JP2003521453A - Fructoseamine oxidase: antagonists and inhibitors - Google Patents

Abstract

(57) Abstract: A method for reducing the consequences of macrovascular and microvascular damage in diabetic patients expecting suppression and / or antagonism of fructoseamine oxidase. Copper chelators, substrate analogs, or hydrazine compounds can be used for inhibition and / or antagonism.

Description

Detailed Description of the Invention

The present invention relates to a therapeutic method using an inhibitor of fructoseamine oxidase enzyme, a pharmaceutical composition containing the inhibitor, in a dosage regimen for medical treatment, manufacture of a pharmaceutical composition, and treatment. The present invention relates to a dosage form of the inhibitor, a method of using the inhibitor, and compounds, methods and products related thereto.

Diabetes is a common disease that affects about 16 million Americans. For example, Report of the Expert Committee on Diagnosis and Classification of Diabetes, Diabetes Care, Volume 20, 118.
See pages 3-1197 (1997). People with diabetes are more likely to have complications, putting both quality of life and life expectancy at great risk. Almost half of the people diagnosed with diabetes before age 31 usually die of cardiovascular or renal complications before they reach age 50. Moreover, before dying, the illness is often crippled and debilitated over the years. Decker T
., Prussian J., Larsen M., Diabetologia, Volume 14, pp.363-370 (1978
Year). People with diabetes have a 25x increased risk of blindness, a 20x increased risk of renal failure, and a 20x increased risk of limb amputation as a result of gangrene. The risk of developing arterial heart disease and ischemic brain damage is 2 to 6 times higher. Klein R., Klein B., Moss S., Davis M., Domez D., Dia
See betes Care, Volume 8, pages 311-315 (1985).

It is estimated that the cost of treating diabetes in the United States reached $ 98 billion in 1997, mainly due to these long-term complications. The breakdown is $ 44 billion for inpatients and outpatients as direct costs, and $ 54 billion for indirect costs such as lost income, lost productivity and premature death. Innovative things in medicine that can slow the progression of diabetes can have enormous implications for treatment and costs. American Diabetes Society, "Economic Impact of Diabetes in the United States, 1997," Diabetes Care, 21: 296-309 (199).
8 years).

At present, elevated blood glucose levels are considered to be the cause of diabetic complications based on the results of “Diabetes Complications and Controlled Trials (DCCT)” and “Predicting Diabetes in the United Kingdom (UKPDS)”. Has been done. N. Eng. J. Med., Volume 379, pages 977-985 (
1993) and Lancet, 352, 837-853 (1998). DCCT
Both UKPDS and UKPDS show that increased diabetic complications are associated with the extent of hyperglycemia and that rigorous treatment improves future outcomes. For example, if blood glucose and glycated hemoglobin levels in capillaries can be maintained below 150 mg / dL and below 7.0%, respectively, the prognosis will be dramatically improved.

The mechanism by which glucose is toxic in the tissues of diabetic patients is not yet known. Glucose condenses with free amino groups on structural and functional proteins to form Schiff bases, which in turn undergo a series of transformations, producing a dark brown Maillard product. The mechanism that diabetic complications are caused by cross-linking of proteins without involvement of enzymes has been previously proposed. See Cerami A, Ulrich PC, Brownley M., U.S. Pat. No. 4,758,583 (1988). However, although increased protein cross-linking is observed in tissues of people with long-standing diabetes, it is unclear whether the Maillard product is one of the disease factors. Wolf SP,
Jean ZY, Hunt JV, Free Rad. Biol. Med., Volume 10, pp. 339-352 (
1991).

The Amadori rearrangement is the most important Maillard transformation. This is because the product of this transformation, fructose amine, is the precursor to any brown product. I have isolated a novel extracellular enzyme that acts as a catalyst for the removal of fructose amines from glycated proteins. A survey of the literature around the world reveals that the enzyme was not previously recognized to exist. Based on its high specificity for glycosylated protein substrates and its use of oxygen as an acceptor, it can be classified as fructose amine oxidase 1.5.3. See. See Enzyme Nomenclature and Recommendations of the United Nations Commission on Biochemistry, Academic Press, London, pages 19-22 (1979).

[0007] Fructose amine oxidase is a metalloenzyme having copper and a quinone cofactor, and belongs to the copper amine oxidase group among enzymes isolated from bacteria, mushrooms, yeasts and mammalian serum. The products of the reaction catalyzed by fructose amine oxidase are unglycated free protein, α-dicarbonyl sugar, and superoxide which is an active oxygen species.

When the activity of fructose amine oxidase is increased, fructose amine bound to the basement membrane protein is decomposed and reactive oxygen species are generated as a reaction product, so that many symptoms considered to be complications of diabetes occur. May appear.
For example, superoxide anion increases intracellular calcium and consequently changes NO synthase activity. Nitric oxide has a strong vasodilatory effect, and its involvement in vascular dysfunction in early-stage diabetes has been previously pointed out. Id Y, Kilo C., Williamson JR, Nephrol. Dial. Transplan
t, Vol. 11, Supplement 5, 5, 72-75 (1996). The reactive oxygen species also
Depending on the amount, it caused a dramatic decrease in the de novo synthesis of heparan sulfate proteoglycans, reduced anion sites on the renal glomerular basement membrane, and increased the permeability of cationic plasma proteins such as albumin in the basement membrane. To raise. Casilla N.
, Watanabe Y., Makin H., Wolner EI, Kanwar YS, Proc. Natl.
See Acad. Sci. USA, Vol. 89, pages 6309-6313 (1992). Increased clearance of urinary albumin is an indicator of the risk of diabetic patients with advanced renal disease and premature death, primarily due to coronary heart disease. Matock MB, Barnes DJ, Viverti GC and others, Diabe
tes, Vol. 47, pp. 1786-1792 (1998).

Once the natural antioxidant defenses have been breached, the copper-catalyzed Haber-Weiss reaction can generate hydroxyl groups from superoxide.
See Halliwell B., Gutterridge JMC, Free Radicals in Biology and Medicine, Clarendon Press, Oxford, pp. 136-176 (1989). The hydroxyl group is a highly reactive species that can cause permanent site-specific damage to basement membrane proteins, causing histopathological changes typical of diabetic microvascular disease. Robins SL, Kotran RS,
See Kumar V., Pathological Basis of Disease, Third Edition, WB Saunders, 991-1061 (1984).

In addition, oxidative stress occurs when the period of time in which the activity of fructose amine oxidase is elevated may be long. Oxidative stress may explain the greater risk of macrovascular illness and the 75% higher mortality rate in diabetic patients compared to non-diabetic individuals. Recent studies have fully validated that oxidative changes in low-density lipoprotein (LDL) are associated with the development of atherosclerosis in coronary and peripheral arteries. In fact, it has been found that oxidised LDL is increased in diabetic patients. Wittstam JL, Br. Heart J., Vol. 69 (Supplement), S12-S18
Page (1993), Picard S., Taruso C., Seruskra A. et al., Diabete
s & Metabolism, Vol. 22, pp. 25-30 (1996). In addition, changes such as oxidation of membrane lipids and oxidation of SH groups of membrane proteins may cause abnormal calcium homeostasis in cells and increase sudden death due to heart disease typical of diabetes. Yousel D., Eigdoo S., Cereli S. et al., Clin.
Chem., 44, 148-154 (1998). Summary of the Invention It has been found in the literature around the world that the existence of an enzyme called fructose amine oxidase has not been known so far. This is a novel enzyme. I play a central role in diabetic complications due to the formation of α-dicarbonyl and reactive oxygen free radical species due to glycosylation of basement membrane protein substrates and excess activity of fructose amine oxidase. I think there is.

I also found that this damage was (i) a copper chelator; (ii) a substrate analog; (iii
) It is believed that there may be improvement by administering an inhibitor or antagonist of fructose amine oxidase selected from the group consisting of hydrazine compounds. The definitions of terms used in this specification (including claims) are as follows.・ "Copper chelating agent" means the activity of fructose amine oxidase in the body
It refers to any drug that can be reduced by, for example, depleting the copper stored in the body or by binding the copper molecule to the reaction center of this enzyme to render it inactive. The copper chelator can be administered in an effective amount through any suitable route of administration (eg, oral administration, injection, etc.).
See some examples below. A “substrate analog” is an amino acid substrate that has been chemically modified to render fructose amine oxidase inactive (eg, by irreversibly (preferably substantially) binding to the active center of the enzyme). Or it means a peptide substrate. The substrate analog can be administered in an effective amount via any suitable route of administration (eg, oral administration, injection, etc.). See some examples below. “A hydrazine compound” contains a —NH—NH ₂ moiety and inactivates fructose amine oxidase (eg, by attaching a quinone molecule to the reaction center of this enzyme to inactivate it). Means any drug. The hydrazine compound can be administered in an effective amount through any suitable route of administration (eg, oral administration, injection, etc.). See some examples below. • “At least regularly” includes from one-time administration to continuous administration. • “Macrovascular damage and microvascular damage” both refer to common types of damage, but the consequences and / or the likelihood of damage to either macrovascular damage or microvascular damage. It is possible to mean only the general type of damage to one side, if it is expected to minimize or improve. “Comprises” can be used to mean “include”. -"And / or" means both "and" and "or". • “In combination” does not necessarily mean the result of simultaneous administration or self-administration (eg, administration can be sequential and may be delayed from one administration to the next). It is possible to administer triene between meals and another medication at the same time as meal). “Triethylenetetramine dihydrochloride” or “triene” refers to any pharmacologically acceptable analog or a derivative thereof that inhibits and / or antagonizes the fructose amine oxidase enzyme for the intended mammalian species or humans. A metabolite of an enzyme (eg an acetylated derivative), either alone or as a separate inhibitor and / or antagonist of the fructose amine oxidase enzyme (from toxicity concerns with respect to the level required for effective inhibition and / or antagonism) It is preferable that it is not contraindicated) and can be administered or self-administered, and can effectively suppress and / or antagonize.

According to one aspect of the invention, symptoms caused by macrovascular and microvascular damage in a patient (human or other mammal) prone to and / or having diabetes (eg, Therapeutic methods aiming at minimizing the early onset of atherosclerosis, blindness, renal failure, neuropathy, etc. are provided. This therapy includes any therapy for controlling blood glucose levels, as well as treatments that at least periodically inhibit or antagonize the activity of the fructose amine oxidase enzyme in the patient.

This inhibition or antagonism preferably occurs as a result of the administration or self-administration of at least one inhibitor or antagonist of the reaction product of fructose amine oxidase. Such inhibitors or antagonists are preferably selected from the group consisting of (i) copper chelators, (ii) substrate analogues, (iii) hydrazine compounds.

The above-mentioned inhibitors or antagonists are preferably administered orally. The inhibitors or antagonists described above are preferably administered orally in whole or in part as part of a dosing regimen. Note that controlling the blood glucose level is also included in the medication plan. According to another aspect of the invention there is provided a pharmacological composition suitable for use in such a method, preferably oral administration, wherein the inhibitor or antagonist of fructose amine oxidase is There is provided a composition comprising with a suitable base for

According to yet another aspect of the present invention, there is provided a pharmacological composition for reducing macrovascular and microvascular damage in a diabetic patient (mammal including human), which comprises fructoseamine. Compositions are provided that include an inhibitor or antagonist of an oxidase and a suitable base for the inhibitor or antagonist.

The base is preferably in the form of a diluent or excipient, and the pharmacological composition is preferably administrable or self-administering orally or by any other method. The present invention also contemplates a form in which the active ingredient is released over a long period of time. According to yet another aspect of the invention, a method of using an inhibitor or antagonist of fructose amine oxidase, which comprises a pharmacological composition comprising the inhibitor or antagonist and a suitable base for the inhibitor or antagonist. Used in the manufacture of an article, the composition treats a patient (human or other mammal) suffering from diabetes (preferably by the method of the invention) to reduce macrovascular and microvascular damage. A use is provided that is characterized as useful for.

According to yet another aspect of the invention, there is provided a method of combining a dosing regimen with the agents described above for use in the dosing regimen. The drugs can be packaged in multiples, or prescribed at the same time, but need not be. According to yet another aspect of the present invention, there is provided a method of treating a patient (human or other mammal) prone to and / or having diabetes, wherein fructose amine There is provided a method of treatment comprising inhibiting or antagonizing the activity of an oxidase enzyme, preferably with one or more agents that are not contraindicated to the patient. Specific examples of inhibitors or antagonists are listed below, but are not limited thereto.

According to one embodiment, the drug is a copper chelate compound, and it is preferred that the drug is administered to the patient or the patient self-administers it. Specific examples of suitable copper chelating compounds include triethylenetetramine dihydrochloride (triene), penicylamine, sarcosine, diaminosarcosine,
Examples include ethylenediaminetetraacetic acid, o-phenanthroline, histidine and the like.

[0019] In another embodiment, the agent is a substrate analog compound comprising an amino acid or peptide fragment having an amino group that is blocked at the N-terminus, the agent being administered to the patient or the patient It is preferable to administer. Specific examples of suitable substrate analog compounds include N-acetyl cysteine, captopril, lisinopril, enalapril and the like.

In another embodiment, the agent is a hydrazine compound having a moiety —NHNH ₂ , and it is preferred that the agent be administered to a patient or that the patient self-administers. Specific examples of suitable hydrazine compounds include diaminoguanidine, hydralazine, carbidopa and the like.

According to yet another aspect of the invention, there is provided a dosing regimen for practicing the methods of the invention and / or a dosing regimen using the dosage units of the invention. According to yet another aspect of the invention, a dosage unit or a pharmacological composition useful for treating a patient (human or other mammal) having diabetes to reduce macrovascular and microvascular damage. A method of using a pharmacologically acceptable compound comprising at least one of a substrate analog, a hydrazine compound and a copper chelating agent in the production of

According to another aspect of the present invention, either or both of the substrate analog and the hydrazine compound having a moiety of —NHNH ₂ (preferably fructose amine.
An effective dosage unit or pharmacological composition for a patient in the method of treatment of the present invention is provided which comprises a sufficient amount, alone or in total, to effectively inhibit or antagonize the reaction product of oxidase. .

Preferably, the dosage unit or the pharmacological composition also comprises one (or more) (preferably another) compound consisting of one (or more) copper chelating agent. . The above-mentioned dosage unit or the above-mentioned pharmacological composition is preferably in a form that can be orally administered, and if necessary, in addition to the base or excipient, other activators (for example, means for lowering blood glucose level). Even can be included.

According to yet another aspect of the present invention, a method for sufficiently suppressing and / or antagonizing fructose amine oxidase in a diabetic patient or a patient suspected of having diabetes to reduce damage to macro and microvessels. , A copper chelator and a triene dosage regimen, dosage unit, or pharmacological composition is provided. According to still another aspect of the present invention, in a diabetic patient or a patient suspected to have diabetes, fructose amine oxidase is sufficiently suppressed and / or antagonized to reduce damage to macro and microvessels. There is provided a captopril dosing schedule, dosage unit, or pharmacological composition that is (at least somewhat) effective, or appears to be effective, in controlling a patient's blood pressure.

According to still another aspect of the present invention, a method for sufficiently suppressing and / or antagonizing fructose amine oxidase in a diabetic patient or a patient suspected of having diabetes to reduce damage to macro and microvessels. , A hydrazine compound dosing schedule, dosage unit, or pharmacological composition is provided. According to yet another aspect of the present invention, an inhibitor and / or an antagonist of (i) acetylcysteine and (ii) other at least one fructose amine oxidase for a diabetic patient or a patient suspected of having diabetes. The dosing regimen, dosage unit, or pharmacological composition of claim 1, wherein the mixture of (i) and (ii) is sufficient to inhibit and / or antagonize fructose amine oxidase in a patient to macrovascular and microvascular There is provided a dosing schedule, dosage unit, or pharmacological composition characterized by reduced vascular damage.

According to yet another aspect of the invention, for a diabetic or suspected diabetic, (i) hydralazine and (ii) at least one other inhibitor of fructose amine oxidase and / or Or an antagonist regimen, dosage unit, or pharmacological composition, wherein the mixture of (i) and (ii) sufficiently suppresses and / or antagonizes fructose amine oxidase in a patient A dosing regimen, dosage unit, or pharmacological composition is provided which is characterized by reducing microvascular damage.

[0027] According to yet another aspect of the invention, in a patient (human or other mammal) prone to and / or suffering from diabetes, acetylcysteine and hydralazine are used to treat that patient. Therapeutic methods are provided that include treatments that inhibit and / or antagonize the activity of fructose amine oxidase enzyme in the body. According to still another aspect of the present invention, a dosing schedule, a dosage unit, or a pharmacological composition for a diabetic patient or a suspected diabetic patient, which comprises acetylcysteine and hydralazine. , Dosage units, or pharmacological compositions are provided.

According to yet another aspect of the invention, there is provided a method of co-administering or sequentially administering acetylcysteine and hydralazine to reduce macrovascular and microvascular damage in a mammal. It is preferred that the mammal has diabetes. According to still another aspect of the present invention, as a method for treating diabetic cataract and / or reducing the possibility of developing diabetic cataract in a mammal, at least the activity of fructose amine oxidase enzyme in the body of the mammal is Methods are provided that include periodic suppressive and / or antagonizing treatments.

This method preferably comprises the step of administering or self-administering an effective amount of triethylenetetramine dihydrochloride (triene). According to another aspect of the invention, a method of treating diabetic cardiomyopathy and / or reducing the likelihood of developing diabetic cardiomyopathy in a mammal comprises at least routinely determining the activity of a fructose amine oxidase enzyme in the mammal. Methods are provided that include therapeutically suppressing and / or antagonizing treatments.

The method preferably comprises the step of administering or self-administering an effective amount of triethylenetetramine dihydrochloride (triene). For all of the above indications, triethylenetetramine dihydrochloride (triene) is co-administered and / or self-administered with one or more other inhibitors and / or antagonists to fructose amine oxidase It is preferable.

The other inhibitors and / or antagonists described above are effective in treating or ameliorating macrovascular and microvascular damage in such patients or mammals, and the other inhibitors and / or antagonists are administered to the patient. It is preferred that administration, or self-administration by the patient, elicit a pharmacological effect on another indication in addition to the effect of triene.

This specification claims my PCT application filed at the same time as this application (claiming priority based on New Zealand Patent Application No. 332085 filed on September 25, 1998)
Are incorporated herein by reference in their entirety. this
Disclosed in the PCT application is monitoring the status of fructose amine oxidase inhibition and / or antagonism in a patient's body to screen and / or screen a patient for risk of damage to blood vessels, particularly microvessels. How to determine and identify those who would benefit from treatment with inhibitors and / or antagonists of fructose amine oxidase, how to measure fructose amine oxidase levels in mammals, and whether diabetic patients or suspected diabetes mellitus , A method for measuring plasma fructose amine oxidase levels in patients, an in vitro assay for serum or plasma fructose amine oxidase, a method for identifying or testing candidate substances, and other related methods.

In vitro measurements are preferably performed on the superoxide reaction product of fructose amine oxidase (or any other oxygen free radical product). Detailed Description of the Invention (i) Extraction of Holoenzymes Fructose amine oxidase in plasma is mostly in the enzyme-substrate complex bound to peptides and proteins (Fig. 1). To maximize the yield of active holoenzyme, the pH of the medium is made alkaline, preferably with phosphate buffer, the sulfhydryl reagent is added, the mixture with the prooxidant is incubated and saccharified. The molecule needs to be generated. Activation is most effective with cupric salts.

Fructose amine oxidase holoenzyme is separated from inactive apoenzyme by affinity absorption chromatography. Suitable saccharified affinity supports are alkylamine beads or beaded cross-linked agarose with amino residues attached to the ends by spacer arms of 6-10 atoms (Pierce). (Available from Bio-Rad, Pharmacia). The affinity support is saccharified by incubation with 400 mM potassium phosphate buffer pH 7.4 containing 50 mM glucose and 0.01% sodium azide at 37 ° C. for 7 days. Since the holoenzyme binds tightly to the glycated amino residue, the residual copper is easily removed by washing with water. The active holoenzyme is eluted with 800 mM NaCl in 50 mM sodium acetate buffer, pH 4.8. The active fractions are pooled and the proteins are precipitated with cold 50% acetone solvent. The protein pellet is collected with a minimal amount of water or saline and lyophilized for long term storage.

Extracts from serum collected and pooled from diabetic and non-diabetic people 35
A clear colorless preparation was obtained from mL. The absorption peaks are at 196 nm and 264 nm,
It was a typical absorption spectrum of fructose amine oxidase (Fig. 2).
. For comparison, the absorption spectrum of the fructose amine oxidase enzyme from Enterobacter erogenes, which has absorption peaks at 196 nm and 255 nm, is also shown in this figure. The activity of the enzyme and the relative activity are as follows.

[0036]

[Table 1]

* The extract from the enzyme is 37 ° C. in 0.05 M TES buffer pH 7.4 containing 1 mM DMF substrate.
It was pre-cultured for 5 minutes. The enzyme activity was measured using 10 μM ferric cytochrome c. The reaction was initiated with 50 μM fructose amine substrate as g-BSA and ΔA _{550 nm} was measured over 5 minutes. + Protein concentrations were determined from A _{210 nm} -A _{220 nm} as compared with BSA standards. (Ii) Identification of Cofactor A p-nitrophenylhydrazine (NPH) adduct of the enzyme of Enterobacter erogenes with A _max 399 nm was obtained as described above. Parsic MM, Janes
See SM, Meth. Enzymol., 258, pp. 34-38 (1995). NPH
-When the enzyme adduct was diluted in 2M KOH, a red shift in the absorption spectrum was observed, giving an A _{max of} 438 nm. Such shifts in the absorption spectrum are typically found in the quinone cofactor of copper amine oxidase.

Example 1: Identification of inhibitors of fructose amine oxidase The purpose of this example was to identify and classify candidate inhibitors of fructose amine oxidase in the theme of PCT International Application NZ No. 332085 filed concurrently. Is to show how to use the fructose amine oxidase assay. This method takes into account drug activity in human serum substrates in vitro.
Enzyme inhibitors have wide application in clinical medicine as therapeutic tools for various metabolic disorders. For example, inhibitors of angiotensin converting enzyme have long been used in the treatment of hypertension. See Harris EE, Patchet AA, Tristorum EW, Wivratt MJ, Amino Acid Derivatives as Antihypertensives, US Pat. No. 4,374,829 (1983). Similarly, 3-hydroxy-3-
Inhibitors of the methylglutaryl-coenzyme A (HMG-CoA) reductase enzyme have long been used in the treatment of hypercholesterolemia. Hoffman WF, Smith
See RL, Lee TJ, Novel Inhibitors of HMG-CoA Reductase, US Pat. No. 4,866,090 (1989). Inhibitors of fructose amine oxidase are substrates that bind to quinone cofactors (hydrazine compounds) and copper cofactors (copper chelating agents) and inhibit these cofactors, or substrates that are similar to normal substrates for enzymes (substrate analogs). ) You can choose from.

Methods: Candidate inhibitors of fructose amine oxidase were screened for human serum or human plasma (separately, using the fructose amine oxidase activity assay described in detail in New Zealand Patent Application No. 332085). Tested in combination). The irreversible inhibition of the enzyme means that the activity of the enzyme gradually decreases over time, and eventually, even when the concentration of the inhibitor is extremely low, the amount of the inhibitor is lower than the amount of the enzyme present. It can be seen from the fact that when the amount is excessive, the activity of the enzyme is completely suppressed.

Results: Table 2 shows the relative activity of various hydrazine compounds, copper chelators, and substrate analogs that are inhibitors of fructose amine oxidase. In some cases, it may span two classes. For example, some hydrazine compounds are also copper chelators. To clarify this point, some potential copper chelators are marked (β). The effect of inhibitors is represented not only by the equilibrium constant, but also by the rate constant (K). This is because the rate constant determines the proportion of enzyme suppressed by a given concentration of inhibitor within a given time. The specificity of the inhibitor for the active center of the enzyme is expressed as the concentration of inhibitor that inactivates 50% of the enzyme (IC ₅₀ ).

[0041]

[Table 2]

¹ Fresh human serum was added with 0.05 M TE at pH 7.4 with 0-1.000 μM inhibitor.
Incubated in S buffer at 37 ° C for 5 minutes. Enzyme activity was measured using 10 μM ferricytochrome c. The reaction was initiated with 50 μM fructose amine substrate as g-BSA and ΔA _{550 nm} was measured over 5 minutes. ² Rate constants were calculated from the results of reacting fructose amine oxidase with 1.0 μM inhibitor or 10.0 μM inhibitor (*). ³ copper chelator candidate (beta) is copper was determined based on the ability to remove copper by dialysis from BSA substrates saturated. Conclusion: 1. Irreversible inhibition of fructose amine oxidase is possible. 2. Inhibitors can be broadly classified into three classes. That is, a hydrazine compound, a substrate analog, and a copper chelating agent. 3. The activity of fructose amine oxidase in human plasma can be lost by micromolar concentrations of inhibitors. 4. Many of the active inhibitors are drugs that have already been medicated in humans to treat other diseases (other than diabetes).

Example 2 Clinical Usefulness of Inhibiting Fructose Amine Oxidase: First Preclinical Study The purpose of this example is how to be a standard model animal for diabetes. To show whether streptozotocin diabetic rats (STZ rats) can be used to examine the clinical efficacy of candidate inhibitors of fructose amine oxidase. In this method, the bioavailability of the drug, the activity of the drug and its metabolites, any undesired effects of the drug, and the toxic factors of the drug are considered.

Method: 48 Wistar rats aged 6-8 weeks and weighing 200-300 g were randomly divided into the following groups: • Group 1 non-diabetic control rats • Group 2 diabetic control rats • Group 3 hydralazine-treated diabetic rats • Group 4 EDTA-treated diabetic rats • Group 5 hydralazine and acetylcysteine-treated diabetic rats • Group 6 acetylcysteine Treated diabetic rat streptozotocin (60 mg / kg body weight) was administered to the lateral vein of the tail. Non-diabetic control rats were injected with buffer as a sham injection. Diabetes was confirmed after one week by the measured value of blood glucose in venous blood exceeding 15 mmol / L, and the diabetic rats were maintained for 3 to 5 days a week to maintain growth, Ultralente insulin was subcutaneously injected (4 U per injection). The drug was administered at a rate of 50 mg / L in drinking water for 8 months. Urine and venous plasma samples were taken monthly.

Results: (a) Control of blood glucose level: The proportion of rats that became diabetic by intravenous administration of STZ exceeded 95%. Intravenous STZ induced severe insulin-dependent diabetes mellitus, which persisted for the entire 8-month study period. Despite insulin replacement therapy, glycemic control failed. This can be seen from the levels of mean ± standard deviation of glucose (4 weeks) and HbA _1c (32 weeks) shown in Table 3.

[0046]

[Table 3]

(B) Survival: The mortality of untreated STZ rats was extremely high. Administration of inhibitors of fructose amine oxidase significantly improved survival (Table 4).

[0048]

[Table 4]

* Chi-square test compared to untreated STZ rats (Group 2) The survival curves for STZ rats are shown in Figure 4 compared to non-diabetic control rats. It was estimated that the rat died from a complication of cardiovascular abnormalities. Overall, renal function remained normal. (C) Weight gain: The study on STZ diabetic rats was stopped at 32 weeks, but non-diabetic control rats gained weight gradually over the 32 weeks of the study. When the study was stopped after 32 weeks, the mean change in body weight of surviving rats was + 74.6% in group 1, -21.0% in group 2, -11.0% in group 3, and +1.2 in group 4. %, Group 5 was + 16.0%, Group 6 was -8.1% (Fig. 5)
). Inhibitors of fructose amine oxidase resulted in weight gain roughly proportional to the activity of the inhibitors when compared to untreated diabetic control rats (Table 2). That is, acetyl cysteine / hydralazine>EDTA> acetyl cysteine> hydralazine. (D) Clinical pharmacokinetics: hydralazine : The bioavailability of hydralazine after oral administration in humans was 26.
~ 55%. However, only 2.0-3.6% of this drug is excreted unchanged in the urine within 24 hours after oral administration. Most of this drug is recovered as an inactive acetylated product. Tulces T., Eur. J. Clin. Pharmacol., Volume 10, 395-401 (1976) and Tulses T., Clin. Pharmacol. Ther., 21.
Vol. Pp. 715-720 (1977). This fact may explain the reduced effect of hydralazine, which functions in this study as an inhibitor of fructose amine oxidase. Furthermore, the dose of the drug to each STZ rat was 12.5 mg / day of hydralazine, or 35 mg / day, based on a daily water consumption of 250 mL, assuming an average body weight of 350 g. kg
Was calculated. This dose for rats is 200 mg / day, which is the recommended maximum dose of hydralazine for humans (assuming an average human body weight of 70 kg is 3
much more than mg / kg). EDTA : The bioavailability of EDTA after oral administration is very low (less than 5%). Because it is poorly absorbed from the intestine, its effectiveness in humans is limited to parenteral administration or irrigation. Win JE et al., Toxicol. Appl. Pharmacol.,
See Volume 16, pages 807-817 (1970). Acetylcysteine : Acetylcysteine is rapidly absorbed from the intestine with a bioavailability of 6-10% in humans. Borgstrom L. et al., Eur. J. Clin. P
See harmacol., Vol. 31, pp. 217-222 (1986). However, this drug is rapidly degraded by the detachment of the acetyl moiety in the liver. See Hordines MR, Clin. Pharmacokinet., 20: 123-134 (1991). The observation that the effect of the drug was gradually lost after 12 weeks in this study could be explained by the fact that the liver enzyme was induced.

Conclusion: 5. Streptozotocin causes severe insulin-dependent diabetes mellitus in rats with high mortality. 6. Survival of STZ rats was increased by treatment with an inhibitor of fructose amine oxidase in proportion to the activity of that inhibitor measured in vitro. 7. Weight gain in STZ rats was promoted by treatment with an inhibitor of fructose amine oxidase. 8. The effect was seen in the simultaneous administration of acetylcysteine and hydralazine. This suggests a synergistic effect between different classes of inhibitors of fructose amine oxidase. 9. Based on the results of these studies using the rat living body, the effect of a candidate inhibitor of fructose amine oxidase in humans was determined by the bioavailability of the candidate, the degradation status of the candidate in vivo, and the tolerance of the candidate. It seems to be affected by the maximum dose. Example 3 Clinical Utility of Inhibiting Fructose Amine Oxidase: Second Preclinical Study The purpose of this example is how to treat streptozotocin diabetic rats, a standard model animal for diabetes. (STZ rats) will be used to show the possibility of investigating the clinical efficacy of fructose amine oxidase inhibitor candidates alone or in combination. In this method, the bioavailability of the drug, the activity of the drug and its metabolites, the interactions between the drugs, any undesired effects of the drug, and the toxic factors of the drug are considered.

Method: Eighty Wistar rats aged 6-8 weeks and weighing 200-300 g were randomly divided into the following groups.・ Group 1 non-diabetic control rats ・ Group 2 diabetic control rats ・ Group 3 diabetic rats treated with captopril (substrate analog) ・ Group 4 diabetic rats treated with carbidopa (hydrazine compound) ・ Group 5 with triene (copper chelator) Treated diabetic rats-Group 6 diabetic rats treated with captopril and triene-Group 7 diabetic rats treated with captopril and carbidopa-Group 8 diabetic rats treated with triene and carbidopa Streptozotocin (60 mg / kg body weight) injected intraperitoneally I had diabetes. Non-diabetic control rats were injected with buffer as a sham injection. Diabetes was confirmed one week later by measuring the blood glucose level of venous blood above 15 mmol / L, and the diabetic rats were treated with Ultralente 3 days a week to maintain growth. -Treatment with subcutaneous injection of insulin (4 U per injection) was performed. The drug was administered at a rate of 50 mg / L in drinking water for 6 months. Urine and venous plasma samples were taken monthly. Rats were killed and necropsied at the end of the study.

Results: (a) Control of blood glucose level: The ratio of rats that became STB-induced diabetic was 80.
%Met. I tried to control glycemia over the six months I did the study, but it didn't work. This can be seen from the mean value ± standard deviation of the levels of HbA1c (4 weeks, 12 weeks, 24 weeks) (Table 5).

[0053]

[Table 5]

(B) Survival rate: When STZ was administered intraperitoneally, the degree of diabetes was reduced and the mortality rate was also lower than when STZ was administered intravenously. Finished research 24
As of the week, the mortality rate of the rats studied was 0% in group 1, 14.3% in group 2, 0% in group 3, 0% in group 4, 0% in group 5, 12.5 in group 6. %, Group 7 was 0%, and group 8 was 0%. There were no significant differences between groups due to the small number of events. (C) Weight gain: STZ diabetes significantly reduces body weight in diabetic rats compared to non-diabetic control rats. Table 6 shows the average weight gains from the start of the study to the 24th week after the completion of the study in the rats studied.

[0055]

[Table 6]

* Student's t-test compared to untreated STZ rats (group 2) Triene (group 5) was more effective than captopril (group 3) and carbidopa (group 4) for overall health. Looks like it brought. The efficacy of triene is shown when this drug is administered in combination with captopril (
It decreases in group 6) or when administered together with carbidopa (group 8). There is no evidence of synergism between different classes of inhibitors of fructose amine oxidase. (D) Cataract formation: Cataract is recognized as a long-term complication that occurs when diabetes is poorly controlled. By 24 weeks when the study was completed, it is shown that large cataracts were formed in STZ rats compared to non-diabetic control rats (Table 7).

[0057]

[Table 7]

* Chi-square test compared with untreated STZ rats (group 2) was not significant at the level of P = 0.05, but the effect of suppressing the formation of large cataracts was that triene was more effective than captopril or captopril. Looks larger than carbidopa. There is no evidence of synergism between different classes of inhibitors of fructose amine oxidase. (E) Diabetic cardiomyopathy: Cardiomyopathy is recognized as a long-term complication that occurs when diabetes is poorly controlled. Macroscopically, STZ rat hearts were distended due to thinning of the ventricular wall. The sections are hematoxylin, eosin,
When stained with Masson's trichrome stain, normal structures were lost in the myocardium of both ventricles and the lesions became pale. In severe rats, lesions started in the subendocardial and subepicardial areas and spread throughout the ventricular wall. In addition, fibrous connective tissue significantly infiltrated between myocytes, and fibrous connective tissue increased inside the arterial wall within the ventricular wall. These findings are consistent with the growing cardiomyopathy. By 24 weeks after the study was completed, severe myocardial fibrosis was found in STZ rats compared to non-diabetic control rats (Table 8).

[0059]

[Table 8]

* Chi-square test compared to untreated STZ rats (Group 2) Triene appears to be very effective in suppressing the progression of diabetic cardiomyopathy. There is no evidence of synergism between different classes of inhibitors of fructose amine oxidase. (F) Clinical Pharmacokinetics Triene: The bioavailability of triene is less than 10%. Most of this unchanged drug is excreted in the urine as acetyl derivatives within the first 6 hours after oral administration. This fact suggests that it is necessary to administer the drug 3 or 4 times a day or use a sustained release drug. Kodama H. et al., Life Sci., No.
Vol. 61, pages 899-907 (1997). Furthermore, plasma levels in non-fasted rats are significantly lower than those observed in fasted rats, so that uptake of triene from the intestinal brush border is suppressed by competing with other amine compounds. It Tanabe R. et al., J. Pharm. Pharmacol.,
See Volume 48, pages 517-521 (1996). This fact indicates that it is most preferable to administer the triene during the fasting period. The differences seen between the triene-treated groups (Groups 5, 6, and 8) may be explained by interference with drug absorption from the intestinal brush border. Finally, in this study, each STZ rat consumed approximately 250 mL of water daily (1 triene per rat).
Equivalent to 12.5 mg a day). Assuming an average rat body weight of 350 g, this dose of triene corresponds to 35 mg / kg. The dose of triene previously used to treat humans under other conditions (not diabetic) is 1.2-2.4 g (17-35 mg / kg assuming a mean human weight of 70 kg). Walsey JM, Lancet, Volume 8273, 643-6
See page 47 (1982). This fact indicates that it is safe to elicit the inhibitory and / or antagonizing effects of fructose amine oxidase by administering to the diabetic patients targeted by this specification in the same amount as that administered to rats in this study. It means that there will be. Captopril : The bioavailability of captopril is about 65% after oral administration.
However, this drug binds to albumin and other plasma proteins almost completely in vivo, forming an inactive disulfide mixture with endogenous thiols. Therefore, the levels of active drug in plasma can be very low. The elimination half-life of unchanged captopril is about 2 hours. Duchin KL et al., Clin. P
See harmacokinet., Vol. 14, pp. 241-259 (1988). These observations may explain the reduced effect of captopril in STZ rats compared to the in vitro results. In addition, each STZ rat consumed about 12.5 mg of captopril per day. This corresponds to 35 mg / kg, assuming an average rat body weight of 350 g. This dose is 150 mg / day, which is the recommended maximum dose of captopril for humans (the average human body weight is 70 kg).
2 mg / kg), which is much higher than that. Carbidopa : In a study in Beagle dogs, oral absorption of carbidopa was almost complete, with an absolute bioavailability of 88%. The biological half-life was 5 hours. See Obak R. et al., J. Pharm. Pharmacol., 36, 415-416 (1984). However, carbidopa is an unstable compound, and spontaneously decomposes in a short time. Leave the solution exposed to light at room temperature for 50 hours in 50 hours.
% Decomposes by oxidation. Papert EJ and others, Movement Disorders, Volume 12, 608
See page 623 (1997). The reduced efficacy of carbidopa in this study could be explained, in part, by the fact that the bioavailability was reduced due to oxidative degradation of this active drug both before and after ingestion by rats. See. Finally, each STZ rat consumed about 12.5 mg of carbidopa daily. This corresponds to 35 mg / kg, assuming an average rat body weight of 350 g. This dose is the recommended maximum dose of carbidopa for humans of 200 mg /
Much more than a day (3 mg / kg assuming an average human body weight of 70 kg).

Conclusion: 10. Intraperitoneal injection of streptozotocin in rats results in a lower mortality rate than intravenous injection of streptozotocin. 11. Weight gain was accelerated in STZ rats treated with the copper chelator triene. Captopril and carbidopa had no effect. 12. Trienes may slow the progression of cataracts. The effects of triene are reduced when the drug is administered in combination with captopril or carbidopa. 13. Treatment with triene prevented the progression of diabetic cardiomyopathy. The effects of triene are reduced when the drug is administered in combination with captopril or carbidopa. 14. Oral administration of triene suppresses the progression of complications (cataracts, cardiomyopathy, premature death) in rats, but its dose, when converted to body weight, was previously under different conditions (not diabetic) in humans. Equal to the dose used for treatment. 15. The previously accepted dose of 1.2-2.4 g / day of triene for humans was 1
Trienes may be an effective means of treating the long-term complications of diabetes when given in 3 or 4 divided doses daily or as a sustained release drug.

Example 4: Clinical utility of inhibiting fructose amine oxidase: a double-blind, placebo-controlled clinical trial The purpose of this example was to determine how to treat diabetic patients. , To show the clinical efficacy of candidate inhibitors of fructose amine oxidase. The detailed protocol based on this proposal has been approved by the Auckland Regional Ethics Committee. In this method, the bioavailability of the drug, the activity of the drug and its metabolites, the interaction between the drugs, any undesired effects of the drug, the toxic factors of the drug, the change from rat treatment to human treatment Consider “scale-up” factors. OBJECTIVES: A pilot study to determine whether triene slows the progression of kidney disease and its associated microvascular complications in patients with diabetic kidney disease caused by non-insulin dependent diabetes mellitus (NIDDM). Patient population: 60 men and women suffering from diabetic kidney disease caused by NIDDM but with little control of blood glucose levels, aged 40 to 70 years. Study design and study period: A double-blind study in which a placebo-treated control was randomly selected. The study period consisted of the following five. -Screening period (finding candidates who meet the criteria of the study);-Recruitment period (ensure informed consent and measurement of the criteria for comparison);-Persuasion period (research protocol and study medication) Attempt to get the law accepted; ・ Maintenance period (treatment with drug / placebo and monitoring of efficacy / safety); ・ Follow-up period (whether there is any adverse effect when discontinuing medication) detection).

In addition to the currently administered antihypertensive drug and blood glucose lowering drug, a blinded drug (triene 400m
g or placebo) 3 times daily 30 minutes before meals. This study randomized all patients for a minimum of 6 months (maintenance period)
Sometimes ends. Of all randomized patients, those who discontinue medication for any reason other than death will be followed up throughout the study. Patients undergoing kidney transplantation or dialysis will be followed up only for life and death.

Results: Effect : The first criterion for evaluating the results obtained is the rate of decrease in renal function that can be seen from the measured values of renal glomerular filtration rate (creatine clearance). The second criterion for evaluating the obtained results is the progress of diabetic retinopathy, diabetic peripheral neuropathy, and diabetic autonomic neuropathy. Safety : • Factors to consider in assessing safety are undesired phenomena and clinical abnormalities. These will be evaluated from time to time by treatment history, physical examination, laboratory analysis, and comparison between groups. Statistical considerations: The sample size in this study is such that the expected reduction in creatine clearance (1 mL / min) in patients with NIDDM with diabetic neuropathy (creatin clearance <90 mL / min) It is estimated by making the first assumption that it will be reduced by the treatment used. This study showed a 6 mL / min change in creatine clearance for 4 readings every 2 months (ie, assuming a 10% loss to follow-up at a 5% level of significance). The ability to detect (80%) over 6 months at 0, 2, 4, 6 months).

Conclusion: 16. It would be confirmed that trienes are effective in treating microvascular complications in patients with NIDDM. 17. The safety of long-term administration of triene to patients with diabetic neuropathy due to NIDDM but poorly controlled blood glucose levels will be confirmed. 18. It also provides a means to determine if D-penicylamine, sarcosine, diaminosarcosine, etc., as copper chelates, another inhibitor of fructose amine oxidase, are clinically effective (i.e., triene is placed in placebo in subsequent clinical trials). Could be used instead of).

[Brief description of drawings]

FIG. 1 is a diagram showing in detail the mechanism of the reaction in which fructose amine and Maillard products are produced from glucose and protein. Fructose amine oxidase decomposes fructose amine in a two-step reaction. That is, first the α-dicarbonyl sugar is produced and then the enzyme / protein complex is oxidized to produce the free non-glycated protein. The reduced copper cofactor is oxidized in the body by molecular oxygen. The product of the oxidation is superoxide.

FIG. 2 is an absorption spectrum (A) of a fructoseamine oxidase enzyme extracted from stored human serum and an absorption spectrum (B) of a fructoseamine oxidase enzyme extracted from a microorganism called Enterobacter erogenes. )
Is.

FIG. 3 is a spectrum (A) of a p-nitrophenylhydrazine (NPH) adduct of the enzyme of Enterobacter erogenes and an absorption spectrum of the NPH-enzyme adduct diluted with 2 M KOH in red. It shows that it is shifting to one direction.

FIG. 4 compares survival curves of untreated STZ-rats with diabetic rats treated with inhibitors of fructose amine oxidase.

FIG. 5 is a graph showing how treated STZ-rats and untreated STZ-rats grew monthly compared to non-diabetic rats.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 31/401 A61K 31/401 31/4172 31/4172 31/4375 31/4375 31/498 31/498 A61P 3/10 A61P 3/10 (31) Priority claim number 334471 (32) Priority date March 3, 1999 (1999. 3.3) (33) Priority claiming country New Zealand (NZ) (31) Priority claim number 337042 (32) Priority date August 3, 1999 (August 3, 1999) (33) Priority country New Zealand (NZ) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ) , TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI. , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG , US, UZ, VN, YU, ZA, ZWF F terms (reference) 4C084 AA17 MA01 MA02 NA05 NA14 ZC352 ZC7 51 4C086 AA01 AA02 BC07 BC38 BC41 CB09 MA01 MA02 NA05 NA14 ZC35 ZC75 4C206 AA01 AA02 FA01 FA53 FA55 HA31 MA01 MA02 MA04 NA05 NA14 ZC35 ZC75

Claims (35)

[Claims] 1. A symptom (eg, atherosclerosis, blindness) caused by macrovascular and microvascular damage in a diabetic-prone and / or diabetic patient (human or other mammal). , Renal insufficiency, early onset of neuropathy, etc.) in addition to all therapies for controlling blood glucose levels, at least the fructose amine oxidase enzyme activity of the patient Therapies that include treatments that are regularly suppressed or antagonized. 2. The method of claim 1, wherein said inhibition or antagonism occurs as a result of administration or self-administration of at least one inhibitor or antagonist of fructose amine oxidase reaction product. 3. The inhibitor or antagonist as described above,   (I) copper chelating agent,   (Ii) a substrate analog,   (Iii) Hydrazine compound The treatment method according to claim 2, wherein the treatment method is selected from the group consisting of: 4. The therapeutic method according to claim 2, wherein the inhibitor or antagonist is orally administered. 5. The oral administration of all or a portion of the inhibitor or antagonist as part of a regimen, which regimen also includes controlling blood glucose levels. The treatment method described in. 6. Suitable for use in a therapeutic method aimed at minimizing the symptoms caused by macrovascular and microvascular damage in a mammal prone to and / or having diabetes. A pharmaceutical composition comprising an inhibitor or antagonist of fructose amine oxidase,
A composition comprising together with a suitable base material for the inhibitor or antagonist. 7. A pharmacological composition for reducing macrovascular and microvascular damage in a patient suffering from diabetes (including mammals including human), which comprises an inhibitor or antagonist of fructose amine oxidase, and the inhibitor or A composition comprising a suitable base for the antagonist. 8. A method of using an inhibitor or antagonist of fructose amine oxidase, which comprises producing a pharmacological composition comprising the inhibitor or antagonist and a suitable base for the inhibitor or antagonist. Utilization, wherein the composition is useful for treating a patient suffering from diabetes (human or other mammal) to reduce macro- and micro-vascular damage. 9. A pharmacological composition in which the pharmacological composition according to claim 6 or 7 is combined with a pharmacological composition having an effect of lowering blood glucose level. 10. A method for treating a patient (human or other mammal) prone to and / or suffering from diabetes, wherein the activity of fructose amine oxidase enzyme in the body of the patient is A method of treatment comprising treatment that is suppressed or antagonized, preferably with one or more agents that are not contraindicated to the patient. 11. The method of claim 10, wherein the agent is a copper chelate and the agent is administered to the patient or the patient self-administers. 12. The method according to claim 11, wherein the copper chelate compound is triethylenetetramine dihydrochloride (triene), ethylenediaminetetraacetic acid, o-phenanthroline, or histidine. 13. The drug is a substrate analog compound containing an amino acid or a peptide fragment having an amino group that is blocked at the N-terminus, and the drug can be administered to a patient or self-administered by the patient. The method of claim 10 characterized. 14. The method according to claim 13, wherein the substrate analog compound is N-acetylcysteine, captopril, or enalapril. 15. The method according to claim 10, wherein the drug is a hydrazine compound having a moiety of —NHNH ₂ , and the drug is administered to a patient or the patient self-administers it. . 16. The method according to claim 15, wherein the hydrazine compound is diaminoguanidine, hydralazine, or carbidopa. 17. A substrate analog and / or a hydrazine compound having a moiety of —NHNH ₂ (preferably an amount sufficient to effectively inhibit or antagonize the reaction product of fructose amine oxidase). , Alone or in total), a dosage unit or pharmacological composition effective for the patient in the treatment of claim 10. 18. The dosage unit or the above-mentioned pharmacological composition is in a form that can be administered orally, and if necessary, a carrier, an excipient, or other activator (for example, means for lowering blood glucose level). 18. The dosage unit or pharmacological composition according to claim 17, characterized in that it also comprises: 19. In order to sufficiently suppress and / or antagonize fructose amine oxidase in diabetic patients or patients suspected of having diabetes to reduce macrovascular and microvascular damage (control of blood pressure in diabetic patients. A captopril regimen, dosage unit, or pharmacological composition that is (at least somewhat) effective, or appears to be effective. 20. For a diabetic patient or a patient suspected of having diabetes, (i) a hydrazine compound, and (ii) at least one other inhibitor and / or antagonist of fructose amine oxidase, and a dosage unit, a dosage unit. , Or a pharmacological composition, wherein the mixture of (i) and (ii) sufficiently suppresses and / or antagonizes fructose amine oxidase in a patient to reduce macrovascular and microvascular damage. A dosing regimen, dosage unit or pharmacological composition, characterized in that: 21. For a diabetic or suspected diabetic patient, (i) acetylcysteine and (ii) at least one other fructose amine oxidase inhibitor and / or antagonist dosing regimen, dosage unit. , Or a pharmacological composition, wherein the mixture of (i) and (ii) sufficiently suppresses and / or antagonizes fructose amine oxidase in a patient to reduce macrovascular and microvascular damage. A dosing regimen, dosage unit or pharmacological composition, characterized in that: 22. A dosing schedule, dosage unit of (i) hydralazine and (ii) at least one other inhibitor and / or antagonist of fructose amine oxidase for a diabetic patient or a patient suspected of having diabetes. Or a pharmacological composition, wherein the mixture of (i) and (ii) sufficiently suppresses and / or antagonizes fructose amine oxidase in a patient to reduce macrovascular and microvascular damage. A dosing schedule, dosage unit, or pharmacological composition characterized by: 23. A method for treating a patient (human or other mammal) prone to and / or suffering from diabetes, which comprises (a) acetylcysteine and hydralazine, or (b) acetylcysteine. And a method for inhibiting and / or antagonizing fructose amine oxidase enzyme activity in the body of a patient using carbidopa. 24. A dosing schedule, dosage unit, or pharmacological composition for a diabetic patient or a patient suspected of having diabetes, which comprises (i) acetylcysteine and hydralazine, or (ii) acetylcysteine and carbidopa. A dosing regimen, dosage unit or pharmacological composition comprising. 25. A method for simultaneous or sequential administration of acetylcysteine and hydralazine to reduce damage to macro and micro vessels in mammals. 26. The mammal having diabetes mellitus.
The method described in 25. 27. A method for simultaneous or sequential administration of acetylcysteine and carbidopa to reduce damage to macro and micro blood vessels in a mammal. 28. The mammal having diabetes mellitus.
The method described in 27. 29. A dosing regimen, dosage unit, or pharmacological composition for a diabetic or suspected diabetic patient, wherein the copper chelating agent, triene, provides sufficient fructose amine oxidase for the patient. A dosing schedule, a dosage unit, or a pharmacological composition characterized by suppressing and / or antagonizing the above to reduce damage to macro and microvessels. 30. A method of treating diabetic cataract and / or reducing the likelihood of developing diabetic cataract in a mammal, wherein the activity of fructose amine oxidase enzyme in the mammal is at least periodically suppressed. And / or antagonizing treatment. 31. Triethylenetetramine dihydrochloride (triene)
31. A method comprising administering an effective amount of or self-administering
The method described in. 32. A method of treating diabetic cardiomyopathy and / or reducing the likelihood of developing diabetic cardiomyopathy in a mammal, wherein the activity of the fructose amine oxidase enzyme in the body of the mammal is at least regular. A method comprising the treatment of inhibiting and / or antagonizing 33. Triethylenetetramine dihydrochloride (triene)
32. The method comprises the step of administering or self-administering an effective amount of
The method described in. 34. Triethylenetetramine dihydrochloride (triene)
Is administered and / or self-administered in combination with one or more other inhibitors and / or antagonists to fructose amine oxidase. Method. 35. The other inhibitor and / or antagonist is effective in treating or ameliorating macrovascular and microvascular damage in a patient or mammal as described above, wherein the other inhibitor and / or antagonist is administered to the patient or mammal. 35. The method of claim 34, wherein the method is administered to the patient or self-administered by the patient to elicit a pharmacological effect on another indication in addition to the effect of triene.