GB2490547A - Tissue transglutaminase inhibitors for use in the treatment of angiogenesis - Google Patents

Abstract

Inhibitors of tissue transglutaminase (ttgase) find use in the treatment of angiogenesis in a subject. In particular, the ttgase inhibitor compounds are those of Formula (I), wherein R1, R2 and X are as defined therein. The said compound are of 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).

Description

NOVEL COMPOUNDS AND METHODS FOR USE IN MEDICINE

Field of the Invention

The present invention provides tissue transglutaminase inhibitor and methods for use in the inhibition of angiogenesis in a subject. In particular, 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).

Introduction

Transglutaminases (TGases) are an important class of protein crosslinking enzymes that catalyse protein aggregation reactions in blood coagulation (Greenberg, CS., et a!., 1991, FASEB J. 5, 3071-3077), skin maturation (Thacher, S. M. & Rice, ft H., 1985, Cell 40, 685-695) and the clotting of seminal secretions (Dubbink, H.J., et 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. Like the well-characterised plasma TGase (blood coagulation factor Xllla) (Greenberg, C.S., et aL, 1991, FASEB J. 5, 3071-3077) and keratinocyte TGase (Thacher, S. M. & Rice, R. H., 1985, Cell 40, 685-695), tTGases are calcium-dependent enzymes that catalyse the formation of crosslinks proteins via c(y-glutamyl) isopeptide bonds and the incorporation of polyamines at certain glutamine residues (Greenberg, C.S., et aL, 1991, FASEB J. 5, 3071-3077). However, 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., et aL, 1995, J Immunot Methods 180, 67-79).

Tissue TGase (tTGase or TG2) is predominantly located in the cytosol, although tTGase

I

has also been reported to exist in the nucleus (Lesort, M., et at, 1998, J Blot Chem. 273, 11991-11994), at the cell surface and in the extracellular matrix (Martinez, J., et at, 1994, Biochemistry 33, 2538-2545). The enzyme is highly expressed in endothelial cells (Greenberg, C. S., et at, 1987, Blood 20, 702-709) and its activity at the surface of such cells is thought to enhance basement membrane stabilisation, cell spreading and cell adhesion (Martinez, J., et at, 1994, Biochemistry 33, 2538-2545; Greenberg, C. S., et at, 1987, Blood 20, 702-709; Kinsella, M. G. & Wight, T. N., 1990, J Blot Chem. 265, 17891-17896; Jones, R.A., et at, 1997, J Cell Sci. 110, 2461-2472; Gaudry C. A., et at, 1999, Exp. Cell Res. 252, 104-113). However, the overall significance of the high amount of enzyme in this cell type and its biological function is poorly understood.

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). For example, tTGase-mediated protein modification has been shown in occur in fibrosis and scarring (Johnson et at, 1999, J Am. Soc. Neph. 10:2146-2157), neurodegenerative diseases including Huntingdon's disease and Alzheimer's disease (Citron et at, 1999, J. Blot Chem. 276:3295-3301), coeliac disease (Marzari et at, 2001, J lmmunot 166:4170- 4176), thrombosis (Ariens et at 2002, Blood 100, 743-754), cancer (Van Groningen et at, 1995, mt. J. Cancer 60:383-387; Mehta, 1994, J Cancer 58:400-406; Mehta et at, 2002, J Natt Cancer Inst. 94:1652-1654), AIDS (Amendola et at, 2002, J lmmunot Methods 265:149-159), psoriasis and inflammatory diseases of the joints (Johnson et at, 2001, Am. J Pathol. 159:149-163).

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). However, the relationship between tTGase activity and angiogenesis appears from the existing scientific literature to be complex. For example, Greenberg at at have reported both an inhibition and an enhancement of angiogenesis in response to increased tTGase in separate studies (see Haroon et at, 1996, FASEB J, Abstract No. 2403, page a1416 and Haroon at at, 1999, FASEB J 13:1787-1795). In another study, Jones et al., 2006, Cell Death Differ. 13(9):1442-1453 report a suppression of endothelial tube formation in two in vitro models of angiogenesis following administration of active tTGase. More recently, Caja at at, 2010, Scand. J Gastroenterot 45:421-7 report a significant negative correlation between endothelial cell angiogenesis and tTGase activity in coeliac disease patients.

Several classes of transglutaminase inhibitor compounds are known in the art, including competitive amine inhibitors, competitive glutamine inhibitors and irreversible inhibitors.

Competitive amine inhibitors include dansylcadaverines (Lorand et at, 1966, Biochem. Biophys. Res. Commun. 25, 629; Lorand et at, 1968, Biochemistry 7, 1214) and N-phenyl-N'-(@-aminoalkyl)thioureas (Lee at at, 1985, J Blot Chem. 260, 14689).

Competitive glutamine inhibitors include aliphatic amides (Gross & Folk, 1973, J Blot Chem. 248, 1301), dipeptides (Gross & Folk, 1973, J Blot Chem. 248, 6534) and polypeptides (Gorman & Folk, 1984, J BioL Chem. 259, 9007). Irreversible inhibitors include iodoacetamide (Gross & Folk, 1973, J Blot Chem. 248, 6534; Folk & Cole, 1966, J Blot Chem. 241, 5518), phenol-containing halomethyl ketones (Folk & Gross, 1971, J Blot Chem. 246, 6683), alkyl isocyanates (Gross et at, 1975, J Blot Chem. 250, 7693), cc-halomethylcarbonyl inhibitors (Reinhardt, 1980, App!. Biochem. 2, 495), dihydroisoazoles (US 4,912,120), azoles, azolium salts (US 4,968,713, thiadiazoles (Keillor, 2001, Biorg. Med. Chem. 9, 3231), and epoxides (Keillor, 2002, Biorg. Med. Chem. 10, 355).

More recently, Pluira et at (1992) J Enzyme Inhibition 6, 181-94 reported irreversible inhibition of transglutaminases by sulfonium methylketones (see also US 4,912,120).

The present invention seeks to provide novel uses and methods for the inhibition of angiogenesis.

Summary of the Invention

According to a first aspect of the invention, there is provided a tissue transglutaminase inhibitor for use in inhibiting angiogenesis in a patient.

By "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 viva). Numerous examples of such inhibitors are well known in the art (for example, see review by Siegel & Khosla, 2007, Pharmacot Ther.

115(2):232-245, the disclosures of which are incorporated herein by reference).

In one embodiment, the tissue transglutaminase inhibitor is a reversible inhibitor, such as a competitive substrate inhibitor. For example, the inhibitor may be selected from the group consisting of: (a) monodansylcadaverine; (b) tTGase cofactors and analogues thereof, such as GIP, GDP, GTPyS and GMP-PCP; (c) Ca2 chelators, such as EDTA; (d) Zn2 metal ions; and (e) acylideneoxoindole compounds (see KlOck et a!., 2011, Bioorg. Med. Chem. LetL 21:2692-2696).

In an alternative embodiment, the tissue transglutaminase inhibitor is an irreversible inhibitor. For example, the inhibitor may be selected from the group consisting of: (a) peptide compounds (for example, see Formula I below); (b) imidazole compounds (for example, see Compound 283 [R2831 below; see also US 5,030,644); (c) dihydroisoxazole compounds, such as KCAO75 and KCCOO9 (for example, see Hausch et al., 2003, Chem. BioI.1O:225-31); (d) cinnamoyl inhibitors (for example, see US 20100204280 to Keillor et a!.); (e) thieno[2,3-d} pyrimidine-4-one acylhydrazide derivatives (for example, see Duval et aL, 2005, Slog,Med Chem.Lett 15:1885); and (f) iodoacetamide (for example, see Folk & Cole, 1966, J Chem. Biol. 241:3238-3240).

In a still further alternative embodiment, the tissue transglutaminase inhibitor is an antibody which binds tiGase and inhibits (at least in part) its transamidation activity.

By "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. Preferably, 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. VH and VL domains) and domain antibodies (dAbs, including single and dual formats [Le. dAb-linker-dAb]). Preferably, a human or humanised antibody is utilised.

Methods for making and testing such antibodies are well known in the art. For example, suitable monoclonal antibodies to tTGase may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Lola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J 0 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.

ln one preferred embodiment, the tissue transglutaminase inhibitor is a dipeptide compound.

Thus, the tissue transglutaminase inhibitor may be a compound of Formula I: (CH2) C021-1 0

I

wherein: X' represents an amino acid group; n' is an integer between 1 and 4; R1' represents benzyl, t-butyl or 9-fluorenylmethyl; and R2' represents

R

wherein R3, R4, R5 and R6 each independently represent lower alkyl or -SR7R8, wherein R7 and R8 each independently represent lower alkyl or a pharmaceutically and/or veterinarily acceptable derivative thereof.

It will be appreciated by persons skilled in the art that X' denotes an a/pha-amino acid moiety, such that the compound has the following general structure (where Raa represents an amino acid side chain, such as H for glycine): Advantageously, X is an L-amino acid moiety.

Preferably, 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. Thus, preferred compounds of the invention include N-benzyloxycarbonyl-L-glutamyl-y- isopropylamide-6-dimethyl-sulfonium-5-oxo-L-norleucine bromide salt and N-benzyloxycarbonyl-L-glutamyl-y-piperidinamide-6-dimethylsulfonium-5-oxo- L-norleucine bromide salt.

In a preferred embodiment of the first aspect of the invention, n' is 2.

Advantageously, CR1' is benzyl.

Conveniently, R2' represents: \+ -S-<'j( Preferably, R2' represents -SR7R8, wherein R7 and R8 each independently represent lower alkyl.

The term "lower alkyl" is intended to include linear or branched, cyclic or acyclic, C1-C5 alkyl, which may be saturated or unsaturated. Lower alkyl groups which R3, R4, R5, R6, R7 and/or R5 may represent include C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C5 alkyl, C3-C5 alkyl, C4-C5 alkyl, CrC4 alkyl, C2-C3 alkyl and C3-C4 alkyl. Preferred lower alkyl groups which R3, R4, R5, R6, S7 and/or R may represent include C1, 02, C3, C4 and C5 alkyl.

Preferably, R3, 54, 55, R5, 57 and/or R8 are -CH3 or -CHCH2. More preferably, R, 54, R, R6, 57 and/or R are -CH3.

In a preferred embodiment of the first aspect of the invention, the compound is selected from the group consisting of: o/c3 Jj H (OH,CH3 2)2+ 0 COH 0 OH3 (a) N-Benzyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine ("Compound 281") 0 NH2 0/ H (OH) ,CH3 flo N(N 22+ L) 0 CO2H 0 OH3 (b) N-Benzyloxycarbonyl-L-glutaminyl-6-dimethylsulfonium-5-oxo-L-norleucine ("Compound 285') o H,CH3 + (OH2)2 O CO2H 0 OH3 (c) N-Benzyloxycarbonyl-L-isoleucinal-6-dimethylsulfonium-5-oxo-L-norleucine ("Compound 286') o ( H,CH3 (OH2)3 o CO2H 0 "OH3 (d) N-Benzyoxycarbonyl-L-phenylalanyl-7-dimethyl-sulfonium-6-oxo-heptanoic acid ("Compound 288") o ( H,0H3

O OH

(e) N-Benzyloxycarbonyl-L-pheriylalanyl-L-5-dimethylsulfonium-4-oxo-norvaline ("Compound 289") o H,CH3 (OH2)2 rs 0 COH 0 CH3 (f) N-Benzyloxycarbonyl-L-alaninal-6-dimethylsulfonium-5-oxo-L-norleucine (" Compound 291") H,CH3 + N(N (CH2)2 0 COH 0 CH3 (g) N-Benzybxycarbonyl-L-glycinal-6-dimethylsulfonium-5-oxo-L-rjorleucjne ("Compound 292')

OH /CH3

(OH2)2 0 CO2H 0 CH3 (h) N-Benzyloxycarbonyl-L-tyrosinal-6-dimethylsulfonium-5-oxo-L-norleucjne ("Compound 293") 0 OH (i) N-Benzyloxycarbonyl-L-prolinyl-6-dimethytsulfonium-5-oxo-L-norleucine ("Compound 294")

OH

(j) N-Benzyloxycarbonyl-L-serinyl-6-dimethylsulfonium-5-oxo-L-norleucine ("Compound 295) 0 OH (k) N-Benzyloxycarbonyl-L-glutaminyl-6-dimethylsulfonium-5-oxo-L-norleucine ("Compound 296") NH2 F3CO2H (I) N-ct-Benzyloxycarbonyt-N-s-trifluoroacetate-L-Iysinyl-6-dimethylsulfonium- 5-oxo-L-norleucine ("Compound 297')

O N

(m) N-a-Benzyloxycarbonyl-y-piperidinyl-L-glutaminyl-6-dimethylsulfonium-5-oxo -L-norleucine ("Compound 298")

N OANN7

(n) N-cc-Benzyloxycarbonyl-y-propyl-L-glutaminyl-6-dimethylsulfonium-5-oxo-L-n orteucine ("Compound 299")

OAN H

OOH

(o) N-Benzyloxycarbonyl-L-phenylalanyl-6-diethylsulfonium-5-oxo-L-norleucine ("Compound 3ocf') H 0 f 0 OOH / (p) N-a-Benzyloxycarbonyl-L-phenylalanyl-6-tetra-methylmercaptoimidazole-5-oxo -L-norleucine ("Compound 301")

HO / \ 0 OH

(q) N-9-Fluorenytmethyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L- norleucine ("Compound 302") (r) N-ct-tert-butyloxycarbonyl-. L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine ("Compound 303') Oy0+ (s) N-cc-Benzyloxycarbonyl-L-prolinyl-6-tetra-methylmercaptoimidazole-5-oxo-L- norleucine ("Compound 304") It will be appreciated by persons skilled in the art that pharmaceutically, and/or veterinarily, acceptable derivatives of the compounds of formula I, such as salts and solvates, are also included within the scope of the invention. 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.

Thus, 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. Other suitable counter-anions will be well known to persons skilled in the art.

in one embodiment, the compound is a bromide salt.

It will be further appreciated by skilled persons that the compounds of formula l may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

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, ag. 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.

Alternatively 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.

Preferably, 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.

By "inhibiting angiogenesis" we mean that administration of the compound is capable of reducing, at least in part, the formation of new blood vessels in viva Thus, 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 (Le. chronic) administration of the compound.

In one embodiment, the compound is for use in the treatment and/or prevention of an eye disease or disorder associated with angiogenesis.

By "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).

In one embodiment, 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.

For example, the eye disease or disorder associated with angiogenesis may be a retinopathy.

Thus, 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.

In a further embodiment, the eye disease or disorder associated with angiogenesis is a diseases or disorder of the anterior eye.

Thus, 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 (Le. growth of blood vessels on iris and into anterior chamber angle; robeosis iridis).

It will be appreciated by persons skilled in the art that the 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.

By 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 (La a TGase inhibitor compound as described above) with the carrier which constitutes one or more accessory ingredients. In general, 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.

For treatment of diseases of the eye, the compound may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for injection into the eye.

Thus, 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.

Typically, compositions for injection are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilising agent and a local anaesthetic such as lignocaine to ease pain at the site of the injection. Generally, 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. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, 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.

In another embodiment, 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 is per day. However, the compounds may also be formulated as irrigating solutions that are applied to the affected eye during surgical procedures.

Alternatively, 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.

It will be appreciated by persons skilled in the art that 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 3° 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 (ImOsm/kgu), but will preferably be about 300 mOsmfkg.

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. The use of 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 surlactant or other appropriate co-solvent in the composition. Such co-solvents include polysorbate 20, 60, and 80, polyoxyethylene/polyoxypropylene surfactants (e.g., Pluronic F-68, F-84 and P-103), cyclodextrin, or other agents known to those skilled in the art.

Typically such co-solvents are employed at a level of from 0.01% to 2% by weight.

The use of 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. Such 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.

In another embodiment, 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.) In yet another embodiment, the TGase inhibitor compounds as described herein can be delivered in a controlled release system. In one embodiment, 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. EngI. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, 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 andfor veterinarily acceptable derivative thereof, in the preparation of a medicament for inhibiting angiogenesis in a patient.

Thus, the tissue transglutaminase inhibitor may be selected from any of those described above in relation to the first aspect of the invention.

For example, the 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. For example, 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.

The tissue transglutaminase inhibitor may be selected from any of those described above in relation to the first aspect of the invention.

For example, the tissue transglutaminase inhibitor may be compound of Formula I. It will be appreciated that the compound should be administered in a therapeutically effective amount to inhibit angiogenesis (at least in part), A therapeutically effective amount', or effective amount', or therapeutically effective', as used herein, 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.

Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a subject. As is appreciated by those skilled in the art, 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. In the methods and use for manufacture of compositions of the invention, 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.

In one embodiment, 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 (Le. 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. For example, detection of the isodipeptide c('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. Hence, a reduction of the isodipeptide in the body fluid provides an indirect measure of reduced protein crosslinking (see Nemes at at, 2002, Mineria Biotechnology 14, 183).

Alternatively, a tissue biopsy may be taken and analysed, for example by ion exchange or reversed phase I-IPLC 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 at, 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. For example, 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).

It will be appreciated by those skilled in the art that treatment may be prophylactic and/or therapeutic. For example, 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. Alternatively, or in addition, the compounds of the invention may be used to reduce or eradicate the symptoms of a disease/disorder in the subject being treated.

It will be further appreciated by those skilled in the art that the compound of the invention may be administered by any route known or developed in the art. Thus, 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.

In one embodiment, the compound is administered systemically, for example intravenously. Alternatively, 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. Advantageously, 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.

It will be appreciated by persons skilled in the art that a subject treated using the method according to the third aspect of the invention may be any mammal. Preferably, the subject is human. Alternatively, the subject may be a dog, cat, horse, or other domestic or farm mammalian animal.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures in which: Figure 1 shows a synthesis route for the production of an exemplary compound according to the first aspect of the invention, namely N-Benzyloxycarbonyt-L-phenylalanyl-6- dimethylsulfonium-5-oxo-L-norleucine bromide salt (Compound 281'). In step (i) the N-ct- CBZ-protected amino acid N-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 N-benzyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt.

Reagents and conditions for each step are as follows: (i) Triethylamine (TEA), THF, H20; (ii) HBr, ethyl acetate; and (iii) Dimethyl sulphide.

Figures 2 to 21 show the effect of increasing concentrations of exemplary compounds of the invention (and prior art compound I,3-dimethyl-2-(2-oxopropylsulfanyl)-31-l-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 pM, along the x-axis. The compounds tested are as follows: Figure Compound tested 2 N-Benzyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 281") 3 1,3-dimethyl-2-(2-oxopropylsulfanyl)-3H-1,3-diazol-1-ium-chloride ("Compound 283") 4 N-Benzyloxycarbonyl-L-glutaminyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 285') N-Benzyloxycarbonyl-L-isoleucinal-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 286") 6 N-Benzyoxycarbonyl-L-phenylalanyl-7-dimethyl-sulfonium-6-oxo-heptanoic acid bromide salt ("Compound 287") 7 N-Benzyloxycarbonyl-L-phenylalanyl-L-5-dimethylsulfonium-4-oxo-norvaline bromide salt ("Compound 289") 8 N-Benzyloxycarbonyl-L-alaninal-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 291") 9 N-Benzyloxycarbonyl-L-glycinal-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 292") N-Benzyloxycarbonyl-L-tyrosinal-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 293") 11 N-Benzyloxycarbonyl-L-prolinyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 294") 12 N-Benzyloxycarbonyl-L-serinyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 295') 13 N-Benzyloxycarbonyl-L-glutaminyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 296") 14 N-a-Benzyloxycarbonyl-N-c-trifluoroacetate-L-lysinyl-6-dimethylsulfonium-5 -oxo-L-norleucine bromide salt ("Compound 297") N-a-Benzyloxycarbonyl-y-piperidinyl-L-glutaminyl-6-dimethylsulfonium-5-oxo -L-norleucine bromide salt ("Compound 298") 16 N-ct-Benzyloxycarbonyl-y-propyl-L-glutaminyl-6-dimethylsulfonium- 5-oxo-L-norleucine bromide salt ("Compound 299") 17 N-Benzyloxycarbonyl-L-phenylalanyl-6-diethylsulfonium-5-oxo-L-norleucine bromide salt ("Compound 300') 18 N-Benzyloxycarbonyl-L-phenylalanyl-6-tefra-methylmercaptoimidazole-5-oxo-L -norleucine bromide salt ("Compound 301") 19 N-9-Fluorenylmethyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L- norleucine bromide salt ("Compound 302") N-cc-tert-butyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norl eucine bromide salt ("Compound 303") 21 N-Benzyloxycarbonyl-L-prolinyl-6-tetra-methylmercaptoimidazole-5-oxo-L-nor leucine bromide salt ("Compound 304") Figure 22 shows SDS-PAGE data demonstrating inhibition of tTGase-mediated crosslinking of fibronectin following treatment with exemplary compounds of the invention (see Example 3). Key: tTG' = tissue transglutaminase, degr. fragments' = degradation fragments, Fn' = fibronectin, Polymers' = cross-linked fibronectin polymers, 281' = N-Benzyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt, 285'l'Rob285' = N-Benzyloxycarbonyl-L-glutaminyl-6-dimethyl-sulfonium-5-oxo-L-norleucine bromide salt.

Figure 23 shows (a) representative Masson's Trichrome stained sections at 1 OOx magnification and (b) collagen Ill stained sections at 200x magnification from kidneys of rats treated for 84 days with inhibitor N-Benzyloxycarbonyl-L-phenylalanyl-6- dimethylsulfonium-5-oxo-L-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. These animals 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 SNJx' 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 Ill staining in the kidney sections from 90 day animals following treatment with inhibitor N-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 N-Benzyloxycarbonyl-L-phenylalanyl-6-dimethyl- sulfon-ium-5-oxo-L-norleucine bromide salt (designated SNx + 281') and I,3-dimethyl-2- (2-oxopropylsulfanyl)-3H-1,3-diazol-1 -ium-chloride (desig-nated SNx + 283'). Data show emission from Leica confocal laser microscope from TRITC-extravidin bound to IGase incorporated biotin cadaverine. 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 14C-putrescine incorporation into N, N'-dimethyl casein at day 84 in kidney homogenates of SNx rats treated with the inhibitors N-Benzyloxycarbonyl-L-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 N-Benzyloxycarbonyl-L-phenylalanyl-6-di-methylsulfonium-5-oxo-L-norleucine bromide salt (designated SNx + 281') and I,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 FXlll, 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 (500pM), or (C) R294 (500pM) was added to the cultures, and changed every two days for 12 days. Cells were fixed and immuno-labeled with CD3I antibody. Cells were photographed using bright-field in a microscope equipped with xlO objective (A, B, C upper panels). In duplicate experiments, at day 13, cells were incubated with FITC-cadaverine for 12 hours (in-situ TG activity), fixed in ethanol, immuno-stained with TRITC-CD3I (specific for endothelial cells) and nuclei counterstained with DAPI. Images were obtained with a fluorescence microscope equipped with a 40X objective (A, B, C lower panels). (D) Co-cultures treated with exogenous VEGF and (E) the inhibitor suramin, were used as positive and negative controls, respectively.

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 pM Calcein AM for 15 mm at 37 °C in 5% C02 and were photographed using 484 nm excitation and 520 nm emission filter on a fluorescent microscope equipped with x 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 (5OpM) with vehicle DM50 (0,01%) (A, negative control), or in presence of the TO inhibitor R294 (5OpM) from day 6 to day 12 of the co-culture (B, active sprouting cells), or from day I to day 12 (C, positive control in presence of inhibitor suramin for day I 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 (DM50 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 102 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.

Panel B Vascular outgrowth in cm after 10 days of treatment with 294 (100 pM) or PBS.

Bars represent the average outgrowth in cm ± S.E.M. (N3). * p«=0.001 using a T-student test.

EXAMPLES

EXAMPLE I -SYNTHESIS OF EXEMPLARY TOASE INHIBITORS

General procedures Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR instrument.

IH spectra were recorded on a JEOL E-270 instrument at 270 Mz. 13C spectra were recorded on the same instrument at 67.8 MHz. All NMR samples were prepared in deuteriochloroform unless otherwise stated. Chemical shifts are reported relative to the internal standard tetramethylsilane and quoted as ppm. Mass spectra were recorded on a micromass platform ESI -MS machine.

Tetrahydrofuran (THF) was freshly distilled from sodium benzophenone ketyl before use.

Ether was distilled from lithium aluminium hydride and stored over sodium wire. Methanol and ethanol were distilled and stored over 5 A molecular sieves. N,N-dimethylformamide 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.

Synthesis of 6-diazo-5-oxo-L-norleucine (DON) The intermediate 6-diazo-5-oxo-L-norleucine, DON, was prepared as previously described in Coutts & Saint (1998) Tetrahedron LetL 39:3243.

Synthesis of 6-bromo-5-oxo-L-norleucine derivatives The following intermediates were synthesised:

KQ o o OOH

1 (279) ONH2 o OOH

OOH 0 0

OOH 4 9 Q9 Os

0 Z 0 0 00 >=0:>< -a >=o -)cno

OO O a N)

CD 0 0

O 0H -t -t 2 92

C C C C

-

-C,) 0 C C

C C C C C

C CH:hC I)

xC z C -1 -s _5 -5 9 999 0 0 0 0 C\ -xz -s F) :>KzzK:D C

C z 0 -t -S

Q

OANHNBr To an ice-cold solution of the appropriate N-a-protected (CBZ, FMOC or BOO) amino acid N-hydroxysuccinimide ester and DON (1 eqv.) in a 1:1 mixture of THF/water (O.5M) was added triethylamine (1.5 eqv.). The reaction mixture was stirred for 2 h. at 0°C and the solvent removed under high vacuum at room temperature. The residue was dissolved in ethyl acetate and treated with a 1:1 mixture of HBr and acetic acid dropwise until gas evolution ceased. The resulting mixture was stirred for a further 10 mm. and ethyl acetate added and the organic layer washed with water (x3) brine (xl) and dried over MgSO4.

Removal of the solvent in vacuo afforded a colourless solid which was recrystallised from an appropriate solvent to give the product in typically 70-80% yield.

(a) N-cx-Benzyloxycarbonyl-L-phenylalanyl-6-bromo-5-oxo-L-norleucine N-ct-Benzyloxycarbonyl-L-phenylalanyl-6-bromo-5-oxo-L-norleucine(see 1' above) was prepared from DON and N-cc-CBZ-L-phenylalanine N-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. C23H25BrN2O6 requires C, 54.66; H, 4.99; N, 5.54%.); Vmax (KBr)/cm1 3294, 1719, 1689, 1655; 3H (d6 acetone) 1.9, 2.2 and 2.7 (4 H, m), 2.9-3.2 (2 H, m), 4.2 (2 H, s), 4.5 (2 H, m), 5.0 (2 H, s), 6.6 (1 H, d), 7.3 (10 H, ArH), 7.6 (1 H, d); S (d6 acetone) 27.0, 36.0, 36.3, 38.6, 51.8, 57.2, 66.7, 127.3, 128.5, 128.6, 129.1, 129.2, 130.2, 137.9, 153.5, 172.4, 173.7, 201.0.

(b) N-a-Benzyloxycarbonyl-L-glutaminyl-6-bromo-5-oxo-L-norleucine N-cc-Benzyloxycarbonyl-L-glutaminyl-6-bromo-5-oxo-L-norleucine (see 2' above) was prepared from DON and N-cx-CBZ-L-glutamine N-hydroxysuccinimide ester (Bachem cat. no. C-1625) m.p. 161-163°C (iso-propanol, dec.), Vmax (KBr)/cm1 3423, 3346, 1702, 1684, 1638; 5H (d4 methanol) 1.8, 1.9, 2.2 and 2.6 (8 H, m), 3.9 (2 H, s), 4.1 (1 H, m), 4.3 (1 H, m), 5.0 (2 H, s), 7.3 (5 H, ArH), 7.6 (1 H, d); 50 (d3 methanol) 26.8, 28.9, 32.5, 35.6, 36.5, 52.6, 55.9, 67.7, 128.8, 129.0, 129.5, 130.2, 136.9, 154.5, 171.9, 174.5, 202.6. MS: mlz Calcd for C19H24BrN3O7: 485 (M-Br = 406). Observed 406.

(c) N-a-Benzyloxycarbonyl-L-isoleucinyl-6-bromo-5-oxo-L-norleucine N-ct-Benzyloxycarbonyl-L-isoleucinyl-6-bromo-5-oxo-L-norleucine (see 3' above) was prepared from DON and N-cc-CBZ-L-isoleucine N-hydroxysuccinimide ester (Novabiochem cat. No. 04-12-0560) m.p. 182-184°C (ethyl acetate, dec.), Vmax (KBr)/cm1 3296, 1720, 1684, 1660; 5H (d3 methanol) 0.9, 1.2 and 1.5 (6 H, m), 1.9, 2.2 and 2.7 (4 H, m), 3,9 (1 H d), 4.0 (2 H, s), 4.4 (2 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); S (d3 methanol) 11.2, 15.9, 25.9, 26.8, 35.6, 36.4, 37.9, 52.3, 61.1, 87.6, 128.7, 129.0, 129.5, 138.1, 154.6, 174.4, 174.5, 202.8.

MS: m/z Calcd for C20H27BrN2O6: 470 (M-Br = 391). Observed 473, 471, 391.

(d) N-cz-Benzyloxycarbonyl-L-alaninyl-6-bromo-5-oxo-L-norleucine N-cz-Benzyloxycarbonyl-L-alaninyl-6-bromo-5-oxo-L-norleucine (see 4' above) was prepared from DON and N-ct-CBZ-L-alanine N-hydroxysuccinimide ester (Novabiochem cat. No. 04-12-0512) m.p. 82-85°C (DCM/ether, dec.), Vmax (KBr)/cm1 3294, 1719, 1686, 1660; 5H (d6 acetone) 1.4(3 H, d), 1.9, 2.2 and 2.6(4 H, m), 4.1, (2 N s), 4.2 (2 H, m), 4.4 (1 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); 8c (d5 acetone) 26.3, 28.9, 34.5, 51.3, 51.9, 66.7, 68.6, 128.7, 129.0, 129.5, 138.1, 154.6, 173.2, 174.4, 202.6. MS: rn/z Calcd for C17H21BrN2O6: 428 (M-Br 349). Observed 429, 349.

(e) N-cx-Benzyloxycarbonyl-L-gtycinyl-6-bromo-5-oxo-L-norleucine N-ct-Benzyloxycarbonyl-L-glycinyl-6-bromo-5-oxo-L-norleucine (see 5' above) was prepared from DON and N-ct-CBZ-L-glycine N-hydroxysuccinimide ester (Novabiochem cat. No. 04-12-0511) and used without further purification.

(f) N-a-Benzyloxycarbonyl-L-tyrosinyl-6-bromo-5-oxo-L-norleucine N-a-Benzyloxycarbonyl-L-tyrosinyl-6-bromo-5-oxo-L-norleucine (see 6' above) was prepared from DON and N-ct-CBZ-L-tyrosine 4-nitrophenyl ester (Fluka cat. No. 97300) and used without further purification.

(g) N-a-Benzyloxycarbonyl-L-prolinyl-6-bromo-5-oxo-L-norleucine N-a-Benzyloxycarbonyl-L-prolinyl-6-bromo-5-oxo-L-norleucine (see 7' above) was prepared from DON and N-a-CBZ-L-proline N-hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0577).

m.p. 129131°C, Vmax (KBr)/cm1 3241, 1725, 1690, 1664; 5H (d3 methanol) 1.9, 2.2, 2.5 and 2.8 (8 H, m), 3.5 (2 H, m), 4.0 (2 H, s), 4.3 (1 H, m), 4.5 (1 H, m), 5.0 (2 H, s), 7.3(5 H, ArH); S (d3methanol) 21.0, 23.2, 27.6, 31.7, 32.6, 48.6, 57.9, 64.4, 124.9, 125.1, 125.4, 125.9, 134.3, 153.0, 170.9, 171.5, 199.2 MS: mlz Calcd for C19H23BrN2O6: 455 (M÷Na =477, M+H = 457). Observed 477, 457.

(h) N-ct-Benzyloxycarbonyl-L-serinyl-6-bromo-5-oxo-L-norleucine N-ct-Benzyloxycarbonyl-L-serinyl-(O-t-butyl)-6-bromo-5-oxo-L-norleucine (see 8' above) was prepared from DON and N-cc-CBZ-L-serine (O-t-butyl) N-hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0585).

Vmax (film)/crri1 3240, 1719,1664; 5H (CDCI3) 1.1 (9 H, s), 1.9, 2.3, and 2.7 (4 H, m), 3.4 (2 H, m), 3.9 (2 H, s), 4.3 (1 H, m), 4.6 (1 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); 5c (CDCI3) 26.4, 27.2, 34.2, 35.5, 51.4, 53.4, 61.5, 67.2, 74.3, 128.2, 128.5, 135.7, 156.3, 171.1, 175.4, 201.0 MS: m/z Calcd for C211-{29BrN2O7: 501 (M+Na = 523, 525). Observed 523, 525.

N-ct-Benzyloxycarbonyl-L-serinyl-6-bromo-5-oxo-L-norleucine (see 9' above) was prepared via removal of the t-butyl protecting group using trifluoroacetic acid and triethylsilane as reagents, Mehta et a!. (1992) Tetrahedron Lett., 33, 5441. The crude product obtained was used without further purification.

Vmax (film)/cm1 3409, 3242, 1720,1668; 5H (d3 methanol) 1.9, 2.3, and 2.7 (4 H, m), 3.4 (2 H, m), 4.0 (2 H, s), 4.2 (1 H, m), 4.5 (1 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); 5c (d3 methanol) 26.4, 35.6, 36.4, 52.6, 57.7, 61.5, 67.8, 128.8, 129.0, 129.1, 129.4, 129.5, 138.1, 158.5, 173.0, 173.8, 202.9 (i) N-ct-Benzyloxycarbonyl-L-glutaminyl-6-bromo-5-oxo-L-norleucine N-ct-Benzyloxycarbonyl-L-glutaminyl-(O-t-butyl)-6-bromo-5-oxo-L-norleucine (see 10' above) was prepared from DON and N-a-CBZ-L-glutamic acid (O-t-butyl) N-hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0551).

Vmax (film)/cm1 3240, 1722, 1714, 1700, 1664; 8H (CDCI3) 1.3 (9 H, s), 1.9, 2.3, and 2.7 (8 H, m), 3.8 (2 H, m), 3.9 (2 H, s), 4.2 (1 H, m), 4.5 (1 H, m), 5.0 (2 H, s), 7.3 (5 H, ArH); 8c (CDCI3) 25.8, 27.9, 31.5, 34.4, 35.5, 51.4, 54.2, 61.5, 67.1, 81.3, 127.9, 128.0, 128.1, 128.5, 136.0, 156.4, 172.3, 173.0, 174.3, 201.3 MS: mlz Calcd for C23H31BrN2O5: 543 (M+Na = 565, 567). Observed 565, 567.

N-a-Benzyloxycarbonyl-L-glutaminyl-6-bromo-5-oxo-L-norleucine trifluoroacetic acid salt (see 11' above) was prepared via removal of the t-butyl protecting group using trifluoroacetic acid and triethylsilane as reagents, Mehta et a!. (1992) Tetrahedron Lett., 33, 5441. The crude product obtained was used without further purification.

Vmax (film)/cm1 3404, 3242, 1720, 1700,1680; 8H (d3 methanol) 1.9,2.2, 2.3, 2.4, and 2.7 (8 H, m), 4.0 (2 H, s), 4.1 (1 H, m), 4.4 (1 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); 6 (d3 methanol) 26.7, 28.3, 31.1, 35.7, 36.4, 52.5, 55.7, 67.7, 128.7, 129.0, 129.5, 138.1, 158.4, 174.5, 174.6, 176.5, 202.8 (j) N-ct-Benzyloxycarbonyl-L-lysinyl-6-bromo-5-oxo-L-norleucine N-a-Benzyloxycarbonyl-L-lysinyl-(N'-BOC)-6-bromo-5-oxo-L-norleucine (see 12' above) was prepared from DON and N-ct-CBZ-L-lysine (N'-BOC) N-hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0526).

8H (CDCI3) 1.4, 1.6 and 1.8 (6 H, s), 1.9, 2.1, and 2.7 (4 H, m), 2.9 (2 H, m), 3.8 (2 H, s), 4.1 (1 H, m), 4.4 (1 H, m), 5.0 (2 H, s), 7.3 (5 H, ArH); sc (CDCI3) 17.9, 22.3, 25.7, 28.3, 31.5, 34.8, 35.6, 51.4, 53.4, 54.8, 58.1, 66.9, 79.4, 127.8, 127.9, 128.0, 128.4, 136.1, 156.5, 172.9, 174.0, 175.5, 201.5 N-ct-Benzyloxycarbonyt-L-lysinyl-6-bromo-5-oxo-L-norleucine trifluoroacetic acid salt (see 13' above) was prepared via removal of the t-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.

(k) N-ct-Benzyloxycarbonyl-'y-piperidinyl-L-glutaminyl-6-brorno-5-oxo-L. -norleucine N-a-Benzyloxycarbonyl-y-piperidinyl-L-glutamic acid (see 14' above) was prepared using the methods of Molina, T.M., et a! (1993) Tetrahedron 49, 3801-3808, BIas, J., et al (2000) Tetrahedron Lett. 41, 4567-4571 and Antonjuk, D.J., et a! (1984) J Chem. Perk/n Trans. 11989-2003.

Vm (fiIm)Icm1 3312, 2940, 1722, 1715, 1698, 1664; 3H (CDCI3) 1.5(6 H, m), 2.0, 22, 2.4 and 2.6 (4 H, m), 3.3 (2 H, m), 3.5 (2 H, m), 4.3 (1 H, q), 5.1 (2 H, s), 6.0 (1 H, d), 7.3 (5 H, ArH); 5 (CDCI3) 24.1, 25.3, 26.2, 28.3, 29.5, 43.2, 46.8, 53.5, 66.7, 127.8, 127.9, 128.4, 136.2, 156.1, 171.5, 174.0.

N-ct-Benzyloxycarbonyl-'y-piperidinyl-glutaminyl-6-bromo-5-oxo-L-norleucin e (see 15' above) was prepared from DON and N-ct-Benzyloxycarbonyt-y-piperidinyl-L-glutamic acid using N-hydroxysuccinimide ester activation.

V (film)/cm1 3325, 2938, 1719, 1689, 1664; 8H (d6 DMSO) 1.4 and 1.5 (6 H, m), 1.8, 2.0, 2.3 and 2.6 (8 H, m), 3.3 (2 H, m), 3.4 (2 H, m), 4.0 (2 H, s), 4.2 (1 H, m), 4.3 (1 H, m), 5.0 (2 H, s), 7.3 (5 H, ArH); 8c (d6 DMSO) 24.1, 25.3, 26.1, 28.8, 33.8, 35.4, 36.8, 41.9, 45.7, 47.6, 54.1, 65.4, 67.5, 127.7, 127.8, 128.3, 137.0, 155.9, 169.6, 173.1, 173.3 200.9.

(I) N-a-Benzyloxycarbonyt-y-propyt-L-glutaminyl-6-bromo-5-oxo-L-norleucine N-ct-Benzyloxycarbonyl-y-propyl-L-glutamic acid (see 16' above) was prepared using the methods of Molina, T.M., et a! (1993) Tetrahedron 49, 3801-3808, BIas, J., et al (2000) Tetrahedron Lett. 41, 4567-4571 and Antonjuk, D.J., et a! (1984) J Chem. Perk/n Trans. 11989-2003.

Vmax (film)/cm1 3328, 2965, 1706, 1702, 1698, 1653; 6H (ODd3) 0.8 (3 H, t), 1.4 (2 H, m), 2.0 and 2.2 (4 H, m), 3.1 (2 H, m), 4.3 (1 H, q), 5.1 (2 H, s), 6.0 (1 H, d), 6.5 (1 H, m), 7.3 (5 H, ArH); S (CDCI3) 11.2, 22.4, 28.6, 32.4, 41.6, 53.4, 67.1, 127.9, 128.1, 128.2, 128.5, 136.0, 156.6, 173.4, 173.9.

N-ct-Benzyloxycarbonyl-y-propyl-glutaminyl-6-bromo-5-oxo-L-norleucine (see 17' above) was prepared from DON and N-a-Benzyloxycarbonyl-y-propyl-L-glutamic acid using N-hydroxysuccinimide ester activation.

Vmax (film)/cm1 3325, 2938, 1719, 1689, 1664; 6H (d5 DM80) 1.4 and 1.5 (6 H, m), 18, 2.0, 2.3 and 2.6 (8 H, m), 3.3 (2 H, m), 3.4 (2 H, m), 4.0 (2 H, s), 4.2 (1 H, m), 4.3 (1 H, m), 5.0 (2 H, s), 7.3 (5 H, ArH); S (d6 DM80) 11.7, 23.5, 25.2, 26.0, 28.8, 33.0, 34.7, 38.1, 52.0, 56.0, 67.7, 128.7, 128.8, 129.3, 137.0, 155.9, 169.6, 174.7, 174.8 200.8.

(m) N-a-9-Fluorenylmethyloxycarbonyl-L-phenylalanyl-6-bromo-5-oxo-L-norleucine N-ct-9-Fluorenylmethyloxycarbonyl-L-phenyalanyl-6-bromo-5-oxo-L-norleucine (see 18' above) was prepared from DON and N-ct-FMOC-L-phenylalanine N-hydroxysuccinimide ester (Bachem cat. no. B-1415).

m.p. 142A43°C, Vmax (KBr)/cm1 3354, 1714, 1700, 1686; 5F1 (d5 DM80) 1.9, 2.2 and 2.7 (4 H, m), 2.9-3.1 (2 H, m), 4.2 -4.4 (5 H, m), 7.3 (9 H, ArH), 7.6 (2 H, ArH), 7.8 (2 H, ArH); 5c (d6 DM80) 25.5, 35.5, 36.6, 37.4, 46.6, 50.9, 56.1, 64.4, 120.0, 124.8, 125.1, 127.2, 127.5, 127.6, 128.7, 129.3, 137.0, 140.7, 143.7, 155.9, 171.9, 173.1, 200.7 (n) N-a-tert-butyloxycarbonyl-L-phenylalanyl-6-bromo-5-oxo-L-norleucine N-u-tert-butyloxycarbonyl-L-phenyalanyl-6-bromo-5-oxo-L-norleucine (see 19' above) was prepared from DON and N-ct-BOC-L-phenylalanine N-hydroxysuccinimide ester (Novabiochem cat. no. 04-12-0074).

Vmax (film)/cm1 3380, 2930, 1722,1699, 1684; 5H (d3 methanol) 1.2 (9 H, s), 1.8, 2.1 and 2.6 (4 H, m), 2.7-3.0 (2 H, m), 4.0 (2 H, s), 4.2 (1 H, m), 4.3 (2 H, m), 4.8 (2 H, s), 7.1 (5 H, ArH); 5c (d3 methanol) 27.0, 28.7, 35.6, 36.4, 39.0, 52.4, 57.3, 80.6, 127.7, 129.4, 130.4, 138.2, 157.6, 174.4, 174.5, 202.7 Preparation of amino acid derived TGase inhibitors Sulfonium salts of the above intermediates were prepared using a modification of procedures previously reported by Pliura et a!. (1992) J Enzyme inhibition 6, 2768 and Shaw (1988) BioL Chem., 263, 2768.

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. O NH2

H

NNSBr 0 0 CO2H ONYNY?Br 0 CO2H H 0 CO2FI 286 011 0 0 /\

UI

0 CO2H OAN1 SBr 292 0 CO2H O>O 0 \JH

OH 0 OH

C C C C a

C C C C

C C

C ci C\ / i-i

x C C C -C,')----&D--Cl)--Cl)--1 -t -t oo oo -

C oI 0 0 a N) 0 0 0 - 0

CD /L--t

-I

(a) N-Benzyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (see 281' above) m.p. 90-92°C, (Found: C, 52.89; H, 4.89; N, 5.05. C25H31BrN2O6S requires C, 52.91; H, 5.51; N, 4.94%.); Vmax (KBr)/cm1 3296, 1715, 1700, 1661; 5H (d5 acetone) 1.9, 2.2 and 2.7 (4 H, m), 2.9-3.1 (2 H, m), 3.2 (6 H, s), 4.6 (2 H, m), 5.0 (2 H, s), 5.4 (1 H, d), 7.3 (10 H, ArH), 7.4 (1 H, d); (d6 acetone) 25.2, 36.1, 38.5, 41.5, 51.9, 56.3, 60.6, 66.3, 128.4, 128.6, 129.0, 129.2, 130.3, 138.2, 138.6, 153.3, 168.7, 173.2, 202.0.

(b) N-Benzyloxycarbonyl-L-glutaminyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (see 285' above) m.p. 100°C (dec.), Vmax (KBr)/cm1 3423, 3346, 1702, 1684, 1638; 8H (DMSO-D6) 1.7, 1.9, 2.1 and 2.6 (8 H, m), 2.8 (6 H, s), 3.3 (1 H, br), 3.9 (2 H, s), 4.2 (1 H, m), 4.7 (2 H, m), 5.0 (2 H, s), 6.7 (1 H, s), 7.3 (5 H, ArH), 7.4 (1 H, d), 8.2 (1 H, d); (DMSO-D6) 24.5, 25.2, 27.7, 31.5, 37.5, 50.6, 53.5, 54.3, 65.4, 127.6, 127.8, 128.3, 128.4, 136.9, 155.9, 172.0, 172.9, 173.8, 201.4. MS: rn/z Calcd for C21H33BrN3O7S: 547.09878 (M-Br = 468.18045). Observed 468.17769.

(c) N-Benzyloxycarbonyl-L-isoleucinal-6-dimethylsulfonium-5-oxo-L-.norleucine bromide salt (see 286' above) m.p. 111-114°C (dec.), Vmax (KBr)/cm 3424, 1717, 1700, 1664; 8H (DMSO-D6) 0.9, 1.2 and 1.5 (6 H, m), 1.9, 2.2 and 2.7 (4 H, m), 3.2 (6 H, s), 3,9 (1 H d), 4.1 (2 H, s), 4.4 (1 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); (DMSO-D8) 11.2, 15.9, 25.9, 26.8, 35.6, 36.4, 37.9, 52.3, 61.1, 67.6, 128.7, 129.0, 129.5, 138.1, 154.6, 174.4, 174.5, 202.8.

MS: m/z Calcd for C22H33BrN2O6S: 532 (M-Br = 453). Observed 453.

(d) N-Benzyloxycarbonyl-L-alaninal-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (see 291' above) m.p. 100-102°C (dec.), Vmax (KBr)/cm1 3426, 1716, 1698, 1660; 8H (d6DMSO) 1.2(3 H, d), 1.8, 2.0 and 2.7 (4 H, m), 2.9 (6H, s), 4.0, (1 H, m), 4.2 (1 H, m), 4.8 (2 H, m), 5.0 (2 H, s), 7.3 (5 H, ArH); S (d6 DMSO) 24.5, 37.4, 49.8, 50.6, 52.0, 53.5, 65.3, 66.3, 127.7, 127.8, 128.3, 137.0, 155.7, 172.8, 172.9, 201.4. MS: m/z Calcd for C19H27BrN2O6S: 490 (M-Br = 411). Observed 411.

(e) N-Benzyloxycarbonyl-L-glycinal-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (see 292' above) m.p. 96-99 °C (dec.), Vmax (KBr)/cm1 3424, 1714, 1701, 1663; 8H (d6 DMSO) 1.8, 2.1 and 2.6 (4 H, m), 2.9 (6H, s), 3.6, (2 H, m), 4.3 (1 H, m), 4.7 (2 H, d), 5.0 (2 H, s), 7.3 (5 H, ArH); 3c (d5DMSO) 24.5, 37.4, 43.2, 50.6, 53.5, 65.4, 66.3, 127.6, 127.8, 128.3, 137.0, 156.5, 169.2, 172.9, 201.4. MS: mlz Calcd for C19H25BrN2O5S: 476 (M-Br = 397). Observed 397.

(f) N-Benzyloxycarbonyl-L-tyrosinal-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (see 293' above) m.p. 111-113 °C (dec.), max (KBr)1cm1 3426, 1716, 1700, 1666; 3H (d6 DMSO) 1.9 and 2.1 (2 H, m), 2.6 (4 H, m), 2.9 (6H, s), 4.2, (2 H s), 4.7 (2 H, d), 4.9 (2 H, d), 6.7 (2H, d, ArH), 7.1 (2 H, d, ArH), 7.3 (5 H, ArH); 6c (d6 DMSO) 24.4, 24.8, 36.5, 37.5, 50.8, 53.5, 56.3, 65.2, 114.8, 127.3, 127.7, 128.0, 128.3, 130.1, 137.07 155.7, 172.0, 172.9, 201.4. MS: m/z Calcd for C25H31BrN2O7S: 582 (M-Br 503). Observed 503.

(g) N-Benzyloxycarbonyl-L-prolinyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (see 294' above) m.p. 93-97°C (dec.), Vmax (KBr)Icm1 3244, 2952, 1720, 1693, 1669; 6H (d3 methanol) 1.9 (4 H, m), 2.2 (2 H, m), 2.7 (2 H, m), 2.8 (6 H, s), 3.6 (2 H, m), 4.3 (1 H, m), 4.5 (1 H, m), 4.8 (2 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); S (d3 methanol) 25.3, 25.4, 26.3, 28.1, 31.1, 38.2, 52.1, 55.9, 67.6, 128.6, 129.1, 129.6, 138.2, 158.5, 174.4, 174.7, 201.8. MB: mlz Calcd for C21H29BrN2O6S: 517 (M-Br = 437). Observed 437.

(h) N-Benzyloxycarbonyl-L-serinyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (see 295' above) m.p. 105°C (dec.), Vmax (KBr)/cm1 3242, 2950, 1719, 1700, 1664; 5H (d3 methanol) 1.9 (2 H, m), 2.3 (2 H, m), 2.9 (6 H, s), 3.8 (2 H, m), 4.2 (1 H, m), 4.4 (1 H, m), 4.8 (2 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); 6c (d3 methanol) 24.6, 25.4, 26.3, 31.6, 32.5, 38.0, 48.3, 51.8, 61.6, 68.0, 128.4, 128.8, 129.1, 129.6, 138.2, 156.7, 174.3, 175.3, 201.8.

MB: m/z Calcd for C19H27BrN2O7S: 507 (M-Br = 427). Observed 427.

(I) N-Benzyloxycarbonyl-L-glutaminyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (see 296' above) m.p. 102-106°C (dec.), Vmax (KBr)/cm1 3329, 2926, 1719, 1698, 1664; 8H (d3 methanol) 1.9, 2.1, 2.4 and 2.7 (8 H, m), 2.9 (6 H, s), 4.1 (1 H, m), 4.5 (1 H, m), 4.8 (2 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); 5c (d3 methanol) 25.3, 25.4, 26.3, 28.1, 31.1, 38.2, 52.1, 55.9, 67.6, 128.6, 129.1, 129.6, 138.2, 158.5, 174.4, 174.7, 201.8. MB: mlz Calcd for C21H29BrN2O8S: 549 (M-Br = 469). Observed 469.

(j) N-ct-Benzyloxycarbonyl-N-c-trifluoroacetate-L-lysinyl-6-dimethylsulfonium- 5-oxo-L-norleucine bromide salt (see 297' above) m.p. 110°C (dec.), Vmax (KBr)/cm1 3334, 1722, 1688; 8H (d3 methanol) 1.4 and 1.7 (6 H, m), 1.9, 2.1 and 2.7 (6 H, m), 2.9 (6 H, s), 4.1 (1 H, m), 4.4 (1 H, m), 4.8 (2 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH). MB: m/z Calcd for C24H35BrF3N3O8S: 662 (M-Br-F3C2O2H = 468). Observed 468.

(k) N-ct-Benzyloxycarbonyl-y-piperidinyl-L-glutaminyl-6-dimethylsulfonium-5-ox o-L-norleucine bromide salt (see 298' above) m.p. 92-94°C (dec.), Vmax (KBr)/cm1 3415, 2933, 1719, 1700, 1664, 1618; 8H (d3 methanol) 1.5, 1.7 (6 H, m), 2.0, 2.3 and 2.7 (4 H, m), 2.9 (6 H, s), 3.4 and 3.5 (4 H, as m), 4.1 (1 H, m), 4.5 (1 H, m), 4.8 (2 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); 5c (d3 methanol) 22.3, 22.4, 23.2, 24.5, 25.4, 25.6, 27.1, 35.2, 38.2, 41.1, 44.8, 49.0, 53.0, 64.6, 125.7, 126.1, 126.6, 155.4, 169.5, 171.2, 171.8, 199.0. MS: m/z Calcd for C26H35BrN3O7S: 616 (M-Br+H = 537). Observed 537.

(I) N-cx-Benzyloxycarbonyl-y-propyl-L-glutaminyl-6-dimethylsulfonium-5-oxo-L-n orleucine bromide salt (see 299' above) m.p. 82-85°C (dec.), Vmax (KBr)/cm1 3414, 2927, 1720, 1698, 1668, 1636; 5H (d3 methanol) 0.9 (3 H, t), 1.5 (2 H, q), 1.9, 2.1 and 2.3 (4 H, m), 2.9 (6 H, s), 3.2 (2 H, t), 4.1 (1 H, m), 4.5 (1 H, m), 4.8 (2 H, m), 5.1 (2 H, s), 7.3 (5 H, ArH); 80 (d3 methanol) 11.7, 23.5, 25.4, 26.0, 28.9, 33.0, 34.7, 38.1, 42.3, 52.0, 56.0, 67.7, 128.7, 128.8, 129.1, 129.6, 138.2, 158.4, 174.4, 174.6, 174.9, 201.8. MS: m/z Calcd for C24H36BrN3O7S: 590 (M-Br+l-l = 511). Observed 511.

Is (m) N-Benzyloxycarbonyl-L-phenylalanyl-6-diethylsulfonium-5-oxo-L-norleucine bromide salt (see 300' above) m.p. 86-89°C (dec.), Vmax (KBr)/cm1 3296, 2932, 1716, 1700, 1661, 1636; 8H (d3 methanol) 1.4 (6 H, t), 1.9, 2.3 and 2.7 (4 H, m), 2.9-3.1 (2 H, m), 3.4 (4 H, q), 4.4 (1 H, m), 4.5 (1 H, m), 4.9 (2 H, m), 5.0 (2 H, s), 7.3 (5 H, ArH); 8 (d3 methanol) 9.4, 26.5, 34.9, 35.1, 38.2, 38.8, 52.0, 57.9, 67.5, 127.8, 128.5, 129.0, 129.4, 129.5, 130.3, 138.4, 158.3, 174.2, 174.4, 174.9, 201.6. MS: tn/z Calcd forC27H35BrN2O6S: 594 (M-Br 515) observed 515 (n) N-Benzyloxycarbonyl-L-phenylalanyl-6-tetra-methylmercaptoimidazole-5-oxo-L -norleucine bromide salt N-cz-Benzyloxycarbonyl-L-phenylalanyl-6-tetra-methylmercaptoimidazole-5-ox o-L- norleucine bromide salt (see 301' above) was prepared from N-cz-Benzyloxycarbonyl- L-phenylalanyl-6-bromo-5-oxo-L-norleucine and 1, 3,4,5-tetramethylimidazoli ne-2-thione, which was prepared by the method of Kuhn and Kratz (1993) Synthesis, 561, using the method of Freund eta!. (1994) Biochemistry, 33, 10109.

m.p. 116-118°C (dec.), Vmax (KBr)1cm1 3414, 3237, 1720, 1657, 1638, 1617; 8H (d3 methanol) 1.7, 2.2 and 2.5 (4 H, m), 2.3 (6 H, s), 2.9-3.1 (2 H, m), 3.9 (6 H, s), 4.3 (2 H, m), 4.8 (2 H, m), 5.0 (2 H, s), 7.2 (5 H, ArH), 7.3 (5 H, ArH); 3 (d3 methanol) 9.1, 26.9, 34.3, 38.7, 39.3, 46.5, 51.9, 58.0, 67.5, 127.8, 129.1, 129.5, 129.6, 130.3, 130.9, 138.9, 138.3, 138.4, 158.2, 174.2, 174.4, 201.8. MS: mlz Calcd for C30H37BrN4O6S: 661 (M-Br+H = 582). Observed 582.

(o) N-9-Fluorenylmethyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L- norleucine bromide salt (see 302' above) m.p. 95-98°C (dec.), Vmax (KBr)/cm1 3415, 3310, 2926, 1720, 1648; 8H (d6 DMSO) 1.8, 2.1 and 27 (4 H, m), 2.8 (6 H, s), 2.9-3.0 (2 H, m), 4.1-4.3 (5 H, m), 4.7 (2 H, m), 7.3 (9 H, ArH), 7.6 (2 H, ArH), 7.8 (2 H, ArH). MS: m/z Calcd for C32H35BrN2O6S: 654 (M-Br575) Observed 575 (p) N-cx-tert-butyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norl eucine bromide salt (see 303' above) m.p. 105-109°C (dec.), Vmax (KBr)/cm1 3413, 2930, 1720, 1663; 8H (d6 DMSO) 1.3 (9 H, s), 1.8, 2.1 and 2.7 (4 H, m), 2.8 (6 H, s), 2.9-3.0 (2 H, m), 4.2 (2 H, m), 4.7 (2 H, m), 7.3 (5 H, ArH). MS: m/z Calcd for C22H33BrN2O5S: 531 (M-Br 453) observed 453 (q) N-Benzyloxycarbonyl-L-prolinyl-6-tetra-methylmercaptoimidazole-5-oxo-L-nor leucine bromide salt N-ct-Benzyloxycarbonyl-L-prolinyl-6-tetra-methylmercaptoimidazole-5-oxo-L- norleucine bromide salt (see 304' above) was prepared from N-ct-Benzyloxycarbonyl-L-prolinyl-6-bromo-5-oxo-L-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. 110°C (dec.), Vmax (KBr)1cm1 3426, 2927, 1700, 1630, 1638, 1617; 6H (d3 methanol) 1.7-2.1 (4 H, m), 2.2 (2 H, m), 2.3 (6 H, s), 2.6 (2 H, m), 3.3 (2 H, m), 3.7 (6 H, s), 3.9 (2 H, m), 4.2 (1 H, m), 4.3 (1 H, m), 5.0 (2 H, m), 7.3 (5 H, ArH); 6c (d3 methanol) 8.7, 23.9, 25.2, 31.1, 33.7, 36.4, 40.4, 44.0, 46.6, 59.5, 65.8, 127.5, 127.8, 128.2, 128.9, 129.0, 136.9, 153.9, 172.1, 173.0, 202.9. MS: m/z Calcd for C26H35BrN4O6S: 610 (M-Br 531) observed 531 Synthesis of N-Benzyloxycarbonyl-L-phenylalanyl-L-2-amino-5-dimeth-ylsulfonium-4-oxo-no rvaline bromide salt (Compound 289') -ccb 7 0 co)L Cx)r Compound 5' is made by reaction of commercially available N-a-CBZ-L-phenylalanine N- hydroxy-succinimide ester (Novabiochem Cat. No. 04-12-0573) and L-aspartic acid 3-t-butyl ester (Novabiochem Cat. No. 04-12-5000) in water/THE (1:1) in the presence of 1.5 equivalents of triethylamine.

(a) N-Benzyloxycarbonyl-L-phenylalanyl-L-2-amino-5-diazo-4-oxo-norvaline tert-butyl ester (see 6' above) N-Methylmorpholine (0.63 ml, 5.75 mmol) followed by n-butyl chloroformate (0.74 ml, 5.75 mmol) were added to a cold (-78°C) solution of N-a-benzyloxycarbonyl-L-phenylalanyl-aspartic acid 3-t-butyl ester (see 5' above) (2.35g, 5 mmol) in THE (100 ml) in an atmosphere of nitrogen. The reaction was stirred for 0.5 h. and an ethereal solution of diazomethane, prepared from N-methyl-N-nitroso-4-toluenesulfonamide (6.23 g, 29 mmol), was added, dropwise, and the reaction left to warm to room temperature overnight. Saturated ammonium chloride solution (100 ml) was added and the mixture stirred vigorously for 5 mm., then the layers were separated.

Removal of the solvent in vacuo gave a solid residue which was recrystallised from cyclohexane/DCM to yield the product as a pale yellow solid (1.91 g, 77%).

m.p. 122-124°C, (Found: C, 62.88; H, 6.38; N, 11.01. C26H30N406 requires C, 63.15; H, 6.11; N, 11.33%.); Vmax (KBr)/cm1 3296, 2105, 1736, 1689, 1655; 6H (CDCI3) 1.4 (9 H, s), 2.8 and 3.2 (2 H, m), 3.6 (2 H, s), 4.4 (1 H, m), 4.6 (1 H, m), 5.1 (2 H, s), 5.2 (1 H, d), 6.9 (1 H, d), 7.3 (1OH, ArH); 6 (CDCI3) 27.8, 36.2, 38.3, 49.6, 55.8, 67.2, 82.8, 127.3, 128.5, 128.6, 129.1, 129.2, 130.2, 136.0, 136.6, 153.5, 169.4, 170.7, 191.5.

(b) N-Benzyloxycarbonyl-L-phenylalanyl-L-2-amino-5-bromo-4-oxo-norvaflne tert-butyl ester (see 7' above) To a cold (0°C) solution of N-benzyloxycarbonyl-L-phenylalanyl-L-2-amino-5-diazo-4-oxo-norvaline tert-butyl ester (see 6' above) (1 g, 2 mmol) in ethyl acetate (40 ml) was added a 1:1 solution of 48% HBr/acetic acid (2 ml) dropwise. The mixture was stirred for a further 10 mm. and the organic was washed with water (10 ml x3), brine (lOmI) and dried over MgSO4. Removal of the solvent in vacuo gave a solid residue which was recrystallised from cyclohexane to give a white solid (0.915 g, 84%).

m.p. 126-127°C; Vmax (KBr)/crTf1 3294, 1738, 1690, 1654; 6H (CDC13) 1.4 (9 H, s), 2.8 and 3.2 (2 H, m), 3.6 (2 H, s), 3.8 (2 H, s), 4.4 (1 H, m), 4.6 (1 H, m), 5.1 (2 H, s), 5.6 (1 H, d), 6.9 (1 H, d), 7.3 (ION, ArH); 3c (CDCI3) 27.8, 33.7, 38.2, 41.4, 49.3, 55.9, 82.9, 127.3, 128.5, 128.6, 129.1, 129.2, 130.2, 136.0, 136.6, 153.5, 169.4, 170.7, 199.8.

(c) N-Benzyloxycarbonyl-L-phenylalanyl-L-2-amino-5-bromo-4-oxo-norvaline (see 8' above) To a solution of the t-butyl ester (see 7' above) (0.55 g, I mmol) in DCM (10 ml) was added trifluoroacetic acid (0.95 ml, 12.5 mmol) and triethylsilane (0.4 ml, 2.5 mmol).

The reaction was stirred for I.5 h. and the volatiles removed under vacuum. The resulting residue was triturated with ether to give the product as a colourless solid (0.38 g 78%), m.p. 134-136°C, (Found: C, 53.57; H, 4.70; N, 5.65. C22H23BrN2O6 requires C, 53.78; H, 4.72; N, 5.70 %.); Vmax (KBr)/cm1 3295, 1718, 1689, 1654; 8H (CDCI3) 2.8 and 3.2 (2 H, m), 3.3 (2 H, m), 3.7 (2 H, s), 4.5 (I H, m), 4.7 (I H, m), 5.1 (2 H, s), 5.8 (1 H, d), 5.9 (1 H, d), 6.8 (1 H, br), 7.3 (ION, ArH); 5c (CDCI3) 33.7, 38.2, 41.4, 49.3, 55.9, 67.2, 1270, 127.8, 128.1, 128.4, 129.3, 136.1, 136.6, 156.5, 171.4 176.7, 200.1.

(d) N-Benzyloxycarbonyl-L-phenylalanyl-L-2-amino-5-dimethyl-sulfonium-4-oxo-no rvaline bromide salt (see 289' above) The sulfonium salt, N-benzyloxycarbonyl-L-phenylalanyl-L-2-amino-5-dimethyl-sulfonium-4-oxo-no rvaline 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, 18%).

m.p. 98°C (dec.); Vmax (KBr)/cm1 1716, 1689, 1669; 8H (d4 methanol) 2.8 and 3.2 (2 H, m), 3.2 (6 H, s), 3.8 (2 H, s), 4.7 (1 H, m), 4.9 (1 H, m), 5.1 (2 H, s), 5.9 (1 H, d), 6.5 (1 I-I, d), 7.3 (10 H, ArH). 3c (d4 methanol) 25.4, 38.8, 43.9, 49.8, 57.8, 67.5, 68.1, 127.8, 128.5, 128.9, 129.5, 130.3, 136.1, 136.6, 158.3, 173.1, 174.1, 200.1.

MS: m/z Caic. for C24H29Br N205S 552 (M-Br=473) Observed: 473 Synthesis of a higher homologue of N-Benzyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine bromide salt (see Compound 287' below) The acid was prepared from 6-diazo-N-(9-fluorenylmethyloxycarbonyl)-5-oxo-L-norleucine ethyl ester (2.53 g, 6 mmol) by the method of Coutts et at Yield after flash column chromatography (ethyl acetate 100%) 1.64 g, 66%.

(a) N-(-9-Fluorenylmethyloxycarbonyl)-L-2-amino-7-diazo-6-oxo-heptanoic acid-I -ethyl ester To a cold (0°C) solution of the acid (0.94 g, 2.3 mmol) in DCM (24 ml) was added oxalyl chloride (1.725 ml of a 2M solution in DCM, 3 3.45 mmol) dropwise. The reaction was warmed to room temperature and stirring continued for a further 40 mm.

The reaction was again cooled to 0°C and oxalyl chloride (1.725 ml of a 2M solution in DCM, 3.45 mmol) added dropwise. The reaction was warmed to room temperature and stirring continued for a further 2 ft The volatiles were removed under reduced pressure to give a yellow solid. The solid was dissolved in TI-IF/acetonitrile (1:1 24 ml) and cooled in an ice bath under a blanket of nitrogen. To the solution was added trimethylsilyldiazomethane (4.6 ml of a 2M solution in hexane, 9.2 mmol) dropwise and the reaction stirred at 0°C for 11/2 h. To the mixture was added saturated ammoniurn chloride solution and the phases separated. The organic was washed with 10% Na2CO3 (5 ml, x3), brine (5 ml) and dried over Na2SO4. Removal of the solvent in vacuo gave an orange oil which was purified by flash column chromatography (3:2 petrol/ethyl acetate) to afford the product as a pale yellow solid (0.65 g, 65%).

m.p 123-124°C (CCL, dec.) Vmax (KBr)/cm1 3354, 2103, 1739, 1686, 1635; 3H (CDCI3) 1.3 (3 H, t), 1.7, 1.9 and 2.3 (6 H, m), 4.2 (3 H, m), 4.4 (3 H, m), 5.2 (1 H, s), 5.4 (1 H, d), 7.3 (4 H, m, ArH), 7.6 (2 H, ArH), 7.8 (2 H, m, ArH); S (CDCI3) 14.1, 20.6, 32.0, 40.1, 47.1, 53.5, 54.5, 61.6, 67.0, 120.0, 125.1, 127.0, 127.7, 141.2, 143.8, 155.9, 172.2, 192.4.

(b) L-2-amino-7-diazo-6-oxoheptanoic acid 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) Vmax (KBr)/cm1 3436, 2108, 1630 (Weygand at at, Chem. Ber. 91, 1037-40).

(c) N-Benzyloxycarbonyl-L-phenylalanyl-7-bromo-6-oxo-heptanoic acid The dipeptide was prepared by the method previously described to give a colourless solid (93 mg, 66%).

m.p. 108-110 °C (ethyl acetate), Vm (KBr)/crri1 3296, 1715, 1700, 1661; 8H (d3 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 H, s), 7.3 (10 H, ArH), 7.4 (1 1-1, d); 8c (d3 methanol) 21.0, 31.8, 39.7, 53.2, 57.7, 56.3, 67.5, 127.7, 128.6, 128.9, 129.4, 130.4, 138.1, 138.5, 158.2, 174.2, 174.8, 203.4.

MS: m/z Calcd for C24H27BrN2O6: 518 (M-Br 439). Observed 519, 439.

(d) N-Benzyloxycarbonyi-L-phenylalanyl-7-dimethylsulfonium-6-oxo-heptanoic acid bromide salt (Compound 288') The sulfonium salt was prepared as previously described, to give a colourless hygroscopic solid (48 mg, 57%).

m.p. 94-96°C (dec.), Vmax (KBr)/cm1 3296, 1715, 1700, 1661; 5H (d5 acetone) 1.7, 1.9 and 2.7 (6 H, m), 2.8-3.1 (2 H, m), 2.9 (6 H, s), 4.4 (2 H, m), 4.9 (2 H, d), 5.0 (2 H, s), 7.3 (10 H, ArH); S (d5 acetone) 20.5, 25.3, 31.7, 39.0, 41.6, 53.1, 53.2, 57.7, 67.5, 127.7, 128.5, 128.9, 129.4, 129.5, 130.4, 138.2, 138.5, 158.2, 174.3, 174.7, 202.3.

MS: mlz Calcd for C26H33BrN2O6S: 580 (M-Br = 501). Observed 501.

EXAMPLE 2-INHIBITION OF TGAsE ACTIVITY The efficacy of 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 N,N'-dimethyl casein (DMC).

Experiments were performed as follows: Inhibition of guinea pig liver TG (TGase) was tested using an enzyme-linked sorbent assay (ELSA) based on the incorporation of biotin cadaverine (BTC) into N, N'-dimethylcasein (DMC). Microtitre plates (96-well) were coated with 100 p1 of 10 mg/mt DMC in 10 mM Iris p1-I 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 CaCl2, 5 mM DII and 0.132 mM BTC in 50 mM Tris pH 7.4. The mix was prepared so that the appropriate final concentrations would be achieved upon addition of j.xl of 200 ig/ml TOase to 990 p1 of mix to start the reaction. TGase inhibitors were initially prepared as 100 mM stock solutions in H20 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 mM CaCl2.

Following addition of IGase, 100 p1 of solution was pipetted into B replicate wells per sample, and the reaction was allowed to proceed for 1 hour at 37°C. The reaction was terminated by removal of the solution and the addition of 100 p1 of 10 mM EDTA in PBS pH7.4. Plates were again washed twice with TBS-Tween pH 7.4, once with lBS pH 7.4, and blocked by incubation with 100 p.1 per well of 3%(w/v) bovine serum albumin (BSA) in PBS pH7.4 for 1 hour at room temperature. Incorporated BTC was detected by incubation with 100 p1 per well of Extravidin peroxidase (EXAP) solution, diluted I in 5000 in blocking buffer for 1 hour at 37°C. Plates were washed as before and prior to development, plates were preincubated for 5 minutes in 0.05M phosphate-citrate buffer pH5.0 containing 0.014% (v/v) H202. The solution was removed and replaced with 100 p.1 per well of the same buffer containing 75 p.g/ml tetramethylbenzidine (TMB). The developing reaction was allowed to proceed at room temperature for 5-15 minutes and was terminated by the addition of 50 p1 of I N H2S04. The absorbance of the resulting colour was measured on a microtitre plate reader at 450 nm.

The data shown indicate a representative experiment using eight replicate samples. The mean absorbance 450nm ± SD is shown.

The effect of exemplary compounds of the invention (and control compounds) on tissue transglutaminase activity in vitro are shown in Figures 2 to 21.

EXAMPLE 3-INHIBITION OF IGASE-MEDIATED PROTEIN CROSS-LINKING Assay method 1. Preactivate TGase in 3mM DTT (where applicable) on ice for 1 hr.

2. Crosslink TGase with fibronectin in 40Mm Tris/lOOmM NaCI at 50 pg/mI final Concentration each, according to Table I below. Include non-activated Tgase/ preactivated Igase controls in the presence and absence of the inhibitors to investigate potential homodimer formation.

3. Incubate at 37 °C for 2hr to allow crosslink formation to take place.

4. Solubilise crosslink in 2x Laemmli buffer. VorteX and spin down insoluble material.

5. Load 20 pg of total protein (Tgase+ Fn) on a 7% acrylamide SDS PAGE gel. Run gel at 100 my until dye escapes from the bottom of the gel.

6. Stain with coomassie brilliant blue for 1 hr at RT.

7. De-stain in 30% methanol/I 0% acetic acid at RT.

TABLE I

ci) ci) a a a a U) U) (U (U 0 0 0 0 (flU U) (D a)Ct) cuL C C C0 C r rç o o"1 004 0C'J .S LL LL. LL LL ..»= »= o + +g ÷a +a 2Sc EtS 2t 2 Ecu c c *.1 t o etc e' e' e-ect cO O Component oo oE o2 Qfl o O O OZEQO.. Uo..-OLL OF-I-+ F-+ t-+ Tris pH 7.4 40 40 40 40 40 40 40 40 (mM) ___ ___ ___ ___ ___ ___ ___ ___ NaC1 100 100 100 100 100 100 100 100 (mM) ____ ____ ____ ____ ____ ____ ____ ____ TGase 50 50 50 50 50 50 50 50 (pg/mi) ______ ______ ______ ______ ______ ______ ______ Fibronectin 50 50 50 50 50 50 Jpg/mj) ____ ____ ____ ____ ____ ____ ____ ____ Compound 250 281 (pM) ______ ______ ______ ______ ______ ______ ______ ______ Compound 250 283 (pM) ______ ______ ______ _____ ______ ______ ______ ______ Compound 250 250 285 (pM) ______ ______ ______ ______ ______ ______ ______ ______ Compound 281 = N-Benzyioxycarbonyi-L-phenyialanyl-6-dimethyi-suifonium-5-oxo-L-norleucine bromide salt Compound 283 = 1,3-dimethyl-2-(2-oxopropylsulfanyi)-3H-1,3-diazoi-1 -ium-chioride (as disclosed in US 4,968,713) Compound 285 = N-Benzyloxycarbonyi-L-glutaminyl-6-dimethylsuifonium-5-oxo-L-norleucine bromide salt SDS-PAGE data showing tTGase-mediated crosslinking of fibronectin following treatment with exemplary compounds of the invention is shown in Figure 22.

EXAMPLE 4-INHIBITION OF KIDNEY FIBROSIS IN RATS Method for inhibitor delivery using osmotic minipumps Male Wistar rat of approximately 300g weight was anaesthetised using 5% halothane and maintained at 3% for the duration of the surgical procedure. The rat was subjected to a 5/6th subtotal nephrectomy (SNx) by ligation of the left renal artery and vein followed by complete nephrectomy of the left kidney. The right kidney had both the upper and lower poles ligated followed by excision of both poles. A 9-cm cannula (0.32mm bore) was sealed on one end and fenestrated between 3 and 12 mm from the sealed end. This was then inserted through the parenchyma (cut to cut I pole to pole) of the kidney so that the blunt end was just visible through one of the cut ends. This was then sealed in position using tissue glue on both ends of the kidney such that the fenestrated area was within the remnant kidney. The cannula was passed through the muscle wall, which was then stitched using reabsorbable sutures. The cannula was then attached to the regulator of a 2 ml osmotic minipump (Azlet osmotic minipump (2m14), Charles Rivers, UK) that was loaded (primed for 15 hrs at RI) 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 p1 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 eta!., 1997, 99:2950-2960) or collagen Ill staining.

For collagen Ill staining, paraffin embedded sections (4pm) were first dewaxed and hydrated by standard protocol (xylene 10 mm, 100% ethanol 5 mm, 90% ethanol 5 min,75% ethanol 5 mm, 50% ethanol 5 mm, water 10 mm) washed in PBS for 10 mm and any endogenous peroxides quenched by treatment with 3% H202 in methanol for 10 mm.

After washing in PBS for 10 mm sections were treated with the epitope revealing agent TUF (ID Labs Inc. Cat no BP1122) on a water bath at 92 C for 10 mm then allowed to cool to room temperature. Sections were washed with PBS for 10 mm and then trypsin (Zymed Labs Cat No 00-3008) digested (trypsin diluted 1:3) for iü mm at 37 C followed by two washes in PBS for 5 mm each. Sections were then blocked in goat serum (Vector Labs Cat No S1000) incubated at 37 C for 30 mm. The primary collagen Ill antibody (Goat anti-human type Ill collagen, Soutern Biotech Assocs diluted I in 10 in 0.1% bovine serum albumin [BSAJ in PBS) is then added and incubated overnight at 4 C in a humidity chamber. The samples are then washed twice with 0.1% Nonidet in PBS for s mm followed by two washes in PBS for 5 mm. The secondary antibody (rabbit anti goat which is biotinylated from DAKO Cat No E0466) diluted I in 400 in 0.I%BSA/PBS is then added and incubated for 30 mm at 37 C. The sections are then washed twice in 01% 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 30mm at T 370 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 5-30mm). After washing twice with water for 5 mm and then twice with PBS for 5 mm the samples are counterstained with haematoxylin (diluted I in 10 from Thermo Shandon, Gill-2 haematoxylin Cat No 6765007) for 5 mm, washed twice with water for 5 mm, washed with PBS once and then sections mounted using glycergel prior to viewing under a light microscope.

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 at, 1999, J. Am. Soc. Nephrot 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 Ill 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. For Masson's Trichrome staining, 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 ÷1-S.E.M. The composite diagram showing staining in Figure 12(a) shows I field from each animal. For Collagen Ill staining the relative amounts of collagen Ill present (stained brown) were determined by systematically acquiring data from 10 overlapping cortical fields at 200x magnification and expressed as Mean values ± SEM.

in situ TGase activity in kidney cryostat sections Rat kidneys treated in vivo with TGase inhibitors were snap-frozen in liquid nitrogen and 14 pm 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% (wlv) rabbit serum, 10 mM EDTA, 0.01% (vlv) Triton X-100 in 50 mM Tris pH7.4, containing EXAP (diluted I in 200) to block endogenous biotin. Following rehydration, slides were washed twice in PBS p1-17.4, and sections were incubated for 1 hour at 37°C with a reaction mix containing 5 mM CaCI2, 5 mM DTT and 0.5 mM BTC in 50 mM Tris pH 7.4. Negative controls consisted of mixes that did not contain BTC, and where 10 mM EDTA was substituted for 5 mM CaCl2. A positive control was also included that contained 20 pg/mI TGase. Following incubation, slides were washed once in PBS pH 7.4 containing 10 mM EDTA, fixed in ice-cold acetone for 5 minutes and allowed to air-dry. Dried sections were blocked in 3%(wlv) BSA in PBS pH7.4 overnight at 4°C, and incorporated BTC was revealed by incubation with Streptavidin-Cy5, diluted I in 100 in the same buffer for 2 hours at 37°C. Slides were viewed on a Leica TCSNT confocal microscope equipped with excitation and emission filters for Cy5, and emitted fluorescence was quantified with the software supplied by the manufacturer. Figure 25a shows semi-quantitative analysis of the emission from Leica confocal laser microscope from TRITC-extravidin bound to TGase incorporated biotin cadaverine in cryostat sections taken from kidneys of SNx rats treated for 28 days with the inhibitors 281 and 283. 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 +1-SEM taken from 5 separate kidneys.

Analysis of 14C putrescine incorporation A second method of assaying TGase activity, 14C putrescine incorporation into N,N'-dimethylcasein using tissue homogenates of kidneys from SNx rats treated with the inhibitors for 84 days, confirmed the effect of treatment with compounds 281 and 283 on Tgase activity (see Figure 25b).

Putrescine incorporation experiments were performed as described in Skill et at, 2001, Lab. Invest 81:705-7 16 and Lorand eta!., 1972, Anal Biochem 50:623-631 Analysis of proteinurea, creatinine clearance, serum creatinine, urine creatinine and urine urea 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.

TABLE 2

Proteinuria Creatinine Serum creatinine (mg/24 h) clearance (mM/L) ________ ________ (mI/mm) ________ ________ Expenment Group Mean -SE Mean SE Mean SE Control (SNc) 129 14 1.72 0.18 46 0.6 SNx 672 140 0.44 0.2 224 36 SNx+283 835 -93 0.86 0.05 114 16 SNx+281 503 63 0.94 0.09 208 106 Urine creatinine Serum urea Urine urea (mMIL) (mM/L) -(mM/L) Experiment Mean SE Mean SE Mean SE Group _______ _______ _______ _______ _______ _______ Controf(p) 12399 2538 6.22 1.1 1160 226 SNx 2551 695 31.4 4.6 244 29 SNx+ 283 3138 185 16.3 2.2 272 20 SNx+281 3999 560 22.32 4.5 372 50 Proteinuria, creatinine clearance, serum clearance, urine creatinine and urine urea were carried out by standard clinical chemistry techniques (Johnson et at, 1997, J Clln. Invest 99:2950-2960). Creatinine and urea were measured by the standard autoanalyser technique and proteinura by the Biuret method (Johnson et at, 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.

EXAMPLE 5 -INHIBiTION OF ANGIOGENESIS -IN VITRO

introduction

Angiogenesis is the formation of new capillaries from already existing vessels and is a normal and vital process in growth and development. During 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].

The correct interactions among endothelial cells with the pericytes (fibrocytes) and surrounding cells and their association with the extracellular matrix (ECM) and the vascular basement membrane (BM) are crucial for the angiogenic process in health and disease[2]. Angiogenesis occurs under numerous physiological conditions, such as during wound healing, reproduction-associated neovascularisation, development of collateral circulation following tissue grafting and ischemic episode. 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. In initiating this process, 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 Iaminin 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]. 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 (SM) 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. In addition, 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 SM 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].

Different cells secrete different patterns of matrix proteins and likewise not all vascular basement membranes are the same.

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]. In fibrotic conditions, increased TG2 expression not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins paralleled by a NF-kappaB-dependent increase in the expression of transforming growth factor (TGF) beta I, a major regulator of the ECM turnover and cell proliferation [II]. More recently, transglutaminase 2 (TG2) has been identified as one of the key mechanisms of recruitment of this cytokine by crosslinking of the large-latent TGF-beta to the ECM [12].

Here, we provide compelling evidences for an active role of transglutaminases in angiogenesis, notably through its crosslinking activity. Data presented here demonstrate the utility of specific tissue transglutaminase inhibitors as anti-angiogenic compounds.

Materials And Methods Reagents Inhibitors R283 (positive control) and R294 (Le. compound 294' above) were synthesized as described before [13]. The lyophilized compounds were dissolved in DMSO (20%) at a stock concentration of 100 mM. For the different treatments the final concentration of inhibitors used was 500, 100 or 50 pM, 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 CD3I 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-I (R3-IGF-I), heparin, and gentamicin sulphate amphotericin-B (GA-100) (EGM-2 bulletkit, Lonza Wokingham, England, UK). Cells were maintained subconfluent at 37°C in a humidified incubator at 5% C02 atmosphere and media was changed every two days. In the different experiments, cells were used after 3 passages and up to 6.

Co-culture angiogenesis assay I Pooled Human endothelial cells (HUVEC) and dermal foreskin fibroblast were obtained from TCS Cellworks (V2a AngioKit, TCS Cellworks, Buckingham, UK) in 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 (500pM) , or the in house site-directed synthesized TG inhibitor R294 (Compound 294,500pM), every two days [13]. Control experiments were performed using the inhibitors' vehicle DM50 (0.1%), or the VEGF angiogenesis promoter (2 ng/mL) as positive control, and the Suramin compound (50 pM), which blocks the binding of various growth factors and was used as negative control.

At day 12, cells were fixed in cold Ethanol (70%) at room temperature for 30 minutes, and washed three times with PBS (1 %BSA) for 5 minutes. To visualize the tubule development in the co-cultures cells were incubated for 60 minutes at 37°C with a mouse anti-human CD3I primary antibody (V2a Cellworks kit) in Dulbecco's Phosphate-Buffered saline supplemented with 1% BSA. After this period the primary antibody solution was removed and wells washed three times with PBS (1%BSA) before incubation with the secondary antibody (goat anti-mouse lgG alkaline phosphatase conjugate, V2a Cellworks kit) for another 60 minutes at 37°C. 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 In duplicate experiments, co-cultures were incubated with FITC-Cadaverine (8 pM) for 12 hours prior to cell fixation. Cells were stained with CD3I antibody as described above, but using as secondary antibody TRITC-labelled goat anti mouse immunoglobulins (DAKO, Denmark).

Co-culture angiogenesis assay /1 To evaluate the effect of R294 in the mid stage of tubulogenesis, the co-culture of HUVEC and dermal foreskin fibroblast from V2a AngioKit (TCS Cellworks) was 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. Afterwards, DMSO was replaced in the growth media by the R294 inhibitor (50 pM) up to day 12. Controls consisted on vehicle (DMSO, 0,01%), and R294 (50 pM) added starting from day 1, at every change of media and for all the period that the co-culture lasted (12 days). In the different settings, cells were fixed at Day 6 or Day 12, stained for CD3I expression and quantified for tubule formation with TCS Cellworks AngiosSys Image Analysis Software as described above.

Co-culture angiogenesis assay Ill In parallel experiments, the endothelial differentiation of HUVEC cells in the co-culture system V2a-AngioKit was performed using V2a growth media alone, or further supplemented with VEGF (2 ng/mL). The effect of R294 on VEGF-stimulated co-culture was evaluated by adding R294 (5OpM) or R294's vehicle (DMSO 0.01%) concomitantly with VEGF (2ng/mL) at every change of media, starting from day I up to day 12. Cells were fixed at Day 12, stained for CD3I 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 pL loaded per well in 96-well plates. Plates were transferred to a cell culture incubator and incubated at 37°C for 30 mm to allow the basement membrane to gel. Exponentially growing 1-IUVEC cells, nearly confluent (80%), were trypsinized and i.5x105.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% CO2 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 pM Calcein AM at 37°C and in 5% CO2 for 15 mi observed and photographed using a fluorescent inverted microscope with 520 nm emission filter.

To study the dynamic behaviour of endothelial cells in presence or absence of the R294 TG2 inhibitor, 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-i 00 as described above, was complemented with the compounds to test: TG2 inhibitor R294 (500pM), Suramin (5OpM) as negative control, and vehicle DMSO (0.1 %), respectively added before seeding cells into the wells.

Statistical analysis All the quantitative data were analyzed using the commercial program GraphPad PRISM 4.0 (GraphPad Prism, San Diego, CA, USA). All values are given as means ± S.D. For statistical evaluation comparison between groups were analysed by ANOVA, followed by Tukey's Multiple Comparison post-test. The P value 0.05 and lower was considered significant.

Results Active site-directed irreversible inhibitors of transglutaminase inhibit in vitro differentiation of human umbilical vascular endothelial cells (HUVEC) in both, a coculture angiogenesis assay and in a Matrigel system.

1.1 In vitro angiogenesis assay using a co-culture system 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 SuM for Factor XIII greater than 200uM) than for Factor XIII (plasma transglutaminase), whereas R283 has equal potency (IC 50 approx 4uM)for both enzymes.

R283 6:294 The regular addition of either R283 or R294 inhibitors to the growth medium of co-cultured endothelial and fibroblast cells at every change of media and for a 12 day period that the co-culture lasted resulted in a dramatic inhibition of the tubule formation capacity of endothelial cells (Figure 27 A, B, C upper panels). This effect was accompanied by the inhibition of the incorporation of FITC-cadaverine (a fluorescently labelled amine substrate of TG that gets incorporated into available peptide bound gamma-glutamyl residues) along the periphery of the endothelial formed tubular structures (Figure 27 A, B, C lower panels). Experiments conducted with the addition of either increased vascular endothelial growth factor (VEGF), a known promoter of angiogenesis, or suramin, an inhibitor of several growth factor receptors, were used as positive and negative control, respectively (Figure 27 D,E). Since R294 inhibited the incorporation of FITC-cadaverine, this indicates that the TG activity observed in the absence of the inhibitor is extracellular.

1.2 In vitro angiogenesis assay using Matrigel as basement membrane The in vitro formation of capillary-like tubes by endothelial cells in presence of transglutaminase inhibitors was also evaluated using a single cell culture angiogenesis system. Differentiation of endothelial cells into tubular structures was induced by seeding HUVEC cells on a gelled basement membrane matrix (Matrigel) which is a basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma cells.

This in vitro assay is considered as a powerful and rapid method to screen for various factors that promote or inhibit angiogenesis.

In control experiments, the Matrigel-seeded endothelial cells initially attached in the first hour and then migrate toward each other over the next 2 hours, formation of capillary-like tubes was already observed after 4 h and they were mature by 6 hours (Figure 28 A).

Interestingly, when cells are treated with R294 (500pM) a reduced capacity to align and form anatomising tubes was observed (Figure 28 B and C, respectively). Since the doubling time of the cells is approximately 30 hours, we can rule out any effect of the inhibitor on cell proliferation.

All together, these observations suggest a relevant role of the extracellular tissue transglutaminase crosslinking activity in angiogenesis. In the co-culture system, we found the extracellular TG2 to be highly active as measured by FITC-cadaverine incorporation and limited to the area that surrounds the endothelial capillary-like structures. Together with data obtained from the Matrigel angiogenesis assay, the active extracellular enzyme is likely to be delivered by the endothelial cells, or at least in a more relevant and higher extent than what the peripheral fibroblasts could supply. This assumption is made by the fact that the differentiation of HUVEC cells in the absence of fibroblasts, when seeded on a gelled extracellular matrix, can be also affected by the presence of the R294 cell-impermeable TG2 inhibitor.

The TG2 inhibitor R294 affects established active sprouting eridothelial cells in co-cultured angiogenesis assay affecting angiogenesis Active sprouting endothelial cells in the co-culture system were exposed to R294 (5OpM) 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 I to day 12. After treatment, endothelial cells were stained for the CD3I marker and the tubule formation analysed. The initial normal course of the tubule formation (day I to day 6) was consistently affected by the addition of R294 in the late stage of the co-culture (day 6 to day 12) in terms of the number of tubule junctions and tubules formed (Table 3).

Apparently, a regression or destabilization of outgrowing sprouts has occurred due to the presence of the inhibitor. The net formation of luminar structures or anastomosis was also significantly reduced with respect to control groups without the inhibitor. The positive control group where the addition of R294 was initiated since Day I showed a massively reduced tube formation at SOpM The potent pro-angiogenic effect of VEGF on endothelial cells can be partially reversed by P294 in the co-culture model of angiogenesis The most potent pro-angiogenic factor described to date are the vascular endothelial growth factors, in particular VEGF-A, capable of inducing proliferation, sprouting and tube formation of endothelial cells [16]. VEGF, either cleaved and liberated from the ECM by plasmin and MMPs or actively secreted by surrounding cells, can bind to endothelial cells and signal through kinase tyrosine receptors VEGFR and co-receptors including heparin sulphate proteoglycans and neutrofilins [17, 18].

The inhibitor R294 was added to the growth media of the co-culture system, alone or in combination with VEGF (2 nglmL). 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 (overleaf) shows the effect of tissue transglutaminase inhibitor R294 on angiogenesis in HUVEC and Dermal Foreskin fibroblast co-culture assay. Co-cultures treated with the solvent of the inhibitor (DMSO 0.01%) were considered as control group.

Treatments consisted of addition of the inhibitor R294 (5OpM) from Day I 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. In the different experimental conditions cells from day 6 and cells from day 12 of treatment, were fixed in ethanol, stained for CD 31 antigen as described in Materials and Methods and tube formation was scored according to the tubule Area, the number of junctions, the total number of tubules, the total tubule length and the mean tubule length.

Table 3

Effect of tissue transglutaminase inhibitor R294 on angiogenesis in HUVEC and Dermal Foreskin fibroblast co-culture assay Day 6 A Number of Number of Total tubule Mean tubule rea x / junctions tubules length (x103) length Control 81±3,8 68±2,3 162±9,5 8,2±0,1 51±2,7 R294* 84±3,2 71±2,8 157±5,7 7,8±0,1 50±1,2 R294** 58±4,0 34±4,8 107±12 7,9±0,1 51±12 Suramin 29±4,6 25±5,1 87,5±9,2 2,8±0,5 35±1,5 Day 12 Number of Number of Total tubule Mean Area (xl O) junctions tubules length (xl Q3) Control 162±1,4 118±37 286±47 14±3,8 50±7 R294* 127±2,3 79±19 164±19 11±1,4 60±9 R294** 46±8,2 16±11 63±15 3,8±0,6 50±5,3 Suramin 22±7,6 7,5±3,1 48±7,6 1,2±0,1 33±1,4 Calibration 1 pixel * treatment with R294 (5OpM) from Day 6 to Day 12 (during the absence of treatment vehicle, DMSO 0.01%, was used) ** treatment with R294 (5OpM) from Day ito Day 12.

Table 4 (below) 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 (DMSO 0.01%) were considered as control group. Treatments consisted of addition of the angiogenesis promoter VEOF (2ng/mL), or inhibitor R294 (50 pM) or combination of both, VEGF and R294, from Day I to Day 12 of the co-culture. At Day 12 cells were fixed in ethanol, stained for CD 31 antigen as described in Materials and Methods and tube formation was scored for the tubule Area, the number of junctions, the total number of tubules, the total tubule length and the mean tubule length.

Table 4

Influence of tissue transglutaminase inhibitor R294 on VEGF stimulated angiogenesis in HUVEC and Dermal Foreskin fibroblast co-culture assay.

Area Number of Number of Total tubule Mean tubule (x103) junctions tubules length (x103) length Control 126±4,7 116±27 262±31 15±1,2 51±4,2 VEGF 254±3,3 415±68 796±115 26±2,5 33±4,2 R294 + VEGF 207±7,2 281±32 593±43 20±2,3 39±8,3 R294 52±10 22±10 76±19 5,7±1,1 49±7,5 -References 1. Jam, R.K., Molecular regulation of vessel maturation. Nat Med, 2003. 9(6): p. 685-93.

2. Yancopoulos, G.D., S. Davis, N.W. Gale, J.S. Rudge, S.J. Wiegand, and J. Holash, Vascular-specific growth factors and blood vessel formation. Nature, 2000.

407(6801): p. 242-8.

3. Jam, R.K., Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy Science, 2005. 307(5706): p. 58-62.

4. Hughes, C.C., Endothelial-stromal interactions in angiogenesis. Curr Opin Hematol, 2008. 15(3): p. 204-9.

5. Rhodes, J.M. and M. Simons, The extracellular matrix and blood vessel formation: not just a scaffold. J Cell Mol Med, 2007. 11(2): p. 176-205.

6. Davis, G.E. and D.R. Senger, Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stab ilization. Circ Res, 2005. 97(11): p. 1093-107.

7. Darland, D.C. and P.A. D'Amore, Blood vessel maturation: vascular development comes of age. J Clin Invest, 1999. 103(2): p. 157-8.

8. Yurchenco, P.D., P.S. Amenta, and B.L. Patton, Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol, 2004.

22(7): p. 521-38.

9. Jones, R.A., P. Kotsakis, 1.8. Johnson, D.Y. Chau, S. All, G. Melino, and M. Griffin, Matrix changes induced by transglutaminase 2 lead to inhibition of angiogenesis and tumor growth. Cell Death Differ, 2006. 13(9): p. 1442-53.

10. Fisher, M., R.A. Jones, L. Huang, J.L. Haylor, M. El Nahas, M. Griffin, and T.S.

Johnson, Modulation of tissue transglutaminase in tubular epithelial cells alters extracellular matrix levels: a potential mechanism of tissue scarring. Matrix Biol, 2009. 28(1): p. 20-31.

11. Telci, D., R.J. Collighan, H. Basaga, and M. Griffin, Increased TG2 expression can result in induction of transforming growth factor beta 1, causing increased synthesis and deposition of matrix proteins, which can be regulated by nitric oxide. J Biol Chem, 2009. 284(43): p. 29547-58.

12. Huang, L., J.L. Haylor, M. Fisher, Z. I-Iau, A.M. El Nahas, M. Griffin, and T.S.

Johnson, Do changes in transglutaminase activity alter latent transforming growth factor beta activation in experimental diabetic nephropathy? Nephrol Dial Transplant. 25(12): p. 3897-910.

13. Griffin, M., A. Mongeot, R. Collighan, R.E. Saint, R.A. Jones, 1G. Coutts, and D.L.

Rathbone, Synthesis of potent water-soluble tissue transglutaminase inhibitors.

Bioorg Med Chem Left, 2008. 18(20): p. 5559-62.

14. Arnaoutova, I. and H.K. Kleinman, In vitro angiogenesis: endothellal cell tube formation on ge/led basement membrane extract Nat Protoc. 5(4): p. 628-35.

15. Baumgartner, W., N. Golenhofen, A. Weth, 1. Hiiragi, R. Saint, M. Griffin, and D. Drenckhahn, Role of transglutaminase I in stabilisation of intercellular junctions of the vascular endothelium. Histochem Cell Biol, 2004. 122(1): p. 17-25.

16. Ferrara, N., Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev, 2004. 25(4): p. 581-611.

17. Ferrara, N., H.P. Gerber, and J. LeCouter, The biology of VEGF and its receptors.

Nat Med, 2003. 9(6): p. 669-76.

18. Park, J.E., G.A. Keller, and N. Ferrara, The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithellal extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell, 1993. 4(12): p. 1317-26.

EXAMPLE 6 -INHIBITION OF ANGIOGENESIS -IN VIVO Methods and Materials The chorioaallantoic membrane (CAM) assay was used to test the effects of the compounds on angiogenesis in vivo.

Fertilized I 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 tight 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 lOml 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. Once 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). After removal of the needle 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 (lOOuM) or PBS in DMEM medium containing 10% (vlv) 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.

Treatment with the IGase inhibitor Compound 294 resulted in a pronounced inhibition of angiogenesis, as evidenced by the significant reduction in vascular outgrowths in the egg.

EXAMPLE 7 -EXEMPLARY PHARMACEUTICAL FORMULATIONS The following examples illustrate pharmaceutical formulations according to the invention in which the active ingredient is a compound of the invention.

Example A: Tablet

Active ingredient 100 mg Lactose 200 mg Starch 50 mg Polyvinylpyrrolidone 5 mg Magnesium stearate 4 mg 359 mg Tablets are prepared from the foregoing ingredients by wet granulation followed by compression.

Example B: Ophthalmic Solution Active ingredient 0.5 g Sodium chloride, analytical grade 0.9 g Thiomersal 0.001 g Purified water to 100 ml pH adjusted to 7.5 Example C: Tablet Formulations The following 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.

Formulation A

mg/tablet mg/tablet

Active ingredient 250 250 LactoseB.P. 210 26 Povidone B.P. 15 9 Sodium Starch Glycolate 20 12 Magnesium Stearate 5 3 500 300 Formulation B mci/tablet mci/tablet Active ingredient 250 250 Lactose 150 -Avicel Fl-I lOl® 60 26 Povidone B.P. 15 9 Sodium Starch Glycolate 20 12 Magnesium Stearate 5 3 500 300 Formulation C

mg/tablet

Active ingredient 100 Lactose 200 Starch 50 Povidone 5 Magnesium stearate 4 359 The following 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.

Formulation D mg/capsule Active Ingredient 250 Pre-gelatinised Starch NF15 150 400 Formulation E mqlcapsule Active Ingredient 250 Lactose 150 Avicel® 100 Formulation F (Controlled Release Formulation) 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.

rngiiablet Active Ingredient 500 Hydroxypropylmethylcellulose 112 (Methocel K4M Premium)® Lactose B.P. 53 Povidone B.P.C. 28 Magnesium Stearate 7 Drug release takes place over a period of about 6-8 hours and was complete after 12 hours.

Example D: Capsule Formulations Formulation A 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.

Formulation B mg/capsule (a) Active ingredient 250 (b) Lactose B.P. 143 (c) Sodium Starch Glycolate 25 (d) Magnesium Stearate 2 Formulation C mg/capsule (a) Active ingredient 250 (b) Macrogol 4000 BP 350 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 Active ingredient 250 Lecithin 100 Arachis Oil 100 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) The following 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.

mg/capsule (a) Active ingredient 250 (b) Microcrystalline Cellulose 125 (c) Lactose BP 125 (d) Ethyl Cellulose 13 Example E: Injectable Formulation Active ingredient 0.200 g Sterile, pyrogen free phosphate buffer (pH7.0) to 10 ml The active ingredient is dissolved in most of the phosphate buffer (35-40°C), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.

Example F: Intramuscular injection Active ingredient 0.20 g Benzyl Alcohol 0.10 g Glucofurol 75® 1.45 g Water for Injection q.s. to 3.00 ml 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).

Example G: Syrup Suspension Active ingredient 0.2500 g Sorbitol Solution 1.5000 g Glycerol 2.0000g Dispersible Cellulose 0.0750 g Sodium Benzoate 0.0050 g Flavour, Peach 17.42.31 69 0.01 25 ml Purified Water q.s. to 5.0000 ml The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In 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: Suppositoiy mg/suppository Active ingredient (63 pm) 250 Hard Fat, BP (Witepsol HIS -Dynamit Nobel) 1770 One fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45EC maximum. The active ingredient is sifted through a 200 pm sieve and added to the molten base with mixing, using a silverson filled with a culling head, until a smooth dispersion is achieved.

Maintaining the mixture at 45°C, the remaining Witepsol HiS 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.

Example I: Pessaries

mqlpessarv Active ingredient 250 Anhydrate Dextrose 380 Potato Starch 363 Magnesium Stearate 7 The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.

Claims (25)

1. CLAIMS1. A tissue transglutaminase inhibitor for use in inhibiting angiogenesis in a patient.
2. 2. A tissue transglutaminase inhibitor according to Claim I wherein the inhibitor is irreversible.
3. 3. A tissue transglutaminase inhibitor according to Claim 2 wherein the inhibitor is selected from the group consisting of: (a) peptide compounds (for example, see Claim 8 below); (b) imidazole compounds (for example, see Compound 283 [R283] below; see also US 5,030,644); (c) dihydroisoxazole compounds, such as KCAO75 and KCCOO9 (for example, see Hausch et al., 2003, Chem. BiotlO:225-31); (d) cinnamoyl inhibitors (for example, see US 20100204280 to Keillor et a!.); (e) thieno[2,3-dJ pyrimidine-4-one acylhydrazide derivatives (for example, see Duval eta!., 2005, Biog,Med Chem.Lett 15:1885); and (f) iodoacetamide (for example, see Folk & Cole, 1966, J Chem. Blot 241:3238-3240).
4. 4. A tissue transglutaminase inhibitor according to Claim I wherein the inhibitor is reversible.
5. 5. A tissue transglutaminase inhibitor according to Claim 4 wherein the inhibitor is selected from the group consisting of: (a) monodansylcadaverine; (b) tTGase cofactors and analogues thereof, such as GTP, GDP, GTPyS and GMP-PCP; (c) Ca2 chelators, such as EDTA; (d) Zn2 metal ions; and (e) acylideneoxoindole compounds (see KIOck et at, 2011, Bioorg. Med. Chem. Left. 21:2692-2696).
6. 6. A tissue transglutaminase inhibitor according to Claim I wherein the inhibitor is an antibody which binds tiGase and inhibits its transamidation activity.
7. 7. A tissue transglutaminase inhibitor according to Claim I wherein the inhibitor is dipeptide compound.
8. 8. A tissue transglutaminase inhibitor according to Claim 7 wherein the inhibitor is a compound of Formula I: RIJC (CH2)ny R2 CO2H 0Iwherein: X' represents an amino acid group; n' is an integer between I and 4; RI' represents benzyl, t-butyl or 9-fluorenylmethyl; and R2' represents R4-Swherein R3 R4, R5 and R6 each independently represent lower alkyl or -SR7R8, wherein R7 and R8 each independently represent lower alkyl or a pharmaceutically and/or veterinarily acceptable derivative thereof.
9. 9. A tissue transglutaminase inhibitor according to Claim 8 wherein X is an L-amino acid group.
10. 10. A tissue transglutaminase inhibitor according to Claim 8 or 9 wherein X is selected from the group consisting of phenylalanine, glutamine (or an N-substituted derivative thereof), isoleucine, alanine, glycine, tyrosine, proline, serine, lysine and glutamic acid.
11. 11. A tissue transglutaminase inhibitor according to any one of Claims 8 to 10 wherein n' is 2.
12. 12. A tissue transglutaminase inhibitor according to any one of Claims 8 to 11 wherein R1 is benzyl.
13. 13. A tissue transglutaminase inhibitor according to any one of Claims 8 to 12 wherein R2 represents \+
14. 14. A tissue transglutaminase inhibitor according to any one of Claims 8 to 13 wherein R2 represents -SR7R8, wherein R7 and R5 each independently represent lower alkyl.
15. 15. A tissue transglutaminase inhibitor according to any one of Claims 8 to 14 wherein R3, R4, R5, R6, R7 and/or R5 are -Cl-I3 or -CHCH2.
16. 16. A tissue transglutaminase inhibitor according to Claim 8 having theAN( H,CH3 (CH2)2 0 COH 0 following formula:
17. 17. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: O,NH2 o H CH3 (CH2)2+' o CO2H 0 CH3
18. 18. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: OH CH3 oANN (CH2)2+' 0 CO2H 0 CH3
19. 19. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: o/c3 Jj H CH,CH3 N( 2)3s+ 0 COH 0 CH3
20. 20. A tissue transglutaminase inhibitor according to Claim 8 having the following formula:
21. 21. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: (CH2)2/CH3 0 CO2H 0 Cl-I3
22. 22. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: (CH2)2+' 0 COH 0 CH3
23. 23. A tissue transglutaminase inhibitor according to Claim 8 having theOH ciSH (CH,CH3 2)2s+ 0 COH o CH3 following formula:
24. 24. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: o OH
25. 25. A tissue transglutaminase inhibitor according to Claim 8 having the following formula:OH26. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: 0 OH 27. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: NH2 F3CO2H 28. A tissue transglutaminase inhibitor according to Claim 8 having the following formula:ONQO OH29. A tissue transglutaminase inhibitor according to Claim 8 having the following formula:O NH J'ON Fl 0 OH30. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: H o( 0 OH 31. A tissue transglutaminase inhibitor according to Claim B having the following formula: H 0ON /32. A tissue transglutaminase inhibitor according to Claim 8 having the following formula:HH 0 OH33. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: 34. A tissue transglutaminase inhibitor according to Claim 8 having the following formula: 35. A tissue transglutaminase inhibitor according to any one of Claims 8 to 34 in the form of a bromide salt.36. A tissue transglutaminase inhibitor according to any one of Claims I to 35 for use in the treatment and/or prevention of an eye disease or disorder associated with angiogenesis.37. A tissue transglutaminase inhibitor according to Claim 36 wherein the eye disease or disorder associated with angiogenesis is a diseases or disorder of the posterior eye.38. A tissue transglutaminase inhibitor according to Claim 37 wherein the eye disease or disorder associated with angiogenesis is a diseases or disorder of the retina and/or choroid.39. A tissue transglutaminase inhibitor according to Claim 38 wherein the eye disease or disorder associated with angiogenesis is a retinopathy.40. A tissue transglutaminase inhibitor according to Claim 38 or 39 wherein the eye disease or disorder associated with angiogenesis is 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.41. A tissue transglutaminase inhibitor according to Claim 36 wherein the eye disease or disorder associated with angiogenesis is a diseases or disorder of the anterior eye.42. A tissue transglutaminase inhibitor according to Claim 41 wherein the eye disease or disorder associated with angiogenesis is 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).43. 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.44. The use according to Claim 43 wherein the inhibitor is reversible.45. The use according to Claim 44 wherein the inhibitor is selected from the group consisting of: (a) monodansylcadaverine; (b) tTGase cofactors and analogues thereof, such as GTP, GDP, GTPyS and GMP-PCP; (c) Ca2 chelators, such as EDTA; (d) Zn2 metal ions; and (e) acylideneoxoindole compounds (see KlOck et aL, 2011, Bioorg. Med. Chem. LetL 21:2692-2696).46. The use according to Claim 43 wherein the inhibitor is irreversible.47. The use according to Claim 46 wherein the inhibitor is selected from the group consisting of: (a) peptide compounds (for example, see Formula I below); (b) imidazole compounds (for example, see Compound 283 [R283] below; see also US 5,030,644); (c) dihydroisoxazole compounds, such as KCAO75 and KCCOO9 (for example, see Hausch et al., 2003, Chem. BioLlO:225-31); (d) cinnamoyl inhibitors (for example, see US 20100204280 to Keillor et a!.); (e) thieno[2,3-dl pyrimidine-4-one acylhydrazide derivatives (for example, see Duval eta!., 2005, Biog,Med Chem.Lett 15:1885); and (0 iodoacetamide (for example, see Folk & Cole, 1966, J Chem. Blot 241:3238-3240).48. The use according to Claim 43 wherein the inhibitor is an antibody which binds tiGase and inhibits its transamidation activity.49. The use according to Claim 43 wherein the inhibitor is dipeptide compound.50. The use according to Claim 49 wherein the tissue transglutaminase inhibitor is a compound of Formula I: RIJL (CH2)ny R2 CO2H 0Iwherein: X' represents an amino acid group; n' is an integer between I and 4; R1' represents benzyl, t-butyl or 9-fluorenylmethyl; and R2' representsRwherein R3, R4, R5 and R5 each independently represent lower alkyl or -SR7R8, wherein R7 and R5 each independently represent lower alkyl.51. The use according to Claim 49 wherein the compound is as defined in any one of Claims 6 to 33.52. The use according to Claim 49 or 50 wherein the medicament is for the treatment and/or prevention of an eye disease or disorder associated with angiogenesis 53. The use according to Claim 52 wherein the eye disease or disorder associated with angiogenesis is a diseases or disorder of the posterior eye.54. The use according to Claim 53 wherein the eye disease or disorder associated with angiogenesis is a diseases or disorder of the retina and/or choroid.55. The use according to Claim 54 wherein the eye disease or disorder associated with angiogenesis is a retinopathy.56. The use according to Claim 54 or 55 wherein the eye disease or disorder associated with angiogenesis is 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.57. The use according to Claim 50 or 51 wherein the eye disease or disorder associated with angiogenesis is a diseases or disorder of the anterior eye.58. The use according to Claim 57 wherein the eye disease or disorder associated with angiogenesis is 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).59. 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.60. A method according to Claim 59 wherein the inhibitor is reversible.61. A method according to Claim 60 wherein the inhibitor is selected from the group consisting of: (a) monodansylcadaverine; (b) tTGase cofactors and analogues thereof, such as GTP, GDP, GTPyS and GMP-PCP; (c) Ca2 chelators, such as EDTA; (d) Zn2 metal ions; and (e) acylideneoxoindole compounds (see Klbck et aL, 2011, Bioorg. Med. Chem. Lett. 21:2692-2696).62. A method according to Claim 58 wherein the inhibitor is irreversible.63. A method according to Claim 62 wherein the inhibitor is selected from the group consisting of: (a) dipeptide compounds (for example, see Formula I below); (b) imidazole compounds (for example, see Compound 283 [R283] below; see also US 5,030,644); (c) dihydroisoxazole compounds, such as KCAO7S and KCCOO9 (for example, see Hausch et al., 2003, Chem. BioIAO:225-31); (d) cinnamoyl inhibitors (for example, see US 20100204280 to Keillor et a!.); (e) thieno[2,3-d] pyrimidine-4-one acylhydrazide derivatives (for example, see Duval et at, 2005, Biog,Med Chem.Lett 15:1885); and (f) iodoacetamide (for example, see Folk & Cole, 1966, J Chem. Blot 241:3238-3240).64. A method according to Claim 59 wherein the inhibitor is an antibody which binds tTGase and inhibits its transamidation activity.65. A method according to Claim 59 wherein the inhibitor is dipeptide compound.66. A method according to Claim 65 wherein the inhibitor is a compound according of formula I: R1A (CH2)ny R2 CO2H 0Iwherein: LX' represents an amino acid group; n' is an integer between I and 4; R1' represents benzyl, t-butyl or 9-fluorenylmethyl; and R2' representsRwherein R3, R4, R5 and R5 each independently represent lower alkyl or -StR7R8, wherein R7 and R8 each independently represent lower alkyl.67. A method according to Claim 66 wherein the compound is as defined in any one of Claims 8 to 35.68. A method according to any one of Claims 59 to 67 wherein the compound or formulation is administered in an amount sufficient to inhibit, at least in part, angiogenesis.69. A method according to any one of Claims 59 to 68 wherein the medicament is for the treatment and/or prevention of an eye disease or disorder associated with angiogenesis 70. A method according to Claim 69 wherein the eye disease or disorder associated with angiogenesis is a diseases or disorder of the posterior eye.71. A method according to Claim 70 wherein the eye disease or disorder associated with angiogenesis is a diseases or disorder of the retina and/or choroid.72. A method according to Claim 71 wherein the eye disease or disorder associated with angiogenesis is a retinopathy.73. A method according to Claim 71 or 72 wherein the eye disease or disorder associated with angiogenesis is 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.74. A method according to any one of Claims 69 wherein the eye disease or disorder associated with angiogenesis is a diseases or disorder of the anterior eye.75. A method according to Claim 74 wherein the eye disease or disorder associated with angiogenesis is 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).76. A method according to any one of Claims 59 to 75 wherein the subject is human.77. A method according to any one of Claims 59 to 76 wherein the compound is administered repeatedly.78. A method according to any one of Claims 59 to 77 wherein compound is administered systemically.79. A method according to any one of Claims 59 to 78 wherein the compound or formulation is administered at or near a site of angiogenesis.80. A tissue transglutaminase inhibitor for use in inhibiting angiogenesis substantially as described herein with reference to the description.81. Use of a tissue transglutaminase inhibitor substantially as describedherein with reference to the description.82. A method of treating a subject in need of treatment with an angiogenesis inhibitor substantially as described herein with reference to thedescription.