WO2004084903A1

WO2004084903A1 - Use of a trioxopyrimidine for the treatment and prevention of ocular pathologic angiogenesis

Info

Publication number: WO2004084903A1
Application number: PCT/EP2004/003206
Authority: WO
Inventors: Jean-Michel Foidart; Francis Frankenne; Hans-Willi Krell; Vincent Lambert; Agnes Noel; Jean-Marie Rakic
Original assignee: F. Hoffmann-La Roche Ag; Université de Liège
Priority date: 2003-03-27
Filing date: 2004-03-26
Publication date: 2004-10-07

Abstract

The invention provides the use of trioxopyrimidine compound having an inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 defined as a) IC50 value of less 5 µM for MMP-2, MMP-9 and MMP-14 each; b) a ratio of more than 100 for the IC50 values of MMP-1:MMP-2, MMP-1: MMP-9, MMP-1:MMP-14 and c) a ratio of more than 10 for the IC50 values of MMP-3:MMP-2, MMP-3: MMP-9 MMP-3:MMP-14, for the manufacturing of a medicament for the treatment or prevention of ocular pathologic angiogenesis.

Description

Use of a trioxopyrimidine for the treatment and prevention of ocular pathologic angiogenesis

The present invention relates to the use of a trioxopyrimidine compound for the treatment and prevention of ocular disorders associated with pathologic angiogenesis such as age- related macular degeneration, diabetic retinopathy or corneal neovascularization.

Introduction

Angiogenesis is the process of new blood vessel development and formation and can be found in numerous diseases. In general, angiogenesis is caused by infiltration of the basal lamina by vascular endothelial cells in response to angiogenic growth signals, migration of the endothelial cells, and subsequent proliferation and formation of the capillary tube. Blood flow through the newly formed capillary is initiated after the endothelial cells come into contact and connect with a preexisting capillary. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are caused by abnormal neovascularization, including solid tumor growth and metastases, arthritis, some types of eye disorders like age-related macular degeneration (AMD), and psoriasis (see, e.g., reviews by Moses, M.A., and Langer, R., Biotech. 9 (1991) 630-634; Folkman, J., N. Engl. J. Med. 333 (1995) 1757-1763; Auerbach, R., et al., J. Microvasc. Res. 29 (1985) 401-411; Folkman, J., Advances in Cancer Research, eds. Klein and Weinhouse, 1985, Academic Press, New York, pp. 175-203; Patz, A., Am. J. Opthalmol. 94 (1982) 715-743; Folkman, J., et al., Science 221 (1983) 719-725; and Rastinejad, F., et al., Cell 56 ( 1989) 345-355. At present, more than 250 angiogenesis inhibitors are described and approximately half of them display activity in in vivo models (Bouma-ter Steege, J.C., et al., Crit. Rev. Eucaryot. Gene Expr. 11 (2001) 319-334).

The abrupt and often definitive loss of visual function resulting from choroidal neovascularization that occurs in the exudative form of AMD is a worldwide health problem with the ageing population (Fine, S.L., et al., N. Engl. J. Med. 342 (2000) 483-492). It is estimated that by their 90s, one in four people will have lost vision from AMD (Van Newkirk, M.R., et al., Ophthalmology 107 (2000) 1593-1600).

While the primary stimulus for the development of retinal neovascularization is hypoxia, molecular signals involved in the appearance and growth of pathological choroidal neovascularization (CNV) are not well defined (Campochiaro, P.A., J. Cell. Physiol. 184

(2000) 301-310). The net balance between molecules that have positive and negative regulatory activity controls angiogenesis (Carmeliet, P., and Jain, R.K., Nature 407 (2000) 249-257). Vascular endothelial factor (VEGF) and pigment-epithelium derived factor (PEDF) are hence expressed and play an important role in choroidal neovascular membrane formation (Schwesinger, C, et al., Am. J. Pathol. 158 (2001) 1161-1172; Ogata, K, et al., Invest. Ophthalmol. Vis. Sci. 43 (2002) 1168-1175; Dawson, D.W., et al., Science 285 (1999) 245-248). This idea is further supported by the previous demonstration that either inhibition of the VEGF system or overexpression of PEDF was an efficient way of CNV inhibition (Ferrara, N., Semin. Oncol. 6 Suppl. 16 (2002) 10-14; Krzystolik, M.G., et al., Arch. Ophthalmol. 120 (2002) 338-346; Mori, K., et al, Invest. Ophthalmol. Vis. Sci. 43 (2002) 2428-2434). However, angiogenesis is also associated with an important extracellular remodeling involving different proteolytic systems among which the matrix metalloproteinases system (MMPs) plays an essential role (Egeblad, M., and Werb, 2., Nat. Rev. Cancer 2 (2002) 161-174; Overall, CM., and Lopez-Otin, C., Nat. Rev. Cancer 2 (2002) 657-672).

Matrix metalloproteases (MMPs) are a family of zinc- and calcium-dependent proteases that are capable of degrading the extracellular matrix (ECM) and basement membrane (Egeblad, M., and Werb, Z., Nat. Rev. Cancer 2 (2002) 161-174; Overall, CM., and Lopez- Otin, C, Nat. Rev. Cancer 2 (2002) 657-672). They are believed to have pivotal roles in embryonic development and growth (Holmbeck, K., et al., Cell 99 (1999) 81-92; Vu, T.H., et al., Cell 93 (1998) 411-422) as well as in tissue remodeling and repair (Shapiro, S.D., Curr. Opin. Cell Biol. 10 (1998) 602-608; Lund, L.R., et al., EMBO J. 18 (1999) 4645-4656). Excessive or inappropriate expression of MMPs may therefore contribute to the pathogenesis of many tissue-destructive processes, including tumor progression (Rgeblad, M., and Werb, Z., Nat. Rev. Cancer 2 (2002) 161-174; Overall, CM., and Lopez-Otin, C, Nat. Rev. Cancer 2 (2002) 657-672) and aneurysm formation (Carmeliet, P., et al., Nat. Genet. 17 (1997) 439-444). MMP effects are far from being restricted to ECM degradation (Chang, C, and Werb, D., Trends Cell Biol. 11 (2001) S37-43). Peptide growth factors that are sequestered by ECM proteins become available once degraded by MMP-9 (Man<es, S., et al, J. Biol. Chem. 274 (1999) 6935-6945). MMPs can increase the bioavailability o- f VEGF (Bergers, G., et al., Nat. Cell Biol. 2 (2000) 737-744) but also generate angiogenesis inhibitors such as angiostatin by cleavage of plasminogen (Dong, Z., et al., Cell 8S- (1997) 801-810).

At the ocular level, a possible involvement of matrix metalloproteinases (MMPs) fcas also been suggested in the progression of both retinal and choroidal neovascularization and mutations in the TIMP-3 gene (tissue inhibitor of MMPs, type 3) are the cause α»f a rare familial form of macular dystrophy associated with subretinal neovascularization (Das, A., et al., Invest. Ophthalmol. Vis. Sci. 40 (1999) 809-813; Kadonosono, K., et al., Am. J. Ophthalmol. 128 (1999) 382-384; Weber, B., et al., Nat. Genet. 8 (1994) 352-356. It was recently demonstrated in an experimental murine model of laser- induced choroidal neovascularization that inflammatory cells-driven MMP-9 secretion contributed to the development of pathological choroidal angiogenesis (Lambert, V., et al., Am. J. Pathol. 161 (2002) 1247-1253). The magnitude of angiogenesis inhibition consecutive to MMP-9 deficiency was however much less important than that observed previously in the same model applied on mice deficient for plasminogen activator type I (PAI-1) (Lambert, V., et al., FASEB J. 15 (2001) 1021-1027). This suggests that MMP-9 is not the only pathway for rendering angiogenic factors bioavailable, which occurs in other experimental settings (Bergers, G., et al., Nat. Cell Biol. 2 (2000) 737-744). After laster light induced choroidal neovascularization in rats, MMP-2 mRNA expression was induced whereas MMP-9 mRNA expression was generally low and did not increase following laser treatment (Kvanta, A., et al., Curr. Eye Res. 21 (2000) 684-690). It is further known that in MMP-2-deficient mice there occurs reduced choroidal neovascular membrane formation (Berglin, L., et al., Invest. Ophthalmol. Vis. Sci. 44 (2003) 403-408). It is further discussed that MMP-1 and MMP-14 are involved in corneal degradation (Collier, S.A., Clin., Exp. Ophthalmol. 29 (2001) 340- 344).

Inhibition of MMPs, either with the naturally occurring Tissue Inhibitors of Metalloproteases (TIMPs), or with low molecular weight inhibitors, resulted in impressive anti-tumor and anti-metastatic effects in animal models (Brown, P.D., Med. Oncol. 14 (1997) 1-10). Most of the low-molecular weight inhibitors of MMPs are derived from the hydroxamic acid compound class and inhibit MMPs in a broad manner, being not selective for MMP-2 and MMP-9, the key MMPs in tumor invasion, metastatic spread, and angiogenesis. However, MMP inhibiting molecules from another structural class, the trioxopyrimidines, have been described, e.g. in WO 97/23465 and WO 01/25217. This class of compounds is extremely potent, and highly selective, with an almost exclusive specificity for MMP-2, MMP-9, while sparing most other members of the MMP family of proteases.

Several MMP inhibitors, predominantly of the hydroxamic acid substance class with broad substrate specificity were, and in part still are, in clinical testing for anti-tumor treatment. All of the published clinical results with these inhibitors were disappointing, showing little or no clinical efficacy (Fletcher, L., Nat. Biotechnol. 18 (2000) 1138-1139). The reason for this lack of efficacy in the clinic most likely is the fact that patients could not be given high enough doses for anti-tumor or anti-metastatic activity because of the side effects associated with these broadly acting inhibitors. These dose-limiting side effects were predominantly arthralgias and myalgias (Drummond, A.H., et al., Ann. N.Y. Acad. Sci. 878 (1999) 228-235). As a possible way to circumvent this problem, the combination of MMP inhibitors with classical cytostatic/cytotoxic compounds was evaluated in animal studies. Indeed, in these experiments, MMP inhibitors, in combination with cytostatic/cytotoxic drugs, showed enhanced tumor inhibiting efficacy (Giavazzi, R., et al., Clin. Cancer Res. 4 (1998) 985-992). In addition, International Patent Application No. PCT/EP02/04744 shows the combination of trioxopyrimidine based gelatinase inhibitors and cytotoxic/cytostatic compounds such as cisplatin, Paclitaxel, Gemcitabine or Etoposide.

There have been made a lot of attempts to identify compounds which prevent or inhibit macular degenerative diseases. WO 98/16503, WO 98/16506, WO 98/16514, and WO 98/16520 describe the use of orthosulfonamido heteroaryl hydroxamic acids as matrix metalloproteinase inhibitors which might be useful for the treatment of macular degeneration. However, there exists a need for highly potent substances which can be used for the treatment or prevention of macular degenerative diseases.

Description of the Invention

It was surprisingly found that, on the basis of a new in vivo model which is characteristic for ocular pathologic angiogenesis, trioxopyrimidine-based MMP inhibitors which are highly selective for MMP-2, MMP-9 and MMP-14 are useful for the treatment or prevention of ocular pathologic angiogenesis.

The invention therefore provides the use of a trioxopyrimidine compound having an inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 defined as

a) an IC₅₀ value of less than 5 μM for MMP-2, MMP-9 and MMP-14 each; b) a ratio of more than 100 for the IC₅₀ values of MMP-l:MMP-2, MMP-1: MMP-9,

MMP-l:MMP-14; and c) a ratio of more than 10 for the IC₅₀ values of MMP-3:MMP-2, MMP-3: MMP-9,

MMP-3:MMP-14,

for the treatment of prevention of ocular pathologic angiogenesis.

IC₅₀ values are measured by an in vitro assay for MMP enzymatic activity. Such an assay is described by Stack, M.S., and Gray, R.D., J. Biol. Chem. 264 (1989) 4277-4281. This assay is based on the determination of MMP enzymatic activity on a dinitrophenol substrate and fluorescence measurement of the substrate after cleaving by MMPs.

The invention further provides the use of such trioxopyrimidine compounds for the manufacturing of a medicament for the treatment or prevention of ocular pathologic angiogenesis.

Matrix metalloproteinases are well-known in the state of the art and are defined, e.g., by their EC numbers (MMP-1 EC 3.4.24.7; MMP-2 EC 3.4.24.24; MMP-3 EC 3.4.24.17, MMP-9 EC 3.4.24.35, MMP-14 EC 3.4.24).

Trioxopyrimidines useful for the invention are compounds from a well-known structural class. Such compounds are described in, for example, US Patent Nos. 6,242,455 and 6,110,924; WO 97/23465, WO 98/58915, WO 01/25217, which are incorporated herein by reference, and Grams, F., et al., Biol. Chem. 382 (2001) 1277-1285, and are effective and highly selective for MMP-2, MMP-9, and MMP-14.

According to the invention, the following compounds are particularly preferred:

5-Biphenyl-4-yl-5-[4-(4-nitro-phenyl)-piperazin-l-yl]pyrimidine-2,4,6-trione (Compound I)

5-(4-Phenoxy-phenyl)-5-(4-pyrimidin-2-yl-piperazin-l-yl)-pyrimidine-2,4,6-trione (Compound II)

5-[4-(4-Chloro-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)-pyrimidine-2,4,6- trione

(Compound III)

5-[4-(3,4-Dichloro-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)-pyrimidine-

2,4,6-trione

(Compound IV)

5-[4-(4-Bromo-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)-pyrimidine-2,4,6- trione (Compound V). Ocular pathologic angiogenesis is especially a macular degeneration, such as age-related macular degeneration, diabetic retinopathy and corneal neovascularization.

The invention therefore further relates to a method for treating or preventing corneal neovascularization. Corneal neovascularization leads to vision loss in eyes after extensive injury of the limbus. The limbus is a specialized tissue between the cornea and the conjunctiva. After injury or destruction of the limbus, corneal neovascularization due to the inflammation occurs. The use of MMP inhibitors according to the invention inhibits corneal neovascularization and is therefore useful as a therapeutic agent for the treatment of corneal neovascularization, e.g. associated with limbal injury. The disease and current treatments are described by, e.g., Chang, J.H., et al., Curr. Opin. Ophthalmol. 12 (2001) 242-249.

The invention therefore further relates to a method for treating or preventing age-related macular degeneration (AMD). In such a disease, the retinal macula degenerates or becomes disfunctional as a consequence of decreased growth of cells in the macula, increased death or rearrangement of the macular cells. The disease and current treatments are descibed by, e.g., Gottlieb, J.L., JAMA 288 (2002) 2233-2236.

The invention therefore further relates to a method for treating or preventing diabetic retinopathy. Retinal microvascular disfunction in diabetes is a major component of diabetic retinopathy. The disease and current treatments are described by, e.g., Harper, C.A., Clin. Exp. Optom. 82 (1999) 98-101.

According to the invention the trioxopyrimidine-based inhibitors have to be administered to the patient over several months or years (in case of prevention), to the patient in need of such a therapy. The trioxopyrimidine compounds are administered preferably with intravitreal and/or periocular injections, with non-retinotoxic doses ranging between micro and nanomolar concentrations.

The exact dosage of the MMP inhibitors will vary, but can be easily determined. In general, the daily dosage of the inhibitors will range between 1 nmol/kg and day to 1 mmol/kg and day.

The pharmaceutical compositions are aqueous compositions having physiological compatibility. The compositions include, in addition, auxiliary substances, buffers, preservatives, solvents and/or viscosity modulating agents. Appropriate buffer systems are based on sodium phosphate, sodium acetate or sodium borate. Preservatives are required to prevent microbial contamination of the pharmaceutical composition during use. Suitable preservatives are, for example, benzalkonium chloride, chlorobutanol, methylparabene, propylparabene, phenylethyl alcohol, sorbic acid. Such preservatives are used typically in an amount of 0.01 to 1% weight/volume.

Suitable auxiliary substances and pharmaceutical formulations are described in Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of a pharmaceutically acceptable substances include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.

The invention further comprises double knockout (MMP-2-/-; MMP-9-) mouse model and its use for the identification of substances inhibiting ocular pathologic angiogenesis. The mouse model includes artificial choroidal neovascularization induced by laser burns at the optic disk of the eyes. In this model, neovascularization was estimated by the ratio of the thickness from the bottom of the pigmented choroidal layer to the top of the neovascular membrane to the thickness of the intact-pigmented choroid adjectant.

The following examples, references, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Description of the Figures

Figure 1 Absence of MMP-2 and MMP-9 prevents the development of experimental choroidal neovascularization. Hematoxylin-eosin staining of a representative area of choroidal neovascularization at the site of laser-induced trauma in control (A) or in mice deficient for MMP-9 (B), MMP-2 (C) and both MMP-2,9 (D). Almost complete absence of neovascularization is visible in mice deficient for both MMP-2 and MMP-9 when vessels were immunostained with anti- mouse PECAM antibody (immunostained in orange with AEC) compared to other conditions, confirming thereby the reduced incidence of neovascularization calculated before sacrifice by fluorescein angiography evaluation of the number of leaking laser spots (F). The neovascular reaction was determined with computer-assisted image analysis by evaluating the B/C ratio as described previously (Lambert, V., et al, Am. J. Pathol. 161 (2002) 1247-1253 and FASEB J. 15 (2001) 1021- 1027) at day 14 after laser injury of the Bruch's membrane in WT and deficient mice (E). The neural retina (ret), and choroidal layer (ch) are indicated, and the neovascular area is arrowed. *** p<0.001; error bars = SE. Original magnification 200X .

Figure 2 Zymographic analysis of MMP-2 and MMP-9 in knockouts and WT mice.

At day 5 after the induction of choroidal neovascularization by laser, animals were sacrificed and posterior segment extract samples (5 ng/lane) from eyes of WT and MMP-2 or MMP-9 deficient mice were analyzed by gelatin zymography (A). As positive control, medium conditioned by human HT1080 cells was included ("HT"). Data represent the results of a single experiment, which was one of three with similar results. In WT mice, a kinetic zymographic evaluation was also performed at different intervals after laser-induced CNV demonstrating a temporal increase in pro-MMP-9 and the appearance of active forms of MMP-2 (B). In situ zymography with fluorescein-conjugated gelatine demontrated the concentration of gelatinolytic activity at the level of the neovascular membrane growing under the retina (C), with limited residual activity at the top of the membrane in double MMP-2,9 deficient mice (D). The neural retina (ret), and choroidal layer (Ch) are indicated, and the neovascular area is arrowed. Original magnification 200X and 400X.

Figure 3 Kinetic evaluation of MMP expression by semiquantitative R.T-PCR during the development of experimental CNV. The histograms correspond to the densitometric quantification of MMP-

2 (A), MMP-9 (B) and MT1-MMP (C) mRNA normalized to the 28S signal at different endpoints. The evaluation was performed on thte entire posterior segment after the induction of multiple wounds to Bruch's membrane. Unlike MMP-2 and TIMP-2, which remained relatively constant, MMP-9 and MT1-MMP mRNA expression appeared to be induced during the early phases of CNV development. Representa- ive gels are displayed with RT-PCR products expected size (bp) at right. Figure 4 Immunohistochemical staining for fibrinogen/fibrin at the site of laser- induced wound in WT and deficient mice.

Frozen ocular sections sections from wild-type (A), MMP-9 -/- (B) and MMP-2 -/- (C) mice reveal the presence of limited amount of fibrin (stained in orange with AEC) in WT mice or single gene deficient mice contrasting with the accumulation of fibrin at the site of restricted choroidal reaction observed in double gene MMP-2,9 deficient animals (D). Original magnification, 400X.

Figure 5 Decreased CNV formation with MMP inhibitors.

The evaluation of pharmacological inhibition of MMPs on choroidal pathological angiogenesis demonstrated that compound I administered with daily intraperitoneal injections at day 0 (DO) or at day 5 (D5) after laser induction was much more efficient (p<0.001) than a broad spectrum inhibitor (Batimastat, BB-94) (F). The neural retina (ret), and choroidal layer (Ch) are indicated, and the neovascular area is arrowed. *** p<0.001; ** p<0.01; *p<0.05; error bars = SE. Original magnification 200X.

Methods

Genetically modified mice

MMP-2 -/- mice (Itoh, T., et al., J. Biol. Chem. 272 (1997) 22389-22392) and MMP-9 -/- mice (Vu, T.H., et al., Cell 93 (1998) 411-422) were crossed with each other to obtain MMP-2 +/- MMP-9 +/- mice. Then these mice were crossed with each other to obtain MMP-2 -/- MMP-9 -/- (double knockout also designed as MMP2,9 KO), and control wild- type (WT) mice. All mice were genotyped using PCR. Animal experiments were performed in compliance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research. The animals were maintained with a 12-h light/12-h dark cycle and had free access to food and water.

Experimental choroidal neovascularization

Choroidal neovascularization (CNV) was induced in mice by four burns (usually at the 6, 9, 12, and 3 o'clock positions around the optic disc) using a green argon laser (532 nm; 50 μm diameter spot size; 0.05 sec duration; 400 mW) as previously described (Lambert, V., et al., Am. J. Pathol. 161 (2002) 1247-1253; Lambert, V., et al., FASEB J. 15 (2001) 1021- 1027). Mice with haemorrhage or not developing an evident bubble at the site of every laser impact (the sign of ruptured Bruch's membrane) were excluded from further analysis. Included animals (five or more in each condition) were sacrificed at day 14 (except for kinetic mRNA profiles). Before sacrifice, fluorescein angiograms (intraperitoneal injection of 0.3 ml of 1% fluorescein sodium -Ciba) were performed to confirm that laser burns were developing late phase increasing hyperfluorescent spots (corresponding to the leakage of fluorescein from newly formed permeable capillaries). The eyes were then enucleated and either fixed in buffered 3.5% formalin solution for routine histology or embedded in Tissue TeK (Miles Laboratories, Naperville, Illinois) and frozen in liquid nitrogen for cryostat sectioning. Choroidal neovascularization was quantified as previously described (Lambert, V., et al., Am. J. Pathol. 161 (2002) 1247-1253; Lambert, V., et al., FASEB J. 15 (2001) 1021-1027). Briefly, frozen serial sections were cut throughout the entire extent of each burn, and the thickest region (minimum of 5/lesion) selected for the quantification. Using a computer-assisted image analysis system (Olympus Micro Image version 3.0 for Windows 95/NT, Olympus Optical CO. Europe GmBH), neovascularization was estimated by the ratio (B/C) of the thickness from the bottom of the pigmented choroidal layer to the top of the neovascular membrane (B) to the thickness of the intact-pigmented choroid adjacent to the lesion (C). A mean B/C ratio value was attributed to each laser impact.

Gelatin zymography

Choroidal neovascularization was induced in mice by multiple burns (n=30) as described above. Animals were sacrificed at different endpoints and the eyes were enucleated. The posterior segments were cut out and immediately snap frozen in liquid nitrogen. Frozen tissues were then pulverized in liquid nitrogen, homogenized in buffer (0.1 M Tris-HCl pH 8.1, 0.4% Triton X100) and centrifuged for 20 minutes at 5000 X g. The pellets were discarded. Aliquots of supernatants were mixed with SDS sample buffer and electrophoresed directly as previously described by Munaut, C, et al., Invasion Metastasis 15 (1995) 169-178.

In situ zymography

In situ zymography was performed by incubating cryosections (7μm) with 40 μg/ml fluorescein-conjugated gelatine (Molecular Probes, Eugene, OR) in 50 mM Tris-HCl pH 7.5, 10 mM CaCl2, 150 mM NaCl and 0.05% Brij-35 (Calbiochem, CA, USA) for 12 h at 37°C; sections were washed 3 times with water and mounted with Vectashield. Gelatinase activity was visualized using fluorescent microscopy (Pagenstecher, A., et al., Am. J. Pathol. 157 (2000) 197-210).

Immunohistochemistry

Cryostat sections (5mm thick) were fixed in paraformaldehyde 1% in 0.07M phosphate buffered saline (PBS) pH 7.0 for 5 min or in acetone for 10 min at room temperature and then incubated with the primary antibody. Antibodies raised against mouse PECAM (rat monoclonal, PharMingen, San Diego, California; diluted 1/20), mouse MAC-3 (BD Pharming, San Diego, California) and murine fibrinogen/fibrin (goat polyclonal, Nordic Immunological, Tilburg, The Netherlands; dilutedl/400) were incubated for 1 hr at room temperature. The sections were washed in PBS (3X 10 min) and appropriate secondary antibody conjugated to peroxydase (HRP) were added: rabbit anti- goat IgG (Dako, Glostrup, Denmark, diluted 1/100) and rabbit anti- rat IgG ( Sigma -Aldrich; diluted 1/40) were applied for 30 min. For staining, a drop of AEC+ (Dako, 3-amino-9-ethylcarbazole) was added and sections were counterstained for 1 minute in haematoxylin. Specificity of staining was assessed by substitution of nonimmune serum for primary antibody.

For MMP inhibition, WT mice were IP injected either with Compound I or Batimastat. The daily injections started either the same day as CNV induction (DO), or at D5.

RT-PCR analysis

To evaluate the kinetic of PAI-1 mRNA expression by semiquantitative RT-PCR, choroidal neovascularization was induced in mice by multiple argon laser burns and animals were sacrificed at day 3, 5, 10, 14, 20, 40. The posterior segments (RPE-choroid complex vithout neural retina) were cut out and immediately frozen in liquid nitrogen. rTth reverse transcriptase RNA PCR kit (Applied Biosystems, Foster City, California) and two pairs of primers (Eurogentec, Liege, Belgium; oligonucleotides sequences, number of cycles and expected PCR product size are shown in Table 1). The frozen murine tissues w«re first pulverized using a Dismembrator (B. Braun Biotech International, GmbH, MeLsungen, Germany) and total RNA were extracted with the RNeasy extraction kit (Quiagen, Paris, France) according to the manufacturer's protocol. 28S rRNA were amplified vith an aliquot of 10 ng of total RNA using the GeneAmp Thermostable mRNA. Reverse transcription was performed at 70°C for 15 min followed by 2 min incubation at 95°C for denaturation of RNA-DNA heteroduplexes. Amplification started by 15 sec at 94°C, 20 sec at 60°C and 10 sec at 72°C RT-PCR products were resolved on 2% agarose gels and analyzed using a Fluor-S Multilmager (BioRad) after staining with ethidium bromide (FMC BioProducts).

Statistical analysis

Data were analyzed with GraphPad Prism 3.0 (San Diego, CA). The chi-square and Mann- Whitney tests were used to determine if there were significant (p<0.01) differences between WT and deficient mice.

Table 1 RT-PCR parameters

1-16 SEQ ID NOS: 1-16 Example 1

Induction of experimental CNV in WT, MMP-9KO, MMP-2KO and MMP-2,9KO mice.

Neovascularization was estimated at day 14 after induction by immunostaining with anti- PECAM antibodies. This demonstrated a strong inhibition of neovascular progression in MMP2,9 KO mice (Figure ID) compared to single gene deficient (Figure 1B-C) or WT (Figure 1A) animals. This confirmed fluorescein angiography data performed before sacrifice (Figure 1 F) showing a significant reduction (p<0.001) in the number of leaking spots (corresponding to newly formed immature microvessels with leakage of fluorescein). In sites developing a neovascular membrane, choroidal pathological reaction was quantified by measuring, on serial sections, the maximal height of the lesion above the choroidal layer observed in neighbouring intact zones. This was performed by determining the B/C ratio between total thicknesses of lesions ("B", from the bottom of the choroid to the top of the neovascular area) to the thickness of adjacent normal choroid ("C") according to Lambert, V., et al., Am. J. Pathol. 161 (2002) 1247-1253 and FASEB J. 15 (2001) 1021-1027. A significant reduction of the B/C ration was observed in MMP-9 (33 %), MMP-2 (44%) and MMP-2,9 (56%) deficient mice compared to their corresponding WT (p<0.001, Figure IE).

Example 2

Presence of MMP-2 and MMP-9 proteins in the posterior segments of WT and different deficient mice at day 5, and temporal profile during the formation of CNV.

Ocular posterior segment proteins prepared from WT, MMP-9 KO, MMP-2 KO and MMP-2,9 KO mice were analyzed by gelatin zymography (Figure 2A). As expected, no MMP-2 and MMP-9 activity was detected in MMP-2 KO, MMP-9 KO, and (not shown) MMP-2,9 KO mice, respectively. WT mice expressed pro-MMP-2 and -MMP-9 as well as processed MMP-2. The zymograms suggest a compensatory increase in the ocular expression of MMP9 in the MMP-2 knockout mice. However, despite that compensation, MMP-2KO mice were significantly protected from the development of severe CNV. Both MMP-2 and MMP-9 were increasingly processed during the early stages of CNV formation, with the appearance of active forms of MMP-2 (Figure 2B).

In situ zymography revealed that gelatinase activation in the posterior ocular segments was predominantly present in the area developing choroidal neovascularization (Figure 3C). Although gelatinolytic activity was strongly decreased in double MMP2.9 deficient mice, residual activity was still detectable at the top of the choroidal pathological reaction (Figure 3D).

a) Kinetics of MMP expression

To evaluate the level of regulation, the temporal profiles of MMP2 and MMP9 expression were analyzed by semiquantitative RT-PCR. MMP9 expression was upregulated during early phases of CNV formation, while MMP2 (constitutively expressed) showed no significant modulation (Figure 3A-B). The presence of active forms of MMP-2 could theoretically correspond either to a decrease of TIMP expression, or to an increase in the expression of activators (MT1-MMP). RT-PCR analysis demonstrated a constant expression of TIMP-2, but a significant upregulation of MT-l-MMP mRNA (Figure 3C- D). Effect of MMP deficiency on fibrinolytic activity. Plasminogen activators/plasmin play an important role in choroidal neovascularization (Lambert, V., et al., Am. J. Pathol. 161 (2002) 1247-1253) and it has been previously demonstrated that, at least some MMPs might modulate fibrinolysis through a plasminogen-dependent or independent mechanism (Lijnen, H.R., Biochemistry (Mosc) 67 (2002) 92-98; Pepper, M.S., Arterioscler. Thromb. Vase. Biol. 21 (2001) 1104-1117; Hiraoka, N., et al., Cell 95 (1998) 365-377. This was evaluated through the immunohistochemical staining of fibrinogen/fibrin, as an endpoint of fibrinolytic activity. The results demonstrated the presence of similar fibrinogen/fibrin deposits in WT and MMP-2 or MMP-9 deficient mice (Figure 4, A-C). However, there was an accumulation of fibrinogen/fibrin in double MMP-2,9 deficient animals suggesting thereby that the absence of both gelatinases impaired fibrinolytic activity in the choroidal neovascular membrane (Figure 4).

b) MMP inhibitors decrease the development of CNV

There were evaluated the consequences of broad spectrum or selective MMP-2/9 inhibition on CNV development by treating WT mice with daily systemic injections of either

Batimastat or Compound I. The two treatments significantly reduced the CNV formation.

(Figure 5F). However, Compound I was much more efficient (p<0.001) than Batimastat.

Surprisingly, selective MMP-2/9 inhibition treatment started five days after laser induction significantly inhibited the development of choroidal angiogenesis (40% inhubition) indicating thereby a potential for drug-induced regression (Figure 5). The comparison of the inhibitory effects of a daily treatment with either Compound I or with Batimastat showed that Compound I inhibited significantly more the choroidal neovascularization than Batimastat (Figure 5).

Example 3 Determination of MMP enzymatic activity

Inhibitors were tested in a modified fluorescence-assay as described by Stack, M.S., and Gray, R.D., J. Biol. Chem. 264 (1989) 4277-4281. Human MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 are commercially available (e.g. Calbiochem). The pro-enzymes were activated with 1 mM APMA (incubation for 30 min at 37°C) immediately before testing. Activated enzyme is diluted to 100 ng/ml in incubation buffer (50 mM Tris, 100 mM NaCl, lOmM CaCl2, pH 7.6). The compounds were dissolved in 100% DMSO. For IC₅₀ determination a minimum of 8 dilution steps between 0.5 - 1000 nM have been prepared. DNP-substrate (Bachem M1855, 255μM) was dissolved in incubation buffer.

The test tube contains 970μl incubation buffer, lOμl inhibitor solution and lOμl enzyme solution. The reaction was started by adding the lOμl substrate solution.

Kinetics of activity were determined using excitation at 280 nm and emission at 346 nm measured on a FluoroMax™ (Spex Industries Inc., Edison, NJ, USA) over 120 sec. DMSO has been used as control instead of inhibitor solution.

IC₅₀'s are defined as the concentration of inhibitor that gives a signal that is 50% of the positive enzyme control.

IC₅₀ values (nM) are shown in Table 2.

Table 2

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Claims

Patent Claims

1. Use of a trioxopyrimidine compound having an inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 defined as

a) an IC₅₀ value of less than 5 μM for MMP-2, MMP-9 and MMP-14 each; b) a ratio of more than 100 for the IC₅₀ values of MMP-1 :MMP-2, MMP-1: MMP-

9, MMP-l:MMP-14; and c) a ratio of more than 10 for the IC₅₀ values of MMP-3:MMP-2, MMP-3: MMP-

9, MMP-3:MMP-14,

for the manufacturing of a medicament for the treatment or prevention of ocular pathologic angiogenesis.

2. Use according to claim 1, characterized in that said trioxopyrimidine compound is selected from the group consisting of

5 ■Biphenyl-4-yl-5-[4-(4-nitro-phenyl)-piperazin-l-yl]pyrimidine-2,4,6-trione 5-((44--Phenoxy-phenyl)-5-(4-pyrimidin-2-yl-piperazin-l-yl)-pyrimidine-2,4,6- t irriioonnee

5-[4-(4-Chloro-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)- pyrimidine-2,4,6-trione 5-[4-(3,4-Dichloro-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)- pyrimidine-2,4,6-trione

5-[4-(4-Bromo-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)- pyrimidine-2,4,6-trione.

3. Use of a trioxopyrimidine compound having an inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 defined as

a) an IC₅₀ value of less than 5 μM for MMP-2, MMP-9 and MMP-14 each; b) a ratio of more than 100 for the IC₅₀ values of MMP-l:MMP-2, MMP-1: MMP- 9, MMP-l:MMP-14; and c) a ratio of more than 10 for the IC₅₀ values of MMP-3:MMP-2, MMP-3: MMP- 9, MMP-3:MMP-14,

for the treatment or prevention of ocular pathologic angiogenesis. Use according to claim 3, characterized in that said trioxopyrimidine compound is selected from the group consisting of

5-Biphenyl-4-yl-5-[4-(4-nitro-phenyl)-piperazin-l-yl]pyrimidine-2,4,6-trione 5-(4-Phenoxy-phenyl)-5-(4-pyrimidin-2-yl-piperazin-l-yl)-pyrimidine-2,4,6- trione

5-[4-(4-Chloro-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)- pyrimidine-2,4,6-trione

5-[4-(3,4-Dichloro-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)- pyrimidine-2,4,6-trione 5-[4-(4-Bromo-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)- pyrimidine-2,4,6-trione.