CN101152179A - Application of isoniazide as histone deacetylase inhibitors - Google Patents
Application of isoniazide as histone deacetylase inhibitors Download PDFInfo
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- CN101152179A CN101152179A CNA2006101134367A CN200610113436A CN101152179A CN 101152179 A CN101152179 A CN 101152179A CN A2006101134367 A CNA2006101134367 A CN A2006101134367A CN 200610113436 A CN200610113436 A CN 200610113436A CN 101152179 A CN101152179 A CN 101152179A
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Abstract
The invention discloses isoniazid indicated in formula (I), which has a new purpose of being used as histone deacetylase inhibitor. The isoniazid is used to prepare medicine to prevent and/or treat a plurality of diseases related to histone deacetylase.
Description
Background technology
The present invention relates to the new purposes of isoniazid as inhibitors of histone deacetylase, isoniazid is used to prepare the medicine that prevents and/or treats the multiple disease relevant with the histone deacetylase enzyme.
Background technology
Histone is and the chromatinic base substance of the common formation of genomic DNA.Histone forms eight aggressiveness in nucleus, chromosomal DNA Double helix molecule twines on it repeatedly, constitutes chromatin.Chromatin twines the change of tightness degree, is that related gene expression is opened and one of regulatory mechanism of closing.The regulation and control of acetylation of histone level are bases of this regulatory mechanism.During several lysine residue acetylations of histone amino terminal, its positive charge is neutralized, and reduces with the DNA electrostatic attraction of negative charge, and chromatin is in the state of loosening, and makes the transcription factor can be near genomic DNA, and genetic transcription is active state.Histone deacetylase enzyme catalysis lysine residue deacetylation, chromatin is in compact state, and genetic transcription is suppressed.
The inactivation of antioncogene is the key factor of tumor development.The histone deacetylase endonuclease capable suppresses the transcriptional expression of antioncogene, and inhibitors of histone deacetylase then can recover the transcriptional expression of downtrod antioncogene.Some inhibitors of histone deacetylase have clear and definite anti-tumor activity in vitro and in vivo, and toxicity is less usually; Clinical experiment has also confirmed their pharmacological action (Kelly WK, O ' Connor OA, Marks PA.Histone deacetylaseinhibitors:from target to clinical trials.Expert Opin Investig Drugs2002; 11:1695-1713).Inhibitors of histone deacetylase is as the new antitumor drug of a class, has efficient, low toxicity, broad-spectrum characteristics.Tumor pharmacother based on inhibitors of histone deacetylase is called as " tumor is transcribed treatment ".
Recently find that the antitumor action of inhibitors of histone deacetylase can occur in a plurality of links.Some transcription factor such as p53, E2F, c-Myb etc. and some structural protein such as tubulin, also be substrate (Stemer DE, the Berger SL.Acetylation ofHistones and Transcription-Related Factors.Microbiol Mol Biol Rev2000 that acetylation is regulated; 64:435-459), inhibitors of histone deacetylase promotes their acetylation, and its function is had direct influence.Inhibitors of histone deacetylase also has a direct impact (Fuino L to the active and expression of heat shock protein HSP90, EGF receptor, BaliP, Wittmann S, et al.Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizeshuman breast cancer cells to trastuzumab, taxotere, gemcitabine, andepothilone B.Mol Cancer Ther 2003; 2:971-984), they are important known antitumor drug target spots.Except that tumor cell being had the direct lethal effect, inhibitors of histone deacetylase also can suppress the vascularization process of tumor tissues, thereby suppresses growth of tumor and transfer.The vascularization inhibitory action of inhibitors of histone deacetylase may with its secretion (Sawa H to VEGF, Murakami H, Ohshima Y, et al.Histone deacetylaseinhibitors such as sodium butyrate and trichostatin A inhibit vascularendothelial growth factor (VEGF) secretion from human glioblastoma cells.Brain Tumor Pathol 2002; 19:77-81) and the inhibition (DeroanneCF of signal transduction process, Bonjean K, Servotte S, et al.Histone deacetylases inhibitors asanti-angiogenic agents altering vascular endothelial growth factor signaling.Oncogene 2002; 21:427-436) relevant.
In addition, inhibitors of histone deacetylase also has therapeutical effect to other multiple disease.The neural degeneration that HDACI causes oxidative stress has significant protective effect (Ryu H, Lee J, Olofsson BA, et al.Histone deacetylase inhibitors prevent oxidativeneuronal death independent of expanded polyglutamine repeats via anSpl-dependent pathway.PNAS 2003; 100:4281-4286); In the animal model of HuntingtonShi disease and Duchenne-Arandisease, inhibitors of histone deacetylase can obviously improve functional lesion (the Hockly E of animal, Richon VM, Woodman B, et al.Suberoylanilidehydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficitsin a mouse model of Huntington ' s disease.PNAS 2003; 100:2041-2046; Minamiyama M, Katsuno M, Adachi H, et al.Sodium butyrate amelioratesphenotypic expression in a transgenic mouse model of spinal and bulbarmuscular atrophy.Hum Mol Genet 2004; 13:1183-1192).Inhibitors of histone deacetylase also can reduce the brain tissue damage that cerebral ischemia causes, dwindle cerebral infarct size (Ren M, Leng Y, Jeong M, et al.Valproic acid reduces brain damage induced bytransient focal cerebral ischemia in rats:potential roles of histonedeacetylase inhibition and heat shock protein induction.J Neurochem2004; 89:1358-1367).Inhibitors of histone deacetylase has regulating action to the various kinds of cell factor, mice is taken inhibitors of histone deacetylase, can suppress lipopolysaccharide-induced inflammatory mediator such as TNF-α, IL-1-β, the generation of IL-6 and IFN-γ, and can reduce hepatocyte injury (the Leoni F that injection ConA causes, Zaliani A, Bertolini G, et al.The antitumor histonedeacetylase inhibitor suberoylanilide hydroxamic acid exhibitsantiinflammatory properties via suppression of cytokines.Proc Natl AcadSci USA2002; 99:2995-3000).Inhibitors of histone deacetylase also can suppress the propagation of CD4 type T cell, and suppress the gathering of activated T cell fully, its effect link is different with cyclosporine, can be used as immunological rejection (the Skov S that novel immunosuppressant treatment autoimmune disease and histoorgan are transplanted, Rieneck K, Bovin LF, et al.Histonedeacetylase inhibitors:a new class of immunosuppressors targeting a novelsignal pathway essential for CD 154 expression. Blood2003; 101:1430-1438).In the bone marrow transplantation animal model, inhibitors of histone deacetylase is to the not influence of donor anti-tumor activity, and the anti-host's of inhibition donor rejection (Reddy P, Maeda Y, Hotary K, et al.Histone deacetylase inhibitor suberoylanilidehydroxamic acid reduces acute graft-versus-host disease and preservesgraft-versus-leukemia effect.Proc Natl Acad Sci USA2004; 101:3921-3926).In the systemic lupus erythematosus (sle) mouse model, HDACI has the obvious suppression effect to the expression of some cytokines of sick mice spleen cell such as IL-12, IFN-, IL-6 and IL-10, and the albuminuria level of reduction mice, it is heavy to reduce spleen, improve organ injuries such as glomerulonephritis (Mishra N, Reilly CM, Brown DR, et al.Histone deacetylaseinhibitors modulate renal disease in the MRL-lpr/lpr mouse.J Clin Invest2003; 111:539-552).Inhibitors of histone deacetylase has inhibitory action to the pathologic myocardial hypertrophy, and improve heart failure (the Kook H that myocardial hypertrophy causes, Lepore JJ, GitlerAD, et al.Cardiac hypertrophy and histone deacetylase-dependent transcriptionalrepression mediated by the atypical homeodomain protein Hop.J ClinInvest 2003; 112:863-871).Experiment in vitro is found, inhibitors of histone deacetylase can promote synthesis secretion (Mosley AL, the Ozcan S.Glucose regulates insulingene transcription by hyperacetylation of histone h4.J Biol Chem2003 of insulin; 278:19660-19666), improve picked-up (the Takigawa-Imamura H of muscle cell to glucose, Sekine T, Murata M, et al.Stimulation of glucoseuptake in muscle cells by prolonged treatment with scriptide, a histonedeacetylase inhibitor.Biosci Biotechnol Biochem 2003; 67:1499-1506).Inhibitors of histone deacetylase can be induced the synthetic of fetal hemoglobin, is used for the treatment of anemia disease such as thalassemia, reaping hook cytopathy (Cao H.Pharmacological induction of fetalhemoglobin synthesis using histone deacetylase inhibitors.Hematology2004; 9:223-233).Inhibitors of histone deacetylase also can suppress lipogenesis process (Lagace DC, Nachtigal MW.Inhibition of histone deacetylase activity byvalproic acid blocks adipogenesis.J Biol Chem 2004; 279:18851-18860), promote the injury repairing and regeneration (the Iezzi S of Skeletal Muscle Cell, Di Padova M, Serra C, et al.Deacetylase inhibitors increase muscle cell size by promoting myoblastrecruitment and fusion through induction of follistatin.Dev Cell2004; 6:673-684); The sperm that also can suppress mice forms, cause sterile (the Fenic I of male mice reversibility, Sonnack V, Failing K, et al.In vivo effects of histone-deacetylaseinhibitor trichostatin-a on murine spermatogenesis.J Androl2004; 25:811-818).
The enzyme inhibition of inhibitors of histone deacetylase is not limited to mammalian cell, inhibitors of histone deacetylase also has tangible protozoacide, antifungal and antivirus action (Meinke PT, Liberator P.Histone deacetylase:a target for antiproliferative andantiprotozoal agents.Curr Med Chem 2001; 8:211-235).
In sum, histon deacetylase (HDAC) plays an important role in regulator gene is transcribed, and Antibiotic FR 901228 mediation acetylation of histone also influences gene expression.Thereby for the multiple disease due to the abnormal gene expression, Antibiotic FR 901228 is a kind of effective treatment and prophylactic agent.
The chemical constitution of isoniazid.
The effect of isoniazid in the prior art
Isoniazid is a line antitubercular agent of generally acknowledging, its effect is strong, and good effect is cheap, taking convenience, and oral absorption is fast, safety.Isoniazid mainly is that the antibacterial of growth and breeding phase is effective to mycobacteria, and other antibacterials are not almost had effect.Its mechanism of action is not illustrated as yet, may make cell wall rupture relevant with suppressing the synthetic of sensitive bacterial mycolic acid.Clinically, isoniazid is mainly as the medicine of treatment and prevention tuberculosis.Sometimes, isoniazid also is used for bacillus calmette-guerin vaccine immunization therapy superficial bladder cancer to reduce toxic and side effects (the Al Khalifa M of bacillus calmette-guerin vaccine, Elfving P, Mansson W, et al.The effect of isoniazid on BCG-induced toxicity inpatients with superficial bladder cancer.Eur Urol 2000; 37 Suppl 1:26-30).Experimental studies have found that, isoniazid has certain influence to body's immunity, as suppress activity (Sim E, Gill EW, the Sim RB.Drugs that induce systemic lupuserythematosus inhibit complement component C4.Lancet1984 of complement component; 2:422-424), regulate peripheral blood mononuclear cell proliferation activity (Kucharz EJ.Studies onimmunomodulatory properties of isoniazid.Influence of isoniazid onresponsiveness of peripheral blood mononuclear cells to interleukin-2.CentEur JPublic Health 1995; 3:65-66).United States Patent (USP) 4,082,846 find that psoriasis can be treated by isoniazid local application.Some case reports are found, isoniazid can effectively be treated skin chronic eczema (Kubota Y, Kiryu H, Nakayama J.Generalized lichen nitidussuccessfully treated with an antituberculous agent.Br J Dermatol2002; 146:1081-1083)
Lower limb erythema nodositas skin lesion (Li Bingxu, Cai Jianfeng, Lin Xiaohua. the discussion of lower limb erythema nodositas skin lesion and tuberculosis relation. Chinese skin cypridology magazine 16:165-167), IgA
(De Siati L, Paroli M, Ferri C, et al.ImmunoglobulinA nephropathy complicating pulmonary tuberculosis.Ann Diagn Pathol1999; 3:300-303) and kala azar (Peters W, Lainson R, Shaw JJ, et al.Potentiating action of rifampicin and isoniazid against Leishmaniamexicana amazonensis.Lancet 1981; 1:1122-1124).These effects or be summed up as the tuberculosis effect of isoniazid, or mechanism is not clear.
Summary of the invention
The inhibitors of histone deacetylase that the purpose of this invention is to provide a kind of new construction, i.e. isoniazid, in other words, the application of isoniazid in the preparation inhibitors of histone deacetylase.
Advantage of the present invention is to have found that isoniazid all has the effect of inhibition of histone deacetylase activity, promotion acetylation of histone in vitro and in vivo.As the inhibitors of histone deacetylase of a new construction, isoniazid can be used for preparing the medicine for the treatment of multiple disease.The present invention comprise Antibiotic FR 901228 medicament component; can and prevent as Drug therapy, for example inflammation, diabetes, diabetic complication, the subtype thalassemia of isozygotying, fibrosis, liver cirrhosis, acute myeloblastic leukemia (APL), protozoan infection or similar disease by the disease due to abnormal gene expression.And it also is of great use antineoplastic agent and immunosuppressant, can suppress and prevent following organ-graft refection and autoimmune disease.
Transplant organ or tissue, the rejection that produced of heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestinal, brothers, muscle, nerve, intervertebral disc, trachea, sarcoplast, cartilage or the like for example, the graft that produces after the bone marrow transplantation is to host's reaction, and autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus (sle), struma lymphomatosa, multiple sclerosis, myasthenia gravis, type i diabetes etc. for example; Infection due to the pathogenic microorganism (Aspergillus fumigatus, Fusarium oxysporum, star sample hair moss bacterium etc.).
In addition, following disease can be effectively treated and be prevented in the preparation of the medicament of Antibiotic FR 901228.
1) cutaneous manifestations of inflammatory or hyperplasia dermatoses or immunology mediation (for example skin ulcer is cooked in psoriasis, characteristic dermatitis, contact dermatitis, eczematoid dermatitis, seborrheic dermatitis, lichen planus, class sky, skin ulcer, epidermolysis bullosa, urticaria, vasodilation, vasculitis, erythema, skin hypereosinophilic syndrome, lupus erythematosus, acne, alopecia areata are cooked in kitchen property class sky greatly); Other dermatosis is dermatomyositis, leukoderma vulgaris, photosensitivity and cutaneous T cell lymphoma for example;
2) autoimmune disease of eyes (for example keratoconjunctivitis, spring keratitis, the uveitis sick relevant, keratitis, herpetic keratitis, taper keratitis, taper epithelium malnutrition, scleritis, Graves ' oculopathy, keratoconjunctivitis sicca, little water kitchen, iridocyclitis, sarcoidosis, endocrine ophthalmocace etc.) with white Sai Shi; Other ocular disease (for example cataract, iron storage disease, retinitis, melanoma, senile plaque, vitreous body cicatrization, corneal alkali burn;
3) reversibility air flue obstruction disease-asthma (for example chronic asthma, allergic asthma, intrinsic asthma, dust asthma), especially chronic or obstinate asthma (for example late period asthma and air flue hyperirritability), allergy etc.; Other respiratory disorder is benign lymphogranulomatosis, pulmonary fibrosis and idiopathic interstitial lung inflammation for example;
4) mucosa or vascular inflammation (for example gastric ulcer, ischemic or thrombosis blood vessel injury, ischemic disease, enteritis, necrotizing enterocolitis, the damage of intestines relevant, the disease of leukotriene B4 mediation) with thermal burn;
5) inflammation/allergy of intestinal (for example celiac disease, proctitis, eosinophilic gastroenteritis, Mastocytosis, regional enteritis and ulcerative colitis); The allergic disease (for example migraine, rhinitis and eczema) that the food that shows by non-gastrointestinal symptoms is relevant; Other gastrointestinal disease is the colitis of endotoxin shock, false film colitis, medicine or radiation mediation for example;
6) kidney disease (for example interstitial nephritis, nephrorrhagia syndrome, hemolytic uremic syndrome and diabetic nephropathy); Other kidney disease such as ischemic acute kidney insufficiency, chronic kidney hypofunction;
7) sacred disease (for example polymyositis, Guilain-Barre are combined and levied, Meniere ' s disease, polyneuritis, monomer neuritis, cerebral infarction, Alzheimer, parkinson disease, amyotrophic lateral sclerosis (ALS), radiculopathy);
8) cerebral ischemia disease (for example, brain injury, intracerebral hemorrhage (for example, subarachnoid hemorrhage, intracerebral hemorrhage), cerebral thrombosis, cerebral embolism, asystole, shock, transient ischemic attack (TIA) and hypertensive encephalopathy);
9) endocrinopathy (for example hyperthyroidism, Basedow's disease (thyroid is outstanding), protoferriheme disease (for example pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, anerythroplasia);
10) osteopathia (for example osteoporosis);
11) other disease such as environmental illness (for example arteriosclerosis, aortitis syndrome polyarteritis nodosa and [non-inflammatory] cardiomyopathy); Collagen diseases (for example scleroderma, Wegener granuloma, Sjogren syndrome); Obesity; The eosinophilic gastritis; Periodontal (for example gingiva, periodontal tissue, alveolar bone, Dentinal damage); The nephritic syndrome (for example glomerulonephritis); Male pattern baldness, senile alopecia; Muscular dystrophy; Pyoderma and Sezary syndrome; Chromosomal abnormality diseases related (mongolism); Addison ' s disease; The disease of active oxygen mediation [for example relevant organ injury (for example the ischemic disturbance of circulation of organs such as heart, liver, kidney, digestive tract etc.) with preservation, transplanting or ischemic diseases (for example thrombosis, cardiac infarction etc.); The coronary restenosis of postangioplasty and the prevention of tissue adhesion; Autoimmune disease and inflammation (for example former mucosa edema, autoimmune atrophic gastritis, climacteric in advance, male sterility, juvenile diabete; The uveitis that pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, crystalline lens bring out, the special property sent out leukopenia, active chronic hepatitis, the special property sent out liver cirrhosis, discoid lupus erythematosus, autoimmunity orchitis, arteritis (for example arteritis distortion), or polychondritis; Human immune deficiency (HIV) viral infection, AIDS; Allergic conjunctivitis; The cicatrix and the keloid of the hypertrophy due to wound, burn or the operation.
In addition, the formulation components of this invention is for treatment and prevention of liver disease [for example immunogenicity disease (the spontaneous hepatopathys of chronic immunity such as for example immune spontaneous hepatopathy, primary biliary cirrhosis or sclerosing cholangitis), partially hepatectomized, acute severe hepatitis (for example downright bad due to toxin, viral hepatitis, shock or the anoxia), A type hepatitis, the non-B hepatitis of non-A, liver cirrhosis and liver failure (for example explosive hepatitis, tardy hepatitis and " acute-on-chronic " liver failure (acute failure that chronic hepatopathy causes))].
The form of the formulation components useful formulations preparation of the present invention; for example be active component with the Antibiotic FR 901228 with a kind of organic or inorganic carrier or excipient be mixed with mutually solid, semisolid or solution form, with form administration beyond external, intestinal, the gastrointestinal tract.
Active component can be blended, for example, add commonly used nontoxicly, the available carrier of preparation is made tablet, granule, capsule, suppository, solution, Emulsion, suspension, injection, ointment, liniment, eye drop, gel, emulsifiable paste and other available forms.
Carrier can water, glucose, lactose, Radix Acaciae senegalis, gelatin, D-mannitol, starch paste, corn paste, keratin, colloid silicon, Rhizoma Solani tuber osi paste, carbamide and other are suitable for producing the carrier of preparation, under solid, semisolid or liquid form, make stable, the thickening, solvable, painted of medicine, also can use spice.
This component is applied to man-hour, recommends with vein, intramuscular, part or oral administration.Antibiotic FR 901228 the treatment effect dose depend on patient age and situation and adjust.
Preferred dosage is different with the difference of clinical indication.According to treatment patient's disease, may must make some change, and under any circumstance, all determine the suitable dose of individual patient by the doctor to dosage.The effective dose of chemical compound depends on body weight, physiological situation and selected dosage regimen etc. in the per unit dosage.The chemical compound of unit dose is meant and does not comprise the weight of vehicle weight at the chemical compound of interior (when using carrier).
The route of administration that is used to implement chemical compound of the present invention depends on the position that disease and needs are treated.Because the pharmacokinetics of The compounds of this invention and pharmacodynamic profile have to a certain degree different, the most preferred method that therefore obtains treatment concentration in tissue is to increase dosage gradually and monitor clinical effectiveness.For such therapeutic dose of increase gradually, predose will depend on route of administration.
For any particular patient, concrete treatment effective dose level will depend on multiple factor, comprise activity, route of administration, this concrete dosage form of the disease of being treated, disease severity, used concrete dosage form removing speed, treatment persistent period, with this concrete dosage combination or the well-known factors in medical science field such as concrete medicine, patient's age, body weight, sex, diet and general health situation of use simultaneously.Dosage level is generally about 1-20mg/kg/ days, is preferably about 2-5mg/kg/ days.
Description of drawings
Fig. 1. isoniazid is to the inhibiting amount effect curve of histone deacetylase enzymatic activity.
Fig. 2. isoniazid is to the acetylizad influence of colon cancer cell HCT-8 histone H 4 component of In vitro culture.
Fig. 3. isoniazid is in the acetylizad influence of body mouse boosting cell histone H 4 component.
Fig. 4 .Lewis lung cancer in mice lung metastasis counting is (each data point is every mouse lung metastasis counting) relatively.
The specific embodiment
The concrete demonstration of following embodiment application of the present invention.But present embodiment does not limit the scope of application of the present invention.
Embodiment 1: isoniazid is to the inhibitory action of histone deacetylase enzyme
Histone deacetylase activity determination method reference literature (Heltweg B, Jung M.Amicroplate reader-based nonisotopic histone deacetylase activity assay.Anal Biochem 2002; 302:175-183) and done partly to change.
The preparation of histone deacetylase enzyme crude preparation by using: get healthy Kunming mouse liver, shred, add PBS and on 100 eye mesh screens, grind filtration, the centrifugal back of the cell suspension that obtains is cleaned 2 times with PBS, cell precipitation adds equal-volume PBS, vibration mixing, freeze-thaw method cell lysis, centrifugal (4 ℃, 12000rpm 10min), draws supernatant, adding glycerol to final volume is 30%, and protein concentration is 5mg/ml.-20 ℃ of freezing preservations.
With PBS is in the 100 μ l reaction systems of buffer, and the final concentration that adds reacted constituent is respectively: MAL 20 μ mol/L, and enzyme crude preparation by using 30%, Triton 0.1%.Contain isoniazid 1,3.2,10,32 in the reaction, 100mmol/L, and setting does not contain isoniazid sample and two contrasts of not enzyme-added sample.In 37 ℃ of water-baths, react after adding sample.4 repetitions are all done in reaction.After 24 hours, each sample adds 100 μ l 1mol/L HCl cessation reactions, and with 220 μ l ethyl acetate extracting acidified samples, organic facies vacuum evaporation with 40% dissolve with methanol residual solids, moves into 96 hole fluoroscopic examination plates.Go up detection fluorescence: excitation wavelength 325nm, emission wavelength 390nm at fluorescence detector (SpectraMax GeminiXS, Molecular Devices).
Suppression ratio calculates: the fluorescence inhibition with not enzyme-added sample contrast is 100% suppression ratio, does not contain the negative contrast of isoniazid sample (suppression ratio is 0), calculates the suppression ratio of each sample variable concentrations.
Studies show that, isoniazid when 1-100mmol/L concentration, to the histone deacetylase enzymatic activity have dose dependent inhibitory action (Fig. 1. shown in).The calculating isoniazid is 44mmol/L to the IC50 of histone deacetylase enzyme inhibition.
Embodiment 2: isoniazid is to the influence of cultured cell acetylation of histone
Cell culture and drug treating: the colon cancer cell HCT-8 of In vitro culture is inoculated in the 90mm culture dish of the RPMI1640 complete culture solution that contains 10% calf serum, places 37 ℃, 5%CO
2Cultivate in the incubator.24 hour cells grow to the logarithmic growth after date, and adding isoniazid to final concentration is 100mM, continue to cultivate 16 hours.
Histone extracts: press document (Yoshida, M., Kijima, M., Akita, M., Beppu, T. (1990) J.Biol.Chem.265,17174-17179) method extraction groups of cells albumen.After cell is hatched end, discard culture fluid, scrape cell, move into the Eppendorf pipe with cell scraper, 4 ℃ 1500 rev/mins centrifugal 5 minutes collecting cells, ice-cold PBS washing through containing the 5mmol/L sodium butyrate 2 times adds 100 μ l cell pyrolysis liquid (1xPBS, 5mmol/L sodium butyrates, 0.5%TritonX-100,5mM PMSF, 0.2%NaN3), 4 ℃ of cell lysis 10 minutes.4 ℃ 2000 rev/mins centrifugal 8 minutes collecting cells nuclears through ice-cold lysate washing 2 times, add 100 μ l 0.3NHCl, vibration mixing, ice bath 1 hour.4 ℃ 12000 rev/mins centrifugal 10 minutes, collect supernatant, add 1ml acetone, mixing is put-20 ℃ and is spent the night.Next day, 4 ℃ 12000 rev/mins centrifugal 10 minutes, washing with acetone precipitation with 0.3N HCl dissolving histone, is measured protein concentration.
Histone electrophoresis: press document (Sourlingas T.G.; Tsapali D.S.; Kaldis A.D.; Sekeri-Pataryas K.E.Eur J Cell Biol 2001.80; 726-732) method prepares acid carbamide Triton (AUT) gel; get 40-50ug histone electrophoresis to separate the acetylation and the non-acetylized component of histone H 4; gel dyes with Coomassie brilliant blue R-250, and makes densitometric scan.
The result shows, isoniazid when 100mmol/L concentration, to the acetylation of cultured cell histone have facilitation (Fig. 2. shown in).
Healthy Kunming mouse, lumbar injection isoniazid 100mg/kg or equal-volume PBS.After 4 hours, the cervical vertebra dislocation is put to death.Get mouse spleen, shred, add PBS and on 100 eye mesh screens, grind filtration, the cell suspension that obtains is centrifugal after contain the ice-cold PBS of 5mmol/L sodium butyrate and clean 2 times, adds 500 μ l cell pyrolysis liquid (1xPBS, 5mmol/L sodium butyrates, 0.5%Triton X-100,5mM PMSF, 0.2%NaN3), 4 ℃ of cell lysis 10 minutes.4 ℃ 2000 rev/mins centrifugal 8 minutes collecting cells nuclears through ice-cold lysate washing 2 times, add 100 μ l 0.3N HCl, vibration mixing, ice bath 1 hour.4 ℃ 12000 rev/mins centrifugal 10 minutes, collect supernatant, add 1ml acetone, mixing is put-20 ℃ and is spent the night.Next day, 4 ℃ 12000 rev/mins centrifugal 10 minutes, washing with acetone precipitation with 0.3N HCl dissolving histone, is measured protein concentration.-20 ℃ of freezing preservations.Get 40-50ug histone AUT electrophoresis, to separate the acetylation and the non-acetylized component of histone H 4, gel dyes with Coomassie brilliant blue R-250, and makes densitometric scan.
The result shows, lumbar injection isoniazid 100mg/kg, to the acetylation of mouse spleen histone have facilitation (Fig. 3. shown in).
Embodiment 4: the inhibitory action that the Mice Bearing Lewis Lung Cancer lung is shifted:
The C57BL/6 mice of lotus Lewis lung cancer, the cervical vertebra dislocation is put to death.Fixing back iodine tincture alcohol disinfecting, aseptic condition takes out the tumor piece down, grinding, homogenate, the centrifugal back of the cell suspension that obtains is cleaned 2 times with PBS, and cell precipitation inoculates 0.15ml tumor liquid for every C57BL/6 female mice oxter with PBS 1/10 dilution.With the animal random packet.In inoculation pneumoretroperitoneum injection in 24 hours isoniazid (15) or PBS contrast (16) in 100mg/kg/ days, administration is 5 days weekly.After 4 weeks, two groups of mices all have 5 in inoculation position generation tumor.The cervical vertebra dislocation is put to death, and takes by weighing mouse tumor weight, body weight.As shown in table 1, lumbar injection isoniazid 100mg/kg/ days does not have obvious influence to mice body weight and tumor growth.Mouse lung Bouin ' s is liquid-solid fixed, branch on count kitchen range number under anatomic microscope.As shown in Figure 4, isoniazid obviously reduces the lung transfer incidence rate (Fisher ' s accurately checks, p＜0.05) of mouse tumor when this dosage.
Table 1.Lewis lung cancer in mice body weight and tumor anharmonic ratio than (average ± standard deviation, n=5).
| Body weight | Tumor is heavy | |
| Isoniazid | 22.8±1.5 | 6.3±2.5 |
| Contrast | 23.4±1.9 | 6.5±3.2 |
Claims (6)
1. isoniazid is as the application of inhibitors of histone deacetylase.
Isoniazid the preparation prevention with or the medicine of the treatment human or animal disease relevant with the histone deacetylase enzyme in application.
3. according to the application of claim 2, it is characterized in that the described disease relevant with the histone deacetylase enzyme comprises tumor, inflammation, diabetes and complication thereof, hereditary, fibrosis, sclerosis, histoorgan transplant rejection, autoimmune disease, the subtype thalassemia of isozygotying, fibrosis, liver cirrhosis, acute myeloblastic leukemia (APL), protozoan infection or similar disease.
4. according to the application of claim 3, it is characterized in that described histoorgan comprises heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestinal, brothers, muscle, nerve, intervertebral disc, trachea, sarcoplast, cartilage, bone marrow.
5. according to the application of claim 3, it is characterized in that described autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus (sle), struma lymphomatosa, multiple sclerosis, myasthenia gravis, type i diabetes.
6. according to the application of claim 3, it is characterized in that described protozoon comprises Aspergillus fumigatus, Fusarium oxysporum, star sample hair moss bacterium.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010148572A1 (en) * | 2009-06-26 | 2010-12-29 | Asan Laboratories Co., Ltd. | Method for treating or ameliorating mucocutaneous or ocular toxicities |
| CN105535976A (en) * | 2015-12-31 | 2016-05-04 | 浙江大学医学院附属第一医院 | Application of transcript profile imprinting prediction medicine MS-275 in mice inflammatory bowel disease medicine preparation |
| CN106610434A (en) * | 2016-02-26 | 2017-05-03 | 弗雷米德生物医药技术（天津）有限公司 | ELISA detection kit for HPV16 type E7 protein |
| CN110882250A (en) * | 2018-09-07 | 2020-03-17 | 上海市计划生育科学研究所 | Application of compound in treating asthenospermia |
| CN112195236A (en) * | 2020-10-23 | 2021-01-08 | 武汉大学 | Application of HDAC2 in preparing marker for evaluating liver inflammation progression of acute liver failure |
-
2006
- 2006-09-28 CN CNA2006101134367A patent/CN101152179A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010148572A1 (en) * | 2009-06-26 | 2010-12-29 | Asan Laboratories Co., Ltd. | Method for treating or ameliorating mucocutaneous or ocular toxicities |
| CN102458473A (en) * | 2009-06-26 | 2012-05-16 | 英属开曼群岛商安盛开发药物股份有限公司 | Method for treating or ameliorating mucocutaneous or ocular toxicities |
| AU2009348848B2 (en) * | 2009-06-26 | 2012-10-25 | Sunny Pharmtech, Inc. | Method for treating or ameliorating mucocutaneous or ocular toxicities |
| CN105535976A (en) * | 2015-12-31 | 2016-05-04 | 浙江大学医学院附属第一医院 | Application of transcript profile imprinting prediction medicine MS-275 in mice inflammatory bowel disease medicine preparation |
| CN106610434A (en) * | 2016-02-26 | 2017-05-03 | 弗雷米德生物医药技术（天津）有限公司 | ELISA detection kit for HPV16 type E7 protein |
| CN110882250A (en) * | 2018-09-07 | 2020-03-17 | 上海市计划生育科学研究所 | Application of compound in treating asthenospermia |
| CN112195236A (en) * | 2020-10-23 | 2021-01-08 | 武汉大学 | Application of HDAC2 in preparing marker for evaluating liver inflammation progression of acute liver failure |
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