CN104162163A

CN104162163A - Application of acyl-coenzyme A oxidase as therapeutic target of diabetes

Info

Publication number: CN104162163A
Application number: CN201310185274.8A
Authority: CN
Inventors: 曾嘉
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-05-17
Filing date: 2013-05-17
Publication date: 2014-11-26
Also published as: CN104582730B; CN104582730A; WO2014183483A1

Abstract

The invention discloses application of acyl-coenzyme A oxidase as a therapeutic target of diabetes and discloses an acyl-coenzyme A oxidase inhibitor. The acyl-coenzyme A oxidase inhibitor inhibits the activity of acyl-coenzyme A oxidase in the body of a diabetic patient so as to substantially reduce the oxidative stress level in a target organ or tissue, improve insulin resistance in the body and exert a treatment effect on diabetes.

Description

Acyl coenzyme A oxidase is as the application of the treatment target spot of diabetes

Technical field

The invention belongs to biochemistry and pharmaceutical field, particularly, the present invention relates to the method by suppressing acyl coenzyme A oxidase active treatment diabetes in body, acyl coenzyme A oxidase inhibitor or the acyl coenzyme A oxidase inhibitor precursor purposes in the medicine of preparation treatment diabetes.

Background technology

Diabetes are a kind of due to h and E factor interaction, insulin relatively or definitely lacks and the syndrome of carbohydrate, fat and protein metabolism disorder that target tissue insulin resistant causes, that a kind of lasting hyperglycemia is feature, chronic, general metabolic disease.If be not effectively treated after onset diabetes, can there is blood capillary and macroangiopathy widely, there is the pathological change of the multisystems such as nerve, urinary system, circulation.Diabetes mainly contain 2 types: (1) type 1 diabetes, and beta Cell of islet destroys, and conventionally causes insulin definitely to lack; (2) type 2 diabetes mellitus, is called again non-insulin-dependent diabetes mellitus, and show as whole body insulin resistant companion insulin and relatively lack, or hypoinsulinism companion insulin resistant.In diabetes mellitus in China patient, type 1 diabetes accounts for 5.6% of diabetes, and type 2 diabetes mellitus accounts for 93.7% of diabetes, and other types diabetes only account for 0.7%.

Within 2012, global diabetics reaches 3.5 hundred million people, and its potential maximum increases crowd in Asia, accounts for the more than 65% of overall growth number.Diabetes mellitus in China patient has increased by 2 times in nearly 10 years, and ill total number of persons approaches 100,000,000 people, occupies first, the whole world.

Treat taking insulinize as main for type 1 diabetes at present, but type 1 diabetes patient is often with insulin resistant, after insulinize, blood glucose is still controlled unstable.Treat for type 2 diabetes mellitus, in the time that control body weight all fails effectively to control blood glucose with motion, often treat with oral antidiabetic drug, there are at present two large classes to commonly use orally-taken blood sugar reducing medicine, the first kind is insulin secretion stimulators, as sulfonylureas etc., this class medicine major side effects is easily to cause hypoglycemia, and larger to Liver and kidney toxicity; Equations of The Second Kind is euglycemic agent, mainly contain thiazolidinediones (TZDs) medicine as pioglitazone etc. with biguanides as metformin etc.Thiazolidinediones early application is more, but easily causes obesity, and the apparent side effects such as the risk of bladder cancer are suffered from heart failure and increase, use in recent years fewer and feweri; In biguanides use procedure, easily occur feeling sick, the adverse side effect such as diarrhoea.

In view of limitation and the toxic and side effects of above-mentioned Remedies for diabetes, find the treatment target spot of new diabetes, and development of new more preferably Remedies for diabetes is significant.

Oxidative stress (Oxidative stress) is that body is in the time being subject to various destructive stimulus, activity in vivo oxygen-derived free radicals (Reactive Oxygen Species, and active nitrogen free radical (Reactive Nitrogen Species ROS), RNS) produce too much, body degree of oxidation exceedes its oxidation resistance, causes the damage of tissue.ROS comprises superoxide anion (O ₂ ^-), hydroxy radical ( ^.and hydrogen peroxide (H OH) ₂o ₂).Oxidative stress is the pathogenesis basis of body insulin resistant and diabetes.The target tissue of insulin action is as muscle, in liver and adipose cell, producing excessive ROS causes body oxidative stress level to rise, activate a series of protein kinases as protein kinase C (PKC θ/ε), p38-MAPK, c-jun N terminal kinase (JNK), the signal proteins such as S6kinase1 (S6K1), these signal proteins will cause IRS IRS-1 phosphorylation inactivation (Boura-Halfon S.and Zick Y., Am.J.Physiol.Endocrinol.Metab., 2009, 296, E581-E591), then cause that downstream signal albumen is as phosphatidylinositol 3-kinase (PI3K) and the decline of protein kinase B (PKB/Akt) isoreactivity, cause GLUT4 (GLUT4) in muscle cell endochylema to reduce to cell membrane transposition, liver hepatic glycogen synzyme (glycogen synthase) activity significantly reduces, body produces insulin resistant, cause generation and development (the Styktal J.et.al. of diabetes, Free Radic.Biol.Med., 2012, 52, 46-58, Rains JL and Jain SK., Free Radic.Biol.Med., 2011,50,567-575, Gardner CD.et.al., Exp.Biol.Med.2003,228,836-842).

Therefore, reduce insulin action target tissue as ROS content in muscle, liver, can effectively improve insulin resistant, improve muscle and picked-up and the utilization of liver to glucose, be considered to treat effective means (Houstis N.et.al., the Nature of diabetes, 2006,440,944-948; Henriksen EJ., Free Radic.Biol.Med., 2011,51,993-999; Evans JL.et.al., Antioxid.Redox Signaling, 2005,7,1040-1052).

Fatty acid beta-oxidation mainly completes in mitochondrion and peroxisome, and wherein carbon chain lengths is less than 20 fatty acid and mainly in mitochondrion, completes oxidation energy supply, long-chain and metabolism in peroxisome of very-long-chain fatty acid that carbon chain lengths is greater than 20.Free fatty must activate becomes acyl coenzyme A (acyl-CoA), just can become the substrate of mitochondrion and peroxisome beta-oxidation metabolic enzyme, completes beta-oxidation process.

Acyl coenzyme A oxidase (acyl-CoA oxidase, AOX, EC1.3.3.6), it is the enzyme of catalysis first step reaction in peroxisome fatty acid beta-oxidation process, also be the rate-limiting enzyme of peroxisome fatty acid beta oxidation process, the about 72kDa of molecular weight, per molecule AOX contains a part flavin adenine dinucleotide (FAD) (Flavin adenine dinucleotide, FAD).AOX catalysis acyl coenzyme A carbochain 2-3 position dehydrogenation, forms 2-enoyl coenzyme A (2-enoyl-CoA), and the hydrogen atom of taking off is directly combined with molecular oxygen, forms H ₂o ₂(Reddy JK.and Hashimoto T., Annu.Rev.Nutr., 2001,21,193-230).

There is no about AOX inhibitor at present any report as Remedies for diabetes.

Summary of the invention

The object of the present invention is to provide the purposes of acyl coenzyme A oxidase as the treatment target spot of diabetes, and the Remedies for diabetes of developing accordingly.

In a first aspect of the present invention, provide the purposes of acyl coenzyme A oxidase inhibitor, for the preparation of the pharmaceutical composition of prevention, improvement or treatment diabetes.The oxidasic international zymetology classifying and numbering of described acyl coenzyme A is EC1.3.3.6.

In a preference, described acyl coenzyme A oxidase inhibitor reduces oxidative stress level in target organ or tissue by suppressing acyl coenzyme A oxidase, reduce oxyradical (ROS) content in insulin action target tissue, improve insulin resistant, thus prevention, improvement or treatment diabetes.

In another preference, described diabetes comprise: type 1 diabetes or type 2 diabetes mellitus.

In another preference, described type 1 diabetes is characterized as insulin secretion in body and definitely lacks companion's insulin resistant, gives can not effectively control blood glucose after insulinize; Described type 2 diabetes mellitus is characterized as insulin resistant companion insulin and relatively lacks, or hypoinsulinism companion insulin resistant.

In another preference, described acyl coenzyme A oxidase inhibitor is the material that suppresses acyl coenzyme A oxidase active or expression.

In another preference, described acyl coenzyme A oxidase inhibitor includes, but is not limited to: small organic molecule, micromolecule inorganic matter, anti-acyl coenzyme A oxidase antibody, can and suppress its active nucleic acid fragment and polypeptide with acyl coenzyme A oxidase specific binding.

In another preference, described acyl coenzyme A oxidase inhibitor also comprises inhibitor precursor.

In another preference, described acyl coenzyme A oxidase inhibitor or inhibitor precursor include, but is not limited to: 10,12-, 25 carbon diacetylenic acid (Pentacosadiynoic acid; CAS coding: 66990-32-7, LH-919) or 10,12-, 25 carbon diine acyl coenzyme A(10,12-Pentacosadiynoyl-CoA, LH-919-CoA).

In another preference, described pharmaceutical composition also for:

Suppress acyl coenzyme A oxidase active in patient body;

Improve insulin resistant;

Reduce oxidative stress level in target organ or tissue; Or

Reduce oxyradical (ROS) content in insulin action target tissue.

In another aspect of this invention, provide a kind of for preventing, improve or treat the pharmaceutical composition of diabetes, described pharmaceutical composition comprises: acyl coenzyme A oxidase inhibitor (preferably, for treating the inhibitor of effective dose); And pharmaceutically acceptable carrier or excipient.

In a preference, described acyl coenzyme A oxidase inhibitor is the material that suppresses acyl coenzyme A oxidase active or expression.

In another preference, described acyl coenzyme A oxidase inhibitor includes, but is not limited to: anti-acyl coenzyme A oxidase antibody, can and suppress its active nucleic acid fragment and polypeptide, small organic molecule, micromolecule inorganic matter with acyl coenzyme A oxidase specific binding.

In another preference, described pharmaceutical composition also comprises: medicine, dietary supplement, Halth-care composition.

In another preference, described acyl coenzyme A oxidase inhibitor also comprises inhibitor precursor; Preferably include, but is not limited to: 10,12-, 25 carbon diacetylenic acids (LH-919) or 10,12-, 25 carbon diine acyl coenzyme A (LH-919-CoA).

In another aspect of this invention, a kind of method of preparing prevention, improving or treat the pharmaceutical composition of diabetes is provided, described method comprises: by acyl coenzyme A oxidase inhibitor and pharmaceutically acceptable carrier or mixed with excipients, obtain described pharmaceutical composition.

In another aspect of this invention, provide a kind of method of prevention, improvement or treatment diabetes, described method comprises: acyl coenzyme A oxidase active or expression in the patient body that suppresses to need prevention, improve or treat.

In a preference, described inhibition acyl coenzyme A oxidase active or the method for expression are: use acyl coenzyme A oxidase inhibitor or the acyl coenzyme A oxidase inhibitor precursor of effective dose to the patient who needs prevention, improves or treat.

In another preference, the target organ of described acyl coenzyme A oxidase inhibitor effect or be organized as mammal or the oxidasic organ-tissue of people's expression in vivo acyl coenzyme A, comprises liver organization, muscular tissue, fatty tissue, beta Cell of islet and renal tissue etc.

In another preference, described acyl coenzyme A oxidase inhibitor is LH-919-CoA or its precursor LH-919.Preferably select LH-919 to implement, its application dosage is 0.1-1000 μ g/kg/d; Preferably, application dosage is 5-200 μ g/kg/d.

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Brief description of the drawings

Fig. 1 .LH-919-CoA suppresses the AOX activity analysis of purification.AOX is dissolved in 20mM PBS buffer (pH7.4), concentration 80nM, in enzymatic solution, add respectively 80nM (▼), 240nM (△), 480nM (■), 960nM () LH-919-CoA, and 800nM (zero) LH-919 free acid, matched group adds same volume distilled water (●), at 25 DEG C, hatch respectively after different time, measure AOX residual enzyme activity.

Fig. 2 .LH-919-CoA suppresses AOX Determination of Kinetic Parameters.

Fig. 3 .LH-919-CoA vitro inhibition rat liver peroxisome AOX activity analysis.

Fig. 4. variable concentrations LH-919-CoA vitro inhibition rat liver peroxisome AOX activity analysis.

In Fig. 5 .AOX inhibitor precursor LH-919 body, suppress C57BL mouse liver AOX activity analysis.

In Fig. 6 .AOX inhibitor precursor LH-919 body, suppress Wistar rat liver AOX activity analysis.

The impact of Fig. 7 A.AOX inhibitor precursor treatment on alloxan induction diabetes rat oral glucose tolerance (OGTT).

After the treatment of Fig. 7 B.AOX inhibitor precursor, alloxan induction diabetes rat oral glucose tolerance blood glucose rolls off the production line area (AUC) relatively.

The impact of Fig. 8 .AOX inhibitor precursor treatment on alloxan induction diabetes rat serum triglycerides.

The impact of Fig. 9 .AOX inhibitor precursor treatment on alloxan induction diabetes rat serum free fatty acid.

The impact of Figure 10 .AOX inhibitor precursor treatment on alloxan induction diabetes rat liver AOX activity.

The impact of Figure 11 .AOX inhibitor precursor treatment on alloxan induction diabetes rat serum MDA.

The treatment of Figure 12 .AOX inhibitor precursor is to alloxan induction diabetes rat liver H ₂o ₂the impact of content.

The impact of Figure 13 .AOX inhibitor precursor treatment on alloxan induction diabetes rat liver MDA.

The impact of Figure 14 A.AOX inhibitor precursor treatment on ob/ob mice oral glucose tolerance (OGTT).

Figure 14 B.AOX inhibitor precursor treatment is on area (AUC) impact of rolling off the production line of ob/ob mice oral glucose tolerance blood glucose.

Figure 15 .AOX inhibitor precursor is treated the impact on ob/ob mice serum insulin.

Figure 16 .AOX inhibitor precursor is treated the impact on ob/ob mice serum triglyceride.

Figure 17 .AOX inhibitor precursor is treated the impact on ob/ob mice serum free fatty.

Figure 18 .AOX inhibitor precursor is treated the impact on ob/ob mouse liver AOX activity.

Figure 19 .AOX inhibitor precursor is treated ob/ob mouse liver H ₂o ₂the impact of content.

Figure 20 .AOX inhibitor precursor is treated the impact on ob/ob mouse liver MDA.

Figure 21 .AOX inhibitor precursor is treated the impact on db/db mice serum insulin.

Figure 22 .AOX inhibitor precursor is treated the impact on db/db mice serum triglyceride.

Figure 23 .AOX inhibitor precursor is treated the impact on db/db mice serum free fatty.

Figure 24 .AOX inhibitor precursor is treated the impact on db/db mouse liver AOX activity.

Figure 25 .AOX inhibitor precursor is treated the impact on db/db mouse liver MDA.

Detailed description of the invention

The present invention discloses the purposes of acyl coenzyme A oxidase inhibitor (or inhibitor precursor) in the medicine for the preparation for the treatment of diabetes first.Particularly, in the present invention, disclose by giving acyl coenzyme A oxidase inhibitor, suppressed acyl coenzyme A oxidase active in diabetics body, and then significantly reduced oxidative stress level in target organ or tissue, improve insulin resistant in body, treatment diabetes.

As used herein, described LH-919 refers to 10,12-, 25 carbon diacetylenic acids (10,12-Pentacosadiynoic acid), its CAS coding: 66990-32-7.LH-919 is the precursor of LH-919-CoA, and LH-919 can be converted into LH-919-CoA in vivo.

As used herein, described LH-919-CoA refers to 10,12-, 25 carbon diine acyl coenzyme A(10,12-Pentacosadiynoyl-CoA).

In specific embodiment of the invention scheme, taking liver as example, as the target organ of AOX inhibitor effect, the test AOX inhibitor inhibitory action to liver AOX in vitro, and AOX inhibitor or AOX inhibitor precursor (precursor) inhibitory action to liver AOX in vivo.It needs to be noted, target organ or tissue that AOX inhibitor or AOX inhibitor precursor act on are in vivo not limited to liver, comprise that in mammal or human body, all express the organ of AOX albumen, such as muscular tissue, fatty tissue, beta Cell of islet, renal tissue etc., because the AOX albumen that in body, these Different Organs are expressed is same gene transcription product, consensus amino acid sequence, and there is identical physiologically active.

Particularly, the specific embodiment of the invention comprises following 3 aspects:

The preparation of 1.AOX inhibitor and In-vitro Inhibitory Effect

The inventor has prepared LH-919-CoA.Taking LH-919 acid as raw material, prepare high-purity LH-919-CoA, test its inhibitory action to AOX.Prepared the synthetic method reference literature of acyl coenzyme A by free fatty and implement (Li D.et.al., J.Am.Chem.Soc., 2001,121,9034-9042).

The inventor finds first, and LH-919-CoA can significantly suppress liver AOX activity in vitro.First adopt recombinant expressed and highly purified rats'liver AOX as target protein, measure the inhibition of LH-919-CoA to AOX, result LH-919-CoA can suppress rapidly AOX activity, and this inhibitory action is irreversible.Free LH-919 acid does not have inhibitory action to AOX.

Adopt the complete peroxisome of rat liver as determination object, LH-919-CoA is in vitro to AOX inhibitory action in peroxisome in test, and result LH-919-CoA can suppress rapidly and significantly rat liver peroxisome AOX activity.

In 2.AOX inhibitor precursor body, suppress AOX activity

LH-919-CoA is LH-919 conversion product in vivo.In one embodiment of the invention, give Wistar rat oral gavage by doses LH-919, after absorbing completely, put to death, dissect rapidly rat, win liver, separate liver peroxisome, by rat liver peroxisome inclusions is analyzed, detect and in peroxisome, have LH-919-CoA.Therefore, after LH-919 absorbs in vivo, be converted into LH-919-CoA at liver.

In fact, the mechanism that free fatty activates as acyl coenzyme A in cell has been perfectly clear, taking peroxisome as example, on peroxisomal membrane, there is long-chain/utmost point long-chain fatty acyl-CoA synthetase (acyl-CoA synthetase), be acyl coenzyme A (acyl-CoA) by free fatty (NEFA) activation, after enter peroxisome, enter AOX protein active center (Reddy JK.and Hashimoto T. as catalytic substrate, Annu.Rev.Nutr., 2001,21,193-230).

In one embodiment of the invention, give C57BL mouse gavage by LH-919, after gavage 3h, put to death, dissect rapidly mice, win liver, separate peroxisome, measure AOX enzymatic activity, result is compared with matched group, and perfusion group C57BL mouse liver AOX is active significantly to be reduced, and inhibitory action intensity and perfusion dosage proportional.

In another embodiment of the present invention, give Wistar rat oral gavage by LH-919, after gavage 3h, put to death, dissect rapidly rat, win liver, separate peroxisome, measure AOX activity, result is compared with matched group, and perfusion group Wistar rat liver AOX is active significantly to be reduced, and inhibitory action intensity and perfusion dosage proportional.

Learn by body outer suppressioning experiment, free LH-919 is to AOX unrestraint effect.After LH-919 absorbs in vivo, be converted into LH-919-CoA and suppress AOX activity.

Therefore, LH-919 is the precursor of AOX inhibitor LH-919-CoA, and LH-919 in vivo metabolite LH-919-CoA can suppress AOX activity fast and irreversibly.

3.AOX inhibitor or AOX inhibitor precursor suppress AOX activity in vivo, treatment diabetes

Under diabetic disease states, because body insulin resistant causes glucose utilization obstacle, therefore organize and utilize more body fat oxidation that energy is provided, blood free fatty acid (non-esterified fatty acid, NEFA) content significantly raises, enter liver, muscle, the NEFA of the tissues such as beta Cell of islet also increases thereupon, cause these to organize and participate in metabolic enzyme activities of fatty acid beta oxidation and the equal compensatory of fatty acid beta oxidation level (the Horie S.et.al. that significantly raises in peroxisome, J.Biochem., 1981, 90, 1691-1696, Asayama K.et al., Mol.Cell.Biochem., 1999,194,227-234).

In the embodiment of the present invention, diabetes are large/and the active all compared with normal groups of mouse liver AOX significantly raise.

The inventor finds first, after diabetes produce, by suppressing the body internal target AOX of organ or tissue activity, can reduce H in body internal target organ or tissue ₂o ₂generation, reduce oxidative stress level in these organ or tissues, significantly improve insulin resistant in body, obtain the effect of beyond thought treatment diabetes.

The purposes of AOX inhibitor precursor in the medicine for the preparation for the treatment of diabetes, described AOX inhibitor precursor is converted into AOX inhibitor in mammal or human body, and suppresses AOX activity.For example, in the embodiment of the present invention, LH-919 is AOX inhibitor precursor, and LH-919, after absorbing in body, is converted into LH-919-CoA at liver, can significantly suppress fast AOX activity.

Should be appreciated that AOX inhibitor and the physiologically active of AOX inhibitor precursor in mammal or human body are identical, suppress target organ or organize AOX activity.

AOX inhibitor or AOX inhibitor precursor can be treated type 1 diabetes and type 2 diabetes mellitus.

Wherein said type 1 diabetes is characterized as insulin secretion in body and definitely lacks companion's insulin resistant, gives can not effectively control blood glucose after insulinize; Described type 2 diabetes mellitus is characterized as insulin resistant companion insulin and relatively lacks, or hypoinsulinism companion insulin resistant.

Particularly, in embodiment of the present invention, carried out the test of pesticide effectiveness for a kind of type 1 diabetes animal model and two kinds of type 2 diabetes mellitus animal models.

Embodiment of the present invention, taking a kind of AOX inhibitor precursor LH-919 as example, is carried out the animal test of pesticide effectiveness.LH-919 in vivo conversion product LH-919-CoA is AOX inhibitor, can suppress significantly fast in vivo AOX activity.

In the test of pesticide effectiveness, the effective dose of LH-919 treatment diabetes is 0.1-1000 μ g/kg/d, and better dosage is 10-200 μ g/kg/d.AOX inhibitor precursor LH-919 treatment diabetes have dose dependent.

One embodiment of this invention, first for type 1 diabetes, adopt alloxan induction diabetes rat model, due to alloxan selective destruction beta Cell of islet, cause that insulin definitely lacks, blood glucose sharply raises, and therefore gives alloxan diabetes rats subcutaneous injection low dosage protamine zine insulin, prevents the ketoacidosis causing because blood glucose is too high.After Cheng Mo, diabetes rat fasting glucose significantly raises and with insulin resistant, serum triglycerides and free fatty significantly raise, and liver AOX activity and vivo oxidation stress level all significantly raise.

Give after the treatment of various dose AOX inhibitor precursor, treatment group blood glucose significantly reduces (p<0.01) compared with matched group, oral glucose tolerance (OGTT) ability significantly strengthens, and serum triglycerides and free fatty (NEFA) content significantly reduces (p<0.01).After the treatment of AOX inhibitor precursor, active significantly reduce (p<0.01) of diabetes rat liver AOX, vivo oxidation stress level significantly reduces thereupon, show Serum MDA (malondialdeyde, MDA) content significantly reduces (p<0.01), liver H ₂o ₂significantly reduce (p<0.01) with MDA content.

Therefore, for type 1 diabetes companion Insulin Resistance Animal Model, give after insulinize, because of insulin resistant in body, can not effectively control blood glucose.After AOX inhibitor or the treatment of AOX inhibitor precursor, by suppressing AOX activity in body, significantly oxidative stress level in ameliorate body, improves insulin resistant, reduces blood glucose, improves body sugar tolerance, thus treatment type 1 diabetes.

Another 2 embodiments of the present invention, for type 2 diabetes mellitus, select ob/ob mice, two kinds of animal models of db/db mice, these 2 kinds of animal models are spontaneous type 2 diabetes mellitus animal model, have the features such as insulin resistant, hyperglycemia, hyperinsulinemia and hyperlipidemia.Liver AOX activity also significantly raises simultaneously, and vivo oxidation stress level compared with normal group significantly raises.

After giving respectively these two kinds of type 2 diabetes mellitus animals with the treatment of AOX inhibitor precursor, treatment group blood glucose and serum insulin levels significantly reduce (p<0.01) compared with matched group, insulin resistance index HOMA-IR significantly declines, and oral glucose tolerance (OGTT) significantly strengthens.Serum triglycerides and free fatty (NEFA) content also significantly reduces (p<0.01).After the treatment of AOX inhibitor precursor, ob/ob mice and db/db mouse liver AOX enzymatic activity significantly reduce (p<0.01), thereby vivo oxidation stress level significantly reduces, and shows liver H ₂o ₂content and MDA content all significantly reduce (p<0.01).

Therefore, for type 2 diabetes mellitus animal model, give AOX inhibitor or the treatment of AOX inhibitor precursor, suppress AOX activity in body, significantly oxidative stress level in ameliorate body, reduces blood glucose, improves body sugar tolerance, thus treatment type 2 diabetes mellitus.

Based on the inventor's above-mentioned new discovery, the invention provides the purposes of the inhibitor of a kind of AOX, for the preparation of the medicine of prevention, improvement or treatment diabetes.

As used herein, described AOX inhibitor has comprised time adjustment, blocker, antagonist, blocker etc.

Described AOX inhibitor refers to the activity of any AOX of reduction albumen, the stability that reduces AOX gene or albumen, the expression of lowering AOX albumen, minimizing AOX albumen effective acting time or suppresses the material of transcribing and translating of AOX gene, these materials all can be used for the present invention, as for lowering AOX useful material, thereby can be used for prevention or treatment diabetes.For example, described inhibitor comprises: the antibody that small organic molecule, micromolecule inorganic matter, micromolecular compound, specificity are combined with AOX or part, can and suppress its active nucleic acid fragment and polypeptide etc. with AOX specific binding, specificity disturbs siRNA molecule or the antisense nucleotide etc. of AOX gene expression.

As optimal way of the present invention, described AOX inhibitor is compound L H-919-CoA or its precursor LH-919.The present invention also comprises isomer, racemic modification, pharmaceutically acceptable salt, the hydrate of above-claimed cpd.Described " pharmaceutically acceptable salt " refers to that described compound reacts the salt generating with mineral acid, Organic Acid and Base metal or alkaline-earth metal etc.Compound has one or more asymmetric centers.So these compounds can be used as racemic mixture, independent enantiomer, independent diastereomer, non-enantiomer mixture, cis or transisomer existence.Described " precursor " refer to after taking by suitable method, and the precursor of this compound carries out metabolism or chemical reaction and is transformed into this compound in patient body, or the salt or the solution that are made up of this compound.

As the optional mode of another kind of the present invention, described AOX inhibitor can be the antibody that a species specificity is combined with AOX.Described antibody can be monoclonal antibody or polyclonal antibody.Available AOX protein immune animal, as rabbit, mice, rats etc. produce polyclonal antibody; Multiple adjuvant can be used for strengthening immunoreation, includes but not limited to Freund adjuvant etc.Similarly, expression AOX or its cell with antigenic fragment can be used to immune animal and produce antibody.Described antibody can be also monoclonal antibody, and this type of monoclonal antibody can be utilized hybridoma technology to prepare (to see the people such as Kohler, Nature256; 495,1975; The people such as Kohler, Eur.J.Immunol.6:511,1976; The people such as Kohler, Eur.J.Immunol.6:292,1976; The people such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).

As the optional mode of another kind of the present invention, described AOX inhibitor can be the specific siRNA molecule of a kind of AOX (siRNA).As used herein, described " siRNA (small interfering RNA; siRNA) " refers to a kind of short-movie section double stranded rna molecule, the specific mRNA that can degrade taking the mRNA of homologous complementary sequence as target, this process is exactly that RNA disturbs (RNA interference) process.SiRNA can be prepared into the form of double-strandednucleic acid, and it contains a positive-sense strand and an antisense strand, and these two chains only form double-stranded under the condition of hybridization.A double-stranded RNA complex can be prepared by the positive-sense strand being separated from each other and antisense strand.Therefore, by way of example, complementary positive-sense strand and antisense strand are chemosynthesis, can hybridize by annealing thereafter, produce synthetic double-stranded RNA complex.

The present invention also provides a kind of pharmaceutical composition, described AOX inhibitor or AOX inhibitor precursor that it contains effective dose, and pharmaceutically acceptable carrier.Pharmaceutical composition of the present invention can be used for, by suppressing AOX activity, improving body insulin resistant, treatment diabetes.

As used herein, the composition of " pharmaceutically acceptable " is applicable to people and mammal and without adverse side effect (as toxicity, stimulation and allergy), has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.

As used herein, term " pharmaceutical composition " includes, but is not limited to: medicine, dietary supplement, Halth-care composition, as long as they contain AOX inhibitor of the present invention or AOX inhibitor precursor, as the active component of prevention, improvement or treatment mammal and people's diabetes.

In the present invention, term " contains " and represents that various compositions can be applied in mixture of the present invention or compositions together.Therefore, term " mainly by ... composition " and " by ... form " be included in during term " contains ".

Acceptable carrier on pharmaceutical composition Chinese materia medica of the present invention, comprises (but being not limited to): olive oil, saline, buffer, glucose, water, glycerol and combination thereof.Conventionally pharmaceutical preparation should match with administering mode.Described pharmaceutical composition should be manufactured under aseptic condition.The dosage of active component is treatment effective dose.

The dosage form of pharmaceutical composition of the present invention can be diversified, as long as the dosage form that can make active component effectively arrive in mammal or human body is all fine.Such as being selected from: tablet, capsule, powder, granule, syrup, solution, suspension or aerosol.

The in the situation that of needs, pharmaceutical composition of the present invention can also with other one or more for diabetes or the effective material use in conjunction of metabolism syndrome.For example, in described compositions, also can contain: the medicine that (c) is selected from following one or more: antidiabetic medicine, fat-reducing medicament, slimming medicine, antihypertensive drug or the anticoagulation medicine of other kind.In the time of two or more medication combined administration, generally have and be better than individually dosed effect.For example, the antidiabetic medicine of other kind is selected from: biguanides, sulfonylurea, meglitinide hypoglycemic medicine, alpha-glucosidase inhibitor, euglycemic agent (as Thiazolidinediones), aP2 inhibitor, DPPIV inhibitor, SGLT2 inhibitor, insulin, GLP-1 (GLP-1) or their analog.

Should be appreciated that and the invention is not restricted to concrete grammar described herein, comprise the testing program of employing, analysis test method and reagent etc., these all can change.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, the third edition, Science Press, the condition described in 2002, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.

The preparation of embodiment 1, AOX inhibitor and body outer suppressioning experiment

(1) preparation of AOX inhibitor LH-919-CoA

The preparation method reference literature of LH-919-CoA carries out (Li D.et.al., J.Am.Chem.Soc., 2001,121,9034-9042).Concrete operations: LH-91920mg (Sigma; St.Louis; MO); add 5mL anhydrous tetrahydro furan (tetrahydrofuran; THF) (Acros; Geel; Belgium), in, under nitrogen protection, in solution, add 0.1mmol triethylamine (triethylamine) (Sigma, St.Louis; MO); after mix and blend 10 minutes, at 0 DEG C, in solution, dropwise add 0.1mmol isobutyl chlorocarbonate (isobutyl chloroformate) (Acros, Geel; Belgium), stirring at room temperature 1h.After by 50mg coenzyme A sodium salt (Coenzyme A) (USB; Cleveland; OH) be dissolved in distilled water; add 1mol/L NaOH and adjust pH to 8.0; under nitrogen protection, be added drop-wise in reactant liquor; continue to stir after 20 minutes, slowly drip 1M hydrochloric acid, pH value of solution is adjusted to 5.5～6.0.Under negative pressure, organic solvent is vapored away, the residual organic solvent of twice removal of extracted with diethyl ether for remaining aqueous solution, collect lower floor's water, through centrifugal ultrafiltration pipe (Millipore, Billerica, MA) rear reversed-phase high-performance liquid chromatography (RP-HPLC) the separation and purification LH-919-CoA that passes through of filtration.RP-HPLC separation condition: chromatographic column hypersil C18 post (250mm × 4.6mm, 5 μ m), adopt gradient elution, mobile phase A is 25mM kaliumphosphate buffer (pH5.9), Mobile phase B is 85% (v/v) methanol aqueous solution, and elution program Mobile phase B accounts for mobile phase cumulative volume ratio and is: 0-5min0%B, 5-24min0%-100%B, 24-35min100%B, flow velocity: 0.8mLmin ^-1, detect wavelength 260nm, 30 DEG C of column temperatures, sample size: 100 μ L.LH-919-CoA appearance time, about 27min, is collected LH-919-CoA component, then removes desolventizing through vacuum lyophilization, obtains LH-919-CoA freeze-dried powder.

(2) expression and purification restructuring AOX

The present invention adopts recombinant rat liver AOX to screen its inhibitor as target protein, should be appreciated that restructuring AOX has identical physiologically active with natural A OX.Recombinant expressed and the affinity purification reference literature method of rats'liver AOX in E.coli implemented (Zeng J.et.al., Protein Expression and Purification.2004,37,472-478), AOX enzymatic activity after highly purified is 1.89U/mg, purity of protein >95%.

(3) LH-919-CoA analyzes the recombinant rat liver AOX In-vitro Inhibitory Effect of purification

Above-mentioned highly purified recombinant rat liver AOX is dissolved in 20mM PBS buffer (pH7.4), concentration 80nM, in enzymatic solution, add respectively variable concentrations LH-919-CoA (inhibitor group) again, matched group adds same volume distilled water, at 25 DEG C, hatch respectively after different time, measure inhibitor group and matched group acyl coenzyme A oxidase residual activity.Acyl coenzyme A oxidase active assay method is according to document (Luo Y.et al., Arch.Biochem.Biophys., 2000,384,1-8), change the i.e. generation (ε of 2-enoyl coenzyme A product by Shimadzu UV-1800 ultraviolet/visible spectrophotometric determination 263nm absorbance _263nm=6.7mM ^-1cm ^-1), measure AOX activity.Reaction system comprises 50mmol/L PBS buffer, pH7.4,30 μ M palmitoyl-CoA (Sigma, St.Louis, MO), and 500ng AOX, cumulative volume 500 μ L.

The results are shown in Figure 1, LH-919-CoA and can suppress fast AOX activity, and inhibitory action strengthens with LH-919-CoA concentration.Free LH-919 acid does not have inhibitory action to AOX, LH-919 free acid must activate for acyl coenzyme A be could suppress AOX activity after substrate form.

LH-919-CoA is irreversible to the inhibitory action of AOX, experimental procedure is as follows: AOX is dissolved in 20mM PBS buffer, pH7.4, final concentration 100nM, in enzymatic solution, add 2 μ M LH-919-CoA again, at 25 DEG C, hatch 60min, to ensure that enzyme and inhibitor fully act on, then pack AOX and inhibitor mixed thing in bag filter (MWCO 5000), put into again 2L 20mM PBS buffer (pH7.4), to buffer dialysis 24h, changed during this time a dialysis solution every 4 hours, fully to remove free LH-919-CoA in 4 DEG C.100nM AOX is added respective volume distilled water by matched group, and all the other operations are identical with inhibitor group.After dialysis, inhibitor group and matched group are measured respectively to AOX enzymatic activity, result is as shown in table 1, and inhibitor group is removed after free LH-919-CoA by dialysis, its AOX activity can not be recovered, matched group enzymatic activity does not have significant change, and therefore, LH-919-CoA is irreversible to the inhibitory action of AOX.

Table 1, LH-919-CoA suppress AOX Before and after dialysis specific activity

Note: the percent value of matched group activity before relative activity represents the each group of activity value of measuring and dialyses.

Because LH-919-CoA is irreversible to the inhibitory action of AOX, therefore adopt K _iand k _inactcharacterize inhibition kinetics, reference literature method is carried out (Li D.et.al., J.Am.Chem.Soc., 2001,119,111-133).Experimental procedure is as follows: 50nM AOX is dissolved in 20mM PBS buffer, pH7.4, add respectively 0.5 μ M to enzymatic solution again, 1 μ M, 3 μ M, 5 μ M, 10 μ M, 15 μ M Z-919-CoA, at 25 DEG C, hatch 0min, 5min, 10min, after 15min and 20min, measure respectively each dosage group AOX activity, under each LH-919-CoA concentration, take the logarithm incubation time is mapped with relative residual activity, adopt Sigmaplot12.0 software to carry out linear fit, calculate apparent inhibition speed k under each inhibitor concentration _obs, by k under each concentration _obsto inhibitor concentration double-reciprocal plot, shown in Fig. 2, calculate LH-919-CoA to AOX enzyme inhibition dynamics parameter K _ivalue is 810nM, k _inactvalue is 3.08min ^-1.

(4) LH-919-CoA analyzes rat liver peroxisome AOX In-vitro Inhibitory Effect

Rat liver peroxisome separates reference literature and implements (Small GL.et.al., Biochem.J., 1985,227,205-210).Operating procedure is as follows: accurately take rat fresh liver and organize 0.5g, on ice bath, shred, be suspended in (10% (w/v) sucrose in 9 times of volume homogenate buffers, 3mM imidazoles, 20mM PBS buffer, pH7.4), on ice bath after electronic homogenate, centrifugal 10 minutes of 5000 × g at 4 DEG C, draws supernatant.Lower sediment is suspended in a small amount of homogenate buffer more centrifugal 10 minutes of 5000 × g again, merges supernatant in 35000 × g centrifugal 30 minutes, and lower sediment is peroxisome.

Getting 200 μ g peroxisomes (with protein refractometer) is suspended in 20mM PBS buffer (pH7.4), in solution, add 5 μ M LH-919-CoA (inhibitor group) again, matched group adds respective volume distilled water, at 25 DEG C, hatch 0min, 2min, 5min, after 10min and 20min, measures respectively inhibitor group and control group A OX activity.

The active reference literature of peroxisome AOX is measured (Johnson JK.et.al., Biochemstry, 1992,31,10564-10575; Luo Y.et al., Arch.Biochem.Biophys., 2000,384,1-8), adopt spectrophotography, mensuration AOX catalytic substrate 3-indolepropionyl-CoA (IP-CoA) is oxidized trans-3-indoleacryloyl-CoA (IA-CoA) amount generating and reflects AOX activity, the extinction coefficient epsilon of IA-CoA _367nmfor 26.5mM ^-1cm ^-1.Reactant liquor is containing 50mmol/L PBS buffer, pH7.4,30 μ mol/L IP-CoA (purity >95%), 30 μ mol/L FAD (Sigma, St.Louis, MO), 0.5g/L BSA (Sangon Biotech., Shanghai), 0.02%TritonX-100 (Sangon Biotech.,, and 200 μ g peroxisomes (with protein refractometer) Shanghai).Adopt Shimadzu UV-1800 UV/visible spectrophotometer, measure 367nm light absorption value and change, under analysis condition, the required peroxisome protein content of generation 1 μ mol IA-CoA per minute is 1 unit of enzyme activity (U).

As shown in Figure 3, taking rat peroxisome as surveying the object of living, LH-919-CoA can suppress rat peroxisome AOX activity to result fast.

Another experiment, getting 200 μ g peroxisomes (with protein refractometer) is suspended in 20mM PBS buffer (pH7.4), in solution, add respectively variable concentrations LH-919-CoA (inhibitor group), matched group adds respective volume distilled water, at 25 DEG C, hatch after 2min, measure respectively the each group of inhibitor and control group A OX activity, as shown in Figure 4, LH-919-CoA vitro inhibition peroxisome AOX is active to add and suppresses dosage correlation with institute result.

Embodiment 2, AOX inhibitor precursor suppress AOX function analysis in large/Mice Body

(1) LH-919 is converted into the measuring of LH-919-CoA in animal body

6 of SPF level Wistar rats, male, body weight 220-250g, after fasting 12h, 6 rats are divided into 2 groups, matched group and medicine group, 3 every group, matched group only gives olive oil gavage 0.2mL/, medicine group is dissolved in LH-919 in respective volume olive oil, and gavage LH-919100 μ g/kg puts to death whole rats after 3h, dissect and win liver rapidly, put into immediately liquid nitrogen and preserve.

In liver cell, the mensuration list of references of subcellular fraction unit inclusions acyl coenzyme A is implemented (Melde K.et.al., Biochem.J., 1991,274,395-400).Operating procedure is as follows: accurately take liver organization 0.3g, on ice bath, shred, be suspended in 9 times of volume homogenate buffers on (10% sucrose, 3mM imidazoles, 20mM PBS) ice bath after electronic homogenate, centrifugal 10 minutes of 4 DEG C of 5000 × g, draw supernatant.Lower floor is suspended in a small amount of homogenate buffer more centrifugal 10 minutes of 5000 × g again, merges supernatant in 30000 × g centrifugal 30 minutes, and the precipitation obtaining is peroxisome.First peroxisome is suspended in to (4 DEG C) in 2mL ultra-pure water, then adds 200 μ L 5mM HClO ₄precipitating proteins, and 20nM Lignoceryl-CoA (C24:0) is as reference sample, protein precipitation is in 16000 × g centrifugalize, supernatant K ₂cO ₃regulate pH to 7.0, centrifugal removal KClO ₄precipitation, supernatant is removed solvent through lyophilization, and remaining solid thing is dissolved in 200 μ L ultra-pure waters, detects matched group and the each sample LH-919-CoA of medicine group content by RP-HPLC, finally confirms by mass spectrum (MS).RP-HPLC operating condition, chromatographic column hypersil C18 post (250mm × 4.6mm, 5 μ m), mobile phase A is 25mM kaliumphosphate buffer (pH5.9), and Mobile phase B is 85% methanol aqueous solution, adopts gradient elution, gradient elution program Mobile phase B accounts for mobile phase cumulative volume ratio: 0-5min0%B, 5-24min0%-100%B, 24-35min100%B, flow velocity: 0.8mLmin ^-1, detect wavelength 260nm, column temperature: 30 DEG C, sample size: 25 μ L.

Result shows, in the each sample liver of medicine group peroxisome, LH-919-CoA all detected, in matched group liver peroxisome, LH-919-CoA do not detected.

Therefore, LH-919, through absorbing and enter after liver in body, is converted into LH-919-CoA, enters peroxisome, interacts as substrate and AOX.

(2) AOX inhibitor precursor is to liver AOX the Inhibitory Effects in C57BL mouse body

16 of SPF level C57BL mouses, male female each 8, body weight 20-25g, after mice fasting 12h, 16 mices are divided into 2 groups, matched group and medicine group, 8 every group, male female half and half.Matched group only gives olive oil gavage 0.2mL/; Medicine group is dissolved in LH-919 in respective volume olive oil, gavage LH-91920-80 μ g/kg.After gavage 3h, whole mices are put to death, dissect and win liver rapidly, put into immediately liquid nitrogen and preserve.Accurately take liver 0.3g, homogenate separates and obtains after peroxisome, measures respectively matched group and the each sample AOX of medicine group activity.

Data represent with X ± SEM, carry out statistical procedures with SPSS17.0 software, and between each group, diversity relatively adopts independent sample t inspection.With matched group comparison, * P < 0.05, * * P < 0.01, * * * P < 0.001.

As shown in Figure 5, in its liver organization of medicine group, AOX enzymatic activity significantly reduces compared with matched group result, shows that AOX inhibitor precursor LH-919 is converted into LH-919-CoA in vivo, can significantly suppress C57BL mouse liver AOX enzymatic activity, consistent with experiment in vitro result.AOX inhibitor precursor LH-919 suppresses liver AOX enzymatic activity and has good dose dependent in C57BL mouse body.

(3) AOX inhibitor precursor is to liver AOX the Inhibitory Effects in Wistar rat body

12 of SPF level Wistar rats, male female each 6, body weight 220-250g, after rat fasting 5h, is divided into 2 groups, matched group and medicine group, 6 every group, male female half and half.Matched group only gives olive oil gavage 0.2mL/; Medicine group is dissolved in LH-919 in respective volume olive oil, and gavage LH-91910-80 μ g/kg puts to death whole rats after 3h, dissects and wins liver rapidly, puts into immediately liquid nitrogen and preserves.Accurately take liver 0.3g, homogenate separates and obtains after peroxisome, measures respectively matched group and the each sample AOX of medicine group enzymatic activity.

As shown in Figure 6, in its liver organization of medicine group, AOX enzymatic activity significantly reduces compared with matched group result, shows that AOX inhibitor precursor LH-919 is converted into LH-919-CoA in vivo, can significantly suppress liver AOX activity, consistent with experiment in vitro result.AOX inhibitor precursor LH-919 suppresses rat liver AOX enzymatic activity and has good dose dependent.

Embodiment 3, AOX inhibitor precursor are to the experiment of alloxan induction diabetes rat blood sugar lowering

50 of SPF level Wistar rats, body weight 180-200g, feeds with common rat feed the feed of freely intaking.Rat adapted to feed after one week, and 20 rats are as normal group, all the other 30 rat modelings.30 rat fasting 18h pneumoretroperitoneum injection alloxan (Sigma, St.Louis, MO) 200mg/kg, after 48h, every special protamine zine insulin (Determir in rat skin lower injection ground, Novo Nordisk, Denmark) 4-6U/, 7d continuously, administration time 16:00-16:30, to maintain its basic physiological function, prevent ketoacidosis.5:00am fasting in morning in the 8th day, surveys fasting glucose from rat tail vein blood sampling after 3h, and blood sugar detection adopts blood glucose meter (OneTouch UltraVue, Johnson and Johnson, New Brunswick, NJ) and supporting reagent paper to measure, lower same.After the continuous 7d of alloxan induction diabetes rat subcutaneous injection protamine zine insulin, fasting glucose is elevated to 22-25mmol/L, and blood glucose tends towards stability, and insulin resistant forms, totally 24 Cheng Mo.

After Cheng Mo, diabetes rat is evenly divided into 3 groups by blood sugar level, 8 every group: diabetes rat matched group, gives olive oil gavage 0.2mL/ pcs/day; Diabetes rat low dosage AOX inhibitor precursor treatment group, is dissolved in LH-919 in respective volume olive oil, gives LH-919 gavage 10 μ g/kg/d; Diabetes rat high dose AOX inhibitor precursor treatment group, is dissolved in LH-919 in respective volume olive oil, gives LH-919 gavage 20 μ g/kg/d.Establish in addition 8 of normal rat matched groups, give olive oil gavage 0.2ml/ pcs/day; 8 of normal rat medicine groups, are dissolved in LH-919 in respective volume olive oil, give LH-919 gavage 20 μ g/kg/d.Diabetes rat model group and treatment group administration are organized simultaneously and are continued subcutaneous injection protamine zine insulin, continuous 13 days.During this time respectively at d4, d8, d125:00am fasting, after 3h, tail vein blood is measured fasting glucose.The capable OGTT experiment of d11,5:00am fasting, after 3h, tail vein blood is measured fasting glucose, as basis contrast (0min), then the each treated animal of diabetes rat gives glucose 2.5g/kg gavage, difference 30min after this, 60min, measure each group of blood glucose with 120min tail vein blood, draw blood glucose-time graph of 0-120min, and calculate Area under the curve of blood glucose AUC _0-120min.

After Drug therapy, 5:00am fasting in the 13rd day, plucks eyeball and gets blood, separation of serum after 3h.Measure serum triglycerides content, adopt triglyceride detection kit to measure (the safe clinical diagnosis of Beijing Northization company limited, Beijing), serum free fatty acid content adopts free-fat acid detection kit to measure (Bioengineering Research Institute is built up in Nanjing, Nanjing).

Isolating hepatocytes peroxisome, measures each group of hepatocyte AOX activity, measures taking IP-CoA as substrate.H in each group hepatocyte ₂o ₂content adopts H ₂o ₂detection kit is measured (Beyotime Biotech., Haimen).Serum and liver lipid peroxide contents adopt micro-malonaldehyde (MDA) detection kit to measure (Bioengineering Research Institute is built up in Nanjing, Nanjing).

Data represent with X ± SEM, carry out statistical procedures with SPSS 18.0 softwares, and between each group, diversity relatively adopts independent sample t inspection.With the comparison of normal rat matched group, #P < 0.05, ##P < 0.01, ###P < 0.001.With the comparison of diabetes rat model group, * P < 0.05, * * P < 0.01, * * * P < 0.001.

Diabetes rat gave AOX inhibitor precursor LH-919 treatment after 4 days, and blood glucose significantly declines before compared with administration, and it is stable that decline level maintains always.Blood glucose decline level and dosage correlation, as shown in table 2.OGTT experimental result shows, AOX inhibitor precursor LH-919 can significantly improve the oral glucose tolerance of alloxan induction diabetes rat, sees Fig. 7 A, with model control group comparison, and the AUC of LH-919 high and low dose group _0-120minarea all significantly declines (p<0.01), and AUC _0-120minarea decline degree and dosage correlation, as shown in Figure 7 B.

Table 2, AOX inhibitor precursor LH-919 are to alloxan induction diabetes rat hypoglycemic activity

Between each group, diversity relatively adopts independent sample t inspection.With the comparison of diabetes rat matched group, * P < 0.05, * * P < 0.01, * * * P < 0.001.

After treatment in 12 days, diabetes rat serum triglycerides (Fig. 8) and free fatty (Fig. 9) content significantly decline.Measure hepatocyte peroxisome AOX activity, as shown in figure 10, the active compared with normal group of diabetes rat matched group liver AOX significantly rises result, and after the treatment of AOX inhibitor precursor, treatment group hepatocyte AOX activity significantly reduces compared with matched group.Content of MDA significantly declines (Figure 11) simultaneously, H in hepatocyte ₂o ₂content significantly declines (Figure 12) compared with matched group, liver MDA content significantly decline (Figure 13).

The above results shows, after the treatment of AOX inhibitor precursor, diabetes rat liver ROS significantly reduces, and liver oxidative stress level significantly reduces, and in body, insulin resistant improves, and blood glucose significantly reduces.Therefore AOX inhibitor or AOX inhibitor precursor can be treated the type 1 diabetes taking insulin resistant as feature.

Embodiment 4, AOX inhibitor precursor are tested ob/ob mice blood sugar lowering

16 of the male ob/ob mices of SPF level, body weight 42-45g, as diabetic groups; 16 of C57BL mouses, body weight 20-22g, as normal group.Ob/ob mice and C57BL mouse are all fed with common mouse feed, the feed of freely intaking.Ob/ob mice adapted to feed after 1 week, fasting 3h, and tail venous blood sampling, blood glucose meter is measured fasting glucose, is divided into two groups, 8 every group: ob/ob mice matched group, gives 0.1mL olive oil gavage by blood sugar level; AOX inhibitor for treating group, is dissolved in AOX inhibitor precursor LH-919 in respective volume olive oil, gives LH-919 gavage 50 μ g/kg/d.C57BL mouse is divided into 2 groups at random, 8 every group: C57BL mouse normal group, gives olive oil gavage 0.1ml/ pcs/day; C57BL medicine group, LH-919 is dissolved in respective volume olive oil, gives LH-919 gavage 50 μ g/kg/d.Each group continues gavage 16 days.

After medicine feed the the 4th, 8,12,16d fasting 3h, tail venous blood sampling blood glucose meter is measured fasting glucose.Row OGTT experiment in the 13rd day after gavage, 5:00am fasting, after 3h, tail vein blood is measured fasting glucose, as basis contrast (0min), then the each treated animal of ob/ob mice gives glucose 2.0g/kg gavage, difference 30min after this, 60min, measure each group of blood glucose with 120min tail vein blood, draw blood glucose-time graph of 0-120min, and calculate Area under the curve of blood glucose AUC _0-120min.

Within the 16th day, survey after blood glucose, plucked eyeball and get blood, separation of serum.Insulin ELISA test kit (Millipore, Billerica, MA) is measured serum insulin, and calculate insulin resistance index (HOMA-IR) (Mattews DR.et.al., Diabetologia, 1985,28,412-419).Kit measurement serum triglycerides, free fatty acid content.

Put to death subsequently all ob/ob mices, win liver, preparation liver homogenate, isolating hepatocytes peroxisome, measures each group of hepatocyte AOX activity, measures taking IP-CoA as substrate.H in hepatocyte ₂o ₂content adopts H ₂o ₂detection kit is measured (Beyotime Biotech., Haimen).Hepatic lipid peroxidation level adopts micro-malonaldehyde detection kit to measure (Bioengineering Research Institute is built up in Nanjing, Nanjing).

Data represent with X ± SEM, carry out statistical procedures with SPSS18.0 software, and between each group, diversity relatively adopts independent sample t inspection.With the comparison of C57BL mouse matched group, #P < 0.05, ##P < 0.01, ###P < 0.001.With the comparison of ob/ob mice matched group, * P < 0.05, * * P < 0.01, * * * P < 0.001.

Ob/ob mice gave AOX inhibitor precursor LH-919 treatment after 4 days, and blood glucose significantly declines before compared with administration, and it is stable, as shown in table 3 that decline level maintains always.OGTT experimental result shows, can significantly improve the oral glucose tolerance of ob/ob mice through AOX inhibitor precursor LH-919 treatment, sees Figure 14 A, with the comparison of ob/ob mice matched group, and AOX inhibitor precursor group AUC _0-120minarea significantly declines (p<0.01), as shown in Figure 14B.

Table 3, AOX inhibitor precursor LH-919 are to ob/ob diabetic mice hypoglycemic activity

Between each group, diversity relatively adopts independent sample t inspection.Ob/ob mouse model group compares, * P < 0.05, * * P < 0.01, * * * P < 0.001.

After treatment in 16 days, ob/ob mice treatment group serum insulin content significantly declines (Figure 15), and HOMA-IR significantly declines compared with matched group.Ob/ob mice treatment group serum triglycerides (Figure 16) and free fatty (Figure 17) content significantly decline compared with matched group.Measure hepatocyte peroxisome AOX activity, as shown in figure 18, after the treatment of AOX inhibitor precursor, ob/ob mice treatment group hepatocyte AOX activity significantly reduces compared with matched group result.H in ob/ob mice treatment group hepatocyte ₂o ₂content significantly declines (Figure 19) compared with matched group, liver MDA content significantly decline (Figure 20).

The above results shows, after the treatment of AOX inhibitor precursor, ob/ob mice treatment group liver oxidative stress level significantly reduces, and in body, insulin resistant significantly improves, and blood glucose significantly reduces.Therefore AOX inhibitor or AOX inhibitor precursor can be treated type 2 diabetes mellitus.

Table 4, AOX inhibitor precursor LH-919 are to db/db diabetic mice hypoglycemic activity

Between each group, diversity relatively adopts independent sample t inspection.Db/db mouse model group compares, * P < 0.05, * * P < 0.01, * * * P < 0.001.

Embodiment 5, AOX inhibitor precursor are tested db/db mice blood sugar lowering

16 of the male db/db mices of SPF level, body weight 38-42g; 8 of C57BL mouses, body weight 20-23g, all feeds with common mouse feed the feed of freely intaking.Db/db mice adapted to feed after 1 week, fasting 3h, and tail venous blood sampling, blood glucose meter is measured fasting glucose, is evenly divided into two groups, 8 every group: db/db mice matched group, gives 0.1mL olive oil gavage by blood sugar level; AOX inhibitor precursor treatment group, LH-919 is dissolved in respective volume olive oil, gives db/db mice LH-919 gavage 50 μ g/kg/d.Separately establish 8 of C57BL mouse normal group, give olive oil gavage 0.1ml/ pcs/day.Each group continues gavage 16 days.

The 4th, 8,12,16 days fasting 3h after medicine feed, tail venous blood sampling, blood glucose meter is measured fasting glucose.Within the 16th day, survey after blood glucose, plucked eyeball and get blood, separation of serum.Insulin ELISA test kit (Millipore, Billerica, MA) is measured serum insulin, and calculates insulin resistance index (HOMA-IR).Kit measurement serum triglycerides, free fatty acid content.

Put to death subsequently all db/db mices, win liver, preparation liver homogenate, isolating hepatocytes peroxisome, measures each group of hepatocyte AOX activity, measures taking IP-CoA as substrate.Hepatic lipid peroxidation level adopts micro-malonaldehyde (MDA) to measure test kit and detects (Bioengineering Research Institute is built up in Nanjing, Nanjing).

Data represent with X ± SEM, carry out statistical procedures with SPSS 18.0 softwares, and between each group, diversity relatively adopts independent sample t inspection.With the comparison of db/db mice matched group, * P < 0.05, * * P < 0.01, * * * P < 0.001.

Db/db mice is giving after AOX inhibitor precursor LH-919 treatment, and blood glucose significantly reduces, as shown in table 3.After treatment in 16 days, db/db mice treatment group serum insulin levels significantly declines (Figure 21) compared with matched group, and HOMA-IR significantly declines compared with matched group.Db/db mice treatment group serum triglycerides (Figure 22) and free fatty (Figure 23) content also significantly reduce compared with matched group.Measure hepatocyte peroxisome AOX activity, as shown in figure 24, after the treatment of AOX inhibitor precursor, db/db mice treatment group hepatocyte AOX activity significantly reduces compared with matched group result.Meanwhile, H in db/db mice treatment group hepatocyte ₂o ₂content significantly declines compared with matched group, and liver MDA content also significantly reduces (Figure 25).

The above results shows, after the treatment of AOX inhibitor precursor, in db/db mice treatment group body, ROS content significantly reduces, and in body, insulin sensitivity been significantly enhanced, and blood glucose significantly reduces.Therefore AOX inhibitor or AOX inhibitor precursor can be treated type 2 diabetes mellitus.

All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

1. the purposes of acyl coenzyme A oxidase inhibitor, for the preparation of the pharmaceutical composition of prevention, improvement or treatment diabetes; The oxidasic international zymetology classifying and numbering of described acyl coenzyme A is EC1.3.3.6.

2. purposes as claimed in claim 1, is characterized in that, described diabetes comprise: type 1 diabetes or type 2 diabetes mellitus.

3. purposes as claimed in claim 1, is characterized in that, described acyl coenzyme A oxidase inhibitor is the material that suppresses acyl coenzyme A oxidase active or expression.

4. purposes as claimed in claim 3, it is characterized in that, described acyl coenzyme A oxidase inhibitor comprises: small organic molecule, micromolecule inorganic matter, anti-acyl coenzyme A oxidase antibody, can and suppress its active nucleic acid fragment and polypeptide with acyl coenzyme A oxidase specific binding.

5. purposes as claimed in claim 1, is characterized in that, described acyl coenzyme A oxidase inhibitor also comprises inhibitor precursor.

6. purposes as claimed in claim 5, is characterized in that, described acyl coenzyme A oxidase inhibitor or inhibitor precursor comprise: 10,12-, 25 carbon diacetylenic acids, and its CAS is encoded to 66990-32-7; Or 10,12-, 25 carbon diine acyl coenzyme A.

7. purposes as claimed in claim 1, is characterized in that, described pharmaceutical composition also for:

Suppress acyl coenzyme A oxidase active in patient body;

Improve insulin resistant;

Reduce oxidative stress level in target organ or tissue; Or

Reduce oxyradical content in insulin action target tissue.

8. for preventing, improve or treat a pharmaceutical composition for diabetes, it is characterized in that, described pharmaceutical composition comprises:

Acyl coenzyme A oxidase inhibitor; And

Pharmaceutically acceptable carrier or excipient.

9. pharmaceutical composition as claimed in claim 8, is characterized in that, described acyl coenzyme A oxidase inhibitor is the material that suppresses acyl coenzyme A oxidase active or expression.

10. pharmaceutical composition as claimed in claim 8, it is characterized in that, described acyl coenzyme A oxidase inhibitor comprises: anti-acyl coenzyme A oxidase antibody, can and suppress its active nucleic acid fragment and polypeptide with acyl coenzyme A oxidase specific binding, small organic molecule, micromolecule inorganic matter.

11. pharmaceutical compositions as claimed in claim 8, is characterized in that, described acyl coenzyme A oxidase inhibitor also comprises inhibitor precursor; Preferably comprise: 10,12-, 25 carbon diacetylenic acids, its CAS is encoded to 66990-32-7; Or 10,12-, 25 carbon diine acyl coenzyme A.

12. 1 kinds of methods of preparing prevention, improving or treat the pharmaceutical composition of diabetes, is characterized in that, described method comprises: by acyl coenzyme A oxidase inhibitor and pharmaceutically acceptable carrier or mixed with excipients, obtain described pharmaceutical composition.

The method of 13. 1 kinds of preventions, improvement or treatment diabetes, is characterized in that, described method comprises: acyl coenzyme A oxidase active or expression in the patient body that suppresses to need prevention, improve or treat.

14. methods as claimed in claim 13, it is characterized in that, described inhibition acyl coenzyme A oxidase active or the method for expression are: use acyl coenzyme A oxidase inhibitor or the acyl coenzyme A oxidase inhibitor precursor of effective dose to the patient who needs prevention, improves or treat.