METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF CHOROIDAL NEOVASCULARISATION

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of choroddal neovascularisation. In particular, the present invention relates to a method of treating choroidial neovascularisation in a subject in need thereof comprising administering to the subject of therapeutically effective amount of a mineralocorticoid receptor antagonist.

Claims

1. A method of treating choroidal neovascularisation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a mineralocorticoid receptor antagonist.

2. The method of claim 1 wherein the subject suffers from a disease selected from the group consisting of age-related macular degeneration, myopic choroidal neovascularization, idiopathic choroidal neovascularization, polypoidal chorioretinopathy associated or not to central serous chorioretinoapthy and some inflammatory conditions such as such uveitis posterior, traumatic choroidal rupture, angioid streaks, and ocular histoplasmosis syndrome.

3. The method of claim 1 wherein the choroidal neovascularisation is secondary to age-related macular degeneration.

4. The method of claim 1 wherein the choroidal neovascularisation is secondary to pathological myopia.

5. The method of claim 1 wherein the choroidal neovascularisation is an idiopathic choroidal neovascularisation.

6. The method of claim 1 wherein the subject suffers from polypoidal choroidal vasculopathy.

7. The method of claim 1 wherein the MR antagonist is an epoxy-steroidal mineralocorticoid receptor antagonist or a non-epoxy-steroidal mineralocorticoid receptor antagonist.

8. The method of claim 1 wherein the MR antagonist is administered to the subject in combination with an anti-VEGF agent.

9. The method of claim 1 wherein the subject is refractory to an anti-VEGF treatment.

10. The method of claim 1 wherein the MR antagonist is administered to the subject by intraocular or periocular administration, either directly injected into the vitreous or in a peri-ocular space.

11. The method of claim 1 wherein the MR antagonist is administered to the subject in the form of microspheres that are injected into the subconjunctival space or into the vitreous.

12. The method of claim 10, wherein the peri-ocular space is a sub conjunctival space, a sub tenon space, a peri bulbar space, a reto bular space, an intra scleral space or a supra choroidal space.

Description

FIGURES

[0097] FIG. 1A-K. Subcutaneous administration of spironolactone limits choroidal neovascularisation (CNV) induced by laser in rat eyes.

[0098] Representative early (A-C) and late phase (D-F) fluorescein angiograms (FA) performed at the peak of CNV (ie. 14 days after laser induction) highlight reduced vascular leakage in the spironolactone-treated group (spi) compared to the laser control group. Intravitreous anti-VEGF treatment serves as positive control. Grading of vascular leakage on FA (G) shows significant decrease in angiographic score with spironolactone treatment (−45%) that is comparable to anti-VEGF group (−37%). Results are expressed as mean angiographic score/burn/rat±SEM. In each of both eyes, 6 laser burns were made, n=11 rats for control group, 6 for spironolactone group, and 5 for anti-VEGF group. Kruskal-Wallis test was used followed by Dunn's comparison. *, P<0.05. Combined treatment with intravitreous anti-VEGF+subcutaneous spironolactone shows significant additive effect with −84% of leakage as compared to control (nor shown).

[0099] CNV in retinal pigment epithelium/choroid flatmounts were labelled with FITC-GSL I-Isolectin B4. Spironolactone-treated group (I) shows reduced CNV size compared to the laser control (H). Anti-VEGF treatment (J) is the positive control group. Quantification of the size of CNV (K) show significant decrease in CNV volume in spironolactone and anti-VEGF groups compared to laser control. Results are expressed as mean CNV volume/burn/rat±SEM. Six laser burns per rat eye, n=11 rats for control group, 6 for spironolactone group, and 7 for anti-VEGF group. Kruskal-Wallis test followed by Dunn's multiple comparison was used. **, P<0.01.

[0100] FIG. 2. MR expression in rat retina tissues at different time point after laser induction.

[0101] At day 3 after laser (peak of macrophage infiltration), MR is up-regulated in both retinal pigment epithelium (RPE)-choroid complex (left) and neuroretina (right), compared to control normal rat eyes (ctrl, without laser treatment). At day 16, peak of neovascularisation, MR expression in RPE-Choroid (left) and neuroretina (right) was found to decline to the same level of controls. Results are expressed as mean±SEM. n=6 rats for control, 5 rats for D3 and 4 rats for D16. Kruskal-Wallis test was used followed by Dunn's comparison. *, P<0.05.

[0102] FIG. 3A-E. Spironolactone inhibits macrophage/microglial recruitment and down-regulates pro-inflammatory gene expression in rat retinas 3 days after laser.

[0103] The peak of macrophage/microglial infiltration/activation occurs at day 3 after laser. We labelled these cells with IBA1 on retinal pigment epithelium (RPE)-choroid flatmounts. Round activated IBA1 positive cells were counted in the areas with laser burns. Spironolactone (Spi) decreases IBA1 positive cells in the burns (B) compared to the non-treated eyes (A). Cell quantification shows significant fewer activated macrophage/microglia observed in spironolactone treated eyes than in laser control group (C). Bars in A and B: 100 μm. Results are expressed as mean IBA1 positive cells/burn/rat±SEM. Six laser burns per rat eye, n=7 rats for laser group and 4 rats for laser+Spi group. Mann Whitney U test was used, *P<0.05.

[0104] Quantitative PCR at day 3 show that spironolactone down-regulates the laser-induced gene expression of MCP1, IL1β, IL6 and TNFα in the RPE-choroid complex (D), and MCP1, IL1β and TNFα in the neuroretina (E). However, the expression of pro-angiogenic factors, VEGF-A and P1GF, is not regulated at RNA level (D and E) showing that spironolactone effect is independent from VEGF and P1GF. Results are expressed as mean±SEM, n=5 rats for laser group and 6 rats for spironolactone group. Mann-Whitney U test was used. *P<0.05.

[0105] FIG. 4A-F. Intravitreous poly(lactide-co-glycolide) microspheres releasing Spironolactone Limit Laser-induced CNV in rat eyes.

[0106] Fluorescein angiograms performed at day 14 after laser show reduced fluorescein intensity in eyes injected with spironolactone-loaded microspheres (Spi-MPs, B) compared to eyes injected with non-loaded microspheres (NL-MPs, A). We observed a tendency to decrease in angiographic score in Spi-MPs group compared to NL-MPs group (C), however, the difference is not significant. Results are expressed as mean angiographic score/burn/rat±SEM. In each of both eyes, 6 laser burns were made, n=5 rats in NL-MPs group and 7 rats in Spi-MPs group. Mann Whitney U test was used for statistic analysis.

[0107] CNV in retinal pigment epithelium/choroid flatmounts were labelled with FITC-GSL I-Isolectin B4. We observed a decrease in CNV staining in Spi-MPs group (E) compared to NL-MPs group (D). Quantification of CNV on z-stack confocal images show that CNV volume significantly declines in eyes injected with Spi-MPs compared to NL-MPs injected eyes. Results are expressed as mean CNV volume/burn/rat±SEM. Six laser burns per rat eye, n=8 rats each group. Mann Whitney U test was used, *P<0.05.

[0108] FIG. 5A-H. Laser-induced CNV in vascular endothelium-specific MR knock-out mice (EC-MR-KO) and in monocyte-specific MR knock-out mice (MC-MR-KO).

[0109] Fluorescein angiography (FA) was performed at day 10 after laser, corresponding to the peak of CNV development in mice (A-D). At day 12, after intravenous perfusion with FITC-Dextran, eyes were enucleated. Perfused CNV was observed in retinal pigment epithelium-choroid flatmounts (E-H).

[0110] FA shows reduced fluorescein leakage in the eyes of both tissue-specific MR knock-out mice (B and C), compared to wild-type mice (C57BL/6) (WT, A), which is confirmed by angiographic scoring (D). Results are expressed as mean score/laser burn±SEM. Four laser burns per eye, n=68 burns for WT, 60 for EC-MR-KO and 16 for MC-MR-KO mice. Kruskal-Wallis test followed by Dunn's multiple comparison was used, **P<0.01, ***P<0.001.

[0111] FITC-GSL I-Isolectin B4 labelling shows smaller CNV developed in eyes of EC-MR-KO (F) and MC-MR-KO mice (G) than in WT mice (E). Quantification of CNV on z-stack confocal images finds that CNV size is decreased in both MR knock-out mice compared to WT (H). Results are expressed as mean CNV volume/laser burn±SEM. Four burns per eye, n=80 burns for WT, 68 for EC-MR-KO and 24 for MC-MR-KO mice. Kruskal-Wallis test followed by Dunn's multiple comparison was used, *P<0.05.

[0112] FIG. 6A-B. Experimental designs of rat (A) and mouse (B) models of laser-induced CNV.

EXAMPLE

[0113] Materials & Methods

[0114] Animals

[0115] All experiments were performed in accordance with the European Communities Council Directive 86/609/EEC and approved by local ethical committees (Ce5/2012/113). Six to eight-week old male Brown Norway rats from Janvier breeding Center (Le Genest-Saint-Isle, France) were used to create rat model of CNV. Vascular endothelial cell-specific MR knock-out mice (EC-MR-KO) and monocyte/macrophage-specific MR knock-out mice (MC-MR-KO) were generated in C57BL/6 genetic background mice and were obtained from team of Dr F. Jaisser (UMRS 1138, team 1). EC-MR-KO mice were created by mating transgenic mice expressing Cre recombinase in endothelial cells with mice harbouring MR-floxed alleles (Berger S et al. Loss of limbic mineralocorticoid receptor impairs behavioural plasticity. PNAS USA 2006). MC-MR-KO mice were obtained by mating mice expressing Cre recombinase in monocytes/macrophages (Clausen B E et al. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Trangenic Research 1999) with mice harbouring MR-floxed alleles. Animals were kept in pathogen-free conditions with food, water and litter and housed in a 12-hour light/12-hour dark cycle. Anesthesia was induced by intramuscular ketamine (40 mg/kg for rats, 50 mg/kg for mice) and xylazine (4 mg/kg for rats, 10 mg/kg for mice) Animals were sacrificed by carbon dioxide inhalation or cervical dislocation.

[0116] Experimental Designs

[0117] The experimental designs of rat and mouse models of laser-induced CNV are detailed in the FIG. 6. The treatments given in rat CNV model were considered as prevention.

[0118] Laser Coagulation

[0119] After anesthesia and dilation of the pupils, coverslips were positioned on the cornea as a contact glass. For rats, six to eight burns were performed 2 to 3-disk diameters away from the papillae with an Argon laser (532 nm) mounted on a slit lamp (170 mW, 0.1 second and 50 μm). For mice, four laser burns were induced at the 3, 6, 9 and 12 o'clock positions around the optic disc (250 mW, 0.05 second and 50 μm). The presence of a bubble witnessed the rupture of Bruch's membrane and confirmed the success of the laser impact.

[0120] Treatments

[0121] Treatments were introduced only in rat model of CNV. After laser photocoagulation, rats were divided into 6 groups: 1) non-treated; 2) daily subcutaneous injection of spironolactone diluted in oliver oil and DMSO (25 mg/kg/day) until sacrifice; 3) daily subcutaneous injection of vehicle (oliver oil and DMSO) until sacrifice; 4) intravitreous injection (IVT) of anti-rat VEGF (1.5 μg/μl, 5 μl, RnD system, Lille, France) only at day 0 (Couturier A et al. Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation. Mol Vis 2014;20:908-20); 5) IVT of spironolactone-loaded PLGA MPs only at day 0 (2.2 μg/μl, 5 μl obtained from team of Pr R. Herrero-Vanrell, Complutense University of Madrid). Considering the ex vivo release profile of Spi-MPs, the daily dose of spironolactone released in the vitreous is estimated at 18 μM; 6) IVT of blank PLGA MPs (5 μl) only at day 0.

[0122] Fluorescein Angiography (FA)

[0123] FA was performed 14 days (in rats) or 10 days (in mice) after laser induction. Pupils dilated, fluorescein (0.2 mL (rat) or 0.1 mL (mouse) of 10% fluorescein in saline) was injected intravenously in the tail. Early and late phase angiograms were recorded 1-3 and 5-7 min respectively after fluorescein injection. For each laser-induced lesion, fluorescein leakage was graded qualitatively by evaluating the increase in size/intensity of dye between the early and late phase. Angiographic scores were established by two masked observers according to the following criteria: grade 0 indicates no hyperfluorescence; grade 1 indicates a slight hyperfluorescence with no increase in intensity nor in size; grade 2 indicates a hyperfluorescence increasing in intensity but not in size; grade 3 indicates a hyperfluorescence increasing both in intensity and size. An additional grade 4 was given when the hyperfluorescence size increase is more than 2 diameter of initial laser burn.

[0124] RPE-Choroidal Flatmounts and CNV Quantifications

[0125] Two days after FA examination (time necessary for fluorescein elimination), eyes were enucleated, fixed in 4% paraformaldehyde for 15 min at room temperature and sectioned at the limbus; the cornea and lens were discarded. Retina was separated from RPE-choroid complex. Eight radial incisions were made on RPE-choroid which was then flatmounted and post-fixed with acetone for 15 min at −20° C. After washing with 0.1% Triton x100 in PBS, FITC-GSL I-Isolectin B4 (1:200, Vector) was applied over night at −4° C. After washing with PBS, RPE-choroid was flatmounted and observed with a confocal microscope (Zeiss LSM710, Le Pecq, France). Images of the CNV were captured with a digital video camera coupled to a computer system. Horizontal optical sections (1 μm interval) were obtained from the surface of the CNV. The deepest focal plane in which the surrounding choroidal vascular network connecting to the lesion could be identified was judged to be the floor of the CNV lesion. The area of CNV-related fluorescence on each horizontal section was measured using ImageJ software. The summation of the whole fluorescent area on z-stack images from the top to the bottom of the CNV was used as an index for the CNV volume.

[0126] Mice were perfused with FITC-dextran (molecular weight 2,000,000, Sigma-Aldrich, St-Quentin Fallavier, France) before enucleation. After RPE-choroidal flatmounting, FITC-dextran perfused CNV was examined and analyzed as previously described.

[0127] Immunofluorescence on RPE-Choroidal Flatmounts

[0128] Three days after laser induction (peak of macrophage infiltration), rat RPE-choroidal flatmounts were also prepared for immunofluorescence. Rabbit polyclonal anti-IBA1 (1:400, Wako, Neuss, Germany) was applied over night at −4° C. After washing with 0.1% Triton x100/PBS, flatmounts were incubated with Alexa Fluo® 594 goat anti-rabbit IgG (1:200, Molecular Probes, Leiden, The Netherlands), the nuclei were counterstained with DAPI. IBA1 positive macrophage/microglial cells were counted on stained laser lesions. The average cell number/per impact/rat eye was calculated.

[0129] Quantitative PCR

[0130] Three days after laser induction, rat neuroretinas and RPE-choroid-sclera complexes were carefully dissected from enuleated eyes and snap-frozen in liquid nitrogen and stored at −80° C. until use. Total RNA was isolated from tissues using RNeasy Plus Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. First-strand complementary DNA was synthesized from total mRNA using random primers (Life Technologies, Cergy Pontoise, France) and SuperScript II reverse transcriptase (Life Technologies). Transcript levels of MR, MCP1, IL6, IL1β, TNFα, VEGF-A and P1GF were analyzed by quantitative real-time PCR performed in 7500 Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA) with either SYBR Green or TaqMan detection.

[0131] As preliminary results showed no difference in CNV volume and angiographic score between non-treated laser group and vehicle-treated laser group, we pooled the results of these two groups as controls for subcutaneous spironolactone-treated laser group.

[0132] Statistics

[0133] Results are expressed as mean±SEM. Statistical analyses were carried out using GraphPad Prism 5 for Windows (GraphPad Software Inc., San Diego, Calif., USA). Nonparametric Kruskal-Wallis and Mann-Whitney tests were used to compare continuous data when appropriate. P-values of 0.05 or less were considered significant.

[0134] Results

[0135] Our results show that systemic administration of MR antagonist, spironolactone, limits the CNV development, effect comparable to that of anti-VEGF injected intravitreously in the rat eyes (FIG. 1).

[0136] As MR is up-regulated in early phase of the disease (FIG. 2), and MR antagonism inhibits the accumulation of activated macrophage/microglial cells in the laser burned areas (FIG. 3A-C), MR over-activation may play a role in the early inflammation induced by laser through macrophage/microglial activation and finally contribute to the CNV development. At day 3, laser-induced expression of cytokines (TNFα, Il1β, IL6) and chemokines (MCP1), inflammatory components known to be implicated in the CNV formation (Lavalette Sophie et al. Il1β inhibition prevents CNV and does not exacerbate photoreceptor degeneration. Am J Pathol 2011; Raoul W, CCL2/CCR2 and CX3CL1/CX3CR1 chemokine axes and their possible involvement in age-related macular degeneration. J Neuroinflammation 2011; Kramer M et al. MCP1 in the aqueous humour of subjects with age-related macular degeneration. Clin Experiment Ophthalmol 2012; Miao H et al. Inflammatory cytokines in aqueous humor of subjects with CNV. Mol Vis 2012), is abrogated by spironolactone (FIGS. 3D and E), however the expression of pro-angiogenic factors (VEGF and PGF) is not modified, suggesting that MR antagonist acts through pathways other than that of VEGF to prevent CNV development.

[0137] To limit eventual systemic side effects associated with long-term use of MR antagonist, microspheres controlled releasing spironolactone were developed and injected intravitreously in the rat eyes. They also show beneficial effect to prevent CNV (FIG. 4).

[0138] Macrophages/microglia and vascular endothelial cells (EC) are critical for the pathogenesis of CNV, as they express VEGF and cytokines and chemokines which in turn regulate monocyte and endothelial cell recruitment (Grossniklaus et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in CNV. Mol Vis 2002; Kvanta et al. Subfoveal fibrovascular membranes in AMD express VEGF. IOVS 1996). Macrophages and choroidal EC also express MR. In order to evaluate the role of macrophage MR and endothelial MR in CNV formation, we performed laser coagulation using EC-specific MR knockout mice and monocyte/macrophage-specific MR knockout mice. Both mice show reduced CNV formation (FIG. 5), suggesting EC MR and macrophage MR contribute to CNV development.

REFERENCES

[0139] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.