Think that lysine and other difunctional aminoacid are particularly useful aspect this.Think these aminoacid and early stage glycation product reaction from glucose and albumino reaction.As if the early stage glycation product of describing in this patent is 3DG.

The United States Patent (USP) 5,318,982 of Ulrich etc. has been described with 1,2, and the 4-triazole is made the inhibitory action that inhibitor forms AGE-albumen.The inhibitor that this patent is described comprises diaminourea-substituent, and this substituent can react and complexation with 3DG.This patent is described these chemical compounds and early stage glycation product (this paper is defined as 3DG) reaction.

The United States Patent (USP) 5,272,165 of Ulrich etc. has been described the purposes of 2-alkylidene-aminoguanidine as the inhibitor of AGE-albumen formation.Think that inhibitor and 3DG that this patent is described are highly reactive.The metabolism of the not mentioned inhibition of this patent 3DG forms.

The United States Patent (USP) 5,262,152 of Ulrich etc. is described and is used amidrazone and the formation of derivant inhibition AGE-albumen.The chemical compound that this patent is described is the α-Xiao Ying amine.W.P.Jencks, the third edition, McGraw Hill, New York.The for example 3DG reaction of known this compounds and dicarbonyl compound.

The United States Patent (USP) 5,258,381 of Ulrich etc. is described and is used the formation of 2-replacement-2-imidazoline inhibition AGE-albumen.The chemical compound that this patent is described contains contiguous amino, and they can easily react with 3DG.

The United States Patent (USP) 5,243,071 of Ulrich etc. has been described application 2-alkylidene-aminoguanidine and has been suppressed the formation of AGE-albumen.Chemical compound that this patent is described and 3DG have highly reactive, and its effect is that complexation should the activity toxicity molecule.

The United States Patent (USP) 5,221,683 of Ulrich etc. has been described application diamino-pyridine chemical compound and has been suppressed the formation of AGE-albumen.Think that useful especially described diamino-pyridine chemical compound and 3DG reaction forms the complex that contains 6 yuan of stable rings.

The United States Patent (USP) 5,130,337 of Ulrich etc. has described the application amidrazone and derivant suppresses the formation of AGE-albumen.The inhibitor that this patent is described is the α-Xiao Ying amine, known in the art they will and form stable compound with the 3DG fast reaction.

The United States Patent (USP) 5,130,324 of Ulrich etc. has been described application 2-alkylidene-aminoguanidine and has been suppressed the formation of AGE-albumen.The effect of the chemical compound that this patent is described is and early stage glycation product (3DG) reaction that described early stage glycation product is produced by glucose and proteins react.

The United States Patent (USP) 5,114,943 of Ulrich etc. has been described the pyrimidine of using amino-replacement and has been suppressed the formation of AGE-albumen.Think chemical compound that this patent is described and 3DG fast reaction and make its detoxification.

The above-mentioned patent of neither one proposes and will form the means of the inhibitory action of 3DG as the therapeutic intervention that prevents diabetic complication to metabolic.In fact, even neither one patent prompting in these patents, the generation of 3DG relates to the enzyme approach.

The United States Patent (USP) 5,108,930 of Ulrich etc. has been described the method for detection of biological sample aminoguanidine level.Think this be determined at by measure aminoguanidine the elimination time determine to have potentiality effectiveness in the renal function.The main effectiveness that is used for the assay method that this patent describes be measure aminoguanidine organize level so that in the research of animal and human's class, can keep the dosage that is enough to suppress the formation of AGE-albumen.The risk that 3DG, the 3DF of the not mentioned use urine of this patent or DGA ratio are determined diabetics generation complication.

The United States Patent (USP) 5,231,031 of Szwergold etc. has been described the risk and the method for determining the therapeutic efficiency of these complication of assessment diabetes dependency disease.This patent has been described two kinds of phosphorylation chemical compounds measuring in the diabetics erythrocyte.This patent does not have these two kinds of chemical compounds of chemical identification.Yet two kinds of chemical compounds all are not 3DG or 3DF, measure its level in urine in diagnosis embodiment of the present invention.

The method of controlling by the metabolism of measuring glycosylation dead end product monitoring diabetics is known.Known glycosylation hemoglobin concentration has reflected the average blood sugar concentration in former weeks.The United States Patent (USP) 4,371,374 that is presented to A.Cerami etc. has been described by the glycosylated protein catabolite monitoring method of glucose level in the quantitative urine, and more particularly the catabolite of described glycosylated protein is non-enzymatic glycosylation aminoacid and peptide.This method means utilizes alkaline boric acid affinity forming specific complex with the copline cis that sees the glycosylation dead end product-glycol group, thereby separates and quantitative this class dead end product.

The United States Patent (USP) 4,761,368 that is presented to A.Cerami has been described separation and purification and has been present in for example chromophore of bovine serum albumin and poly-L-Lysine of brown polypeptide.Chromophore 2-(2-furanylcarbonyl)-4 (5)-2 (furanylcarbonyl)-1H-imidazoles (FFI) are the deutero-conjugation heterocycles of amino condensation in 2 molecule glucoses and 2 lysine sources.This patent further describes the method that FFI is used for detecting " wearing out " (degree of advanced glycosylation) of protein sample, wherein by measuring the above-mentioned chromophoric amount in the described sample, relatively this measurement result and standard substance (" age " proteins associated sample of its FFI content and described sample) then, thus determine described sample " age ".

The long-standing needs of not realizing in the existing therapeutic scheme of diabetics are the effective ways that are used for following purpose: identify to have the diabetics that diabetes dependency disease risk takes place; Prevent, reduce or postpone the generation of this class disease and the interests of determining such therapeutic intervention by therapeutic intervention.

Summary of the invention

The present invention comes from and has found to relate to the metabolic pathway that enzyme mediation property makes FL be converted into FL3P and produces higher concentration 3-deoxyglucosone (3DG) at the organ that is subjected to diabetes affects.Further investigation to the biochemical function of this newfound approach subsequently trends towards showing that it has important function in the etiology of diabetic nephropathy.In addition, suspect that this approach may cause taking place various known diabetes dependency diseases.

Find that this discovery has practicality in the present invention, on the one hand, the invention provides a compounds, this compounds has enzyme inhibition activity and effectively suppresses fructose-lysyl oxidase and is converted into fructose-lysine-3-phosphate.By measuring the relevant enzyme inhibition activity of determining The compounds of this invention easily.Described assay method comprises the aqueous solution that fructose-lysine, adenosine triphosphate (ATP), fructose-lysine-3-phosphate kinases source and The compounds of this invention are provided, and wherein the amount of The compounds of this invention is enough to confirm that it suppresses active; Make the solution that is obtained accept to promote as the fructose-lysine-3-phosphate of above-mentioned kinases, fructose-lysine and the interactional product of adenosine triphosphate and the condition of adenosine diphosphate (ADP) formation; And the output that detects at least a such product, the aqueous solution that does not add The compounds of this invention with fructose-lysine, adenosine triphosphate and the fructose-lysine-3-phosphate kinases source of same amount is compared, and The compounds of this invention has reduced the amount of this class product.Just now the assay method of Miao Shuing also belonged to category of the present invention.

According to a further aspect, the invention provides the pharmaceutical formulation that is used to prevent, reduce or postpone diabetics generation diabetic complication, said preparation comprises as the invention described above chemical compound of active component and pharmaceutically acceptable carrier.

According to one side more of the present invention, prevention, reduction are provided or postpone to have the method for the diabetics generation diabetic complication of diabetic complication occurrence risk, this method comprises that giving described patient suppresses the effective dose The compounds of this invention that fructose-lysyl oxidase is converted into fructose-lysine-3-phosphate.This method can be used for preventing or treats the similar morbid state of other cause of disease, sees for details hereinafter.

According to another aspect of the present invention, the invention provides the method for the risk of estimating diabetics generation diabetes dependency disease.This method comprises the saccharifying lysine residue source that gives the amount that described patient produces scheduled volume saccharifying lysine residue; With the 3-deoxyglucosone of normal study subject (being non-diabetic study subject or the study subject that does not have the diabetes clinical symptoms) and the ratio of 3-deoxidation fructose is benchmark, measures the ratio available from 3-deoxyglucosone in described patient's the biological sample and 3-deoxidation fructose.Compare with asymptomatic study subject, the ratio height of 3-deoxyglucosone in the diabetics sample and 3-deoxidation fructose illustrates that the risk of diabetics generation diabetes dependency disease is higher.

The present invention also provides the method for estimating the effectiveness of therapeutic intervention in preventing diabetic complication.This method comprises that detection is before the begin treatment intervention and afterwards available from the concentration of the 3-deoxyglucosone in the biological sample of diabetics, 3-deoxidation fructose and fructose-lysine.3-deoxyglucosone in the more described then sample and 3-deoxidation fructose concentration and with the concentration of fructose-lysine.With detect in the biological sample of before the begin treatment intervention, gathering with respect to the 3-deoxyglucosone of fructose-lysine concentration and 3-deoxidation fructose concentration with compare, this concentration and reduction in the biological sample of gathering after the begin treatment intervention illustrate that therapeutic intervention is effective.

Further aspect of the present invention provides and informs that diabetics may cause taking place the method for the food of diabetes dependency disease.This method comprises the content that detects the saccharifying lysine residue in the food, and this information is for example being informed diabetics in the packaging for foodstuff or on the public publication that diabetics uses.

According to the present invention, find biological sample for example the 3DF level in the urine raise and diabetic complication risk significant correlation take place.Therefore, another embodiment of the present invention provides the method for the risk of estimating diabetics generation diabetes dependency disease, this method is based on being scheduled to baseline values with reference to one or more 3DF, and whether the measurement result that will be present in the 3DF in the diabetics biological sample index of the probability of diabetic complication takes place as described patient.

Another aspect of the present invention comprises the method for the sensitivity that reduces patient's pair cancer relevant with the glycated protein of ingesting.This method comprises and gives Pharmaceutical composition, and said composition contains and fructose-lysyl oxidase is converted into fructose-lysine-3-phosphate has the active reactive compound of inhibition.The present invention also comprises prevention, reduces or postpones that AGE-albumen takes place and forms the method for cancer that causes.This method comprises that the inhibition that gives therapeutic dose produces the medicine of 3-deoxyglucosone.

Means as the molecular mechanism of the further evaluation vicious transformation relevant with containing the glycated protein meals, be provided at the susceptible animal subject and induce method for cancer, this method comprised with the glycated protein meals feed enough time of described animal, made that the 3-deoxyglucosone level in the biological fluid raises 3 times at least.Estimate this class animal with respect to non-processing control animal.

Also provide screening to influence the method for the material of cancer generation according to the present invention.Make biological fluid 3DG level raise at least 3 times by feed animal subject glycated protein meals, thereby bring out cancer.Animal is divided into two groups then, wherein a treated animal is accepted chemical compound to be evaluated, and another treated animal is as negative control.Put to death two treated animals after the suitable time, estimate the carcinoma that two treated animals exist and/or do not have.

At last, in another embodiment of the invention, the screening prevention is provided, reduces or postpones the method for the material of generation cancer.This method may further comprise the steps: with glycated protein feedstuff feed susceptible animal subject, 3-deoxyglucosone (3DG) content that the amount of the described feedstuff of feed and time are enough to keep biological fluid is different from the 3DG content that feed is substantially free of the similar susceptible animal subject of glycated protein feedstuff.To be tried material then and give a part of described animal subject, and another part animal is not tried material.Animal is put to death in the back again, and relatively the tissue slice of the susceptible animal subject of each described part is estimated the described effect that is tried material.

The accompanying drawing summary

Fig. 1 illustrates the initial step that the rapid reaction of the proteic multistep of AGE-that produces irreversible modification comprises.

Fig. 2 illustrates the reaction that lysine recovery approach comprises.

Fig. 3 is the sketch map of urine distribution form, illustrates time dependent 3DF, 3DG and the FL of ingest 2gFL and 24 hours body one by one of continuous monitoring.

Fig. 4 is 7 the time dependent sketch maps of volunteer's secretion of urine 3DF of 2g fructose lysine of ingesting.

Fig. 5 is for control animals with the 3DF of the experimental group that contains 0.3% glycated protein meals of keeping ingesting and the comparison diagram of N-acetyl group-beta-amino glucosidase (NAG).

Fig. 6 illustrates and ingests control feed or be rich in the rat urine 3DF of glycated protein feedstuff and the linear relationship between the 3DG level.

The horizontal 3DG of fasting of Fig. 7 A and 7B diagram normal individual and diabetics urine is to the mapping of the horizontal 3DF of fasting.

Detailed Description Of The Invention

Providing to give a definition helps to understand the present invention, and details are as follows:

1. saccharification lysine residue-statement used herein " saccharification lysine residue " refers to the stable adduct of reduced sugar and the modification lysine residue that contains lysine albumino reaction generation.

Most albumen lysine residue is positioned at protein surface as the positive charge amino acid of expection. Therefore, with albumen that serum or other biological fluid contact on lysine residue can with solution in the glycan molecule free responding. There are a plurality of stages in this reaction. Starting stage comprises that lysine free amine group and the ketone group of sugar form Schiff alkali. Then this head product is reset through Amadori and is produced stable ketoamine compound.

This serial reaction occurs in available multiple sugar. When the sugar that relates to is glucose, initial Scchiff alkali product will participate in aldehyde part on the C-1 of glucose and the imines between the lysine epsilon-amino forms. Amadori resets and forms lysine, the 1-deoxidation-1-(epsilon-amino lysine)-fructose (this paper is called fructose-lysine or FL) that is connected with fructose C-1 carbon.

Available other aldose for example galactolipin carries out similar reaction (Dills, Am.J. Clin.Nutr., 58:S779 (1993)) with ribose. For purpose of the present invention, no matter the accurate structure of modifying glycan molecule how, the connotation of saccharification lysine residue comprises the early stage product of the epsilon-amino residue reaction of any reduced sugar and protein lysine.

In addition, term saccharification lysine residue, glycated protein and glycosylated protein or glycosylation lysine residue can use in this article alternately, and this is consistent with current usage in the common mutual Scientific Magazine that uses such statement.

Fructose-lysine-term " fructose-lysine " (FL) this paper be used to refer to any saccharification lysine, no matter mix the saccharification lysine of albumen/peptide or the saccharification lysine that discharges from albumen/peptide by proteolytic digestion. This term is not particularly limited in the chemical constitution that is commonly referred to fructose-lysine, it is reported that albumen lysine residue and glucose response form fructose-lysine. As mentioned above, lysine amino can react with various sugar. In fact, a report points out that glucose is active minimum sugar (Bunn etc., Science, 213:222 (1981)) in one group 16 kinds (16) different test sugar. Therefore, with glucose seemingly, no matter where mention term fructose-lysine at this specification, include Tagatose-lysine that galactolipin and lysine form, comprise equally all other sugared condensation products, no matter described sugar is natural sugar or synthetic sugar. From this specification as can be known, the reaction of albumen-lysine residue and sugar comprises a plurality of reactions steps. The final step of these reaction series comprises protein-crosslinking and produces the multi-cluster material that is called AGE-albumen that wherein Cucumber is fluorescent material. This class modified protein of proteolytic digestion can not produce and the covalently bound lysine of glycan molecule. Therefore, when the use herein of " fructose-lysine ", the connotation of this term does not comprise the material of these types.

3. fructose-lysine-3-phosphate-by forming this compound from ATP enzyme transfer energy-rich phosphate base to FL. Term fructose-lysine-3-phosphate used herein (FL3P) means to comprise the phosphorylation fructose that all can enzyme formation-lysine part, and is that no matter dissociate or protein bound.

4. fructose-lysine-3-phosphate kinases-this term refers to, as above definition can make the FL enzyme be converted into one or more albumen of FL3P when the energy-rich phosphate source is provided in addition.

5.3-deoxyglucosone-3-deoxyglucosone (3DG) is 1,2-dicarbapentaborane-3-desoxysugar (being also referred to as 3-deoxyhexamethylose aldehyde ketone), forms the 3-deoxyglucosone when cracking FL3P produces free lysine and inorganic phosphate. For the object of the invention, all possible dicarbapentaborane sugar that term 3-deoxyglucosone forms when comprising cracking FL3P, FL3P has widely definition as mentioned above.

Be present in human kidney 6.FL3P lysine reclaims approach-lysine recovery approach, also may be present in other tissue, it is regenerated as the unmodified lysine of free amino acid or the unmodified lysine that polypeptide chain is mixed in regeneration. Explain further to see below that this approach is a key factor that causes diabetic complication.

7.AGE-albumen-refer at Scientific Magazine and term used herein " AGE-albumen " (advanced glycosylation end products modified protein) dead end product (Brownlee of sugar and albumino reaction, Diabetes Care, 15:1835 (1992) and Niwa etc., Nephron, 69:438 (1995)). Obviously, for example albumen lysine residue and glucose response can not stop along with the formation of fructose-lysine. FL can stand a plurality of dehydrations and rearrangement reaction produces non-enzymatic 3DG, and 3DG reacts with free amine group again, causes the crosslinked and brown of relevant albumen. In fact, suitable evidence shows that more than the 3DG of definition is the center intermediate in this modification reaction.

9. " saccharifying meals "-this statement used herein is meant wherein any specific meal of glycated protein instead of part normal protein.Statement " saccharifying meals " and " glycated protein meals " can be used in this article alternately.

To small part, also may all diabetic complications be because due to glucose and other the active sugared covalent modification albumen.M.Brownlee，Diabetes，43：836(1994)。As mentioned above, confirmed the normal individual height of sugar-modified protein concentration of diabetic and animal.In fact, the proteic increase of diabetes dependency AGE-is higher than the increase of blood sugar level.

In the past, people generally believed that 3DG derived from the albuminolysis that contains the saccharifying lysine residue in the body.Generally believe that also these saccharifying lysines can not be used as origin of amino acid.By hereinafter as can be known, this viewpoint of believing in the past is wrong.

10. " susceptible animal subject "-this statement used herein is meant because there is the higher laboratory animal strain of tendentiousness of its vicious transformation of some genetic mutation and formation tumor.In research as herein described, use the Eker rat of epiloia gene (Tsc-2) sudden change.Persons skilled in the art are known high other experimental rat or the mouse species of tendentiousness that forms tumor.Term " similar susceptible animal subject " is meant with the similar animal of genetic background that compares non-processing animal.

As mentioned above, the present invention results from the discovery to former unknown metabolic pathway, and this approach produces 3DG with enzymic catalytic reaction.Can this enzyme approach of enzyme inhibition, therefore reduce the generation of toxicity 3DG.

In a series of research process to the diabetes kidney, the perchloric acid extraction thing of the diabetes rat kidney that streptozotocin (streptozotoxin) brings out ³¹P NMR spectral detection shows the unusual new peak of NMR spectrum.Present inventor's former studies confirm that exists fructose-3-phosphoric acid (A.Petersen etc., Biochem.J., 284:363-366 (1992) in rat lens and human red blood cell; Lal etc., Arch.Biochem.Biophys., 318:191 (1995); Szwergold etc., Science, 247:451 (1990) and Lal etc., InvestigativeOpthalmology and Visual Science, 36 (5): 969 (1995)).Other extraordinary phosphorylation sugar (Szwergold etc., Diabetes, 44:810 (1995) and Kappler etc., Metabolism, 44,1527 (1995)) has been identified in early stage research in rat lens.Therefore, have adequate cause to think, the peak of this new evaluation is another kind of phosphorylation sugar.Further deep experimentation shows that this noval chemical compound is not a kind of simple sugar, but at the fructose-lysine of 3 phosphorylations of fructose component.

Confirmed this evaluation with two kinds of methods.With the synthetic real fructose-lysine-3-phosphate (FL3P) of following embodiment 2 disclosed methods, be presented at ³¹Resonate with diabetes rat kidney peak in the P NMR spectrum.Also acrose-lysine is injected into the non-diabetic rat.The FL3P level of these rat kidney of injection back significantly raises.

Carried out 2 experiments with confirm FL3P with enzymic catalytic reaction directly derived from FL.Synthesize with the fructose-lysine of deuterium, and it is injected into rat at 3 labellings of fructose partial C.Injected back 3 hours, and took out kidney, extract with perchloric acid from these rats.NMR spectroscopy shows, separates and contains deuterium-labeled from the FL3P of these rats material 3 of fructose portion C.In addition, the rat kidney tissue homogenate is presented in the reaction that needs ATP and fructose-lysine simultaneously and can produces FL3P.There is specificity FL3P kinases in the experiment confirm of mentioning at last, because do not form FL3P during with fructose lysine and ATP incubation together following of physiological condition.The further experiment that relates to kidney cortex part confirms that this kinase activity is not to be uniformly distributed in kidney, but concentrates on the renal tubules proximal region, and this district is one of region of anatomy that confirms the earliest human and animal's diabetes kidney damage.

The FL3P aqueous solution is unsettled.It is degraded formation 3DG, lysine and inorganic phosphate fast.This reaction also takes place in the body.Still do not know that at present the FL3P vivo degradation is abiogenous reaction or enzymic catalytic reaction.Yet, suspect that very enzyme catalysis participates in degraded, because produce 3DG by fructose-lysine very fast at intact kidney.

Fig. 2 illustrates FL3P lysine and reclaims the approach reactions steps.In the first step, fructose-lysine and ATP react in the kinase catalytic reaction of FL3P and form fructose-lysine-3-phosphate (FL3P) and ADP.The 3-position phosphorylation of fructose part makes fructose lysine molecule instability.The F3LP of Huo Deing is decomposed to form the free recycling lysine of 3-deoxyglucosone (3DG), inorganic phosphate and unmodified then, and lysine can be used for protein synthesis.Aldehyde reductase makes 3DG be reduced to 3-deoxidation fructose (3DF), thereby makes the 3DG detoxification, and 3DF is secreted in the urine.

Although it is this approach of fructose-lysine that Fig. 2 illustrates with the most general saccharifying lysine, it is evident that very that for those skilled in the art various similar molecules can be through this approach reaction.In fact, see below and explain in detail, the substrate selection that FL3P lysine reclaims approach is very widely, guarantees the extensive definition of above given term.

Other experiment shows that lysine recovery approach sees various animals, comprises sheep, pig, Canis familiaris L., rabbit, cow, mice and chicken.This approach also is present in the mankind.Known lysine is a kind of essential amino acids, and its concentration at most of foods is lower, so can think that FL3P lysine recovery approach is that omnipresence exists.In addition, the lysine residue of significant proportion exists with glycosylated form in the food, will increase the ratio of this modification lysine during cook food.Because these saccharifying lysine residues can not be used for synthetic protein,, and provide selection advantage for the organism that has this approach so lysine recovery approach has great practical value.

Diabetes have 2 effects to lysine recovery approach.Separation from the saccharifying lysine concentration of the blood protein of diabetics than a non-diabetic height.So compare with the non-diabetic individuality, must make more lysines of diabetics enter lysine recovery approach.In addition, according to the preliminary observed result of 3DG in diabetics and the normal individual urine and 3DF ratio as can be seen, diabetics reduces the ability of 3DG detoxification by this approach.These two factors make diabetics urine 3DG concentration increase (to see Fig. 7 together; In addition referring to Lal etc., Arch.Biochem.and Biophys., 342 (1): 254-60 (1997)).

Except kidney,, especially in erythrocyte, crystalline lens and the peripheral nervous tissue, also identified and participated in the factor that lysine reclaims approach at other tissue.Diabetic complication involves all these tissues.For example diabetic retinopathy is relevant with the diabetes microvascular complication in the erythrocyte location, and the kidney location is relevant with diabetic nephropathy, and the peripheral nervous location is relevant with the diabetes peripheral neurophaty.These factors also see pancreas.Experimentizing to determine that there is situation in these factors in skin.Be present in skin if find these factors, then think to prevent the suitable carrier topical therapeutic of available inhibition chemical compound of the present invention collagen cross-linking, and improve skin elasticity thus, thereby alleviate its ill-effect.

The experiment of carrying out often proves, human not only produces 3DG but also produce 3DF by the per os albumen that contains the saccharifying lysine residue of ingesting.Below these experiments of Xiang Ximiaoshuing confirm convincingly, and the mankind exist lysine to reclaim approach.A perplexing phenomenon has also been illustrated in these experiments, and promptly some diabetics produces diabetic complication, even and another part diabetics glycemic control is very bad, such complication can not take place.Know the reason of this phenomenon according to data provided herein.The 3DG detoxification ability of diabetics has nothing in common with each other.Part of diabetes mellitus crowd's aldehyde reductase activity seems than most of glycosuria patient groups' height.Therefore, these individualities can be by making the 3DG detoxification that is higher than normal level effectively, thereby increase the amount that lysine reclaims approach that enters.The impaired patient of other function not quite can make the horizontal detoxification of 3DG of its rising, and the risk that therefore diabetic complication takes place is higher.

The more detailed description that sees below, experiment confirm can stimulate lysine to reclaim approach by using the glycated protein meals.Identical with above-mentioned FL situation, observe FL3P, 3DG and 3DF raises at the animal subject of feed glycated protein meals.

Inhibitor compound block lysine of the present invention reclaims approach, prevents to form toxicity 3DG by FL3P.

Following is that the explanation enzyme inhibitor is applicable to that implementing of the present invention one overlaps complete standard, and determines any some experiment whether inhibitor meets these standards of inferring.Be applicable to that candidate's inhibitors of kinases of the present invention can be the natural product that separates from plant or microorganism.Perhaps they can be the synthetic molecules that obtains according to the correct understanding to enzyme reaction and its mechanism.The also method synthetic inhibitor of available associating.Can produce by starting point at random and to mix the library.And, available integrated processes preparation and the relevant all cpds of identifying in the past of FL3P kinase target inhibitor.

Regardless of the source of inferring inhibitor, can not think that the chemical compound that does not meet the standard of enumerating below all is can suppress lysine to reclaim approach, prevents, reduces or postpone to take place the useful medicine of diabetic complication or relevant etiology disease thus.

1. described inhibitor should be micromolecule and be easy to by cellular uptake.In order to satisfy this standard, the molecular weight of described inhibitor must be less than 2,000 dalton, and about 1,000 or littler better.

2. the necessary competitiveness of described inhibitor, noncompetitive, irreversibility or suicide inhibition FL3P kinases.If described inhibitor is competitiveness or noncompetitive inhibitor, then suppress constant K _iMust be less than about 1mM.It is desirable to, suppressing constant must be less than 100 μ M, and better is about 40 μ m or lower.If the inhibitory action of described inhibitor is suicide or other unidirectional type, then this suppresses the no practical significance of constant requirement.

3. the necessary water soluble solution of described inhibitor, and in the aqueous solution of physiological pH, be stable.Only the concentration that can be equal to or greater than 10 μ M at described inhibitor or inhibitor salt is dissolved in normal saline or serum, just satisfies the dissolubility requirement.Only it active keeps 50% when above after 1 hour at incubation at the inhibitor solution that is dissolved in 37 ℃ of normal saline, just satisfies this stability requirement.It is desirable to, incubation 1 day or 1 day the above inhibitor activity must keep more than 50%.

4. described inhibitor must have acceptable pharmacokinetics.That is, give that its treatment valid density must be maintained to few 1 hour behind the described medicine.It is desirable to, its valid density should keep 8 hours at least.Better is that being administered once every day just is enough to keep the treatment concentration of described inhibitor.This requirement does not also mean that described inhibitor reaches treatment concentration surely with regard to one after the administration first time.Existing many successful exemplary drugs, just can observe medicinal effectiveness as long as prolong administration this moment.The real meaning of this standard is that in case reach valid density, this valid density can be kept more than 1 hour after the administration the last time.This paper has described the experiment of test therapeutic efficacy.

5. described inhibitor must be nontoxic.This standard-required, this inhibitor does not have toxicity to the mankind when giving therapeutic dose.It is desirable to, when inhibitor its toxicity when the level of blood and/or target tissue doubles the therapeutical effect level not obvious.Better is, when 6 times of inhibitor levels to or when more being higher than therapeutic domain, its toxicity is not obvious.Secular inhibitor for treating just can prevent diabetic complication.So avirulence requires to comprise acute toxicity and chronic toxicity that chronic toxicity may just manifest when the life-time service that prolongs.Adopt the good zooscopy of the setting up toxicity of evaluate candidate molecule easily.In the I clinical trial phase, estimate human toxicity.

Be used to implement isomer and the pharmaceutically acceptable salt that chemical compound of the present invention comprises following formula: compound and described chemical compound:

Wherein X be-NR '-or-O-, R ' is selected from H and straight or branched alkyl (C ₁-C ₄) and replacement or unsubstituted aryl (C ₆-C ₁₀) or aralkyl (C ₇-C ₁₀); R is the substituent group that is selected from following group: H, amino acid residue, polyamino acid residue, peptide chain, straight or branched aliphatic group (C ₁-C ₈) (substituent group that does not replace or contained at least one nitrogen or oxygen replaces), straight or branched aliphatic group (C ₁-C ₈) (substituent group that does not replace or contained at least one nitrogen or oxygen replaces, and by at least one-O-,-NH-or-NR "-the part interruption), R " be straight or branched alkyl (C ₁-C ₆) and the aryl (C that do not replace or replace ₆-C ₁₀) or aralkyl (C ₇-C ₁₀), prerequisite be when X represent-NR '-time, the connected nitrogen-atoms of R and R ' also can be represented to replace or the heterocycle of unsubstituted 5-7 annular atoms together, wherein at least one in nitrogen and the oxygen is the unique hetero atom in the described ring, described aryl (C ₆-C ₁₀) or aralkyl (C ₇-C ₁₀) and described heterocyclic substituent be selected from H, alkyl (C ₁-C ₆), halogen, CF ₃, CN, NO ₂With-O-alkyl (C ₁-C ₆); R ₁It is polyol moiety with 1-4 Linear Carbon atom; Y is a carbonyl moiety

Or hydroxy methylene part Z is selected from-H ,-O-alkyl (C ₁-C ₆) ,-halogen ,-CF ₃,-CN ,-COOH and-SO ₃H ₂And optional be-OH.

" R " substituent example nitrogenous or oxygen comprises the substituent group derived from following material: gamma-amino-Alpha-hydroxy butanoic acid ((CH ₂) ₂-CHOH-COOH), 1,2,4-triamido butane ((CH ₂) ₂-CHNH ₂-CH ₂NH ₃), 3,6-diaminourea-5-hydroxyl enanthic acid (CH ₂-CH (OH)-CH ₂-CH (NH ₂)-CH ₂-COOH) etc.

Formula I structure has asymmetric center, can exist with racemate, racemic mixture and various stereoisomer and their mixture, and all such isomeric forms belong to category of the present invention.

Although some chemical compound with following formula I structure is known before being, but believe that other chemical compound is new, such chemical compound belongs to category of the present invention, and all formula I chemical compounds are used to suppress the purposes that enzyme catalysis in the body produces 3DG and also belong to category of the present invention.

For example glucose, galactose, mannose, ribose, xylose or derivatives thereof and aminoacid or other suitable primary amine or secondary amine reaction produce and have carbonyl moiety and (be the inhibitor of Y=-C (=O)-), thereby make the inhibitor of above structural formula can to make suitable sugar.Perhaps make for example NaBH of the factor that Schiff alkali intermediate is reduced to amine at selectivity ₃CN exists down, can make sugar for example glucose, galactose, mannose, ribose, xylose etc. and amino-or the above-mentioned type reactant reaction of hydroxyl-replacements, and therefore to have alcohol moiety (be the inhibitor of Y=-CH (OH)-) in generation.During use, the active part of amino acid reactants can be amido or amido on the amino acid side chain or the hydroxyl on the alpha-carbon atom.Suitable aminoacid comprises essential amino acids.Instantiation includes but not limited to glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, aspartic acid, phenylalanine, tyrosine, histidine and tryptophan.Other suitable reactants is from the more amino carboxylic acid of wide range of types, for example pyroglutamic acid, Beta-alanine, γ-An Jidingsuan, episilon amino caproic acid etc.In case of necessity, also can use above-mentioned N-acyl derivative of amino acid, for example formoxyl lysine.

Other suitable reactants includes but not limited to not replace or substituted aryl (C ₆-C ₁₀) chemical compound, wherein said substituent group can be alkyl (C ₁-C ₃), alkoxyl, carboxyl, nitro or halogen, the alkane that replaces or replace not, wherein said substituent group can be at least an alkoxyl; Perhaps do not replace or substituted nitrogen-containing heterocyclic compound, wherein said substituent group can be alkyl (C ₁-C ₃), aryl (C ₆-C ₁₀), alkoxyl, carboxyl, nitro or halogen.The example of last group reaction thing comprise m-methyl, p-methyl-, m-methoxyl group, o-methoxyl group-and m-nitro-aminobenzene, o-and p-amino benzoic Acid; N-propylamine, n-butylamine, 3 methoxypropyl amine; Morpholine and piperidines.

The typical inhibitor compound of the following formula A that sees attached list.Can be used as the known compound example of implementing inhibitor of the present invention and include but not limited to meglumine (meglumine), sorbitol lysine and mannitol lysine.

Inhibitor compound described herein can form pharmaceutically acceptable salts with various inorganic or organic acid or alkali.Suitable alkali comprises ammonium salt and other amine salt of for example alkali metal salt, alkali salt, ammonium salt, replacement.Suitable acid comprises for example hydrochloric acid, hydrobromic acid and methanesulfonic acid.

The pharmaceutically acceptable salt of formula I chemical compound can make according to method well known to those skilled in the art.

Available various kinase activity assay method detection compound suppresses the kinase whose ability of FL3P.A kind of useful algoscopy comprise make potential inhibitor with fructose-lysine and ATP incubation under the situation that has renal tissue homogenate or other enzyme source.Preparation detected components solution detects solution and generally comprises 1mM or inhibitor compound of the present invention still less, 1-10mM fructose lysine (FL), 0.1-10mM ATP and be enough to make FL to be converted into the enzyme source amount of fructose lysine-3-phosphoric acid.In the 4.5-9.5pH scope, preferably in neutral pH or near carrying out incubation under the neutral pH.Heated culture temperature should for 4-40 ℃ of enzymatic activity coupling.Preferably under physiological temp, carry out incubation.Behind the incubation, add Acid precipitation albumen cessation reaction, use ³¹P-NMR spectroscopy detects the EL3P that produces.Compare with the control sample that does not contain inhibitor, the sample that contains inhibitor compound reduces or eliminates and produces FL3P.

Other algoscopy is applicable to the fast measuring enzyme inhibition.A kind of such algoscopy comprises application fructose-lysine and gamma marker ³²P or ³³P-ATP.Because FL3P is in conjunction with Dow-1, but ATP and other phosphate of great majority but in conjunction with Dow-1, therefore in predetermined reaction time, be generally 10 minutes after, make and detect solution and product FL3P and remaining reaction mixture are separated by the Dow-1 resin column.The solution adding that produces is equipped with in the container of scintillation solution such as Ecoscint A, counts, with the radioactivity amount of determining to be produced.

Because be difficult to obtain a large amount of tissues, be cloned into expression system such as colibacillary recombinant type kinases so preferably use.Can easily obtain to clone kinases from " shot gun method " clone in tissue specificity cDNA library.This class library obtains from commercial source easily.For example they can derive from Clontech, Palo Alto, CA.It is available that (be positioned at San Diego, λ cloning system California) carries out contemplated shotgun cloning available from Stratagen.This clone's test kit comprises the detailed description of its use.

Pharmaceutical formulation of the present invention comprises as one or more above-claimed cpds of active component and pharmaceutically acceptable mounting medium or adjuvant.

These compositions can be prepared into the various forms that is applicable to administration, comprise liquid and solid form.Therefore, the form of described preparation can be tablet, capsule sheet (caplet), pill or lozenge, and perhaps described preparation can be filled in the suitable vessel, capsule for example, and perhaps if suspending agent, but fill is in bottle." pharmaceutically acceptable mounting medium " used herein comprises all solvents, diluent or other liquid vehicle, dispersion or suspension adjuvant, surfactant, isotonic agent, thickening agent or emulsifying agent, antiseptic, solid binder, lubricant etc., as long as be applicable to the particular dosage form that needs.The representative instance of suitable carrier medium comprises gelatin, lactose, starch, magnesium stearate, Pulvis Talci, plant and animal fat and oil, natural gum, polyglycols etc.Remington ' s Pharmaceutical Sciences, the 15th edition, (MackPublishing Co., Easton PA1975) disclose preparation Pharmaceutical composition various carriers that use and the technology that becomes known for its preparation to E.W.Martin.Unless a certain conventional mounting medium and enzyme inhibitor of the present invention do not match, for example produce any unwanted biological effect or interact in harmful mode, otherwise its application belongs to category of the present invention with any other component of pharmaceutical formulation.

In pharmaceutical formulation of the present invention, the content of described active medicine is at least 5% (weight) of described preparation total amount, generally is not higher than 98% (weight), if any, comprises mounting medium and/or adjuvant.The ratio of active medicine is preferably the 65%-95% (weight) of described compositions.

The auxiliary activity medicine is preferably the chemical compound of 3DG in the coalition.This compounds include but not limited to aminoguanidine, amino benzoic Acid and derivant, cysteine and derivant thereof, the amino imidazoles, 1 that replaces, 1 of the dibasic benzene imidazoles of 2-, replacement, 2,4-triazole, diamino-pyridine and derivant thereof, the amino pyrimidine that replaces, alkamine, two amines etc.Pharmaceutical formulation of the present invention also can comprise the antihypertensive drug as the auxiliary activity medicine, particularly including Angiotensin-Converting (ACE) inhibitor.

If necessary or desired, also can add auxiliary substance in the described pharmaceutical formulation, for example protect described reactive compound to exempt from or help described reactive compound to be absorbed into the chemical compound of blood by stomach acids destroy.Such auxiliary substance can comprise for example chelating agent, for example the borate of partial offset gastric acid condition or other salt etc.Transmit described reactive compound with soap and can strengthen absorption (containing at described reactive compound under the situation of one or more basic functionalities).

Any administered dose and any route of administration that can adopt effective inhibition FL3P lysine to reclaim approach give The compounds of this invention and any auxiliary activity composition together.Therefore, statement used herein " treatment effective dose " but be meant the nontoxic enough dosage of described enzyme inhibitor, this q.s is enough to reach the needs treatment and produces 3DG with the metabolic that prevents diabetic complication or suppress due to other medical reasons, for example alleviates aging effect or AGE-albumen and forms and have other human disease states of pathogenic effects.The exact dose that needs can have nothing in common with each other, and depends on patient's type, age and general situation, complication character, concrete enzyme inhibitor and its administering mode etc.

The compounds of this invention preferably is mixed with the dosage form that is easy to administration and dosage homogeneous.Dosage unit form used herein is meant the physics individual of the enzyme inhibitor that is applicable to patient to be treated.Each dosage should comprise to be estimated itself or can produce the amount of active substance of the treatment effect of needs with selected pharmaceutical carrier medium.Usually the dosage unit that gives The compounds of this invention contains about 1mg-2, and the described chemical compound of 500mg (in described weight of formulation) preferably contains about 5mg-250mg.

The character of Zhi Liao diabetic complication as required, The compounds of this invention can be oral or parenteral give, parenteral gives for example to be intramuscular injection, peritoneal injection, venoclysis etc.For the treatment effect that obtains to need, the dosage level that oral or parenteral gives The compounds of this invention is the about 0.7 μ g-20mg of per kilogram weight in patients every day, is preferably about 30 μ g-3.5mg, once a day or repeatedly.

Special preferred oral organized enzyme inhibitor, prerequisite is that oral dose can make inhibitor reach effective treatment level at blood and/or target tissue.Those skilled in the art can easily measure the micromolecular inhibitor level in Deproteinization blood, kidney and other target tissue sample.Can compare inhibitor concentration and the predetermined constant that suppresses in these samples.Tissue water Pingyuan County lacks therapeutic activity far below suppressing constant declaration.If the irreversibility inhibitor can confirm by the FL3P kinases level of measuring respective organization or refute to lack such therapeutic activity.In all cases, can be rich in the food of saccharifying lysine residue or fructose-lysine by the mankind or animal subjects are ingested, and detect 3DG and 3DF amount in its urine before ingesting and after ingesting, estimate therapeutic activity.Compare with secretion level before the same subject inhibitor for treating, the experimenter who has the therapeutic activity inhibitor in its system secretes 3DG and 3DF all reduces, and the fructose of secretion of urine-lysine increases, and sees for details hereinafter.

According to selected definite inhibitor, give The compounds of this invention usually once a day or reach as high as every day 4-5 time.Although preferred dosage regimen once a day makes diabetics get used to keeping a close eye on its morbid state, acceptant so if necessary more frequent dosage regimen is so that transmit above-mentioned daily dose.But the accurate scheme that gives chemical compound described herein and compositions must depend on needs, the type of being treated and the attending doctor's of the individual patients for the treatment of judgement.Term used herein " patient " comprises the human and animal.

Inhibitor compound described herein can be used for resisting diabetic complication, and being particularly useful for resisting influences the diabetics more than 40% and be the diabetic nephropathy of main cause that needs the end stagerenaldisease of dialysis and renal transplantation.In addition, these inhibitor can be used for preventing or treat AGE-albumen forming other disease that causes, for example old and feeble effect of hypertension, apoplexy, neurodegenerative disease such as Alzheimer type alzheimer disease, circulatory diseases, atherosclerosis, osteoarthritis, cataract and senile general.

Preliminary experiment shows that the glycated protein of ingesting for a long time stimulates lysine to reclaim approach health is caused serious adverse effect.Identical with the situation of FL, observe FL3P, 3DG and 3DF raises at the animal subject of feed glycated protein meals.See Table B.Through after 8 months such meals, observe glycated protein meals animal and to be similar to the obvious nephropathy of seeing at diabetes kidney evidence of science, see hereinafter embodiment 10 for details.Also observe 3DG and the instantaneous rising of 3DF level in the urine at the proteic human volunteer of a small amount of saccharifying of ingesting.

Table B glycated protein % FL3P concentration (nM, kidney) 3DG/3DF concentration (μ M, blood plasma)

0??????????????????97??????????????1.4/0.05

1??????????????????295?????????????-

2.5????????????????605?????????????-

5??????????????????937?????????????-

10?????????????????1066????????????3.6/0.12

20?????????????????1259????????????5.2/0.14

30?????????????????1267????????????6.2/0.28

Because stimulate newfound lysine recovery approach to cause systemic 3DG level significantly to raise, whether gestation is produced obviously influence so determine the saccharifying meals.The results suggest of Huo Deing so far, this approach has very strong effect, sees embodiment subsequently.

In addition, well-known is that the meals that rat and mice susceptible strain early stage (wean back) are supplied with have appreciable impact to type 1 diabetes, sickness rate 10-90%.At nearest 10 years, people carried out more research to this phenomenon.Referring to for example, Diabetes, 46 (4): 589-98 (1997) and Diabetes Metab.Rev., 12 (4): 341-59 (1996), and the list of references of wherein quoting.Part present inventor extremely induces the meals of diabetes to study with regard to two kinds by two.(Dyets, the onset diabetes rate that Inc.) causes is minimum, and 3DF/ inosine ratio minimum in the observation of carrying out (1.0) in the urine for AIN-93.The onset diabetes rate that Purina500 causes is the highest, and 3DF/ inosine ratio raises 2.5 times in the urine.Owing to observe FL3P, 3DG and 3DF at pancreas in rat, the metabolite of fructose lysine kinases and this metabolic pathway may participate in the generation of type i diabetes.The immune system abnormality of this type diabetes susceptible animal (the useful model of human insulin's dependent diabetes mellitus or type i diabetes), make it to coming across the unknown antigen sensitivity of pancreas beta cell, cause of the autoimmune sexual assault of described animal autoimmune system its beta cell.Cause pancreas to destroy subsequently, therefore make animal can not produce insulin.It is well-known that 3DG and albumino reaction can produce new antigen site.Therefore, it seems that the antigenic property source of various meals is that pancreas decomposes the 3DG that fructose lysine-3-phosphoric acid produces.

In addition, react with amine,, and have mutagenesis and oncogenic potential and cross-linked proteins so it can interact with DNA because known 3DG is general.

Discovery FL3P lysine recovery approach makes can effectively distinguish diabetic population first, and can determine that diabetic complication may take place the diabetics of what subgroup.Can carry out this detection easily to experimenter's biological fluid such as urine, blood constitutent (especially blood plasma or serum), lymph fluid, interstitial fluid etc.

After the overnight fast, the ingest food of the saccharifying lysine residue that contains higher concentration of human experimenter.For example this food form can be the suitable source of cheese/sugar " cake " (embodiment 5 that sees below is described) or some other saccharifying lysine or acrose-lysine.When use contained the albumen of saccharifying lysine residue, the saccharifying lysine content was preferably the amino acid whose 0.02-10% of total protein, or more preferably about 0.2-0.4%.The total amount of the saccharifying lysine residue of oral dose should about 0.3g.Be preferably in before the saccharifying lysine source of ingesting, other suitable time of 1,3 and 5 hour afterwards or each clinical setting permission collects urine sample.

Detect the 3DG and the 3DF level of urine sample, calculate the ratio of 3DG and 3DF metabolite.The concrete grammar that uses in this detects is learned implementing the present invention optional.Need, can use following examples 5 described GC methods.Perhaps can use colorimetric method or immunoassay, this will be apparent to those skilled in the art.

Obviously, the main hazard factor that diabetics is faced is a glycemic control, and DCCT completely recently clearly confirms this point.But, only can not explain the generation of diabetic complication with blood sugar level; Relatively as seen diabetic complication incidence rate and historical blood sugar level are quite dispersive.

The method of determining the diabetes subgroup crowd that generation diabetic complication risk is the highest is an aspect of particular importance of the present invention.This method comprises before the saccharifying lysine source of ingesting and the back Best Times of ingesting is measured FL, 3DG and 3DF level.

For example, the urine 3DG/3DF ratio of normal subjects's fasting is about 0.025, and this ratio of diabetics is higher, can be up to 5 times even higher.The data acknowledgement of Fig. 7 this point, Fig. 7 shows that the normal person's of blood glucose amount 3DG/3DF ratio is 0.025 (1/39.77), scattering around this value is very tight, and the average ratio of diabetics (meansigma methods 0.069) increases more than 2 times, and is also much bigger around the scattering of this meansigma methods.

The present invention confirms that the 3DG that diabetics produces increases.So the antagonism diabetic complication needs to remove highly effectively this toxic metabolites.The 3DG/3DF ratio that calculates with methods described herein makes people can estimate the efficient of 3DG detoxification pathways.The individuality of low ratio generally is not easy to take place diabetic complication.The risk of higher rate individuality (comprising the individuality of ratio in normal range) is higher, and that ratio is higher than the risk of individuality generation diabetic complication of normal range is high especially.

Recently the blood plasma of 4 kinds of different rat strains and the measurement result of urine fructose lysine (FL) are confirmed that the mode of its corresponding kidney processing blood FL makes a variation very big.In fact, according to the ratio of FL and its metabolite and inosine, in 2 strains (Long Evans, Brown Norway) in 4 strains, filterable all FL of kidney appear in the urine.According to filtering inosine relatively, other 2 strains (Sprague Dawley, Fischer) in, 10-20% plasma F L appears in the urine.These measurement results are pointed out forcefully, and the variability that the mammal kidney is handled FL is big.If known rodent is identical with human kidney's function, then has adequate cause to think, the similar variation of handling FL is present in the mankind.Because FL mainly enters fructose lysine and reclaims approach,, cause in the kidney part and whole machine body produces high-caliber 3-deoxyglucosone (3DG) so whole approach may be subjected to obvious stimulation the human individual who is absorbed lot of F L by ultrafiltrate.This observed result can be used as the basis of diagnostic test, and wherein blood plasma that obtains more simultaneously or blood serum sample and urine sample determine that FL enters the amount of kidney and further appears at part in the urine.This ratio is starkly lower than 1 individuality and has the risk that various kidney pathology take place so, and described kidney pathology includes but not limited to diabetic nephropathy, senile renal failure and renal carcinoma.

Utilization is reclaimed the treatment effectiveness that the test of approach can handy and safe ground mensuration inhibitors of kinases of the present invention to lysine.Testing scheme is identical with the above scheme that just provides, exception be except detecting urine 3DG and 3DF level, also to detect urine fructose lysine level.Before beginning FL3P kinase inhibitor for treating and to carry out this test afterwards all be useful.With the urine 3DG and the 3DF horizontal summation of each time point, and compare with fructose-lysine level of measuring with same sample.

The activity that lysine reclaims approach causes ingesting, and 3DG and the 3DF level in the urine reaches peak value after the food that is rich in the saccharifying lysine residue.The concentration ratio of these metabolite and unreacted fructose-lysine (for the normal components of human urine) reflects the activity of this approach.Suppress lysine recovery approach the amount of draining 3DG and 3DF is reduced, and the drainage level of fructose-lysine increases.Therefore, behind begin treatment, the reduction of detection (3DG+3DF)/fructose-lysine ratio is the treatment effectiveness of inhibitors of kinases quantitatively.It should be noted that volume of urine or metabolite concentration are not the factors of explaining this mensuration, because only consider metabolite ratio.

According to above disclosed content as can be known, the per os food that contains high concentration saccharifying lysine residue of ingesting causes producing kidney and serum 3DG.Should warn nephropathy risk individuality for example diabetics avoid these high concentration food.Can in all sorts of ways and detect saccharifying lysine residue concentration.Hereinafter embodiment 4 has described a kind of such measuring method.Yet, the assay method that exemplifies below any suitable measuring method of accurately measuring saccharifying lysine residue level all can replace.The assay method example of specific design includes but not limited to colorimetry and immunization.

No matter used assay method how, measure the saccharifying lysine residue content of food prepared therefrom and the risk individuality of these measurement results being informed the disorder of generation renal function, make and all belong to category of the present invention for this class individuality high food of saccharifying lysine content of avoiding ingesting.

Provide following examples to describe the present invention in more detail.Provide these embodiment only to be used to illustrate purpose of the present invention, must not be construed as limiting the invention.Except as otherwise noted, otherwise all temperature among the embodiment be degree centigrade.

Embodiment 1

The separation of FL3P and evaluation

The perchloric acid extraction thing of diabetes rat kidney ³¹P NMR the analysis showed that the resonance of new sugared monophosphate is 6.24ppm, does not observe such result at non-renal tissue, and obviously lower in the level of non-diabetic kidney.Make eluent described extract of chromatography on the microcrystalline Cellulose post with 1-butanols-acetic acid-water (5: 2: 3), isolate the chemical compound relevant with the resonance that is observed.Measuring its structure with proton 2D COSY is fructose-lysine 3-phosphoric acid.Inject animal (Finot and Mauson, Helv.Chim.Acta, 52:1488 (1969)) by FL afterwards, and show that direct phosphoric acid turns to FL3P, has confirmed this point with preparation as mentioned above.Confirm that with the deuterated FL of position-3 specificity the phosphoric acid position is a carbon-3.This can be by analyzing connection and disconnected ³¹P NMR spectrum carries out.Normal P-O-C-H connects that to produce J value be that the FL3P of 10.3Hz is bimodal, and P-O-C-D does not connect, and generation connection and disconnected unimodal, and is identical with 3-deuterate FL3P finding.The unique property of FL3P is, when handling with sodium borohydride, it is converted into 2 new resonance 5.85 and 5.95ppm, and they are with respect to mannitol-and sorbitol-lysine 3-phosphoric acid.

Embodiment 2

FL3P's is synthetic

With 50ml MeOH backflow 1mmol dibenzyl-glucose 3-phosphoric acid and 0.25mmol α-carbobenzoxy group-lysine 3 hours.With 100ml water dilute solution, with the pyridine form the Dow-50 post (2.5 * 20cm) go up chromatographies, at first water (200ml), use 600ml buffer (0.1M pyridine and 0.3M acetic acid) eluting then.When water washing end, buffer washing beginning, wash out target compound.Under 20psi hydrogen, remove carbobenzoxy group and benzyl blocking groups with 5%Pd/C, obtain the FL3P of 6% productive rate.

Embodiment 3

Algoscopy by FL and ATP enzyme generation FL3P and screening inhibitor

Begin to use ³¹P NMR confirms the kinase activity of kidney cortex.Contain 150mM potassium chloride, 5 mM DTT, 15mM magnesium chloride with 9ml, the fresh Ren sus domestica cortex sample of 50mM TrisHCl homogenate 3g of pH7.5.With 10, centrifugal 30 minutes of 000g, then with supernatant with 100, centrifugal 60 minutes of 000g.Add ammonium sulfate to 60% saturation.In 4 ℃ after 1 hour, centrifugal collecting precipitate is dissolved in it in former buffer of 5ml.With the 2ml equal portions of this solution and 10mM ATP and 10mM FL (pressing the foregoing description 1 preparation) in 37 ℃ of incubations 2 hours.With 300 μ l perchloric acid quenchers reaction, centrifugal removal albumen is in SephadexG10 (desalination on 5 * 10cm) posts. ³¹P NMR analyze reaction mixture detects the FL3P that forms.

According to thus obtained kinase activity evidence, carry out radiological measuring.Utilize FL3P not design this mensuration in conjunction with the Dow-1 anion exchange resin.Found its this characteristic when attempting to separate FL3P.Because most phosphates is in conjunction with this resin, thus suspect whole chemical compounds that a large amount of and ATP react and any excessive ATP can in conjunction with, FL3P is stayed in the solution.The first step is to determine to remove the amount of resin that the ATP in this mensuration needs.Described mixture is sucked in the 0.9ml aqueous suspension of 200mg Dow-1, vortex is also centrifugal with potting resin, thereby finishes this step.Draw the 0.8ml supernatant thus and be added on the fresh dried resin of 200mg vortex and centrifugal.Draw 0.5ml volume supernatant and add among the 10ml Ecoscint A, and counting.The residue counting is 85cpm.This step is used for described mensuration.To be dissolved in homogenate buffer again in 4 ℃ with the precipitate that 60% ammonium sulfate precipitation cortex homogenate crude product obtains.This is determined at 0.1ml 50mM TrisHCl, contains 10mM γ among the pH7.5 ³³P-ATP (40,000cpm), 10mMFL, 150mM KCl, 15mM magnesium chloride, 5mM DTT.With 1,2 and 4mg albumen triplicate measure 30 minutes in 37 ℃ to determine that FL3P produces the relation between speed and the enzyme concentration.Deduct the blank value that does not have FL of operation simultaneously, and record data.It is about 20nmols/hr./mg. albumen that the activity that observes is equivalent to the FL3P synthesis rate.

Embodiment 4

Meglumine and the inhibitory action of different polyhydric alcohol lysines to formation 3-deoxyglucosone

A. general polyhydric alcohol lysine is synthetic

With sugar (11mmole), α-carbobenzoxy group-lysine (10mmol) and NaBH ₃CN (15mmole) is dissolved in 50ml MeOH-H ₂Among the O (3: 2), stirred 18 hours in 25 ℃.With excessive Dow-50 (H) ion exchange resin treatment solution, with the NaBH of decomposing excessive ₃CN.This mixture (liquid and resin) is transferred to Dow-50 (H) post, and (on 2.5 * 15cm), the water thorough washing is to remove excessive sugar and boric acid.With 5% ammonium hydroxide eluting carbobenzoxy group-polyhydric alcohol lysine.The residue that obtains during with evaporation is dissolved in the water-methanol (9: 1), reduces with hydrogen (20psi) with 10% palladium carbon catalyst.Filter and evaporation acquisition polyhydric alcohol lysine.

B. use the embodiment of sorbitol lysine, mannitol lysine and galactitol lysine reduction urine and blood plasma 3-deoxyglucosone

Collected urine 3 hours from 6 rats.Also obtain plasma sample.Peritoneal injection gives rat 10 μ mol sorbitol lysines, mannitol lysine or galactitol lysine then.Regather urine 3 hours, and when finishing in 3 hours, obtained plasma sample.

As the 3-deoxyglucosone in these samples of mensuration as described in following examples 5, and with different volumes to inosine normalization.Sorbitol lysine makes that urine 3-deoxyglucosone on average reduces by 50%, mannitol lysine on average reduces by 35%, and galactitol lysine on average reduces by 35%.Sorbitol lysine makes blood plasma 3-deoxyglucosone reduction by 40%, mannitol lysine reduce by 58%, and galactitol lysine reduces by 50%.

C. use the 3-deoxyglucosone in the meglumine reduction urine

By above-mentioned b) handle 3 rats, exception be peritoneal injection meglumine (100 μ mol), replace above-mentioned lysine derivative.Injected back 3 hours, the average 3-deoxyglucosone concentration in the urine reduces by 42%.

Embodiment 5

The mankind ingest, and FL, the 3DG in the urine and 3DF raise behind the glycated protein

A. preparation contains the food of glycated protein: 260g casein, 120g glucose and 720ml water are mixed obtaining the homogeneous mixture.This mixture is transferred to metal stamper and cooked 68 hours in 65 ℃.Then the cake that obtains is worn into coarse powder (a course powder).

The Kjeldahl method is measured this powder and is contained 60% protein.

B. measure the saccharifying lysine content: prepare the described dry cake mix of 1g by above step a, with 6N HCl back hydrolysis 20 hours.With sodium hydroxide solution the pH value of solution that obtains is adjusted to 18, and is diluted to 100ml.The product furosine that produces with fructose lysine acid hydrolysis measures the fructose lysine content on amino-acid analyzer.The fructose lysine content that records described cake in this way is 5.5% (w/w).

C. embodiment: allow the volunteer take food not contain the meals 2 days of fructose lysine, the food 22.5g of the preparation as described herein of ingesting then effectively accepts the fructose lysine of 2g dosage thus.Collect urine at interval with 2 hours, collected continuously 14 hours, collect last urine in the time of 24 hours.

D. measure FL, 3DG and 3DF in the urine: measure FL with Waters 996 diode arrays through HPLC to the gradient elution system of acetonitrile-sodium acetate-water (6: 2: 92) with Waters C18 Free Amino Acid post and acetonitrile-methanol-water (45: 15: 40), elution rate is 1ml/min.Mark meglumine in quantitatively using.

Measure 3DF with HPLC after making the sample deionization.Use PA1 post (Dionex) and with the 32mM sodium hydroxide with the 1ml/min eluting, in Dionex DX-500HPLC system, analyze.Undertaken quantitatively by the standard curve that obtains every day with synthetic 3DF.

Measure 3DG by GC-MS after making the sample deionization.PBS derivation 3DG with 10 times of excessive diaminonaphthalenes.Ethyl acetate extraction obtains salt-free component, is translated into trimethyl silyl ether with Tri-Sil (Pierce).On the Hewlett-Packard5890 that selects ion monitoring GC-MS system, analyze.Use following temperature program(me) merge the silicon dioxide capillary column (DB-5, carry out GC on 25mx.25mm): 150 ℃ of 250 ℃ of injection ports, initial column temperature keep 1 minute, be increased to 290 ℃ and kept 15 minutes with 16 ℃/minute then.The selected ion monitoring of mark U-13C-3DG in quantitatively 3DG uses and uses.

Fig. 3 is FL, 3DF and the 3DG that a volunteer ingests and produces in the urine behind the glycated protein.Obviously whole 3 kinds of metabolite all appear in the urine fast.Even 3DF and also all slightly risings of 3DG in the urine after 24 hours.

Fig. 4 forms for the 3DF in each member who is subjected to the examination group of 7 people.Being tried example at all sees similar fitgures and distributes.As shown in Figure 4, occurred 3DF behind the bolus injection FL in about 4 hours and drain the peak, even still can see 3DF in 24 hours behind the bolus injection and slightly raise.

Embodiment 6

The feed experiment

The concentration that diabetics is excreted to the N-acetyl group-β-glycosamine enzyme (NAGase) in the urine raises.It is believed that it is the early sign of tubular injury, but not exclusively understand the pathogeny that NAGase raises in the urine.Proposed NAGase excretion in the diabetics urine increase be since diabetes cause the activation of near-end renal tubules lysosome Excreta in the urine is increased rather than cytoclasis due to.

The result that this embodiment obtains shows, all relatively in, experimental group 3DF and NAGase level all raise with respect to matched group.Therefore, feed glycated protein animal is excreted to excessive N AGase in the urine, is similar to the result who obtains at diabetics.Compare with control animal, the NAGase excretion increases about 50% in the laboratory animal urine.Compared with the control, the 3DF in these animal urines also increases 5 times.By Fig. 5 and 6 as seen, urine 3DF is closely related with 3DG.It seems that two kinds of chemical compounds all remove from blood plasma with glomerular filtration rate, and heavily do not absorb.

Embodiment 7

The proteic sds gel electrophoresis of kidney

Two rats are injected 5 μ mol FL or mannitol (with comparing), continuous 5 days every day.Put to death animal, take out kidney and dissected kidneys cortex and medullary substance.Contain 150mM potassium chloride, 15mM magnesium chloride and 5mM DTT with 5 volumes, the 50mM TrisHCl homogenate tissue of pH7.5.With 10, centrifugal 15 minutes of 000g removes cell debris, and then with supernatant with 150, centrifugal 70 minutes of 000g.On 12% polyacrylamide gel and 4-15% and 10-20% gradient gel, analyze soluble protein through SDS PAGE.In all cases, compare with the animal of injection mannitol, the low-molecular-weight band of the kidney extract of injection FL animal disappears or naked eyes detection low-molecular-weight band weakens.

Embodiment 8

Synthesizing of 3-O-methyl fructose lysine

Anhydrous 3-O-methyl glucoside of backflow 19.4g (0.1mol) and the suspension of 1g sodium sulfite in 30ml methanol and 15ml glycerol 30 minutes add 0.035mol α-carbobenzoxy group-lysine and 4ml acetic acid then.This solution 3 hours refluxes.With 1 this solution of volume water treatment, (4 * 50cm) go up chromatography, and at first the water eluting is used acetic acid pyridine eluting then at the Dowex-50 post with the pyridine form.Merging and evaporation contain the flow point of pure material.The substance dissolves that obtained in 50ml water-methanol (9: 1), is reduced with hydrogen (20psi) with 10% palladium carbon catalyst.Filter and evaporation acquisition 3-O-methyl-fructose lysine.

For example according to the known method of those skilled in the art with the saccharifying agent raw material (can be aminoacid, polyamino acid, peptide etc.) of the selected nitrogenous or oxygen of fructose saccharifying for example, can prepare other particular compound with following formula (I) structure, need, described saccharifying agent can be the saccharifying agent of chemical modification.

Embodiment 9

Other assay method of FL3P kinase activity

A. prepare mother solution

Preparation detects buffer, and it is 100mM HEPES pH8.0,10mMATP, 2mM magnesium chloride, 5mM DTT, 0.5mM PMSF.Fructosyl-spermine the mother solution of preparation 2mM fructosyl-spermine hydrochloric acid.The spermine contrast liquid of preparation 2mM spermine hydrochloric acid.

B. acrose base-spermine

Use known method (J.Hodge and B.Fisher, Methods Carbohydr.Chem., 2:99-107 (1963)) acrose base-spermine.With mol ratio 8: 4: 1 (spermine: glucose: 50ml methanol-water (1: the 1) mixture of preparation spermine (500mg), glucose (500mg) and sodium pyrosulfite (80mg) pyrosulfite), refluxed 12 hours.Water is diluted to 200ml with described product, is splined on DOW-50 post (5 * 90cm).Remove the unreacted glucose with 2 cylinder hydrops, remove described product and unreacted spermine with 0.1M ammonium hydroxide.The described product peak flow point that lyophilizing merges is by measuring the quantitative of described product ¹³The integration of the C-2 fructose base peak of C NMR spectrum (with 45 ° of pulses, 10 seconds relaxation delays and do not have NOE to go connect to collect the NMR data), thus determine fructosyl-spermine concentration.

C. kinases purity analysis

Preparation incubation mixture, it comprises the described enzyme preparation of 10 μ l, 10 μ l detect buffer, 1.0 μ Ci ³³P ATP, 10 μ l fructosyl-spermine mother solutions and 70 μ l water make it in 37 ℃ of incubations 1 hour.When incubation finishes, 90 μ l (2 * 45 μ l) sample point sample on the cellulose phosphate disk (Whatman P-81) of 2 diameter 2.5cm, is allowed its drying.Water thorough washing disk.After the drying, disk is placed scintillation vial and counting.

Measure each enzyme flow point with suitable spermine contrast duplication.

Embodiment 10

The nephropathy Neo-Confucianism that observes at glycated protein meals animal subject

Make 3 rats glycated protein meals (20% total protein of ingesting; 3% glycated protein) 8 months, and compare with the control diet rat that ingests of 9 same age.The main obviously increase of glomerule infringement of finding saccharifying meals animal.The common pathological changes that observes these animals is segmented sclerosis, the distortion of renal tubules wall epithelium and the interstitial fibrosis that is attached to the glomerule clump of glomerular capsule.Whole 3 glycated protein meals animals and only the glomerule infringement of 1 control diet animal surpass 13%.The probability that its opportunistic takes place is less than 2%.Except the pathological change that observes at glomerule, also in renal tubules, observe many transparent (hylinated) cast.Although it is not quantitatively, more in saccharifying meals animal hyaline cast.Also observing the NAGase level saccharifying meals animal raises.

Can find out that by this experimental result the saccharifying meals cause that a series of histologic lesions that observe in kidney of diabetic patients that are similar to take place animal subject.

Embodiment 11

The saccharifying meals are to the influence of filial generation

In preliminary experiment, make 5 pairs of mices saccharifying meals (18% total protein of ingesting; 3% glycated protein), and in 7 months to its breeding 6 times.The young Mus of work that 6 filial generations that produce obtain following number: 17,23,13,0,3 and 0.In view of the young Mus that lives after the breeding for the third time obviously reduces, institute is so that two groups (10 pairs every group) ingest or saccharifying meals (13% total protein; 3% glycated protein) or control diet (13% total protein; 0% glycated protein).So far, two groups of children Mus breeding 4 times obtains similar result at two groups.Gestation produces the young Mus of 49/20 (saccharifying meals/control diet) for the first time; II-para, 18/41; For the third time, 37/27; The 4th time, 20/33.The 5th the present well afoot of gestation.After tested the right hyperglycemia situation of mice.The blood sugar level of experimental group and matched group is respectively 120 and 112mg/dl.

The Preliminary Determination result of mice urine 3DF level shows, positive according to expectation, and when ingesting saccharifying meals described herein, systemic 3DF obviously raise (about 5-10 times).

Embodiment 12

The carcinogenic effect of fructose lysine approach

In order to study the carcinogenic potential of the metabolite that fructose lysine approach forms, test in the high susceptibility rat of renal carcinoma strain.Make 4 rats glycated protein meals of ingesting, 3 rats control diet of ingesting.Ingest after 10 weeks, put to death animal, detect its kidney.The ingest renal carcinoma size of whole 4 rats of saccharifying meals of discovery surpasses 1mm, and does not see big like this renal carcinoma in control rats.The probability that its opportunistic takes place is less than 2%.Data shows, the excessive fructose lysine that glycated protein in the animal's diet produces causes that the 3DG level raises in renal tubular cell (known renal tubular cell is the cell source of most of renal carcinomas), 3DG can interact with cell DNA, causes various sudden changes, finally causes taking place cancer.Possible this process rises in the cancer at human renal carcinoma and other position takes place and makes important function.

Embodiment 13

The glycated protein meals are to the meals effect of susceptible rat kidney cell cancer

In other experiment of the relation of estimating glycated protein meals and renal cell carcinoma, 28 had make its rat be divided into two groups sudden change that the renal carcinoma susceptible takes place.One group of glycated protein meals of ingesting, another group control diet of ingesting.The glycated protein meals are for wherein adding the standard nutritious food of 3% glycated protein.Being prepared as of glycated protein adds entry (2 * dry matter weight) mixed casein and glucose (2: 1), in 60 ℃ of baked mixts 72 hours.Preparation contrast in the same manner, different is water and before baking, casein and glucose not being mixed not.Make the rat described meals of ingesting after the 3 week ages wean immediately, and keep the described meals of feed arbitrarily in 16 weeks afterwards.Put to death animal then, fixedly kidney is made the section of hemotoxylin and eosin.Trained pathologist detects pathological changes.Identified 4 types pathological changes.Comprise: cyst, very little tumor like cell agglomerate (usually less than 10 cells), 0.5mm or littler little tumor and greater than the tumor of 0.5mm.All the ingest animal of control diet of the every type of pathological changes that observes the animal of the saccharifying meals of ingesting is many, shown in seeing the following form.

	Cyst	≤ 10 cells	?≤0.5mm	?＞0.5mm	Amount to
						Contrast	???2	????9	?????9	?????3	???23
The saccharifying meals	???9	???21	????32	?????6	???68

In order to sum up described result, calculate the average pathological changes number of each kidney section of every kind of meals.Control diet and saccharifying meals are respectively 0.82 ± 0.74 and 2.43 ± 2.33.The probability about 2/100,000 that opportunistic takes place.

These results support above-mentioned hypothesis forcefully: discovery of the present invention basis is that mutagenesis is gone back in the effect of lysine recovery approach, also therefore produces carcinogenic effect.These results for exploitation suppress the Therapeutic Method of this approach and medicine with alleviate renal carcinoma and wherein this approach other organ cancer that may have a similar action provide the foundation.

Embodiment 14

Drain 3-deoxidation-fructose explanation type i diabetes patient in the urine Microalbuminuria takes place

As mentioned above, the serum levels of saccharifying intermediate 3 deoxidations of diabetics-glucosone (3DG) and reproducibility detoxification product 3 deoxidations-fructose (3DF) thereof raises.Malleability insulin-dependent diabetes (IDDM) and Microalbuminuria (according to starting from 1990-1993 repeatedly measurement result to baseline values in 2 years) and do not take in one group of 39 individuality among the patient of ACE inhibitor before one group at Joslin diabetes center have been identified the relation of described chemical compound baseline level and Microalbuminuria subsequently (MA) progress.

Detect 3DF and the 3DG baseline values that drips (random spot urine) with urine by HPLC and GC-MS.(n=15) compare with non-progress person (non-progressor), in subsequently 4 years, develop into the baseline values obviously higher (p=0.02) of the log 3DF/ urine inosine ratio of high-level MA or albuminuretic individuality (n=24).The progress person's who records in this research (progressor) inosine baseline values is about 0.24 μ mole/mg, but not the about 0.18 μ mole/mg of progress person's inosine baseline values.The baseline 3DG/ urine inosine of each group is than different.Adjust HgA _IC(major part of glycosylated hemoglobin) baseline values can obviously not change these discoveries.The relevant other evidence that provides takes place for urine 3DF with the diabetes renal complication in these results.

A. quantitative 3-deoxidation fructose

Following processing sample: make 0.3mL equal portions given the test agent by containing the ion exchange column of 0.15mL AG 1-X8 and 0.15ml AG 50W-X8 resin.With the 0.3mL deionized water post is washed 2 times then, inhale and remove unnecessary liquid, filter through 0.45mm Millipore filter.

Processing sample with Dionex DX500 tomographic system analysis injection (50 μ L).Carbopac PA1 anion-exchange column is used the eluent of 16% sodium hydroxide (200mM) and 84% deionized water composition.The apply pulse amperometric is by Electrochemical Detection 3DF.The standard 3DF solution that before and after each unknown sample, all comprises expection 3DF concentration.

B. the measurement of urine inosine

Adopt improvement to be used for plate and read the terminal colorimetric analysis of device (Sigma diagnostic kit 555-A) mensuration urine inosine concentration.Identify that inosine concentration makes volume of urine normalization, to measure the metabolite level that wherein exists.

C. the albuminous measurement of urine

In order to measure experimenter's urine albumin level, collect urine and drip, on the BN100 device, carry out immune nephelometry with N-albumin reagent box (Behring).Be commercially available antialbumin antibody.All the urine albumin level be can measure with any suitable algoscopy, ELISA algoscopy, radioimmunoassay, western blot analysis and dot blotting analysis included but not limited to.

According to the research data that the patient at Joslin diabetes center obtains, urine 3DF level raises relevant with generation diabetes Microalbuminuria as can be known.This observed result provides new Diagnostic parameters for the probability that serious renal complication takes place the evaluation diabetics.

Although above description and/or exemplified some embodiment of the present invention, according to above disclosure, various other embodiments will be apparent to those skilled in the art.So, the invention is not restricted to specific embodiments described and/or that exemplify, but can carry out various changes and improvements under the appended claims scope not departing from.

Claims (18)

1. be used to reduce the chemical compound that patient couple and this patient take in the susceptibility of the relevant cancer of glycated protein, described chemical compound is chemical compound and isomer and the pharmaceutically acceptable salt with following formula structure:

Wherein X be-NR '-or-O-, R ' is selected from H and straight or branched alkyl (C ₁-C ₄) and replacement or unsubstituted aryl (C ₆-C ₁₀) or aralkyl (C ₇-C ₁₀); R is the substituent group that is selected from following group: H, amino acid residue, polyamino acid residue, peptide chain, do not replace or contained the straight or branched aliphatic group (C of the substituent group replacement of at least one nitrogen or oxygen ₁-C ₈), the substituent group that do not replace or contained at least one nitrogen or oxygen replaces and by at least one-O-,-NH-or-NR "-the straight or branched aliphatic group (C of part interruption ₁-C ₈), R " be straight or branched alkyl (C ₁-C ₆) and the aryl (C that do not replace or replace ₆-C ₁₀) or aralkyl (C ₇-C ₁₀), prerequisite be when X represent-NR '-time, the connected nitrogen-atoms of R and R ' also can be represented to replace or the heterocycle of unsubstituted 5-7 annular atoms together, wherein at least one in nitrogen and the oxygen is unique hetero atom of described ring, described aryl (C ₆-C ₁₀) or aralkyl (C ₇-C ₁₀) and described heterocyclic substituent be selected from H, alkyl (C ₁-C ₆), halogen, CF ₃, CN, NO ₂With-O-alkyl (C ₁-C ₆); R ₁It is polyol moiety with 1-4 Linear Carbon atom; Y is a carbonyl moiety

Or hydroxy methylene part Z is selected from-H ,-O-alkyl (C ₁-C ₆) ,-halogen ,-CF ₃,-CN ,-COOH and-SO ₃H ₂,

2. be used to reduce the method that patient couple and this patient take in the susceptibility of the relevant cancer of glycated protein, this method comprises the step of the Pharmaceutical composition that comprises reactive compound, and described reactive compound has the inhibition activity that effective inhibition fructose-lysyl oxidase is converted into fructose-lysine-3-phosphate.

3. according to the method for claim 2, wherein said cancer is a renal cell carcinoma.

4. according to the method for claim 2, wherein said reactive compound has competitiveness or noncompetitive enzyme inhibition, and it suppresses constant (Ki) less than about 1mM.

5. according to the method for claim 2, wherein said reactive compound: (i) molecular weight is less than about 2,000 dalton; (ii) the dissolubility in normal saline or serum is more than or equal to 10 μ M; (iii) incubation its enzyme inhibition activity reservation at least 50% after at least 1 hour in normal saline or serum.

6. according to the method for claim 2, wherein said chemical compound is chemical compound and isomer and the pharmaceutically acceptable salt with following structural formula:

Wherein X be-NR '-or-O-, R ' is selected from H and straight or branched alkyl (C ₁-C ₄) and replacement or unsubstituted aryl (C ₆-C ₁₀) or aralkyl (C ₇-C ₁₀); R is the substituent group that is selected from following group: H, amino acid residue, polyamino acid residue, peptide chain, do not replace or contained the straight or branched aliphatic group (C of the substituent group replacement of at least one nitrogen or oxygen ₁-C ₈), the substituent group that do not replace or contained at least one nitrogen or oxygen replaces and by at least one-O-,-NH-or-NR "-the straight or branched aliphatic group (C of part interruption ₁-C ₈), R " be straight or branched alkyl (C ₁-C ₆) and the aryl (C that do not replace or replace ₆-C ₁₀) or aralkyl (C ₇-C ₁₀), prerequisite be when X represent-NR '-time, the connected nitrogen-atoms of R and R ' also can be represented to replace or the heterocycle of unsubstituted 5-7 annular atoms together, wherein at least one in nitrogen and the oxygen is unique hetero atom of described ring, described aryl (C ₆-C ₁₀) or aralkyl (C ₇-C ₁₀) and described heterocyclic substituent be selected from H, alkyl (C ₁-C ₆), halogen, CF ₃, CN, NO ₂With-O-alkyl (C ₁-C ₆); R ₁It is polyol moiety with 1-4 Linear Carbon atom; Y is a carbonyl moiety

Or hydroxy methylene part Z is selected from-H ,-O-alkyl (C ₁-C ₆) ,-halogen ,-CF ₃,-CN ,-COOH and-SO ₃H ₂

7. according to the method for claim 2, wherein said Pharmaceutical composition orally give.

8. prevent, alleviate or postpone the patient and cause taking place method for cancer because AGE-albumen forms, this method comprises that the described patient of the inhibition that gives described patient treatment effective dose produces the chemical compound of 3-deoxyglucosone.

9. according to the method for claim 8, wherein said cancer is a renal cell carcinoma.

10. according to the method for claim 8, wherein said chemical compound is chemical compound and isomer and the pharmaceutically acceptable salt with following structural formula:

Wherein X be-NR '-or-O-, R ' is selected from H and straight or branched alkyl (C ₁-C ₄) and replacement or unsubstituted aryl (C ₆-C ₁₀) or aralkyl (C ₇-C ₁₀); R is the substituent group that is selected from following group: H, amino acid residue, polyamino acid residue, peptide chain, do not replace or contained the straight or branched aliphatic group (C of the substituent group replacement of at least one nitrogen or oxygen ₁-C ₈), the substituent group that do not replace or contained at least one nitrogen or oxygen replaces and by at least one-O-,-NH-or-NR "-the straight or branched aliphatic group (C of part interruption ₁-C ₈), R " be straight or branched alkyl (C ₁-C ₆) and the aryl (C that do not replace or replace ₆-C ₁₀) or aralkyl (C ₇-C ₁₀), prerequisite be when X represent-NR '-time, the connected nitrogen-atoms of R and R ' also can be represented to replace or the heterocycle of unsubstituted 5-7 annular atoms together, wherein at least one in nitrogen and the oxygen is unique hetero atom of described ring, described aryl (C ₆-C ₁₀) or aralkyl (C ₇-C ₁₀) and described heterocyclic substituent be selected from H, alkyl (C ₁-C ₆), halogen, CF ₃, CN, NO ₂With-O-alkyl (C ₁-C ₆); R ₁It is polyol moiety with 1-4 Linear Carbon atom; Y is a carbonyl moiety

Or hydroxy methylene part

Z is selected from-H ,-O-alkyl (C ₁-C ₆) ,-halogen ,-CF ₃,-CN ,-COOH and-SO ₃H ₂

11. bring out method for cancer at the susceptible animal subject, this method comprises the described animal subject glycated protein of feed meals, and 3-deoxyglucosone (3DG) content that the amount of described feed glycated protein meals and time are enough to keep described animal subject biological fluid is increased at least three times of similar susceptible animal subject biological fluid 3DG content that feed is substantially free of the meals of described glycated protein.

12. according to the method for claim 11, wherein said cancer is a renal cell carcinoma.

13. screening influences the method for the material of cancer generation, this method may further comprise the steps:

(i) followingly bring out cancer at the susceptible animal subject: the described animal glycated protein of feed meals, 3-deoxyglucosone (3DG) content that the amount of described feed glycated protein meals and time are enough to keep described animal organism body fluid are increased at least three times of similar susceptible animal subject same organisms liquid 3DG content that feed is substantially free of the meals of described glycated protein;

(ii) will be tried material and be given a part of described animal subject, and do not given another part animal subject;

(iii) put to death described animal subject; And

The (iv) nuclear cancer of relatively in each described part susceptible animal subject, finding (nucleiccarcinomas).

14. according to the method for claim 13, wherein each described part is about half of described animal subject.

15. according to the method for claim 13, wherein said cancer is a renal cell carcinoma.

16. the method for the material that screening prevention, reduction or delay cancer take place, this method may further comprise the steps: feed susceptible animal subject glycated protein meals, 3-deoxyglucosone (3DG) content that the amount of described feed glycated protein meals and time are enough to keep described animal organism body fluid are increased at least three times of similar susceptible animal subject same organisms liquid 3DG content that feed is substantially free of the meals of described glycated protein; To be tried material and be given a part of described animal subject, and do not given another part animal subject; Put to death described animal subject; And each described part susceptible animal subject tissue slice of comparison.

17. according to the method for claim 16, wherein each described part is about half of described animal subject.

18. according to the method for claim 16, wherein said cancer is a renal cell carcinoma.

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