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WO2012153125A1 - Nouveaux composés et procédés destinés à être utilisés en médecine - Google Patents

Nouveaux composés et procédés destinés à être utilisés en médecine Download PDF

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WO2012153125A1
WO2012153125A1 PCT/GB2012/051007 GB2012051007W WO2012153125A1 WO 2012153125 A1 WO2012153125 A1 WO 2012153125A1 GB 2012051007 W GB2012051007 W GB 2012051007W WO 2012153125 A1 WO2012153125 A1 WO 2012153125A1
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Prior art keywords
tissue transglutaminase
inhibitor
angiogenesis
transglutaminase inhibitor
compound
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Martin Griffin
Mileidys PEREZ
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Aston University
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Aston University
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    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C07K5/06069—Ser-amino acid
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    • C07K5/06—Dipeptides
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    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof

Definitions

  • the present invention provides tissue transglutaminase inhibitor and methods for use in the inhibition of angiogenesis in a subject.
  • the present invention provides compounds and methods for use in the treatment and/or prevention of angiogenic diseases and disorders of the eye, such as diabetic retinopathy and age-related macular degeneration (AMD).
  • AMD age-related macular degeneration
  • Transglutaminases are an important class of protein crosslinking enzymes that catalyse protein aggregation reactions in blood coagulation (Greenberg, C.S., ef a/., 1991 , FASEB J. 5, 3071-3077), skin maturation (Thacher, S. M. & Rice, R. H., 1985, Cell 40, 685-695) and the clotting of seminal secretions (Dubbink, H.J., ef a/., 1999, Lab. Invest. 79, 141-150).
  • the most widespread member of the family is the cellular form of the enzyme, tissue transglutaminase (tTGase), which is expressed in varying amounts in many cell types.
  • tTGases are calcium-dependent enzymes that catalyse the formation of crosslinks proteins via e(v-glutamyl) isopeptide bonds and the incorporation of polyamines at certain glutamine residues (Greenberg, C.S., et a/., 1991, FASEB J. 5, 3071-3077).
  • tTGase is unique in the transglutaminase family of enzymes in that is able to bind and hydrolyze GTP and ATP (Achyuthan, K. E. & Greenberg, C. S., 1987, J. Biol. Chem. 262, 1901-1906), and to bind to fibronectin (Achyuthan, K. E., ef a/., 1995, J. Immunol. Methods 180, 67-79).
  • Tissue TGase (tTGase or TG2) is predominantly located in the cytosol, although tTGase has also been reported to exist in the nucleus (Lesort, M., et a/., 1998, J. Biol. Chem.
  • Protein modification mediated by tissue transglutaminases has been implicated in the pathology and aetiology of numerous diseases and processes (see review by Aeschlimann & Thomazy, 2000, Connective Tissue Research 41(1): 1-27).
  • tTGase-mediated protein modification has been shown in occur in fibrosis and scarring (Johnson et al., 1999, J. Am. Soc. Neph. 10:2146-2157), neurodegenerative diseases including Huntingdon's disease and Alzheimer's disease (Citron et al., 1999, J. Biol. Chem. 276:3295-3301), coeliac disease (Marzari ef a/., 2001, J. Immunol.
  • Tissue TGase has also been implicated in a number of diseases involving angiogenesis, such as the development of solid tumours and rheumatoid arthritis (Folkman, J., 1995, Nat. Med. 1, 27-31).
  • angiogenesis appears from the existing scientific literature to be complex.
  • Greenberg et al. have reported both an inhibition and an enhancement of angiogenesis in response to increased tTGase in separate studies (see Haroon ef al., 1996, FASEB J, Abstract No. 2403, page a1416 and Haroon ef al., 1999, FASEB J 13:1787-1795).
  • transglutaminase inhibitor compounds include competitive amine inhibitors, competitive glutamine inhibitors and irreversible inhibitors.
  • Competitive amine inhibitors include dansylcadaverines (Lorand et a/., 1966, Biochem. Biophys. Res. Commun. 25, 629; Lorand et a/., 1968, Biochemistry 7, 1214) and N- phenyl-N'-(ro-aminoalkyl)thioureas (Lee et at., 1985, J. Biol. Chem. 260, 14689).
  • Competitive glutamine inhibitors include aliphatic amides (Gross & Folk, 1973, J. Biol. Chem.
  • Irreversible inhibitors include iodoacetamide (Gross & Folk, 1973, J. Biol. Chem. 248, 6534; Folk & Cole, 1966, J. Biol. Chem. 241, 5518), phenol-containing halomethyl ketones (Folk & Gross, 1971, J. Biol. Chem. 246, 6683), alkyl isocyanates (Gross ef a/., 1975, J. Biol. Chem.
  • the present invention seeks to provide novel uses and methods for the inhibition of angiogenesis.
  • tissue transglutaminase inhibitor for use in inhibiting angiogenesis in a patient.
  • tissue transglutaminase inhibitor we include any compound, polypeptide or other agent that inhibits, in part or in whole, the transamidating activity of a tissue transglutaminase enzyme (preferably in vivo). Numerous examples of such inhibitors are well known in the art (for example, see review by Siegel & Khosla, 2007, Pharmacol. Ther. 115(2):232-245, the disclosures of which are incorporated herein by reference).
  • the tissue transglutaminase inhibitor is a reversible inhibitor, such as a competitive substrate inhibitor.
  • the inhibitor may be selected from the group consisting of:
  • tTGase cofactors and analogues thereof such as GTP, GDP, GTPyS and GMP- PCP;
  • the tissue transglutaminase inhibitor is an irreversible inhibitor.
  • the inhibitor may be selected from the group consisting of: peptide compounds (for example, see Formula I below);
  • dihydroisoxazole compounds such as KCA075 and KCC009 (for example, see Hausch et al., 2003, Chem. Biol.10:225-31);
  • cinnamoyl inhibitors for example, see US 20100204280 to Keillor et al.
  • the tissue transglutaminase inhibitor is an antibody which binds tTGase and inhibits (at least in part) its transamidation activity.
  • antibody we include substantially intact antibody molecules, as well as chimaeric antibodies, humanised antibodies, human antibodies (wherein at least one amino acid is mutated relative to the naturally occurring human antibodies), single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen binding fragments and derivatives of the same.
  • the antigen-binding fragment is selected from the group consisting of Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab- like fragments (e.g. Fab fragments, Fab' fragments and F(ab) 2 fragments), single variable domains (e.g. V H and V L domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).
  • a human or humanised antibody is utilised.
  • Suitable monoclonal antibodies to tTGase may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982).
  • the ability of such antibodies to inhibit the transamidation activity of tTGase may be tested as described in Example 2 below.
  • the tissue transglutaminase inhibitor is a dipeptide compound.
  • tissue transglutaminase inhibitor may be a compound of Formula I:
  • X represents an amino acid group; is an integer between 1 and 4;
  • 'Ri' represents benzyl, t-butyl or 9-fluorenylmethyl
  • R 3i R4, R5 and F3 ⁇ 4 each independently represent lower alkyl or -S + R 7 R 8 , wherein R 7 and R 8 each independently represent lower alkyl or a pharmaceutically and/or veterinarily acceptable derivative thereof.
  • 'X' denotes an alpha-ammo acid moiety, such that the compound has the following general structure (where R aa represents an amino acid side chain, such as H for glycine):
  • X is an L-amino acid moiety.
  • X is selected from the group consisting of phenylalanine, glutamine (including N-substituted derivatives thereof, such as N-substituted piperidinyl and propyl derivatives), isoleucine, alanine, glycine, tyrosine, proline, serine, lysine and glutamic acid.
  • preferred compounds of the invention include W-benzyloxycarbonyl-L-glutamyl ⁇ y- isopropylamide-6-dimethyl-sulfonium-5-oxo-Z.-norleucine bromide salt and /V- benzyloxycarbonyl-L-gIutamyl-y-piperidinamide-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt.
  • 'n' is 2.
  • 'R ⁇ is benzyl.
  • 'R 2 ' represents:
  • 'R 2 ' represents -S + R 7 R 8 , wherein R 7 and R 8 each independently represent lower alkyl.
  • lower alkyl is intended to include linear or branched, cyclic or acyclic, C ⁇ -Cs alkyl, which may be saturated or unsaturated.
  • Lower alkyl groups which R 3> R4, R 5 , R 6l R 7 and/or R 8 may represent include C C 4 alkyl, C 1 -C3 alkyl, C C 2 alkyl, C 2 -C 5 alkyl, C 3 -C 5 alkyl, C 4 -C 5 alkyl, C 2 -C 4 alkyl, C 2 -C 3 alkyl and C 3 -C 4 alkyl.
  • R 3 , R4, R 5 , Re, R 7 and/or R 8 may represent include C 2 , C 3 , C 4 and C 5 alkyl.
  • R 3, R4, R 5 , Re, R/ and/or R 8 are -CH 3 or -CHCH 2 . More preferably, R 3, R4, R 5 , Re, R 7 and/or R 8 are -CH 3 .
  • the compound is selected from the group consisting of:
  • salts which may be mentioned include: acid addition salts, for example, salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acid, with carboxylic acids or with organo-sulfonic acids; base addition salts; metal salts formed with bases, for example, the sodium and potassium salts.
  • the compounds of formula I may be counterbalanced by counter-anions.
  • exemplary counter-anions include, but are not limited to, halides (e.g. fluoride, chloride and bromide), sulfates (e.g. decylsulfate), nitrates, perchlorates, sulfonates (e.g. methane-sulfonate) and trifluoroacetate.
  • halides e.g. fluoride, chloride and bromide
  • sulfates e.g. decylsulfate
  • nitrates e.g. perchlorates
  • sulfonates e.g. methane-sulfonate
  • trifluoroacetate e.g., trifluoroacetate.
  • Other suitable counter-anions will be well known to persons skilled in the art.
  • the compound is a bromide salt.
  • Compounds of formula I may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism.
  • Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques.
  • the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric esters by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the invention.
  • the compounds of the first aspect of the invention comprise L forms of an alpha-amino acid.
  • the tissue transglutaminase inhibitor compounds of the first aspect of the invention are for use in inhibiting angiogenesis in a patient.
  • inhibiting angiogenesis we mean that administration of the compound is capable of reducing, at least in part, the formation of new blood vessels in vivo.
  • the compound may inhibit angiogenesis in vivo by at least 10% compared to the level of angiogenesis in the absence of the compound, for example by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. It will be appreciated that inhibition may require repeated (i.e. chronic) administration of the compound.
  • the compound is for use in the treatment and/or prevention of an eye disease or disorder associated with angiogenesis.
  • disease or disorder associated with angiogenesis we mean a disease or disorder in which abnormal or otherwise undesirable angiogenesis occurs, such that partial or complete inhibition of angiogenesis provides a beneficial effect to the patient (e.g. alleviates one or more symptoms and/or slows or prevents progression of the disease or disorder).
  • the eye disease or disorder associated with angiogenesis is a diseases or disorder of the posterior eye, such as a disease or disorder of the retina and/or choroid.
  • the eye disease or disorder associated with angiogenesis may be a retinopathy.
  • the eye disease or disorder associated with angiogenesis may be selected from the group consisting of diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, central retinal vein occlusion, sickle cell retinopathy, branch and central retinal vein occlusion and retinal trauma.
  • the eye disease or disorder associated with angiogenesis is a diseases or disorder of the anterior eye.
  • the eye disease or disorder associated with angiogenesis may be selected from the group consisting of chronic inflammation or infection (e.g. HSV infection of the ocular surface resulting in blood vessel formation), corneal scarring, wound repair, pterygium and neovascular glaucoma (i.e. growth of blood vessels on iris and into anterior chamber angle; robeosis iridis).
  • tissue transglutaminase inhibitor compounds of the first aspect of the invention will typically be provided in the form of a pharmaceutical formulation comprising the compound and a pharmaceutically acceptable carrier.
  • 'pharmaceutically acceptable carrier we include a substantially non-toxic, pyrogen-free excipient or adjuvant.
  • the formulation may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (i.e. a TGase inhibitor compound as described above) with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations in suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste. It will be appreciated by those skilled in the art that the compounds for oral administration should preferably be formulated so as to be protected in the gut and to permit bioadsorption.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the compound may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for injection into the eye.
  • the pharmaceutical composition may be for topical ophthalmic use, for example queous eye drops, oily eye drops, eye ointments, eye lotions, ocuserts, hydrogel contact lenses, collagen shields and ophthalmic rods.
  • compositions for injection are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilising agent and a local anaesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Compounds as described herein may also be administered to the affected eye(s) of a subject by transscleral delivery, for example by passive diffusion, controlled release device with or without a remote on-demand delivery system, osmotic pump, or via an implant in the eye, preferably a sustained release implant in the posterior of the eye.
  • the compound is administered by topical application to the eye.
  • the compounds are typically administered to the affected eye by applying one to four drops of a sterile solution or suspension, or a comparable amount of an ointment, gel or other solid or semisolid composition, to the surface of the affected eye one to four times per day.
  • the compounds may also be formulated as irrigating solutions that are applied to the affected eye during surgical procedures.
  • the compounds may be administered systemically.
  • the ophthalmic compositions may contain one or more TGase inhibitor compounds as described herein, one or more anti-inflammatory agents, or combinations thereof in pharmaceutically acceptable vehicles.
  • the ophthalmic compositions may contain one or more TGase inhibitor compounds as described herein in combination with one or a combination of other treatment agents, such as steroid drugs, such as triamcinolone, fluocinolone, anacortave acetate, dexamethasone and combinations thereof; and/or a non-steroidal anti-inflammatory drug, such as celecoxib, VIOXX, flurbiprofen, and aspirin, and combinations thereof.
  • a preferred composition contains both a MCP-1 inhibitory agent and a CCR-2 inhibitory agent, preferably an antibody or functional antibody fragment specific for MCP-1 and CCR-2, respectively, in combination with an anti-inflammatory agent or steroid drug.
  • Additional preferred compositions also contain one or more VEGF inhibitors, such as an anti-VEGF antibody.
  • Topical compositions will typically have a pH in the range of 4.5 to 8.0.
  • the ophthalmic compositions must also be formulated to have osmotic values that are compatible with the aqueous humor of the eye and ophthalmic tissues. Such osmotic values will generally be in the range of from about 200 to about 400 milliosmoles per kilogram of water (“mOsm/kg”), but will preferably be about 300 mOsm/kg.
  • Ophthalmic pharmaceutical products are typically packaged in multidose form. Preservatives are thus included to prevent microbial contamination during use. Suitable preservatives include: polyquaternium-1, benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, or other agents known to those skilled in the art.
  • polyquaternium-1 as the antimicrobial preservative is preferred. Typically such preservatives are employed at a level of from 0.001% to 1.0% by weight.
  • the solubility of the TGase inhibitor compounds as described herein may be enhanced by a surfactant or other appropriate co-solvent in the composition.
  • a surfactant or other appropriate co-solvent in the composition include polysorbate 20, 60, and 80, polyoxyethylene/polyoxypropylene surfactants (e.g., Pluronic F-68, F-84 and P- 03), cyclodextrin, or other agents known to those skilled in the art.
  • co-solvents are employed at a level of from 0.01% to 2% by weight.
  • viscosity enhancing agents to provide the topical compositions with viscosities greater than the viscosity of simple aqueous solutions may be desirable to increase ocular absorption of the active compounds by the target tissues or increase the retention time in the eye.
  • viscosity building agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose or other agents know to those skilled in the art. Such agents are typically employed at a level of from 0.01% to 2% by weight.
  • Local administration to the affected eye(s) may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g. in conjunction with a wound dressing after surgery or via drops or application of a gel or other topical solution, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibres.
  • the TGase inhibitor compounds as described herein can be delivered in a vesicle, in particular a liposome (See Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
  • the TGase inhibitor compounds as described herein can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
  • a controlled release system can be placed in proximity of the eye.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
  • a second aspect of the invention provides the use of a tissue transglutaminase inhibitor or a pharmaceutically and/or veterinarily acceptable derivative thereof, in the preparation of a medicament for inhibiting angiogenesis in a patient.
  • tissue transglutaminase inhibitor may be selected from any of those described above in relation to the first aspect of the invention.
  • tissue transglutaminase inhibitor may be compound of Formula I.
  • the medicament may be for the treatment or prevention of any disease or disorder associated with abnormal or otherwise undesirable angiogenesis.
  • the medicament may be for the treatment or prevention of an eye disease or disorder associated with angiogenesis (as described above in relation to the first aspect of the invention).
  • a third aspect of the invention provides a method of treating a subject in need of treatment with an angiogenesis inhibitor comprising administering to said subject a tissue transglutaminase inhibitor, or a pharmaceutically and/or veterinarily acceptable derivative thereof.
  • tissue transglutaminase inhibitor may be selected from any of those described above in relation to the first aspect of the invention.
  • the tissue transglutaminase inhibitor may be compound of Formula I.
  • a 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective' refers to that amount which provides a therapeutic effect for a given condition and administration regimen (via an inhibition of angiogenesis).
  • This is a predetermined quantity of the compound of the invention calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the subject.
  • a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a subject.
  • the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent.
  • a therapeutically effective amount of the active component is provided.
  • a therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
  • the compound according to the first aspect of the invention is administered in an amount sufficient to inhibit, at least in part, tTGase-mediated protein modification (i.e. cross-linking). More preferably, the compound or formulation is administered in an amount sufficient to inhibit tTGase-mediated protein cross-linking by at least 10%, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. Most preferably, the compound or formulation is administered in an amount sufficient to inhibit completely tTGase-mediated protein cross-linking.
  • TGase-mediated protein modification may be measured by methods known in the art.
  • detection of the isodipeptide s(y-glutamyl)lysine in body fluids can be used as an indirect measure of the frequency of crosslinking in diseases which involve this protein cross link.
  • a reduction of the isodipeptide in the body fluid provides an indirect measure of reduced protein crosslinking (see Nemes et a/., 2002, Minerva Biotechnology 14, 183).
  • tissue biopsy may be taken and analysed, for example by ion exchange or reversed phase HPLC after proteolytic digestion of the material (Griffin & Wilson, 1984, Mol. Cell Biochem. 58:37- 49), or by staining biopsy sections and analysing by immunohistochemistry (Skill et al. , 2001 , 81 :705-716).
  • the method may be for the treatment or prevention of any disease or disorder associated with abnormal or otherwise undesirable angiogenesis.
  • the method may be for the treatment or prevention of an eye disease or disorder associated with angiogenesis (as described above in relation to the first aspect of the invention).
  • treatment may be prophylactic and/or therapeutic.
  • the compounds of the invention may be used to slow and/or to prevent the onset of a disease/disorder in the subject being treated.
  • the compounds of the invention may be used to reduce or eradicate the symptoms of a disease/disorder in the subject being treated.
  • the compound of the invention may be administered by any route known or developed in the art.
  • the compound or formulation may be administered by parenteral injection (e.g. intraoccular, intravenous, intravitreal, subcutaneous or intramuscular), by topical application, by inhalation or nasal administration, or orally.
  • the compound is administered systemically, for example intravenously.
  • the compound or formulation may be administered topically, e.g. at or near a target site where angiogenesis is to be inhibited.
  • Treatment with a compound of the invention may consist of a single dose or a plurality of doses over a period of time.
  • the compound is administered repeatedly.
  • Compounds of the invention may also be administered by a surgically implanted device that releases the compound directly to the required site over a prolonged period of time, for example in the vicinity of a solid tumour.
  • a subject treated using the method according to the third aspect of the invention may be any mammal.
  • the subject is human.
  • the subject may be a dog, cat, horse, or other domestic or farm mammalian animal.
  • Figure 1 shows a synthesis route for the production of an exemplary compound according to the first aspect of the invention, namely A/-Benzyloxycarbonyl-L-phenylalanyl-6- dimethylsulfonium-5-oxo-L-norleucine bromide salt ('Compound 281').
  • step (i) the N- - CBZ-protected amino acid /V-hydroxysuccinimide ester is reacted with 6-diazo-5-oxo-L- norleucine (DON) to produce Z-phenylalaninyl bromomethyl ketone, which is then reacted with dimethylsulphide to produce /V-benzyloxycarbonyl-L-phenylalanyl-6- dimethylsulfonium-5-oxo-L-norleucine bromide salt.
  • DON 6-diazo-5-oxo-L- norleucine
  • Reagents and conditions for each step are as follows:
  • TAA Triethylamine
  • Figures 2 to 21 show the effect of increasing concentrations of exemplary compounds of the invention (and prior art compound 1 ,3-dimethyl-2-(2-oxopropylsulfanyl)-3H-1 ,3-diazol- 1-ium-chloride) on the inhibition of guinea pig liver transglutaminase (tTG), as measured by an enzyme-linked sorbent assay (ELSA) (see Example 2, below).
  • the concentration of the test compound test is given in ⁇ , along the x-axis.
  • the compounds tested are as follows:
  • FIG 22 shows SDS-PAGE data demonstrating inhibition of tTGase-mediated crosslinking of fibronectin following treatment with exemplary compounds of the invention (see Example 3).
  • 1TG' tissue transglutaminase, 'degr.
  • fragments' degradation fragments
  • 'Fn' fibronectin
  • 'Polymers' cross-linked fibronectin polymers
  • '281' ⁇ /- Benzyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-/.-norleucine bromide salt
  • '2857'Rob285' W-Benzyloxycarbonyl-L-glutaminyl-6-dimethyl-sulfonium-5-oxo-L- norleucine bromide salt.
  • Figure 23 shows (a) representative Masson's Trichrome stained sections at 100x magnification and (b) collagen III stained sections at 200x magnification from kidneys of rats treated for 84 days with inhibitor W-Benzyloxycarbonyl-L-phenylalanyl-6- dimethylsulfonium-5-oxo-/--nor-leucine bromide salt (designated 'SNx + 281') or 1 ,3- dimethyl-2-(2-oxopropylsulfanyl)-3H-1,3-diazol-1-ium-chloride (designated * SNx + 283').
  • SNx indicates animals in which a subtotal nephrectomy has been performed.
  • mice either had PBS (SNx) or TGase inhibitor compound 281 or 283 (SNx+281 and SNX+283, respectively) instilled into their kidney.
  • SNc' refers to sham operated animals and 'SNx' refers to animals which have had PBS instilled into their kidneys. Five animals per group were used (see Example 4).
  • Figure 24 shows Quantative Image Analysis of (a) Masson's Trichrome staining and (b) collagen III staining in the kidney sections from 90 day animals following treatment with inhibitor A/-Benzyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (designated 'SNx + 281') and 1,3-dimethyl-2-(2-oxopropylsulfanyl)-3H-1,3- diazol-1-ium-chloride (designated 'SNx + 283').
  • 'Snc' and 'SNx' are referred to as in legend to Figure 12 above. Five animals per group were used (see Example 4).
  • Figure 25 shows the inhibition of TGase activity in kidneys of rats treated with compounds having TGase inhibitor activity.
  • Figure 25 (a) is a histogram showing semi-quantative analysis of in situ TGase activity in cryostat sections taken from kidneys of SNx rats treated for 28 days with the inhibitors /S/-Benzyloxycarbonyl-/.-phenylalanyl-6-dimethyl- sulfon-ium-5-oxo-L-norleucine bromide salt (designated 'SNx + 281') and 1 ,3-dimethyl-2- (2-oxopropylsulfanyl)-3H-1,3-diazol-1-ium-chloride (desig-nated 'SNx + 283').
  • 'SNc' refers to control kidneys obtained from animals on which a sham operation was performed without subtotal nephrectomy.
  • 'SNx' refers to subtotal nephrectomy. Inhibitors were delivered to the kidney by mini-pumps (see Example 5).
  • Figure 25 (b) is a histogram showing TGase activity measured by 4 C- putrescine incorporation into N, N'-dimethyl casein at day 84 in kidney homogenates of SNx rats treated with the inhibitors /V-Benzyloxycarbonyl-/.-phenylalanyl-6-di-methyl- sulfonium-5-oxo-L-norleucine bromide salt (designated 'SNx + 281') and 1 ,3-dimethyl-2- (2-oxopropylsulfanyl)-3H-1 ,3-diazol-1-ium-chloride (designated 'SNx + 283'). Five animals per group were used (see Example 4).
  • Figure 26 shows the effect on renal function in rats of 84 days treatment with the inhibitors A/-Benzyloxycarbonyl-/--phenylalanyl-6-di-methylsulfonium-5-oxo-/.-norleucine bromide salt (designated 'SNx + 281') and 1 ,3-dimethyl-2-(2-oxopropylsulfanyl)-3H-1 ,3-diazol-1- ium-chloride (designated 'SNx + 283'), as determined using measurements of (a) proteinuria and (b) creatinine clearance.
  • 'SNc' refers to control kidneys obtained from animals on which a sham operation was performed without subtotal nephrectomy.
  • 'SNx' refers to subtotal nephrectomy. Five animals per group were used (see Example 4).
  • Figure 27 shows the effect of R283, a cell permeable inhibitor of TG2 and FXIII, and R294, a cell impermeable inhibitor of TG2, on the capacity of HUVEC cells to differentiate into tubular angiogenic structures.
  • a co-culture system of HUVEC and dermal foreskin fibroblast was used here as a model for angiogenesis (see Material and Methods). Mixed cells were defrosted, seeded and allowed to adhere for 24 hours. Afterwards, fresh special media containing (A) DMSO (0.1%) as vehicle, (B) R283 (500 ⁇ ), or (C) R294 (500 ⁇ ) was added to the cultures, and changed every two days for 12 days. Cells were fixed and immuno-labeled with CD31 antibody.
  • Figure 28 shows the dynamics of the behaviour of endothelial cells after plating on a basement membrane substratum (Matrigel) in presence or absence of R294 inhibitor.
  • A Vehicle.
  • B R294.
  • C Suramin. HUVECs were seeded at a concentration of 15,000 cells per well of 96-well plate on top of the gelled reduced growth factor BME and incubated for 2, 4 and 6 hours as indicated, at 37 °C in 5% C02. Cells were labelled with 2 ⁇ Calcein AM for 15 min at 37 °C in 5% C02 and were photographed using 484 nm excitation and 520 nm emission filter on a fluorescent microscope equipped with *10 objective.
  • Figure 29 shows the effect of R294 on active sprouting endothelial cells.
  • HUVEC/Dermal foreskin fibroblast co-cultures were allowed to growth for 12 days in the absence of R294 (50 ⁇ ) with vehicle DMSO (0,01%) (A, negative control), or in presence of the TG inhibitor R294 (50 ⁇ ) from day 6 to day 12 of the co-culture (B, active sprouting cells), or from day 1 to day 12 (C, positive control in presence of inhibitor suramin for day 1 to day 12.
  • Figure 30 shows the effect of R294 on VEGF stimulated angiogenesis. Co-cultures were grown for 12 days in presence of either vehicle (DMSO 0.01%), VEGF+ vehicle, R294, or VEGF+R294. Shown is the appearance of endothelial cell tubes with and without angiogenesis stimulators, alone or in combination with TG2 inhibitor at day 12.
  • vehicle DMSO 0.01%
  • VEGF+ vehicle R294, or VEGF+R294.
  • Shown is the appearance of endothelial cell tubes with and without angiogenesis stimulators, alone or in combination with TG2 inhibitor at day 12.
  • Figure 31 shows vascular growth after 10 days of treatment with "Compound 294" or PBS as control on CAM.
  • Panel A Representative pictures of eggs treated with 294 or PBS after 10 days of culture. Arrows in the panel of the left defined vascular ramifications. The outgrowth on the allantoic membrane is represented by black spots on the right panel.
  • Ether was distilled from lithium aluminium hydride and stored over sodium wire. Methanol and ethanol were distilled and stored over 5 A molecular sieves. /V,/V-dimethyIformamide was distilled from calcium hydride and stored over 5 A molecular sieves. Chloroform, dichloromethane and acetone were dried over granular calcium chloride. Solvents used for flash column chromatography were distilled before use.
  • Flash chromatography was carried out using Fluka silica gel 60, 220-240 mesh size. Thin layer chromatography was carried out using Whatman silica gel 60A F254 pre-coated glass plates.
  • A7-a-Benzyloxycarbonyl-L-phenylalanyl-6-bromo-5-oxo-/.-norleucine(see above) was prepared from DON and /V-a-CBZ-L-phenylalanine A/-hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0573) (see Figure 1) m.p. 132-133°C (ethyl acetate), (Found: C, 54.42; H, 5.14; N, 5.44.
  • A/-a-Benzyloxycarbonyl-L-glutaminyl-6-bromo-5-oxo-L-norleucine (see '2' above) was prepared from DON and A/-a-CBZ-L-glutamine /V-hydroxysuccinimide ester (Bachem cat. no. C-1625) m.p.
  • Ay-a-Benzyloxycarbonyl-L-isoleucinyl-6-bromo-5-oxo-L-norleucine was prepared from DON and A/-a-CBZ-/--isoleucine W-hydroxysuccinimide ester (Novabiochem cat. No. 04-12-0560) m.p.
  • A/-a-Benzyloxycarbonyl-L-glycinyl-6-bromo-5-oxo--.-norleucine was prepared from DON and A/-a-CBZ-L-glycine W-hydroxysuccinimide ester (Novabiochem cat. No. 04-12-0511) and used without further purification.
  • A/-a-Benzyloxycarbonyl-/--tyrosinyl-6-bromo-5-oxo- .-norleucine (see '6' above) was prepared from DON and /V-a-CBZ-L-tyrosine 4-nitrophenyl ester (Fluka cat. No. 97300) and used without further purification.
  • W-a-Benzyloxycarbonyl-L-prolinyl-6-bromo-5-oxo-/--norleucine was prepared from DON and W-a-CBZ-L-proline /V-hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0577). m.p.
  • A/-a-Benzyloxycarbonyl- --serinyl-(0-t-butyl)-6-bromo-5-oxo----norleucine was prepared from DON and A/-a-CBZ-L-serine (O-t-butyl) N- hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0585).
  • A/-a-Benzyloxycarbonyl-L-serinyl-6-bromo-5-oxo-L-norleucine (see '9' above) was prepared via removal of the i-butyl protecting group using trifluoroacetic acid and triethylsilane as reagents, Mehta et al. (1992) Tetrahedron Lett., 33, 5441. The crude product obtained was used without further purification.
  • W-a-Benzyloxycarbonyl-/.-glutaminyl-(0-t-butyl)-6-bromo-5-oxo-L-norleucine was prepared from DON and /V-a-CBZ-Z.-glutamic acid (O-t-butyl) N- hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0551).
  • A/-a-Benzyloxycarbonyl- .-lysinyl-(A/ -BOC)-6-bromo-5-oxo-L-norleucine was prepared from DON and /V-a-CBZ-Z_-lysine ( ⁇ /'-BOC) N- hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0526).
  • a -a-Benzyloxycarbonyl-L-lysinyl-6-bromo-5-oxo-L-norleucine trifluoroacetic acid salt was prepared via removal of the f-butyl protecting group using trifluoroacetic acid and triethylsilane as reagents, Mehta et al. (1992) Tetrahedron Lett., 33, 5441. The crude product obtained was used without further purification.
  • A/-a-Benzyloxycarbonyl-y-piperidinyl-glutaminyl-6-bromo-5-oxo-/.-norleucine (see '15' above) was prepared from DON and W-a-Benzyloxycarbonyl-y-piperidinyl-L-glutamic acid using /V-hydroxysuccinimide ester activation.
  • W-a-Benzyloxycarbonyl-y-propyl-L-glutamic acid was prepared using the methods of Molina, T.M., et al (1993) Tetrahedron 49, 3801-3808, Bias, J., et al (2000) Tetrahedron Lett. 41 , 4567-4571 and Antonjuk, D.J., et al (1984) J. Chem. Perkin Trans. 1 1989-2003.
  • W-a-Benzyloxycarbonyl-y-propyl-glutaminyl-6-bromo-5-oxo-L-norleucine was prepared from DON and A/-a-Benzyloxycarbonyl-y-propyl-Z--glutamic acid using A/-hydroxysuccinimide ester activation.
  • the bromomethyl ketone was dissolved in the minimum amount of dry methanol to achieve solution. Methyl sulfide (2.5-7.5 eqv.) was added and the solution left in a tightly stoppered flask for 24-48 h. until the reaction was judged complete by TLC. Purification was achieved by dissolving the residue in deionised water and extracting the organic soluble impurities with ethyl acetate. Freeze drying the aqueous portion afforded the product salts as colourless solids in typically 80-90% yields.
  • W-a-Benzyloxycarbonyl-L-phenylalanyl-6-feira-methylmercaptoimidazole-5-oxo-/.- norleucine bromide salt was prepared from V-a-Benzyloxycarbonyl- L-phenylalanyl-6-bromo-5-oxo-Z_-norleucine and 1 ,3,4,5-tetramethylimidazoline-2- thione, which was prepared by the method of Kuhn and Kratz (1993) Synthesis, 561, using the method of Freund et al. (1994) Biochemistry, 33, 10109. m.p.
  • Compound '5' is made by reaction of commercially available /S/-a-CBZ-/.-phenylalanine N- hydroxy-succinimide ester (Novabiochem Cat. No. 04-12-0573) and .-aspartic acid ⁇ - ⁇ - butyl ester (Novabiochem Cat. No. 04-12-5000) in water/THF (1:1) in the presence of 1.5 equivalents of triethylamine.
  • the sulfonium salt /V-benzyloxycarbonyl-L-phenylalanyl-L-2-amino-5-dimethyl- sulfonium-4-oxo-norvaline bromide, was prepared as described above from bromomethyl ketone (see '8' above) (0.1 g, 0.2 mmol) and methyl sulfide (0.11 ml, 1.5 mmol). Freeze-drying afforded the product as a colourless hygroscopic solid (20 mg,
  • the acid was prepared from 6-diazo-A/-(9-fluorenylmethyloxycarbonyl)-5-oxo-/.-norleucine ethyl ester (2.53 g, 6 mmol) by the method of Coutts et al. Yield after flash column chromatography (ethyl acetate 100%) 1.64 g, 66%.
  • the diazoketone (0.5 g, 1.15 mmol) was deprotected with piperidine as previously described to give the amino acid as a pale yellow solid (57.2 mg, 54%).
  • m.p. 122-124°C (Lit. m.p. 125-126°C) (KBr)/cm 1 3436, 2108, 1630 (Weygand et a/., Chem. Ber. 91, 1037-40).
  • the dipeptide was prepared by the method previously described to give a colourless solid (93 mg, 66%). m.p. 108-110 °C (ethyl acetate), v max (KBr)/cm 1 3296, 1715, 1700, 1661; ⁇ ⁇ (d 3 methanol) 1.6, 1.8 and 2.6 (6 H, m), 2.9-3.1 (2 H, m), 4.1 (2 H, s), 4.4 (2 H, m) 5.0 (2
  • the sulfonium salt was prepared as previously described, to give a colourless hygroscopic solid (48 mg, 57%).
  • exemplary compounds of the invention in the inhibition of transglutaminase was verified by studying the dose-dependency of their effects on the activity of purified guinea pig liver transglutaminase (gplTGase), using an enzyme-linked sorbent assay (ELSA) based on biotinylated cadaverine (BTC) incorporation into ⁇ /, ⁇ /'-dimethyl casein (DMC).
  • ELSA enzyme-linked sorbent assay
  • BTC biotinylated cadaverine
  • TGase guinea pig liver TG
  • ELSA enzyme-linked sorbent assay
  • BTC biotin cadaverine
  • DMC IM'- dimethylcasein
  • Microtitre plates (96-well) were coated with 100 ⁇ of 10 mg/ml DMC in 10 mM Tris pH 7.4 overnight at 4°C. The following day, plates were washed twice with TBS-Tween pH 7.4, once with TBS pH7.4, and a reaction mix was prepared that contained 5 mM CaCI 2 , 5 mM DTT and 0.132 mM BTC in 50 mM Tris pH 7.4.
  • TGase inhibitors were initially prepared as 100 mM stock solutions in H 2 0 and diluted to the appropriate final concentration in the same reaction solution.
  • Negative control samples for TGase activity consisted of mixes that did not contain BTC, and where 10 mM EDTA was substituted for 5 mM CaCI 2 .
  • the data shown indicate a representative experiment using eight replicate samples.
  • the mean absorbance 450nm ⁇ SD is shown.
  • Compound 281 A/-Benzyloxycarbonyl-L-phenylalanyl-6-dimethyl-sulfonium-5-oxo-
  • Compound 283 1 ,3-dimethyl-2-(2-oxopropylsulfanyl)-3H- ,3-diazol-1 -ium-chloride
  • Compound 285 A/-E3enzyloxycarbonyl-L-glutaminyl-6-dimethylsulfonium-5-oxo-L- norleucine bromide salt
  • the cannula was then attached to the regulator of a 2 ml osmotic minipump (Azlet osmotic minipump (2ml4), Charles Rivers, UK) that was loaded (primed for 15 hrs at RT) with either PBS (SNx) or TGase inhibitor 281 or 283 (SNx+281 and SNx+283, respectively) at a concentration of 50 mM (delivery 1.5 ⁇ per hour).
  • the pump was then positioned subcutaneously on the right upper flank of the animal and the skin sutured.
  • the animal was then switched onto oxygen and allowed to partially regain consciousness before being returned to the cage.
  • the pump was changed every 28 days under halothane anaesthesia. After 83 days, the animal was placed in a metabolic cage to collect a 24-hour urine sample. The animal was then anaesthetised, the remnant kidney recovered and a terminal blood sample collected.
  • Tissue samples were sectioned and then underwent Masson's Trichrome staining (Johnson et a/., 1997, 99:2950-2960) or collagen III staining.
  • paraffin embedded sections (4 m) were first dewaxed and hydrated by standard protocol (xylene 10 min, 100% ethanol 5 min, 90% ethanol 5 min,75% ethanol 5 min, 50% ethanol 5 min, water 10 min) washed in PBS for 10 min and any endogenous peroxides quenched by treatment with 3% H202 in methanol for 10 min. After washing in PBS for 10 min sections were treated with the epitope revealing agent TUF (ID Labs Inc. Cat no BP1122) on a water bath at 92 C for 10 min then allowed to cool to room temperature.
  • TUF epitope revealing agent
  • Sections were washed with PBS for 10 min and then trypsin (Zymed Labs Cat No 00-3008) digested (trypsin diluted 1 :3) for 10 min at 37 C followed by two washes in PBS for 5 min each. Sections were then blocked in goat serum (Vector Labs Cat No S1000) incubated at 37 C for 30 min.
  • the primary collagen III antibody Goat anti-human type III collagen, Soutern Biotech Assocs diluted 1 in 10 in 0.1% bovine serum albumin [BSA] in PBS
  • BSA bovine serum albumin
  • the secondary antibody (rabbit anti goat which is biotinylated from DAKO Cat No E0466) diluted 1 in 400 in 0.1%BSA/PBS is then added and incubated for 30 min at 37 C.
  • the sections are then washed twice in 0.1% Nonidet in PBS and the sections then incubated with the Avidin Biotin Enzyme complex (ABC) kit (Vector Labs Cat No PK-6102) according to the manufacturers instructions for 30min at T 37° C.
  • the samples are then washed twice in PBS and the reagent substrate, 3-amino-9- ethyl carbozole (AEC [Vector Labs Cat No SK4200]) added to allow colour development (approx 5-30min).
  • Figure 23 shows (a) representative Masson's Trichrome stained sections and (b) collagen III stained sections from kidneys of animals in which inhibitor compound 281 (designated 'SNx + 281') and compound 283 (designated 'SNx + 283') were instilled (see Johnson et a/., 1999, J. Am. Soc. Nephrol. 10:2146-2157 for method used to induce subtotal nephrectomy).
  • Figure 24 shows quantative image analysis of (a) Masson's Trichrome stain and (b) collagen III stain in kidney sections from 90 day animals following treatment with inhibitor compounds 281 (designated 'SNx + 281') and 283 (designated 'SNx + 283'). Snc and SNx are referred to as above.
  • analysis was performed by systematically acquiring adjacent overlapping cortical fields at 100 x magnification such that 5 fields encompassed more than 80% of the cortex. Each field was then subject to 3 phase analysis using image analysis and the area of blue (collagen), red (cytoplasm) and white (lumen) determined ensuring greater than 95% coverage.
  • the scarring index was determined by expressing the blue phase as a fraction of the cytoplasmic. Five animals per group were used and data expressed a mean values +/- S.E.M.
  • the composite diagram showing staining in Figure 12(a) shows 1 field from each animal.
  • For Collagen III staining the relative amounts of collagen III present (stained brown) were determined by systematically acquiring data from 10 overlapping cortical fields at 200x magnification and expressed as Mean values ⁇ SEM.
  • Rat kidneys treated in vivo with TGase inhibitors were snap-frozen in liquid nitrogen and 14 im sections were cut using a cryostat and allowed to air-dry. Sections were rehydrated for 10 minutes at room temperature in a solution of 5% (w/v) rabbit serum, 10 mM EDTA, 0.01% (v/v) Triton X-100 in 50 mM Tris pH7.4, containing EXAP (diluted 1 in 200) to block endogenous biotin.
  • SNc refers to control kidneys obtained from animals undergoing a sham operation with subtotal nephrectomy.
  • SNx refers to subtotal nephrectomy.
  • Inhibitors were delivered to the kidney by mini pumps as outlined above. Data are mean values +/- SEM taken from 5 separate kidneys.
  • Table 2 shows levels of proteinurea, creatinine clearance, serum creatinine, urine creatinine and urine urea in 90 day SNx rats in which inhibitor compounds 283 and 281 were instilled into the kidneys.
  • Proteinuria, creatinine clearance, serum clearance, urine creatinine and urine urea were carried out by standard clinical chemistry techniques (Johnson et a/., 1997, J. Clin. Invest. 99:2950-2960). Creatinine and urea were measured by the standard auto analysesr technique and proteinura by the Biuret method (Johnson ei a/., supra). Data represent mean values ⁇ SE, taken from 5 animals per group. Proteinuria and creatinine clearance data are shown in histogram form in Figure 26 (a) and (b), respectively.
  • Angiogenesis is the formation of new capillaries from already existing vessels and is a normal and vital process in growth and development.
  • angiogenesis the capillary plexus is remodelled by sprouting, microvascular growth and fusion into a mature and functional vascular bed. This process is critically dependent on the local extracellular architecture.
  • the extracellular matrix not only serves as structural support for existing and developing vasculature but it is also instrumental in providing information guidance for new capillaries[1].
  • Angiogenesis occurs under numerous physiological conditions, such as during wound healing, reproduction-associated neovascularisation, development of collateral circulation following tissue grafting and ischemic episode.
  • angiogenesis Under pathological conditions, angiogenesis underlies a number of pathological processes that include cancer growth, atherosclerosis, diseases of eye such as diabetic retinopathy, vision loss associated with age-related macular degeneration (AMD), and chronic inflammatory disorders such as acute conjuntival inflammation, rheumatoid arthritis, psoriasis, and periodontitis, among others[2, 3]. Multiple consecutive steps are necessary for successful angiogenesis, all of which require the interaction between cells and the ECM. The first step in advancing toward avascular tissue regions involves the opening of existing capillaries and partial degradation of surrounding ECM in allowing cell infiltration.
  • endothelial cells adopt a proteolytic phenotype and begin to break down the basement membrane by both soluble and cell-bound matrix metalloproteases (MMPs) [4].
  • Endothelial cells resultantly lose their contact with BM laminin and become exposed to interstitial collagen, activating signalling cascades responsible for cytoskeleton reorganization and outgrowth of an endothelial tip cell followed by stalk cell proliferation [5]. Furthermore, these cells become motile, migrating and proliferating in response to VEGF, FGFs, and aligning to form new chords leading to tube formation with an encased lumen sealed by tight cell-cell junctions [6].
  • MMPs matrix metalloproteases
  • a basement membrane is then produced by endothelial cells in cooperation with surrounding cells to provide structural support and stabilize tubes into capillaries and mature vessels.
  • the unsupported endothelial tubes will be pruned at this stage. Once mature vessels are formed, blood flow to the new vascularized area raises local oxygen levels resulting in a decrease in VEGF levels and an end to the angiogenic cycle [7].
  • the basement membrane is a specialized ECM structure composed of sheet-like matrixes that are closely attached to cells. It functions as barriers and is necessary for cell polarization, shaping tissue structures and for guiding migrating cells. Vascular basement membrane not only provides blood vessel endothelial cell support but it actually modifies endothelial cell behaviour supporting adhesion of cells and transducing cellular signalling via adhesion receptors.
  • the ECM is a storage place of angiogenesis promoters and other biologically active molecules and is a rich source of angiogenesis inhibitors.
  • the basement membrane is mainly composed of type IV collagen in a network with other BM proteins such as laminins, nidogens, fibulins, SPARC (secreted protein acidic and rich in cysteine), fibronectin, type XV and XVIII collagens, and heparan sulfate proteoglycans [8].
  • BM proteins such as laminins, nidogens, fibulins, SPARC (secreted protein acidic and rich in cysteine), fibronectin, type XV and XVIII collagens, and heparan sulfate proteoglycans [8].
  • Tissue transglutaminase enzyme TG2 is a multifunctional enzyme found both in the intra- and extracellular compartments.
  • TG2 catalyses the crosslinking of proteins via the formation of highly stable epsilon(gamma-glutamyl) lysine bond, is attracting attention from researchers in the past few years mainly due to its reported negative effects on angiogenesis when in excess and by virtue of its ability to increase the deposition of matrix proteins [9].
  • the enzyme has been reported to contribute to the ECM accumulation by accelerating collagen deposition, and by stabilising the ECM against proteolytic decay [10].
  • TG2 transglutaminase 2
  • TGF transforming growth factor beta 1
  • Inhibitors R283 positive control
  • R294 i.e. compound '294' above
  • the lyophilized compounds were dissolved in DMSO (20%) at a stock concentration of 100 mM.
  • the final concentration of inhibitors used was 500, 100 or 50 ⁇ , as described below.
  • the working solutions were prepared from stock solutions and using cell growth media as diluent. In all cases the final concentration of DMSO solvent does not exceed 0.1% (v/v).
  • Suramin and VEGF were obtained from TCS Cellworks (V2a AngioKit, TCSCellworks, Buckingham, UK.
  • Monoclonal anti-human CD31 and anti-mouse-Alcaline phosphatase antibodies and BCIP/NBT substrate were also obtained from TCS Cellworks.
  • HUVEC Cell Culture Human Umbilical Vein Endothelial Cells (HUVEC, Promocell, Heidelberg, Germany) were cultured in EGM-2 media complemented with 2% fetal bovine serum (FBS), hydrocortisone, recombinant human fibroblast growth factor-B (bFGF), endothelial growth factor vascular human recombinant (VEGF), epidermal growth factor human recombinant (hEGF), ascorbic acid, recombinant long R insulin-like growth factor-1 (R3-IGF-1), heparin, and gentamicin sulphate amphotericin-B (GA-100) (EGM-2 bulletkit, Lonza Wokingham, England, UK).
  • FBS fetal bovine serum
  • bFGF recombinant human fibroblast growth factor-B
  • VEGF endothelial growth factor vascular human recombinant
  • hEGF epidermal growth factor human recombinant
  • VEC Human endothelial cells
  • TCS Cellworks V2a AngioKit, TCS Cellworks, Buckingham, UK
  • V2a AngioKit a specially designed medium optimized to support endothelial cells differentiation onto a fibroblast cells layer.
  • the co-culture was performed following the V2a AngioKit protocol. On day first, cells were defrosted and seeded in a 24-well plate in the V2a Seeding Medium. On day two, medium is replaced by the V2a Growth Medium with the corresponding treatments. Fresh Growth Medium containing the test compounds was changed every two days for a period of 12 days.
  • Co-cultures were treated with the well known site-directed TG inhibitor R283 (500 ⁇ ) , or the in house site-directed synthesized TG inhibitor R294 (Compound 294 ,500 ⁇ ), every two days [13].
  • Control experiments were performed using the inhibitors' vehicle DMSO (0.1%), or the VEGF angiogenesis promoter (2 ng mL) as positive control, and the Suramin compound (50 ⁇ ), which blocks the binding of various growth factors and was used as negative control.
  • Tubules were finally stained using BCIP/NBT substrate for 15-20 minutes at 37°C. After that period of time tubules develop a dark purple colour. Wells were washed with distilled water three times, and leave to air dry before plates were photographed. Tubule formation were scored with the aid of the TCS Cellworks AngiosSys Image Analysis Software
  • HUVEC and dermal foreskin fibroblast from V2a AngioKit were used as angiogenesis model, following the Angiokit protocol, as described above.
  • HUVEC cells were allowed to undergo active sprouting in the presence of vehicle (DMSO 0,01%) until day 6 of the co-culture.
  • DMSO was replaced in the growth media by the R294 inhibitor (50 ⁇ ) up to day 12.
  • Controls consisted on vehicle (DMSO, 0,01%), and R294 (50 ⁇ ) added starting from day 1, at every change of media and for all the period that the co-culture lasted (12 days).
  • cells were fixed at Day 6 or Day 12, stained for CD31 expression and quantified for tubule formation with TCS Cellworks AngiosSys Image Analysis Software as described above.
  • V2a-AngioKit was performed using V2a growth media alone, or further supplemented with VEGF (2 ng/mL).
  • VEGF 2 ng/mL
  • the effect of R294 on VEGF-stimulated co-culture was evaluated by adding R294 (50 ⁇ ) or R294's vehicle (DMSO 0.01%) concomitantly with VEGF (2ng/mL) at every change of media, starting from day 1 up to day 12. Cells were fixed at Day 12, stained for CD31 and quantified as described above.
  • Matrigel angiogenesis assay BD MatrigelTM basement membrane matrix (BD Matrigel matrix, no phenol red, reduced growth factor, BD, ) was used to support attachment and differentiation of HUVEC cells using the angiogenic assay protocol described by Arnaoutova and Kleinman [14]. Matrigel matrix was first defrosted at 4°C and 80 ⁇ _ loaded per well in 96-well plates. Plates were transferred to a cell culture incubator and incubated at 37°C for 30 min to allow the basement membrane to gel.
  • HUVEC cells Exponentially growing HUVEC cells, nearly confluent (80%), were trypsinized and 1.5x10 5 .cells were plated per well, on top of the gelled basement membrane in 1 mL of the corresponding medium (see details for each treatment below). Plates were incubated at 37°C, 5% C0 2 in the cell culture incubator for a period of 2, 4 and 6 hours in time-course experiments, and 6 hours in the other experiments. At the end of each period, cells were incubated with 2 ⁇ Calcein AM at 37°C and in 5% C0 2 for 15 min, observed and photographed using a fluorescent inverted microscope with 520 nm emission filter.
  • cell medium consisting of endothelial growth basal medium-2 (EBM-2) fully complemented with 2% FBS, hydrocortisone, bFGF, VEGF, hEGF, ascorbic acid, R3-IGF, heparin, and GA-100 as described above, was complemented with the compounds to test: TG2 inhibitor R294 (500 ⁇ ), Suramin (50 ⁇ ) as negative control, and vehicle DMSO (0.1%), respectively added before seeding cells into the wells.
  • EBM-2 endothelial growth basal medium-2
  • R283 and R294 The effect of R283 and R294 on angiogenesis was evaluated in a cellular system where human endothelial cells are co-cultured with human foreskin fibroblasts in a specially designed medium.
  • the endothelial cells initially proliferate and form small islands within the culture matrix. They subsequently enter a migratory phase during which they move through the matrix to form threadlike tubule structures. These gradually join up to form a network of anastomosing tubules which closely resembles the capillary bed found in physiological conditions.
  • the site-directed irreversible TG inhibitors (see below) immidazolium-derived inhibitor R283 and the peptidic inhibitor R294 (compound 294), inhibit efficiently the cross linking activity of the tissue transglutaminase in situ.
  • R283 differs from R294 in that is cell permeable whereas R294 is mainly impermeable to cells and its effect is, thus in the main, limited to the inhibition of extracellular enzyme [13, 15].
  • R294 also has a greater specificity for TG2(IC 50 for TG2 approx 5uM for Factor XIII greater than 200uM ) than for Factor XIII (plasma transglutaminase), whereas R283 has equal potency (IC 50 approx 4uM)for both enzymes.
  • VEGF vascular endothelial growth factor
  • suramin an inhibitor of several growth factor receptors
  • the TG2 inhibitor R294 affects established active sprouting endothelial cells in co-cultured angiogenesis assay affecting angiogenesis Active sprouting endothelial cells in the co-culture system were exposed to R294 (50 ⁇ ) at Day 6 of their differentiation process supported by the fibroblast-producing ECM layer. The addition of the inhibitor was then regularly maintained at each change of media up to Day 12. Co-cultures without the addition of the inhibitor were considered as negative controls. Positive controls consisted on co-cultures treated with the inhibitor from day 1 to day 12. After treatment, endothelial cells were stained for the CD31 marker and the tubule formation analysed.
  • VEGF-A vascular endothelial growth factors
  • VEGF-A vascular endothelial growth factors
  • the inhibitor R294 was added to the growth media of the co-culture system, alone or in combination with VEGF (2 ng/mL). Controls consisted of co-cultures in the absence of the inhibitor, with and without VEGF (2 ng/mL). As expected, the VEGF effect on cell culture resulted in an enhanced formation of tubules and anastomosis. Interestingly, we found that endothelial cell differentiation stimulated by VEGF is partially affected by the addition of R294 (Figure 30).
  • the addition of the inhibitor does not reverse completely the pro- angiogenic effect of VEGF as compared to control with no VEGF, but it does result in a lower amount of tubules junctions and tubules area as compare to the treatment with VEGF alone (Table 4).
  • Table 3 shows the effect of tissue transglutaminase inhibitor R294 on angiogenesis in HUVEC and Dermal Foreskin fibroblast co-culture assay (see also Figure 29).
  • Co-cultures treated with the solvent of the inhibitor (D SO 0.01%) were considered as control group.
  • Treatments consisted of addition of the inhibitor R294 (50 ⁇ ) from Day 1 to Day 12, or addition of the inhibitor only from Day 6, when active sprouting was occurring, until Day 12.
  • Suramin compound was used as negative control as it inhibits several growth factor receptors.
  • tissue transglutaminase inhibitor R294 on angiogenesis in HUVEC and Dermal Foreskin fibroblast co-culture assay
  • Table 4 shows the influence of tissue transglutaminase inhibitor R294 on VEGF- stimulated angiogenesis in HUVEC and Dermal Foreskin fibroblast co-culture assay.
  • Co- cultures treated with the solvent of the inhibitor were considered as control group.
  • Treatments consisted of addition of the angiogenesis promoter VEGF (2ng/ml_), or inhibitor R294 (50 ⁇ ) or combination of both, VEGF and R294, from Day 1 to Day 12 of the co-culture.
  • VEGF vascular endothelial growth factor
  • Fertilized 1 day old eggs were place horizontally and incubated at 37°C. After 4 days incubation the eggs are removed egg from incubator a maintaining horizontal position.
  • the embryo is detected via shining a directable light source through the end of the egg at an angle slightly below the central line to locate the embryo. The area should glow red and there should be detectable blood vessels, if not then the embryo may be immature and unsuitable for use. If appropriate, re-incubate for a few more hours/days. Using pencil, a faint circle is drawn around the embryo.
  • the egg is then placed into a sterile hood in an egg box-maintaining its horizontal position at all times and avoiding excess agitation.
  • a pencil line is used to mark the central line and needle-incision point on the egg in pencil.
  • Holding the egg gently but firmly in place within the egg box wipe the area with ethanol and using a blunt (19G) needle and 10ml syringe the shell is pierced just below the central line, slightly off centre using a firm twisting needle motion and angling the needle downwards to avoid the embryo.
  • the needle is inserted it is kept angled downwards at approx "9 o clock" fluid is withdrawn approx 4-6mls of albumin (depending on egg size).
  • the puncture hole is sealed carefully with sterilised masking tape. Using a small glass saw, a small window is cut in the top of the egg, approx 1 by 1.5cm. Hold egg firmly but avoid unnecessary agitation to the egg whilst sawing. The shell “window” is removed carefully using tweezers. There should be obvious blood vessels and a tiny pulsating embryo visible within the egg.
  • the inhibitor (100uM) or PBS in DMEM medium containing 10% (v/v) FCS was then added to the open chamber The hole was then sealed up with clear tape and the egg is then incubated at 37°C for 6 days after which time the chamber is examined and photographed under a stereo microscope Results
  • Figure 31 shows vascular growth after 10 days of treatment with "Compound 294" or PBS as control on CAM.
  • Tablets are prepared from the foregoing ingredients by wet granulation followed by compression.
  • formulations A and B are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.
  • formulations D and E are prepared by direct compression of the admixed ingredients.
  • the lactose used in formulation E is of the direction compression type.
  • the formulation is prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression. mq tablet
  • Lactose BP Lactose BP
  • Drug release takes place over a period of about 6-8 hours and was complete after 12 hours.
  • a capsule formulation is prepared by admixing the ingredients of Formulation D in Example C above and filling into a two-part hard gelatin capsule.
  • Formulation B (infra) is prepared in a similar manner.
  • Capsules are prepared by melting the Macrogel 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule. Formulation D mg/capsule
  • Capsules are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.
  • Formulation E Controlled Release Capsule
  • controlled release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with release-controlling membrane (d) and filled into a two- piece, hard gelatin capsule. mq capsule
  • Example F Intramuscular injection
  • the active ingredient is dissolved in the glycofurol.
  • the benzyl alcohol is then added and dissolved, and water added to 3 ml.
  • the mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1 ).
  • the sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added.
  • the active ingredient is added and dispersed.
  • the glycerol is dispersed the thickener (dispersible cellulose). The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension.
  • Example H Suppository mg/suppositorv
  • Witepsol H15 is melted in a steam-jacketed pan at 45DC maximum.
  • the active ingredient is sifted through a 200 pm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45°C, the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix.
  • the entire suspension is passed through a 250 pm stainless steel screen and, with continuous stirring, is allowed to cool to 40°C. At a temperature of 38°C to 40°C 2.02 g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.

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Abstract

La présente invention porte sur des inhibiteurs de la transglutaminase tissulaire, destinés à être utilisés dans l'inhibition de l'angiogenèse chez un sujet. En particulier, l'invention porte sur des composés de la Formule I destinés à être utilisés dans le traitement et/ou la prévention de maladies et de troubles angiogéniques de l'œil, tels que la rétinopathie diabétique et la dégénérescence maculaire liée à l'âge (AMD). L'invention porte en outre sur des procédés pour inhiber l'angiogenèse chez un sujet.
PCT/GB2012/051007 2011-05-06 2012-05-08 Nouveaux composés et procédés destinés à être utilisés en médecine Ceased WO2012153125A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
KR20170128058A (ko) * 2016-05-12 2017-11-22 강원대학교산학협력단 당뇨성 합병증 예방 및 치료용 약제학적 조성물 및 당뇨성 합병증 예방 또는 치료제 스크리닝 방법
US10155948B2 (en) 2016-05-12 2018-12-18 Kangwon National University University-Industry Cooperation Foundation and Pharmaceutical composition for preventing or treating diabetic complications and screening method for preventive or therapeutic agent for diabetic complications

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004113363A2 (fr) * 2003-06-19 2004-12-29 The Nottingham Trent University Composes et procedes d'utilisation

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0019302D0 (en) * 2000-08-08 2000-09-27 Univ Nottingham Trent Biological materials and the use thereof for the treatment of disease
AU2003275259A1 (en) * 2002-09-26 2004-04-19 Northwestern University Anti-angiogenic fragments of pigment epithelium-derived factor (pedf)
GB0304993D0 (en) * 2003-03-05 2003-04-09 Univ Nottingham Trent Novel screening method
EP2194124A1 (fr) * 2005-09-19 2010-06-09 Kai Pharmaceuticals, Inc. Peptide modulateurs de la protéine kinase c de l'angiogenèse
US8389476B2 (en) * 2007-03-29 2013-03-05 The Johns Hopkins University School Of Medicine Parstatin peptides and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004113363A2 (fr) * 2003-06-19 2004-12-29 The Nottingham Trent University Composes et procedes d'utilisation

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
CHHABRA A ET AL: "Tissue transglutaminase promotes or suppresses tumors depending on cell context", ANTICANCER RESEARCH 2009 INTERNATIONAL INSTITUTE OF ANTICANCER RESEARCH GRC, vol. 29, no. 6, June 2009 (2009-06-01), pages 1909 - 1919, XP002681105, ISSN: 0250-7005 *
EVELYN PNG ET AL: "Hyperosmolarity-mediated mitochondrial dysfunction requires Transglutaminase-2 in human Corneal epithelial cells", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 226, no. 3, 1 March 2011 (2011-03-01), pages 693 - 699, XP055033936, ISSN: 0021-9541, DOI: 10.1002/jcp.22389 *
GRIFFIN M ET AL: "Synthesis of potent water-soluble tissue transglutaminase inhibitors", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, ELSEVIER SCIENCE, GB, vol. 18, no. 20, 15 October 2008 (2008-10-15), pages 5559 - 5562, XP025562106, ISSN: 0960-894X, [retrieved on 20080906], DOI: 10.1016/J.BMCL.2008.09.006 *
HAROON ZISHAN A ET AL: "SU5416 delays wound healing through inhibition of TGF-beta 1 activation.", CANCER BIOLOGY & THERAPY 2002 MAR-APR LNKD- PUBMED:12170771, vol. 1, no. 2, March 2002 (2002-03-01), pages 121 - 126, XP002681075, ISSN: 1538-4047 *
HUANG LINGHONG ET AL: "Transglutaminase inhibition ameliorates experimental diabetic nephropathy", KIDNEY INTERNATIONAL, vol. 76, no. 4, August 2009 (2009-08-01), pages 383 - 394, XP002681106, ISSN: 0085-2538 *
JIN R ET AL: "Tissue transglutaminase and glaucoma", CHINESE OPHTHALMIC RESEARCH 200810 CN, vol. 26, no. 10, October 2008 (2008-10-01), pages 795 - 797, XP008154120, ISSN: 1003-0808 *
LHEUREUX D Z ET AL: "The interaction of angiocidin with tissue transglutaminase", EXPERIMENTAL AND MOLECULAR PATHOLOGY, ACADEMIC PRESS, US, vol. 88, no. 1, 1 February 2010 (2010-02-01), pages 15 - 25, XP026875001, ISSN: 0014-4800, [retrieved on 20091118] *
PRIGLINGER SIEGFRIED G ET AL: "Tissue transglutaminase as a modifying enzyme of the extracellular matrix in PVR membranes.", IOVS, vol. 44, no. 1, January 2003 (2003-01-01), pages 355 - 364, XP002681108 *
SIEGEL ET AL: "Transglutaminase 2 inhibitors and their therapeutic role in disease states", PHARMACOLOGY AND THERAPEUTICS, ELSEVIER, GB, vol. 115, no. 2, 13 July 2007 (2007-07-13), pages 232 - 245, XP022152216, ISSN: 0163-7258, DOI: 10.1016/J.PHARMTHERA.2007.05.003 *
SOHN J ET AL: "Novel transglutaminase inhibitors reverse the inflammation of allergic conjunctivitis", JOURNAL OF CLINICAL INVESTIGATION, AMERICAN SOCIETY FOR CLINICAL INVESTIGATION, US, vol. 111, no. 1, 1 January 2003 (2003-01-01), pages 121 - 128, XP002984889, ISSN: 0021-9738, DOI: 10.1172/JCI200315937 *
TONG ET AL.: "Recent Advances: Transglutaminase in Ocular Health and PathologicalProcesses", J CLINIC EXPERIMENT OPHTHALMOL, 21 December 2011 (2011-12-21), pages 1-12, XP002681109, DOI: 10.4172/2155-9570.S2-002 *
YIZHEN HU ET AL: "Inhibitory effect of tissue transglutaminase (tTG) antisense oligodeoxynucleotides on tTG expression in cultured bovine trabecular meshwork cells.", JOURNAL OF HUAZHONG UNIVERSITY OF SCIENCE AND TECHNOLOGY, vol. 25, no. 6, 1 January 2005 (2005-01-01), pages 729 - 31, 737, XP055034174, ISSN: 1672-0733 *
ZHANG S R ET AL: "Tissue transglutaminase in apoptosis of photoreceptor cells in rat retina.", INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE AUG 1996 LNKD- PUBMED:8759346, vol. 37, no. 9, August 1996 (1996-08-01), pages 1793 - 1799, XP002681107, ISSN: 0146-0404 *

Cited By (3)

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KR20170128058A (ko) * 2016-05-12 2017-11-22 강원대학교산학협력단 당뇨성 합병증 예방 및 치료용 약제학적 조성물 및 당뇨성 합병증 예방 또는 치료제 스크리닝 방법
KR101881662B1 (ko) * 2016-05-12 2018-07-26 강원대학교산학협력단 당뇨성 합병증 예방 및 치료용 약제학적 조성물 및 당뇨성 합병증 예방 또는 치료제 스크리닝 방법
US10155948B2 (en) 2016-05-12 2018-12-18 Kangwon National University University-Industry Cooperation Foundation and Pharmaceutical composition for preventing or treating diabetic complications and screening method for preventive or therapeutic agent for diabetic complications

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