WO2009038289A2 - Pharmaceutical compositions for preventing or treating fibrosis - Google Patents

Pharmaceutical compositions for preventing or treating fibrosis Download PDF

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Publication number
WO2009038289A2
WO2009038289A2 PCT/KR2008/004806 KR2008004806W WO2009038289A2 WO 2009038289 A2 WO2009038289 A2 WO 2009038289A2 KR 2008004806 W KR2008004806 W KR 2008004806W WO 2009038289 A2 WO2009038289 A2 WO 2009038289A2
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Prior art keywords
tgase2
fibrosis
nac
inhibitor
cystamine
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PCT/KR2008/004806
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French (fr)
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WO2009038289A3 (en
Inventor
In-Gyu Kim
Dong-Myung Shin
Sung-Yup Cho
Eui-Man Jeong
Gi-Yong Jang
Sang-Chul Park
Dong-Sup Lee
Keun-Hee Oh
Young-Whan Kim
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Seoul National University Industry Foundation
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Publication of WO2009038289A2 publication Critical patent/WO2009038289A2/en
Publication of WO2009038289A3 publication Critical patent/WO2009038289A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the present invention relates to a novel pharmaceutical composition for preventing or treating fibrosis and a method for screening a therapeutic agent to fibrosis.
  • Tissue is bound to extracellular matrix and includes well-arranged cell population surrounded by blood vessel networks. Fibrillation or fibrosis represents abnormal accumulation of collagen matrix due to injury or inflammation which alter structure and function of various tissues. Irrespective of fibrosis occurring sites, most etiology of fibrosis involves excessive accumulation of collagen matrix substituting for normal tissue. Progressive fibrillation in kidney, liver, lung, heart, bone, bone marrow and skin is a main causative factor of death or pain (Border, et al., New Engl. J. Med 331:1286(1994)). In addition, the development of fibrosis is associated with overexpression and overproduction of TGF ⁇ (transforming growth factor ⁇ ) in tissues. TGF ⁇ affects growth, differentiation and gene expression in various cells.
  • TGF ⁇ transforming growth factor ⁇
  • TGF ⁇ in mammalian is classified into three types of isoforms; TGF ⁇ l, TGF ⁇ 2 and TGF ⁇ 3 (de Martin, et al. EMBO J. 6:3673-3677(1987); and Derynck, et al. EMBO J. 7:3737-3743(1988)).
  • TGF ⁇ Activated TGF ⁇ binds to Type III receptor in cell membrane and in turn Type III receptor transfers TGF ⁇ to Type I and Type II receptors.
  • TGF ⁇ -bound receptor phosphorylates Smad2 or Smad3.
  • Phosphorylated Smad2 or Smad3 bind to Smad4 and then translocated into nucleus, regulating expressions of a multitude of genes.
  • TGF ⁇ increases expression of fibronectin, laminin and collagen as major components of extracellular matrix. Meanwhile, it decreases expression of matrix metalloprotease (MMP) to degrade matrix and increases expression of TIMP (tissue inhibitor of MMP) to inhibit MMP proteins, finally resulting in increase of ECM formation.
  • MMP matrix metalloprotease
  • TIMP tissue inhibitor of MMP
  • the TGF ⁇ downregulation induces atherosclerosis and its upregulation is a cause of various types of fibrosis.
  • the present inventors have made intensive studies to understand molecular mechanism underlying fibrosis development and to develop a new target for a therapeutic agent to fibrosis and a novel fibrosis therapeutics.
  • fibrosis was directly associated with activation of cellular TGase2 mediated by TGF ⁇ 2 (transforming growth factor ⁇ 2) signaling pathway and that the TGF ⁇ 2-TGase2 pathway was effectively blocked by our novel fibrosis therapeutics.
  • a pharmaceutical composition for preventing or treating fibrosis comprising (a) a therapeutically effective amount of a transglutaminase 2 (TGase2) inhibitor or N-acetylcysteine (NAC); and (b) a pharmaceutically acceptable carrier.
  • TGase2 transglutaminase 2
  • NAC N-acetylcysteine
  • the present inventors have made intensive studies to understand molecular mechanism underlying fibrosis development and to develop a new target for a therapeutic agent to fibrosis and a novel fibrosis therapeutics.
  • fibrosis was directly associated with activation of cellular TGase2 mediated by TGF ⁇ 2 (transforming growth factor ⁇ 2) signaling pathway and that the TGF ⁇ 2-TGase2 pathway was effectively blocked by our novel fibrosis therapeutics.
  • the present pharmaceutical composition includes TGase2 inhibitors or NAC as active ingredients.
  • the TGase2 inhibitor is selected from the group consisting of cystamine, cysteamine, monodansyl cadaverine, putrescine, histamine, methyl amine, iodoacetamide, 8-phenyl propionylthiocholine, monoamine, diamine, ⁇ -aminobenzoic acid, l-(5-aminopentyl)-3-phenylthiourea, N-benzyloxy carbonyl, 5-deazo-4-oxonorvaline, p-nitrophenylester, glycine methyl ester, CuSO 4 and tolbutamide.
  • the TGase2 inhibitor is cystamine or cysteamine.
  • Fibrosis or fibrotic disorders prevented or treated by the present invention may be specialized in acute or chronic diseases and show common properties such as excessive collagen accumulation and loss of function caused from substituting fiber tissues for normal tissues.
  • Acute fibrosis includes responses to trauma, infection, operation, burn, radiation and chemotherapeutics.
  • Chronic fibrosis is induced by other chronic disorders occurring virus infection, diabetes, obesity, fatty liver, hypertension, scleroderma and fibrosis.
  • Fibrosis to be prevented or treated by the present invention includes all fibrillation diseases comprising fibrosis from pathological state or diseases, radiation injury and chemotherapy agent ⁇ e.g., bleomycin, chlorambucil, cyclophosphamide, methotrexate, mustine or procarbazine). Fibrosis to be prevented or treated by the present pharmaceutical composition may be present at various positions of body.
  • Fibrosis may be present at kidney and observed in glomerulonephritis (Yoshioka et al., Lab Invest 68:154- 63(1993)), diabetic nephropathy (Yamamoto et al., Proc Natl Acad Sd USA 90:1814- 8(1993)), transplantation rejection (Shihabet al., J Am Soc Nephrol 4:671(1993)), HIV nephropathy (Border et al., J Am Soc Nephrol 4:675(1993)), IgA nephropathy and lupus nephropathy; in liver, cirrhosis (Castilla et al., N Engl J Med 324:933- 940(1991) and Nagy et al., Hepatology 14:269-73(1991)), veno-occlusive disease (Anscher et al., N Engl J Med 328:1592-8(1993)), type C he
  • Fibrosis to be prevented or treated by the present pharmaceutical composition includes fibrosis developed at various sites as described above.
  • fibrosis to be prevented or treated by the present pharmaceutical composition is kidney, liver or lung fibrosis.
  • the present pharmaceutical composition includes not only TGase2 inhibitor or
  • NAC but also its pharmaceutically acceptable salt, hydrate, solvate or prodrug as active ingredients.
  • pharmaceutically acceptable salt refers to a salt of
  • the salt is formed by using inorganic acids such as hydrochloride and hydrobromide and organic acids such as acetate, adipate, alginate, aspartate, benzoate, benzensulfonate, p-toluenesulfonate, bisulfate, sulphamate, sulfate, naphthylate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, decylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, 2-hydroxyethanesulfate, lactate, maleate, methanesulfonate, 2-naphtalenesulfonate, nicotinate, ox
  • pharmaceutically acceptable hydrate refers to a hydrate of TGase2 inhibitor or NAC to possess a desirable pharmacological effect.
  • pharmaceutically acceptable solvate means a solvate of TGase2 inhibitor or NAC to possess a desirable pharmacological effect.
  • the hydrate and solvate may be prepared using the acid as described above.
  • prodrug refers to a derivative of TGase2 inhibitor or NAC to undergo biological conversion before TGase2 inhibitor or NAC exhibits pharmacological effects.
  • the prodrugs may be prepared for improving chemical stability, patient compliance, bioavailability, organ selectivity or formulation convenience, prolonging action duration and diminishing side effect.
  • the prodrug of this invention may be easily prepared using TGase2 inhibitor or NAC according to the method ordinarily skilled in the art (See, Burger's Medicinal Chemistry and Drug Chemistry, 5 th ed., 1:172-178 and 949-982(1995)).
  • the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to.
  • the pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.
  • the pharmaceutical composition according to the present invention may be administered via the oral or parenterally.
  • parenterally When the pharmaceutical composition of the present invention is administered parenterally, it can be done by instillation, intravenous, subcutaneous, intramuscular and abdominal administration and most preferably instillation.
  • a suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition.
  • the pharmaceutical composition of the present invention is administered with a daily dose of 0.001-100 mg/kg (body weight).
  • the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form.
  • Formulation may be oil or aqueous media, resuspension or emulsion, extract, powder, granule, tablet and capsule and further comprise dispersant or stabilizer.
  • the pharmaceutical composition of this invention will prevent or treat fibrosis by blocking effectively TGF ⁇ 2-TGase2 pathway as a fibrosis development mechanism.
  • a method for screening a substance to prevent or treat fibrosis comprising the steps of:
  • the present screening method is based on the discovery that TGase2 is associated with fibrosis.
  • a test substance of interest is contacted to TGase2.
  • TGase2 used herein is present inside or outside a cell.
  • Extracellular TGase2 purified recombinant TGase2 may be obtained according to various recombinant DNA methods ordinarily known in the art. Peptides of TGase2 with its original activity may be also used as extracellular TGase2.
  • test substance screened by the present method may be single compounds or mixtures of compounds ⁇ e.g., natural extract or cell or tissue culture).
  • the test substance may be obtained from libraries of synthetic or natural compounds.
  • the methods to obtain library of such compounds are well known in the art.
  • the library of synthetic compounds may be commercially purchased from Maybridge
  • TGase2 activity in cells treated with the test substance is measured.
  • the test substance is selected as fibrosis therapeutics where TGase2 activity is lower in cells treated with the test substance than in those untreated.
  • the measurement of cellular TGase2 activity may be carried out according to various techniques known in the art, preferably (i) in vitro transamidation analysis or (ii) in situ transamidation analysis.
  • In vitro transamidation analysis determines in vitro TGase2 activity by measuring incorporation of [ 14 C] putrescine into N,N'-dimethylcasein.
  • cell homogenate is collected by lysis of test substance-treated cells and incubated with substrate solution containing [ 14 C] putrescine and N,N'-dimethylcasein.
  • TGase2 activity is determined by measuring radioactivity incorporated into N,N'-dimethylcasein.
  • cells not lysed are treated with affinity substance-bound amine compounds.
  • Biotin is the most preferable affinity substance and amine compound is preferably polyamine and most preferably pentylamine.
  • Cells are treated with biotinylated pentylamine (BP).
  • BP-bound proteins in cells may be measured according to various methods. For example, the BP-bound proteins may be analyzed using a solid microtiter plate method. Cell lysate is collected by disruption of BP-treated cells and used to coat microtiter plate.
  • the signal generating-substance includes, but not limited to, an enzymatic ⁇ e.g., alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, ⁇ -glucosidase and cytochrome P 450 ), a radioactive ⁇ e.g., C 14 , I 125 , P 32 and S 35 ), a fluorescent ⁇ e.g., fluorescein), a luminescent, a chemiluminescent, and a FRET (fluorescence resonance energy transfer) substance.
  • an enzymatic ⁇ e.g., alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, ⁇ -glucosidase and cytochrome P 450
  • a radioactive ⁇ e.g., C 14 , I 125 , P 32 and S 35 a fluorescent ⁇ e.g., fluorescein
  • the BP- bound proteins formed in the final step of this invention may be detected by measuring radioactivity from isotope.
  • the signal generating-substance is labeled with enzymes catalyzing colorimetric reactions
  • the BP-bound proteins formed may be detected by measuring reaction products resulting from addition of substrates used in enzyme reaction.
  • the signal generating-substance is labeled with alkaline phosphatase, bromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-Bl- phosphate and ECF (enhanced chemifluorescence) may be used as a substrate for color-developing reactions.
  • BCIP bromochloroindolylphosphate
  • NBT nitro blue tetrazolium
  • ECF enhanced chemifluorescence
  • BP-bound protein in cell may be quantitatively analyzed by detecting signals from signal generating-substance.
  • BP-bound protein may be analyzed using Western blotting.
  • Western blotting method may generally use conventional method (Refer: Peter B. Kaufma et al. Molecular and Cellular Methods in Biology and Medicine, 108-121, CRC press).
  • Western blotting method comprises the steps of (i) pulverizing test substance-treated cells; (ii) loading cell homogenates on SDS-PAGE; (iii) transferring proteins on SDS-PAGE into NC membrane; and (iv) treating streptavidin (or avidin)-conjugated signal generating-substance to NC membrane.
  • BP-bound protein may be also analyzed by cell staining methods. For example, cells cultured on glass coverslip on plate are labeled with BP. Cells are fixed and permeabilized by suitable surfactant ⁇ e.g., Triton X-IOO). Cells are treated with fluorescent substance-bound streptavidin (or avidin) and observed under confocal microscope.
  • suitable surfactant e.g., Triton X-IOO
  • the fluorescent substance includes fluorescein, FITC (fluorescein isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetra methyl- rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole) and Coumarin.
  • the present screening method is carried out using in situ transamidation analysis. Since the direct measurement of intracellular TGase2 activity represents in vivo activity, it is preferable that in situ transamidation analysis is carried out, not in vitro transamidation analysis.
  • the present screening method comprises the steps of:
  • the test substance is selected as fibrosis therapeutics where signals from the step (b-5) are lower than those from control, test substance-untreated cells.
  • the present invention definitely demonstrates molecular mechanism underlying fibrosis development and provides a novel molecular target of fibrosis therapeutics. Moreover, fibrosis therapeutics of this invention effectively prevent or treat fibrosis with much higher safety.
  • this invention provides a pharmaceutical composition for preventing or treating fibrosis. Also, this invention provides a method for screening a substance to prevent or treat fibrosis. The present invention definitely demonstrates molecular mechanism of fibrosis development and provides a novel molecular target of fibrosis therapeutics. Moreover, fibrosis therapeutics of this invention effectively prevent or treat fibrosis with much higher safety.
  • Fig. 1 represents masson trichrome staining to analyze the relationship between TGase2 and fibrosis.
  • Fig. 2 represents masson trichrome staining to analyze effects of NAC (N- acetylcysteine) or cysteamine inhibiting development of fibrosis.
  • NAC N- acetylcysteine
  • BLM 2.2 indicates dose of 1.5 mg/kg and 2.0 mg/kg bleomycin.
  • NAC N-acetylcysteine
  • CYST cysteamine
  • Figs. 3a-3b represent TGF ⁇ l increases expression of FN (fibronectin) in an amount-dependent manner, but not TGF ⁇ 2.
  • 3a human lung fibroblast was treated with TGF ⁇ l or TGF ⁇ 2 for 24 hrs and then expression of FN or ⁇ -smooth muscle actin ( ⁇ -SMA) as molecular marker to fibrosis was examined.
  • 3b TGF ⁇ l was treated for 48 hrs. Cellular activity of TGase was enhanced by TGF ⁇ l treatment for 48hrs.
  • the numbers (upper) in the gel picture are treating amounts of TGF ⁇ l or TGF ⁇ 2.
  • Figs. 4a-4f represent TGF ⁇ l-induced TGase2 activation accelerates deposition of FN in ECM.
  • NT not treated; Cyst, cystamine.
  • 4a cellular TGase2 activity was measured after treating TGF ⁇ l to cells for 24 hrs or 48 hrs in the presence or absence of cystamine or NAC.
  • 4b effects of cystamine or NAC on TGF ⁇ l-mediated expression of FN and ⁇ -SMA.
  • 1. NT (not treated); 2. treatment of TGF ⁇ l; 3. treatment of TGF ⁇ l and cystamine; 4. treatment of TGF ⁇ l and NAC.
  • 4c-4d FN deposition was analyzed by treatment of TGF ⁇ l in the presence or absence of cystamine or NAC.
  • 4e amount of BP-bound protein was measured to analyze a role of TGase2.
  • 4f deposition of TGase2 persewas measured.
  • Figs. 5a-5b represent cystamine or NAC inhibit TGF ⁇ -induced proliferation of lung fibroblast.
  • the treating amount of each cystamine and NAC is 1 mM and 2.5 mM.
  • Figs. 6a-6b represent cystamine or NAC have no effect on apoptosis of
  • TGF ⁇ l-treated cell Apoptosis of TGF ⁇ l-treated cell was investigated by measuring
  • each TGF ⁇ l, cysteamine and NAC is 1 ng/ml, 1 mM and 2.5 mM.
  • PBS phosphate buffered saline
  • bleomycin 2.0 mg/kg, Dong-A pharmaceutical
  • lung fibrosis induced by bleomycin was observed in normal C57BL/6 mice but not in TGase2 ' ⁇ mouse, representing activity of TGase2 has an essential role in fibrosis development.
  • Bleomycin (1. 5 or 2.0 mg/kg) was intrathecal ⁇ injected in C57BL/6 mice at
  • NAC could partially inhibit fibrosis development induced by bleomycin.
  • Cysteamine to directly inhibit activity of TGase2 could represent much more excellent effect on inhibition of lung fibrosis as compared with NAC.
  • Intracellular TGase2 activity was measured by determining a biotinylated pentylamine (BP, Pierce) incorporated into cellular proteins (Shin, D. M. et al., Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation. J. Biol. Chem. 279:15032-15039(2004)).
  • TGase2 activity expressed as folds of activation compared with the samples without oxidative stress after subtracting the value obtained in the absence of BP which represents endogenous biotin-conjugated proteins.
  • TGase2 activity was also visualized by probing BP incorporated into cellular proteins with streptavidin-HRP (SA).
  • IMR90 cell Human primary lung fibroblast, IMR90 cell (ATCC) was maintained in DMEM (Dulbecco's modified Eagle's medium, GIBCO) supplemented with 10% fetal bovine serum (FBS, Hyclone), penicillin (100 U/ml), streptomycin sulfate (100 ⁇ g/ml) and glutamine (2 mM). IMR90 cells were cultured in DMEM containing 2% FBS for 12 hrs at 37°C and exposed to media containing the indicated concentration of TGF ⁇ l (R&D system). Each cystamine (1 mM, Sigma) and NAC (2.5 mM, Sigma) was used to inhibit TGase2 and TGF ⁇ activity.
  • EXAMPLE 5 Western Blot Analysis
  • Figs. 3a-3b is Western blotting analysis representing TGF ⁇ l increases expression of FN (fibronectin) in an amount-dependent manner, but not TGF ⁇ 2.
  • human lung fibroblast IMR90
  • TGF ⁇ l or TGF ⁇ 2 TGF ⁇ 2
  • expression of FN or ⁇ -smooth muscle actin ( ⁇ -SMA) as a molecular marker to fibrosis was examined.
  • FN or ⁇ -SMA were increased in a concentration-dependent manner.
  • TGF ⁇ 2 was treated for 24 hrs
  • ⁇ -SMA expression was increased while FN expression was not changed.
  • TGase expression was hardly changed by treatment of two TGF ⁇ s for 24 hrs. As shown in Fig. 3b, cellular activity of TGase was increased by treatment of TGF ⁇ l for 48 hrs.
  • EXAMPLE 6 Analysis of Extracellular Matrix To measure the amount of fibronectin (FN) deposition into extracellular matrix
  • ECM human primary lung fibroblast
  • IMR90 cell ATCC
  • DOC-EDTA 0.1% (w/v) sodium deoxycholate-2 mM EDTA
  • DOC-EDTA- unsoluble proteins were blocked in milk-Tween-PBS before the addition of either 0.2 g/ml of the monoclonal anti-TGase antibody CUB7042 or a 1:1000 dilution of rabbit anti-fibronectin (clone IST-I, Sigma) at 4 0 C overnight.
  • the deposition amount of each protein was determined by the reaction with O-phenylenediamine dihydrochloride (Sigma). Assays were quantitated by measuring the absorbance at 490 nm on microplate spectrophotometer (Molecular Devices).
  • Figs. 4a-4f represent TGF ⁇ l-induced TGase2 activation accelerates deposition of FN in ECM.
  • cellular TGase2 activity was induced by treatment of TGF ⁇ l for 48 hrs (not 24 hrs). Enhancement of TGase2 activity was eliminated by treatment of cystamine (TGase inhibitor, Sigma) or N -acetylcysteine (NAC, 2.5 mM).
  • cystamine TGase inhibitor, Sigma
  • NAC N -acetylcysteine
  • cystamine or NAC inhibited TGF ⁇ l-induced activation of FN and ⁇ - SMA, demonstrating that activation of TGase2 affects expression of fibrous components.
  • NT not treated
  • treatment of TGF ⁇ l 3.
  • treatment of TGF ⁇ l and cystamine 4.
  • treatment of TGF ⁇ l and NAC 4.
  • TGase2 has been known to crosslink between ECM molecules such as collagen I, III and IV, and fibronectin, resulting in aggregation of proteins with resistance for protein-degradaing enzymes.
  • ECM molecules such as collagen I, III and IV
  • fibronectin resulting in aggregation of proteins with resistance for protein-degradaing enzymes.
  • amount of fibronectin was measured in detergent insoluble matrix. As shown in Fig. 4c, treatment of TGF ⁇ l (1 ng/ml) increased amount of FN bound to detergent insoluble matrix.
  • Fig. 4d represents deposition of FN by treatment of TGF ⁇ l (4 ng/ml).
  • Fig. 4e demonstrates a role of TGase2 by measuring amount of BP-bound proteins and its amount was decreased by cystamine or NAC. As shown in Fig. 4f, it was not observed that TGase2 per se was bound to ECM.
  • TGF ⁇ l-induced activation of TGase2 causes fibrous deposition of ECM molecules using tansamidation response.
  • the increase of cell number was assessed by MTT analysis kit (Roche) according to the manufacturer's protocol and was expressed as folds of increase compared with the samples without TGF ⁇ l.
  • Cell proliferation assay was performed according to the manufacturer's protocol using Brd-U incorporation kit (Roche).
  • the present inventors measured sub-Gl fraction by FACS analysis or caspase activity. Caspase activity was determined using chromogenic substrates, Ac- DEVD-pNA for caspase 2 and Ac-LEVD-pNA (A.G. Scientific, Inc) for caspase 9, respectively.
  • Total cell extracts were prepared using freeze-thaw process in lysis buffer (100 mM HEPES, pH 7.5, 0.1% CHAPS, 0.1% Triton X-100, 100 mM EDTA), followed by centrifugation (12,000 x g, 10 min, 4°C).
  • Cell extracts (30 ⁇ g) were added to assay buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1 % CHAPS, 10 mM DTT) containing chromogenic substrates (200 ⁇ M) and incubated for 4 hrs at 37°C.
  • Caspase activity was quantitated by measuring absorbance at 490 nm and represented as 'relative activity' compared to that of the untreated cells (negative control).
  • Figs. 5a-5b represent cystamine or NAC suppress TGF ⁇ -induced proliferation of lung fibroblast. Increased proliferation of fibroblast after injury is a principal factor of fibrosis. As shown in Fig. 5a, cell number of IMR90 was increased by treatment of TGF ⁇ l. The effect of TGF ⁇ l to cell proliferation could be clearly observed at 48 hrs post-treatment. The number of fibroblast was not affected by treatment of TGF ⁇ 2. In Fig. 5b, IMR90 cells were subjected to TGF ⁇ l in the presence or absence of cystamine or NAC. Both cystamine and NAC strongly inhibited proliferation of fibroblast induced by TGF ⁇ l.
  • TGase2 contributes to induce fibrosis via promoting proliferation of lung fibroblast.
  • Figs. 6a-6b represent cystamine or NAC have no effect on apoptosis of TGF ⁇ l-treated cell.
  • Apoptosis of TGF ⁇ l-treated cell was investigated by measuring PARP fragment (Fig. 6a) or sub-Gl fraction (Fig. 6b) in the presence or absence of cystamine or NAC.
  • PARP fragment or sub-Gl fraction had no significant differences in cells treated with TGF ⁇ , cells treated with both TGF ⁇ and cystamine and cells treated with both TGF ⁇ and NAC.
  • cystamine or NAC have no side-effects on cells.

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Abstract

The present invention relates to a pharmaceutical composition for preventing or treating fibrosis comprising (a) a therapeutically effective amount of a transglutaminase 2 (TGase2) inhibitor or N-acetylcysteine (NAC); and (b) a pharmaceutically acceptable carrier, and a method for screening a substance to prevent or treat fibrosis. The present invention definitely demonstrates molecular mechanism underlying fibrosis development and provides a novel molecular target of fibrosis therapeutics. Moreover, fibrosis therapeutics of this invention effectively prevent or treat fibrosis with much higher safety.

Description

PHARMACEUTICAL COMPOSITIONS FOR PREVENTING OR TREATING
FIBROSIS
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical composition for preventing or treating fibrosis and a method for screening a therapeutic agent to fibrosis.
BACKGROUND OF TECHNIQUE
Tissue is bound to extracellular matrix and includes well-arranged cell population surrounded by blood vessel networks. Fibrillation or fibrosis represents abnormal accumulation of collagen matrix due to injury or inflammation which alter structure and function of various tissues. Irrespective of fibrosis occurring sites, most etiology of fibrosis involves excessive accumulation of collagen matrix substituting for normal tissue. Progressive fibrillation in kidney, liver, lung, heart, bone, bone marrow and skin is a main causative factor of death or pain (Border, et al., New Engl. J. Med 331:1286(1994)). In addition, the development of fibrosis is associated with overexpression and overproduction of TGFβ (transforming growth factor β) in tissues. TGFβ affects growth, differentiation and gene expression in various cells.
TGFβ in mammalian is classified into three types of isoforms; TGFβl, TGFβ2 and TGFβ3 (de Martin, et al. EMBO J. 6:3673-3677(1987); and Derynck, et al. EMBO J. 7:3737-3743(1988)).
Activated TGFβ binds to Type III receptor in cell membrane and in turn Type III receptor transfers TGFβ to Type I and Type II receptors. TGFβ-bound receptor phosphorylates Smad2 or Smad3. Phosphorylated Smad2 or Smad3 bind to Smad4 and then translocated into nucleus, regulating expressions of a multitude of genes. TGFβ increases expression of fibronectin, laminin and collagen as major components of extracellular matrix. Meanwhile, it decreases expression of matrix metalloprotease (MMP) to degrade matrix and increases expression of TIMP (tissue inhibitor of MMP) to inhibit MMP proteins, finally resulting in increase of ECM formation. It is also known that TGFβ is implicated in development of various diseases.
The TGFβ downregulation induces atherosclerosis and its upregulation is a cause of various types of fibrosis.
The cause-effect relationship of TGFβ in etiology of fibrillation disease, i.e., fibrosis is well established but the mechanism how TGFβ induces fibrillation disease has hardly been reported. Moreover, fibrillation diseases are commonly associated with oxidative stress but its mechanism remains unknown.
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.
DETAILED DESCRIPTION OF THE INVENTION The present inventors have made intensive studies to understand molecular mechanism underlying fibrosis development and to develop a new target for a therapeutic agent to fibrosis and a novel fibrosis therapeutics. As results, we have discovered that fibrosis was directly associated with activation of cellular TGase2 mediated by TGFβ2 (transforming growth factor β2) signaling pathway and that the TGFβ2-TGase2 pathway was effectively blocked by our novel fibrosis therapeutics.
Accordingly, it is an object of this invention to provide a pharmaceutical composition for preventing or treating fibrosis.
It is another object of this invention to provide a method for screening a substance to prevent or treat fibrosis.
Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.
In one aspect of this invention, there is provided a pharmaceutical composition for preventing or treating fibrosis comprising (a) a therapeutically effective amount of a transglutaminase 2 (TGase2) inhibitor or N-acetylcysteine (NAC); and (b) a pharmaceutically acceptable carrier.
The present inventors have made intensive studies to understand molecular mechanism underlying fibrosis development and to develop a new target for a therapeutic agent to fibrosis and a novel fibrosis therapeutics. As results, we have discovered that fibrosis was directly associated with activation of cellular TGase2 mediated by TGFβ2 (transforming growth factor β2) signaling pathway and that the TGFβ2-TGase2 pathway was effectively blocked by our novel fibrosis therapeutics.
The present pharmaceutical composition includes TGase2 inhibitors or NAC as active ingredients.
According to a preferable embodiment, the TGase2 inhibitor is selected from the group consisting of cystamine, cysteamine, monodansyl cadaverine, putrescine, histamine, methyl amine, iodoacetamide, 8-phenyl propionylthiocholine, monoamine, diamine, γ-aminobenzoic acid, l-(5-aminopentyl)-3-phenylthiourea, N-benzyloxy carbonyl, 5-deazo-4-oxonorvaline, p-nitrophenylester, glycine methyl ester, CuSO4 and tolbutamide. According to a more preferable embodiment, the TGase2 inhibitor is cystamine or cysteamine.
Fibrosis or fibrotic disorders prevented or treated by the present invention may be specialized in acute or chronic diseases and show common properties such as excessive collagen accumulation and loss of function caused from substituting fiber tissues for normal tissues. Acute fibrosis includes responses to trauma, infection, operation, burn, radiation and chemotherapeutics. Chronic fibrosis is induced by other chronic disorders occurring virus infection, diabetes, obesity, fatty liver, hypertension, scleroderma and fibrosis.
Fibrosis to be prevented or treated by the present invention includes all fibrillation diseases comprising fibrosis from pathological state or diseases, radiation injury and chemotherapy agent {e.g., bleomycin, chlorambucil, cyclophosphamide, methotrexate, mustine or procarbazine). Fibrosis to be prevented or treated by the present pharmaceutical composition may be present at various positions of body. Fibrosis may be present at kidney and observed in glomerulonephritis (Yoshioka et al., Lab Invest 68:154- 63(1993)), diabetic nephropathy (Yamamoto et al., Proc Natl Acad Sd USA 90:1814- 8(1993)), transplantation rejection (Shihabet al., J Am Soc Nephrol 4:671(1993)), HIV nephropathy (Border et al., J Am Soc Nephrol 4:675(1993)), IgA nephropathy and lupus nephropathy; in liver, cirrhosis (Castilla et al., N Engl J Med 324:933- 940(1991) and Nagy et al., Hepatology 14:269-73(1991)), veno-occlusive disease (Anscher et al., N Engl J Med 328:1592-8(1993)), type C hepatitis virus infection, alcohol-induced hepatic fibrosis and autoimmune hepatic fibrosis; in lung, idiopathic pulmonary fibrosis (Anscher et al., N EnglJ Med 328: 1592-8(1993) and Brockelmann et al., Proc Natl Acad ScI USA 88: 6642-6(1991)), auto-immune pulmonary fibrosis (Deguchi, Ann Rheum DIs 51: 362-5(1992)) and bleomycin-induced pulmonary fibrosis; in skin, systemic sclerosis (Kulozik et al., J Clin Invest 86: 917-22(1990)), keloid (Peltonen et al., J Invest Dermatol 97: 240-8(1991)), wounds (Ghahary et al., J Lab Clin Med 122: 465-73(1993)) and eosinophilia-myalgia syndrome (Varga et al., Ann Intern Med 116: 140-7(1992)); in central nervous system, intraocular fibrosis (Conner et al., J Clin Invest 83: 1661-6(1989)); in cardiovascular system, vascular restenosis (Nikol et al., J Clin Invest 90: 1582-92(1992)); in nose, nasal nonpolyposis (Ohno et al., J Clin Invest 89: 1662-8(1992)); in bone or marrow (Reith, J. D. et al., Am J Srg Pathol 20(11): 1368-1377(1996)); in endocrine organ (Endocrinology, 3rd Edition, Edited by Leslie J. DeGroot, Vol. 1, pp. 165-177 and pp. 747-751); in gastrointestinal tract (Tahara, E, J. Cancer Res. Clin. Oncol. 116(2): 121-131(1990).
Fibrosis to be prevented or treated by the present pharmaceutical composition includes fibrosis developed at various sites as described above.
Preferably, fibrosis to be prevented or treated by the present pharmaceutical composition is kidney, liver or lung fibrosis. The present pharmaceutical composition includes not only TGase2 inhibitor or
NAC but also its pharmaceutically acceptable salt, hydrate, solvate or prodrug as active ingredients.
The term used herein "pharmaceutically acceptable salt" refers to a salt of
TGase2 inhibitor or NAC to possess a desirable pharmacological effect. The salt is formed by using inorganic acids such as hydrochloride and hydrobromide and organic acids such as acetate, adipate, alginate, aspartate, benzoate, benzensulfonate, p-toluenesulfonate, bisulfate, sulphamate, sulfate, naphthylate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, decylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, 2-hydroxyethanesulfate, lactate, maleate, methanesulfonate, 2-naphtalenesulfonate, nicotinate, oxalate, tosylate and undecanoate.
The term "pharmaceutically acceptable hydrate" as used herein refers to a hydrate of TGase2 inhibitor or NAC to possess a desirable pharmacological effect. The term "pharmaceutically acceptable solvate" means a solvate of TGase2 inhibitor or NAC to possess a desirable pharmacological effect. The hydrate and solvate may be prepared using the acid as described above.
The term used herein "pharmaceutically acceptable prodrug" refers to a derivative of TGase2 inhibitor or NAC to undergo biological conversion before TGase2 inhibitor or NAC exhibits pharmacological effects. The prodrugs may be prepared for improving chemical stability, patient compliance, bioavailability, organ selectivity or formulation convenience, prolonging action duration and diminishing side effect. The prodrug of this invention may be easily prepared using TGase2 inhibitor or NAC according to the method ordinarily skilled in the art (See, Burger's Medicinal Chemistry and Drug Chemistry, 5th ed., 1:172-178 and 949-982(1995)).
In the pharmaceutical compositions of this invention, the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.
The pharmaceutical composition according to the present invention may be administered via the oral or parenterally. When the pharmaceutical composition of the present invention is administered parenterally, it can be done by instillation, intravenous, subcutaneous, intramuscular and abdominal administration and most preferably instillation.
A suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition. Preferably, the pharmaceutical composition of the present invention is administered with a daily dose of 0.001-100 mg/kg (body weight).
According to the conventional techniques known to those skilled in the art, the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form. Formulation may be oil or aqueous media, resuspension or emulsion, extract, powder, granule, tablet and capsule and further comprise dispersant or stabilizer.
The pharmaceutical composition of this invention will prevent or treat fibrosis by blocking effectively TGFβ2-TGase2 pathway as a fibrosis development mechanism.
In another aspect of this invention, there is provided a method for screening a substance to prevent or treat fibrosis comprising the steps of:
(a) contacting a test substance to a transglutaminase 2 (TGase2) protein; and
(b) measuring an activity of TGase2. The present screening method is based on the discovery that TGase2 is associated with fibrosis.
In the first step of the present screening method, a test substance of interest is contacted to TGase2. TGase2 used herein is present inside or outside a cell.
Extracellular TGase2, purified recombinant TGase2 may be obtained according to various recombinant DNA methods ordinarily known in the art. Peptides of TGase2 with its original activity may be also used as extracellular TGase2.
The test substance screened by the present method may be single compounds or mixtures of compounds {e.g., natural extract or cell or tissue culture).
The test substance may be obtained from libraries of synthetic or natural compounds. The methods to obtain library of such compounds are well known in the art. The library of synthetic compounds may be commercially purchased from Maybridge
Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) and the library of natural compounds may be commercially purchased from Pan Laboratories (USA) and MycoSearch (USA).
Finally, TGase2 activity in cells treated with the test substance is measured. The test substance is selected as fibrosis therapeutics where TGase2 activity is lower in cells treated with the test substance than in those untreated. The measurement of cellular TGase2 activity may be carried out according to various techniques known in the art, preferably (i) in vitro transamidation analysis or (ii) in situ transamidation analysis.
In vitro transamidation analysis determines in vitro TGase2 activity by measuring incorporation of [14C] putrescine into N,N'-dimethylcasein. In particular, cell homogenate is collected by lysis of test substance-treated cells and incubated with substrate solution containing [14C] putrescine and N,N'-dimethylcasein.
Following termination of the reaction, the TGase2 activity is determined by measuring radioactivity incorporated into N,N'-dimethylcasein.
According to in situ transamidation analysis, cells not lysed are treated with affinity substance-bound amine compounds. Biotin is the most preferable affinity substance and amine compound is preferably polyamine and most preferably pentylamine. Cells are treated with biotinylated pentylamine (BP). BP-bound proteins in cells may be measured according to various methods. For example, the BP-bound proteins may be analyzed using a solid microtiter plate method. Cell lysate is collected by disruption of BP-treated cells and used to coat microtiter plate. The signal generating-substance includes, but not limited to, an enzymatic {e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, β-glucosidase and cytochrome P450), a radioactive {e.g., C14, I125, P32 and S35), a fluorescent {e.g., fluorescein), a luminescent, a chemiluminescent, and a FRET (fluorescence resonance energy transfer) substance.
Where the radioactive isotope is used for signal generating-substance, the BP- bound proteins formed in the final step of this invention may be detected by measuring radioactivity from isotope. Where the signal generating-substance is labeled with enzymes catalyzing colorimetric reactions, the BP-bound proteins formed may be detected by measuring reaction products resulting from addition of substrates used in enzyme reaction. For example, where the signal generating-substance is labeled with alkaline phosphatase, bromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-Bl- phosphate and ECF (enhanced chemifluorescence) may be used as a substrate for color-developing reactions. In the case of labeled with horseradish peroxidase, chloronaphtol, aminoethylcarbazol, diaminobenzidine, D-luciferin, lucigenin (bis-N- methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex™ Red reagent (10- acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine), ABTS (2,2- Azine-di[3-ethylbenzthiazoline sulfonate]) and o-phenyldiamine (OPD) may be used as a substrate.
BP-bound protein in cell may be quantitatively analyzed by detecting signals from signal generating-substance. In addition to the solid microtiter plate method described above, BP-bound protein may be analyzed using Western blotting. Western blotting method may generally use conventional method (Refer: Peter B. Kaufma et al. Molecular and Cellular Methods in Biology and Medicine, 108-121, CRC press). Preferably, Western blotting method comprises the steps of (i) pulverizing test substance-treated cells; (ii) loading cell homogenates on SDS-PAGE; (iii) transferring proteins on SDS-PAGE into NC membrane; and (iv) treating streptavidin (or avidin)-conjugated signal generating-substance to NC membrane.
BP-bound protein may be also analyzed by cell staining methods. For example, cells cultured on glass coverslip on plate are labeled with BP. Cells are fixed and permeabilized by suitable surfactant {e.g., Triton X-IOO). Cells are treated with fluorescent substance-bound streptavidin (or avidin) and observed under confocal microscope. The fluorescent substance includes fluorescein, FITC (fluorescein isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetra methyl- rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole) and Coumarin.
According to a preferable embodiment, the present screening method is carried out using in situ transamidation analysis. Since the direct measurement of intracellular TGase2 activity represents in vivo activity, it is preferable that in situ transamidation analysis is carried out, not in vitro transamidation analysis.
According to a more preferable embodiment, the present screening method comprises the steps of:
(a) contacting a test substance to a cell comprising TGase2; (b-1) treating the cell with biotinylated polyamine, preferably biotinylated pentylamine (BP);
(b-2) collecting a cell lysate by lysis of polyamine-treated cell;
(b-3) coating a microtiter plate with the cell lysate;
(b-4) treating a streptavidin (or avidin)-conjugated signal generating- substance to the plate; and
(b-5) measuring signals generated from the signal generating-substance.
The test substance is selected as fibrosis therapeutics where signals from the step (b-5) are lower than those from control, test substance-untreated cells.
The present invention definitely demonstrates molecular mechanism underlying fibrosis development and provides a novel molecular target of fibrosis therapeutics. Moreover, fibrosis therapeutics of this invention effectively prevent or treat fibrosis with much higher safety.
As described above, this invention provides a pharmaceutical composition for preventing or treating fibrosis. Also, this invention provides a method for screening a substance to prevent or treat fibrosis. The present invention definitely demonstrates molecular mechanism of fibrosis development and provides a novel molecular target of fibrosis therapeutics. Moreover, fibrosis therapeutics of this invention effectively prevent or treat fibrosis with much higher safety.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 represents masson trichrome staining to analyze the relationship between TGase2 and fibrosis. Abbreviations: B6, C57BL/6 wild type mouse; TGase2nu", TGase2-deficient mouse (TGase2v"); BLM, bleomycin.
Fig. 2 represents masson trichrome staining to analyze effects of NAC (N- acetylcysteine) or cysteamine inhibiting development of fibrosis. Each BLM 1.5 and BLM 2.2 indicates dose of 1.5 mg/kg and 2.0 mg/kg bleomycin. Abbreviations: NAC, N-acetylcysteine; CYST, cysteamine.
Figs. 3a-3b represent TGFβl increases expression of FN (fibronectin) in an amount-dependent manner, but not TGFβ2. 3a, human lung fibroblast was treated with TGF βl or TGF β2 for 24 hrs and then expression of FN or α-smooth muscle actin (α-SMA) as molecular marker to fibrosis was examined. 3b, TGFβl was treated for 48 hrs. Cellular activity of TGase was enhanced by TGFβl treatment for 48hrs. The numbers (upper) in the gel picture are treating amounts of TGFβl or TGFβ2.
Figs. 4a-4f represent TGFβl-induced TGase2 activation accelerates deposition of FN in ECM. NT, not treated; Cyst, cystamine. 4a, cellular TGase2 activity was measured after treating TGFβl to cells for 24 hrs or 48 hrs in the presence or absence of cystamine or NAC. 4b, effects of cystamine or NAC on TGFβl-mediated expression of FN and α-SMA. 1. NT (not treated); 2. treatment of TGFβl; 3. treatment of TGFβl and cystamine; 4. treatment of TGF βl and NAC. 4c-4d, FN deposition was analyzed by treatment of TGFβl in the presence or absence of cystamine or NAC. 4e, amount of BP-bound protein was measured to analyze a role of TGase2. 4f, deposition of TGase2 persewas measured.
Figs. 5a-5b represent cystamine or NAC inhibit TGFβ-induced proliferation of lung fibroblast. The treating amount of each cystamine and NAC is 1 mM and 2.5 mM. Figs. 6a-6b represent cystamine or NAC have no effect on apoptosis of
TGFβl-treated cell. Apoptosis of TGFβl-treated cell was investigated by measuring
PARP fragment (Fig. 6a) or sub-Gl fraction (Fig. 6b) in the presence or absence of cystamine or NAC. The treating amount of each TGFβl, cysteamine and NAC is 1 ng/ml, 1 mM and 2.5 mM.
The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.
EXAMPLES
Experimental Methods EXAMPLE 1: Relationship of TGase2 to Fibrosis The present inventors investigated whether TGase2 was associated with fibrosis, particularly pulmonary fibrosis using C57BL/6 mice at 20 weeks of age (TGase2+/+, Charles River Laboratories) and TGase2-deficient (TGase2"/~) mice (De Laurenzi, V. & Melino, G.. Gene disruption of tissue transglutaminase. MoI. Cell Biol. 21:148-155 (2001)). PBS (phosphate buffered saline) or bleomycin (2.0 mg/kg, Dong-A pharmaceutical) were intrathecal^ injected in wild type C57BL/6 and TGase2"/" mice as negative controls. After 1 month, mouse was sacrificed and lung tissue was extracted and fixed using formalin. Masson trichrome staining was carried out and image of lung tissue was obtained at 400 x magnification.
As shown in Fig. 1, lung fibrosis induced by bleomycin was observed in normal C57BL/6 mice but not in TGase2'Λ mouse, representing activity of TGase2 has an essential role in fibrosis development.
EXAMPLE 2: Inhibitory Effect of NAC or Cysteamine on Fibrosis Development
Bleomycin (1. 5 or 2.0 mg/kg) was intrathecal^ injected in C57BL/6 mice at
20 weeks of age and PBS, NAC (60 mg/kg, Sigma) or cysteamine (50 mg/kg, Sigma) were intra-abdominally injected without delay. After 1 month, mouse was sacrificed and lung tissue was extracted and fixed using formalin. Masson trichrome staining was carried out and image of lung tissue was obtained at 400 x magnification.
As shown in Hg. 2, NAC could partially inhibit fibrosis development induced by bleomycin. Cysteamine to directly inhibit activity of TGase2 could represent much more excellent effect on inhibition of lung fibrosis as compared with NAC.
EXAMPLE 3: Measurement of Intracellular TGase Activity
Intracellular TGase2 activity was measured by determining a biotinylated pentylamine (BP, Pierce) incorporated into cellular proteins (Shin, D. M. et al., Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation. J. Biol. Chem. 279:15032-15039(2004)). TGase2 activity expressed as folds of activation compared with the samples without oxidative stress after subtracting the value obtained in the absence of BP which represents endogenous biotin-conjugated proteins. TGase2 activity was also visualized by probing BP incorporated into cellular proteins with streptavidin-HRP (SA).
EXAMPLE 4: Cell Culture
Human primary lung fibroblast, IMR90 cell (ATCC) was maintained in DMEM (Dulbecco's modified Eagle's medium, GIBCO) supplemented with 10% fetal bovine serum (FBS, Hyclone), penicillin (100 U/ml), streptomycin sulfate (100 μg/ml) and glutamine (2 mM). IMR90 cells were cultured in DMEM containing 2% FBS for 12 hrs at 37°C and exposed to media containing the indicated concentration of TGFβl (R&D system). Each cystamine (1 mM, Sigma) and NAC (2.5 mM, Sigma) was used to inhibit TGase2 and TGFβ activity. EXAMPLE 5: Western Blot Analysis
Whole cell extracts were prepared using sonication in buffer (50 mM Tris-CI, pH 6.8, 6 M urea, 2% SDS, 40 mM dithiothreitol and protease inhibitor cocktail). After centrifugation (12,000 x g, 10 min at 4°C), proteins were quantitated using BCA method. The cell extracts (30 μg) were separated on 8% or 12% SDS-PAGE. Protein expression was probed using monoclonal antibodies specific for actin (Sigma), α-smooth muscle actin (DAKO corporation), fibronectin (Santa Cruz), TGase2 (Shin, D.M. et al. J. Biol. Chem. 279:15032-15039(2004)), PARP (poly(ADP- ribose) polymerase) (Cell signaling) and phosphorylated Smad3 (Wilkes, M. C. et al., MoI. Cell. Biol. 23:8878-8889(2003)), respectively.
Figs. 3a-3b is Western blotting analysis representing TGFβl increases expression of FN (fibronectin) in an amount-dependent manner, but not TGFβ2. As shown in Fig. 3a, human lung fibroblast (IMR90) was subjected to TGFβl or TGFβ2 and then expression of FN or α-smooth muscle actin (α-SMA) as a molecular marker to fibrosis was examined. Where TGFβl was treated for 24 hrs, FN or α-SMA were increased in a concentration-dependent manner. On the contrary, where TGFβ2 was treated for 24 hrs, α-SMA expression was increased while FN expression was not changed. TGase expression was hardly changed by treatment of two TGFβs for 24 hrs. As shown in Fig. 3b, cellular activity of TGase was increased by treatment of TGFβl for 48 hrs. These results demonstrate that TGFβl is a principal isoform to induce FN in lung fibroblast.
EXAMPLE 6: Analysis of Extracellular Matrix To measure the amount of fibronectin (FN) deposition into extracellular matrix
(ECM), human primary lung fibroblast, IMR90 cell (ATCC) was grown on 10-cm tissue culture dish and solubilized with 1 ml of 0.1% (w/v) sodium deoxycholate-2 mM EDTA (DOC-EDTA). DOC-EDTA-soluble fraction was stored for protein analysis (BCA assay) while the insoluble residue, remaining on the plate and predominantly representing the ECM, was used for further steps. For immune-assay, DOC-EDTA- unsoluble proteins were blocked in milk-Tween-PBS before the addition of either 0.2 g/ml of the monoclonal anti-TGase antibody CUB7042 or a 1:1000 dilution of rabbit anti-fibronectin (clone IST-I, Sigma) at 4 0C overnight. The deposition amount of each protein was determined by the reaction with O-phenylenediamine dihydrochloride (Sigma). Assays were quantitated by measuring the absorbance at 490 nm on microplate spectrophotometer (Molecular Devices).
Figs. 4a-4f represent TGFβl-induced TGase2 activation accelerates deposition of FN in ECM. In Fig. 4a, cellular TGase2 activity was induced by treatment of TGFβl for 48 hrs (not 24 hrs). Enhancement of TGase2 activity was eliminated by treatment of cystamine (TGase inhibitor, Sigma) or N -acetylcysteine (NAC, 2.5 mM).
In Fig. 4b, cystamine or NAC inhibited TGFβl-induced activation of FN and α- SMA, demonstrating that activation of TGase2 affects expression of fibrous components. 1. NT (not treated); 2. treatment of TGFβl; 3. treatment of TGFβl and cystamine; 4. treatment of TGF βl and NAC.
TGase2 has been known to crosslink between ECM molecules such as collagen I, III and IV, and fibronectin, resulting in aggregation of proteins with resistance for protein-degradaing enzymes. To investigate whether activated TGase2 might induce deposition of ECM molecule, amount of fibronectin was measured in detergent insoluble matrix. As shown in Fig. 4c, treatment of TGFβl (1 ng/ml) increased amount of FN bound to detergent insoluble matrix.
Fig. 4d represents deposition of FN by treatment of TGFβl (4 ng/ml).
The deposition of FN was inhibited by cystamine or NAC (Fig. 4c and Fig. 4d), suggesting that transamidation activation of TGase2 crosslinks ECM molecules.
Fig. 4e demonstrates a role of TGase2 by measuring amount of BP-bound proteins and its amount was decreased by cystamine or NAC. As shown in Fig. 4f, it was not observed that TGase2 per se was bound to ECM.
As results described above, it could be appreciated that TGFβl-induced activation of TGase2 causes fibrous deposition of ECM molecules using tansamidation response.
EXAMPLE 7: Cell Proliferation and Apoptosis Analysis
The increase of cell number was assessed by MTT analysis kit (Roche) according to the manufacturer's protocol and was expressed as folds of increase compared with the samples without TGFβl. Cell proliferation assay was performed according to the manufacturer's protocol using Brd-U incorporation kit (Roche). For apoptosis assay, the present inventors measured sub-Gl fraction by FACS analysis or caspase activity. Caspase activity was determined using chromogenic substrates, Ac- DEVD-pNA for caspase 2 and Ac-LEVD-pNA (A.G. Scientific, Inc) for caspase 9, respectively. Total cell extracts were prepared using freeze-thaw process in lysis buffer (100 mM HEPES, pH 7.5, 0.1% CHAPS, 0.1% Triton X-100, 100 mM EDTA), followed by centrifugation (12,000 x g, 10 min, 4°C). Cell extracts (30 μg) were added to assay buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1 % CHAPS, 10 mM DTT) containing chromogenic substrates (200 μM) and incubated for 4 hrs at 37°C. Caspase activity was quantitated by measuring absorbance at 490 nm and represented as 'relative activity' compared to that of the untreated cells (negative control).
Figs. 5a-5b represent cystamine or NAC suppress TGFβ-induced proliferation of lung fibroblast. Increased proliferation of fibroblast after injury is a principal factor of fibrosis. As shown in Fig. 5a, cell number of IMR90 was increased by treatment of TGFβl. The effect of TGFβl to cell proliferation could be clearly observed at 48 hrs post-treatment. The number of fibroblast was not affected by treatment of TGFβ2. In Fig. 5b, IMR90 cells were subjected to TGFβl in the presence or absence of cystamine or NAC. Both cystamine and NAC strongly inhibited proliferation of fibroblast induced by TGFβl.
As results described above, it could be demonstrated that TGase2 contributes to induce fibrosis via promoting proliferation of lung fibroblast.
Figs. 6a-6b represent cystamine or NAC have no effect on apoptosis of TGFβl-treated cell. Apoptosis of TGFβl-treated cell was investigated by measuring PARP fragment (Fig. 6a) or sub-Gl fraction (Fig. 6b) in the presence or absence of cystamine or NAC. PARP fragment or sub-Gl fraction had no significant differences in cells treated with TGFβ, cells treated with both TGFβ and cystamine and cells treated with both TGFβ and NAC.
Therefore, it could be appreciated that cystamine or NAC have no side-effects on cells.
Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:
1. A pharmaceutical composition for preventing or treating fibrosis comprising (a) a therapeutically effective amount of a transglutaminase 2 (TGase2) inhibitor or N- acetylcysteine (NAC); and (b) a pharmaceutically acceptable carrier.
2. The composition according to claim 1, wherein the TGase2 inhibitor is selected from the group consisting of cystamine, cysteamine, monodansyl cadaverine, putrescine, histamine, methyl amine, iodoacetamide, 8-phenyl propionylthiocholine, monoamine, diamine, γ-aminobenzoic acid, l-(5-aminopentyl)-3-phenylthiourea, N- benzyloxy carbonyl, 5-deazo-4-oxonorvaline, p-nitrophenylester, glycine methyl ester, CuSO4 and tolbutamide.
3. The composition according to claim 1, wherein the TGase2 inhibitor comprises cystamine or cysteamine.
4. A method for screening a substance to prevent or treat fibrosis comprising the steps of:
(a) contacting a test substance to a transglutaminase 2 (TGase2) protein; and
(b) measuring the activity of the TGase2 protein.
5. The method according to claim 4, wherein the TGase2 protein is present inside or outside a cell.
6. A method for preventing or treating fibrosis comprising administrating to a subject a pharmaceutical composition which comprises a transglutaminase 2
(TGase2) inhibitor or N-acetylcysteine (NAC) as active ingredients.
7. The method according to claim 6, wherein the TGase2 inhibitor is selected from the group consisting of cystamine, cysteamine, monodansylcadaverine, putrescine, histamine, methyl amine, iodoacetamide, 8-phenyl propionylthiocholine, monoamine, diamine, γ-aminobenzoic acid, l-(5-aminopentyl)-3-phenylthiourea, N-benzyloxy carbonyl, 5-deazo-4-oxonorvaline, p-nitrophenylester, glycine methyl ester, CuSO4 and tolbutamide.
8. The method according to claim 6, wherein the TGase2 inhibitor comprises cystamine or cysteamine.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019246298A1 (en) * 2018-06-20 2019-12-26 Axcella Health Inc. Compositions and methods for the reduction or treatment of fibrosis

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998004245A1 (en) * 1996-07-25 1998-02-05 The Victoria University Of Manchester Use of transglutaminase modulators to promote wound healing
KR20050102304A (en) * 2004-04-21 2005-10-26 주식회사 피플바이오 Interaction between transglutaminase and prion protein
US20070049641A1 (en) * 2005-08-24 2007-03-01 Rabindra Tirouvanziam Methods for treating and monitoring inflammation and redox imbalance cystic fibrosis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998004245A1 (en) * 1996-07-25 1998-02-05 The Victoria University Of Manchester Use of transglutaminase modulators to promote wound healing
KR20050102304A (en) * 2004-04-21 2005-10-26 주식회사 피플바이오 Interaction between transglutaminase and prion protein
US20070049641A1 (en) * 2005-08-24 2007-03-01 Rabindra Tirouvanziam Methods for treating and monitoring inflammation and redox imbalance cystic fibrosis

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019246298A1 (en) * 2018-06-20 2019-12-26 Axcella Health Inc. Compositions and methods for the reduction or treatment of fibrosis
CN112839708A (en) * 2018-06-20 2021-05-25 胺细拉健康公司 Compositions and methods for reducing or treating fibrosis

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