Nutlin-3a for treatment of proliferative vitreoretinopathy

Abstract

The invention provides compositions and methods for treatment of proliferative vitreoretinopathy.

Claims

1. A method for inhibiting or reducing the severity of proliferative vitreoretinopathy (PVR) comprising: identifying a subject suffering from PVR; and contacting retinal pigment epithelial cells and/or glial cells in an eye of said subject with a composition comprising a compound comprising the chemical structure ##STR00007## wherein R.sup.1 is independently selected from F, Cl, Br, and I; R.sup.2 is independently selected from F, Cl, Br, and I; R.sup.3 is independently selected from hydrogen, C.sub.1-C.sub.6 alkyl, and C.sub.1-C.sub.6-alkoxy; s is 0, 1, 2, 3, 4, or 5; t is 0, 1, 2, 3, 4, or 5; and u is 0, 1, 2, 3, 4, or 5; or a pharmaceutically acceptable salt thereof, in a sufficient amount that reduces the intra-ocular reduction of the level of p53 or that prevents p53 from interacting with human double min 2 (Hdm2), thereby inhibiting or reducing the severity of PVR.

2. The method of claim 1, wherein R.sup.1 is Cl.

3. The method of claim 1, wherein said Nutlin-3a is administered at a concentration of 0.1 μM, 0.5 μM, 1.0 μM, 2.0 μM, 5 μM, 10 μM, 20 μM, 30 μM, or 50 μM.

4. The method of claim 1, wherein said Nutlin-3a is present in a concentration of 0.1-10% (mg/ml).

5. The method of claim 1, wherein said composition is administered locally to the eye using eye drops.

6. The method of claim 1, wherein said composition is administered intravitreally or subconjunctivally.

7. The method of claim 1, wherein said subject suffering from PVR has undergone rhegmatogenous retinal detachment surgery.

8. The method of claim 1, wherein the subject is older than 24 months of age.

9. The method of claim 1, wherein said composition prevents retinal detachment in said subject.

10. The method of claim 1, wherein said composition reduces the formation of epiretinal membranes in said subject.

11. The method of claim 1, wherein said composition inhibits the contraction of retinal pigment epithelial (RPE) cells in said subject.

12. The method of claim 1, wherein said composition is administered every 48 hours, every 24 hours, every 12 hours, or every 6 hours.

13. The method of claim 1, wherein said composition is administered for 1 day, 3 days, 7 days, 14 days, 30 days, 60 days, 90 days, 120 days, or 365 days.

14. The method of claim 1, wherein said composition further comprises a pharmaceutically acceptable carrier.

15. The method of claim 1, wherein the form of said composition is a solid, a paste, an ointment, a gel, a liquid, an eye drop, an aerosol, a mist, a polymer, a film, an emulsion, or a suspension.

16. The method of claim 1, wherein said subject is a human.

17. The method of claim 1, wherein said subject has not been diagnosed with a retinoblastoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1B include a photomicrograph of a western blot and a bar chart demonstrating that Nutlin-3a prevented both the rabbit vitreous humor (RV)-induced decline in the level of p53 and contraction. FIG. 1A is a photograph of a western blot showing the impact of Nutlin-3a on RV-mediated signaling events. Primary rabbit conjunctival fibroblasts (RCFs) were pre-treated with either Nutlin-3a (10 μM), or vehicle for 30 min and then exposed to either RV (diluted 1:3 in Dulbecco's Modified Eagle Medium (DMEM)), or DMEM for 2 hr. The resulting cell lysates were subjected to western blot analysis using the indicated antibodies. The numbers are a ratio of p-PDGFRα/PDGFRα, p-Akt/Akt or p53/RasGAP (Ras GTPase) activating protein. This data presented is representative of three independent experiments. FIG. 1B is a bar chart showing the impact of Nutlin-3a on RV-mediated contraction. RCFs were subjected to the collagen contraction assay as described above. Nutlin-3a (10 μM) or vehicle was included as indicated. The data were subjected to a paired t test; “*” denoted a statistically significant difference.

(2) FIGS. 2A-2B include a dot plot and a series of photomicrographs showing that Nutlin -3a prevented retinal detachment and elevated p53 in epiretinal membranes. FIG. 2A is a dot plot illustrating the impact of Nutlin-3a on experimental PVR. RCFs were used to induce PVR as described below. The indicated rabbits received no additional injections (none) or 0.1 cc injections of either vehicle, or Nutlin-3a (20 μM) on day 0, 2 and 4. The data were subjected to an unpaired t test or one way Analysis of Variance (ANOVA) test; “*” denoted a statistically significant difference. FIG. 2B is a series of photographs showing that Nutlin-3a enhanced expression of p53 in epiretinal membranes. At the end of the experiment (day 28), the eyes were enucleated, and paraffin sections of whole eyes were subjected to p53 immunohistochemistry. The arrows point to the epiretinal membrane (the arrowheads denote the retina and the brown color indicates p53). The left and right panels are from a Nutlin-3a injected animal, whereas the middle panel is from a rabbit treated with vehicle. The left and middle panels were stained with the p53 antibody, whereas the right panel was stained with non-immune IgG. The scale bar is 50 μm.

(3) FIGS. 3A-3C include a series of western blots and photographs showing that molecularly suppressing expression of p53 rescued the ability of PDGFRα-deficient cells to contract. FIG. 3A is a photograph of a western blot showing the knockdown RCFs. Lentiviruses were used to stably express short hairpin ribonucleic acids (shRNAs) directed against green fluorescent protein (GFP), PDGFRα, or p53 in primary RCFs. The resulting cell lysates were subjected to western blot analysis using the indicated antibodies. The signal intensity was quantified and expressed as a ratio of the loading control (RasGAP). The data presented are representative of two independent experiments. FIG. 3B is a photograph of a western blot showing the expression of PDGFRα potentiated RV-mediated suppression of p53. The cells described in panel A were left resting or exposed to vitreous (diluted 1:3 in DMEM) from normal rabbits (RV) for 2 hr, lysed and total cell lysates were subjected to western blot analysis with the indicated antibodies. The signal intensity was quantified and the data presented are representative of three independent experiments. The data were subjected to a paired t test; “*” denoted a statistically significant difference. FIG. 3C is a bar chart showing RV induced contraction of RCF-loaded collagen gels, which required PDGFRα-mediated suppression of p53. The cells described in panel A were subjected to a collagen contraction assay. RV (diluted 1:3 in DMEM) was added on top of the gels on day 0 and the replenished every 24 hr; the experiments was terminated on day 3. The panel below the bar graph shows photographs of representative gels. The data in the bar graph are triplicates within a single experiment. The data were subjected to a paired t test; “*” denoted a statistically significant difference. The data presented are representative of 3 independent experiments.

(4) FIG. 4 is a dot plot showing that molecularly suppressing expression of p53 restored the ability of PDGFRα-deficient cells to induce retinal detachment. The cells described in FIG. 3A were compared for their PVR potential in a rabbit model of this disease. The insert indicates the type of lenti-short hairpin RNA (shRNA) used to modify the cells. PVR was induced as described below. Each symbol represents the response of an individual rabbit on the indicated day. PVR was graded according to the Fastenberg scale of classification: stage 0, no disease; stage 1, epiretinal membrane; stage 2, vitreoretinal traction without retinal detachment; stage 3, localized retinal detachment (1-2 quadrants); stage 4, extensive retinal detachment (2-4 quadrants without complete detachment); stage 5, complete retinal detachment. The data were subjected to an unpaired t test or one way ANOVA test; “*” denoted a statistically significant difference.

(5) FIGS. 5A-5C include a series of bar charts demonstrating the importance of PDGFRα and suppression of p53 for RV-dependent cell proliferation and protection from apoptosis and senescence. FIG. 5A is a bar chart showing the results of RCFs (sh GFP, sh PDGFRα, sh PDGFRα/sh p53, and sh p53) seeded into a 24-well plate at a density of 5×10.sup.4 cells/well in DMEM+10% fetal bovine serum (FBS). After 6 hours the cells had attached, the medium was changed to either 0.5 ml DMEM, or rabbit vitreous (diluted 1:3 in DMEM). The media were replaced every day. The cells were counted with a hemocytometer on day 3. The mean +/−standard deviation of three independent experiments is shown; * denotes P<0.05 using a paired t-test. FIG. 5B is a bar chart showing the results of RCFs described in A seeded into 60 mm dishes at a density of 100,000 cells/dish in DMEM+10% FBS. After 6 hours the cells had attached, the medium was changed to either 3 ml DMEM, or rabbit vitreous (diluted 1:3 in DMEM). The media were replaced every day. On day 3, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) in an apoptosis assay kit by following the manufacturer's instructions. Cells that were stained with annexin V and/or PI were detected and quantified by flow cytometry in Coulter Beckman XL (Coulter C.). The mean+/−standard deviation of three independent experiments is shown; * denotes P<0.05 using a paired t-test. FIG. 5C is a bar chart showing the results of RCFs described in A seeded into a 12-well plate at a density of 10,000 cells/well in DMEM+10% FBS. After 6 hours the cells had attached, the medium was changed to either 1 ml DMEM, or rabbit vitreous (diluted 1:3 in DMEM). The media were replaced every day. On Day 3, the β-galactosidase activity was measured as outlined in the manufacturer's instructions. Both stained and unstained cells were counted and photographed under an inverted microscope. The mean+/−standard deviation of three independent experiments is shown; * denotes P<0.05 using a paired t-test. The scale bar is 50 μm.

(6) FIG. 6 is a schematic representation showing that RV engaged PDGFRα, which triggered two signaling pathways that drive cellular response intrinsic to PVR. Signaling pathway #1 leads to a reduction in the level of p53, and this is sufficient for protection from apoptosis and senescence. Contraction and proliferation require an additional set of signaling events that constitute pathway #2.

(7) FIGS. 7A-7B include a photograph of a western blot and a bar chart showing p53 attenuated human vitreous humor (HV)-driven collagen gel contraction. FIG. 7A is a photograph of a western blot showing the impact of Nutlin-3a on HV-mediated signaling events. Methods were the same as FIG. 3A, but the RPE cells from a human patient epiretinal membrane were used in place of RCF, and HV (a pool of vitreous from 5 PVR patients diluted 1:3 in DMEM) was used in place of RV. The data presented are representative of three independent experiments. FIG. 7B is a bar chart showing the impact of Nutlin-3a on HV-mediated contraction. This experiments were the same as FIG. 3B with the modification noted in FIG. 7A. The data were subjected to a paired t test. “*” denoted a statistically significant difference. The data shown are representative of three independent experiments.

(8) FIGS. 8A-8B include a series of photographs showing the minimum effective dose and maximum tolerated dose of Nutlin-3a. FIG. 8A is a series of photomicrographs of western blots showing RCFs pretreated with the indicated concentrations of Nutlin-3a for 30 min, and then exposed to RV (diluted 1:3 in DMEM) for 2 hours. Cells were lysed and resulting lysates were subjected to western blot analysis using the indicated antibodies. The data shown are representative of three independent experiments. FIG. 8B is a series of photographs showing RCFs grown to near confluence in DMEM+10% FBS, the medium was replaced with DMEM+5% FBS+the indicated concentration of Nutlin-3a. The medium was replaced every 24 hours, and the photographs were taken on day 3. The data shown are representative of two independent experiments. The scale bar is 50 μm.

(9) FIGS. 9A-9B include a series of photographs showing epiretinal membranes from rabbits subjected to the PVR protocol. Rabbits that underwent a gas vitrectomy were intravitreally injected with the indicated concentration of Nutlin-3a on day 0, 2 and 4. The rabbits underwent a fundus examination on day 1, 3, 5, 7 and 14. Following the last fundus exam rabbits were euthanized, eyes were enucleated and fixed in 10% formalin. Sections were prepared, stained with hematoxylin and eosin, and then photographed. Representative photos of a Nutlin-3a (FIG. 9A) and vehicle (FIG. 9B) injected eye are presented. Arrowheads and arrows point to the retina and epiretinal membrane, respectively; scale bar: 50 μm. The lack of the retinal pigment epithelial cell layer is an artifact related to processing of the tissue.

(10) FIGS. 10A-10B include a series of bar graphs showing that inhibiting PDGFRα kinase activity blocked RV-induced collagen contraction. The indicated RCFs (50,000 cells/ml) (FIG. 10A) or F cells (1 million cells/ml) (FIG. 10B) were subjected to a collagen contraction assays as described below. F cells were immortalized mouse embryo fibroblasts derived from PDGFR knock-out mice that do not express either of the two PDGFR genes, Fα and Fβ cells are F cells in which PDGFRα or PDGFRβ has been re-expressed. After the collagen was solidified (90 minutes), DMEM or RV (diluted 1:3 in DMEM) supplemented with Imatinib (10 μM) or its vehicle was added, and replenished every 24 hours; the experiments were terminated on day 3. The panels below the bar graphs show photographs of representative gels; the data presented are representative of 3 independent experiments. The data were subjected to a paired t test; “*” denoted a statistically significant difference.

(11) FIGS. 11A-11C include a series of photographs of western blots and bar charts showing that PDGFRα did more than suppress p53 to mediate RV-dependent contraction. FIG. 11A is a photograph showing western blot analysis of knockdown RCFs. Lentiviruses were used to stably express shRNAs directed against GFP, PDGFRα, or p53 in primary RCFs. The resulting cell lysates were subjected to western blot analysis using the indicated antibodies. The signal intensity was quantified and expressed as a ratio of the loading control (RasGAP). The data presented are representative of two independent experiments. FIG. 11B is a photograph of a western blot showing that expression of PDGFRα potentiated RV-mediated suppression of p53. The cells described in panel A were left resting or exposed to vitreous (diluted 1:3 in DMEM) from normal rabbits (RV) for 2 hours, lysed and total cell lysates were subjected to western blot analysis with the indicated antibodies. The signal intensity was quantified and the data presented are representative of three independent experiments. FIG. 11C is a bar chart showing RV induced contraction of RCF-loaded collagen gels, which required PDGFRα-mediated suppression of p53. The cells (50,000 cells/ml) described in panel A were subjected to a collagen contraction assay. RV (diluted 1:3 in DMEM) was added on top of the gels on day 0 and the replenished every 24 hours; the experiments were terminated on day 3. The panel below the bar graph shows photographs of representative gels; the data presented are representative of 3 independent experiments. The data were subjected to a paired t test; “*” denoted a statistically significant difference.

(12) FIGS. 12A-12F include a series of photographs showing p53 was undetectable in human PVR membranes. Epiretinal membranes from patients afflicted with PVR subjected to p53 immunohistochemistry. While the positive control (rat colon cancer) demonstrated that this approach could readily detect p53 protein (FIG. 12B), p53 was undetectable in membranes from 7 different patients, three of which are shown (FIGS. 12D-12F). The green/brown pigment is routinely observed in such samples, which contain a large number of retinal pigment epithelial cells; they were also present in the section of a PVR membrane that was stained with an non-immune, isotype-matched control antibody (FIG. 12C). FIG. 12A is a section of rat colon cancer that serves as that negative control for FIG. 12B.

DETAILED DESCRIPTION

(13) Proliferative vitreoretinopathy is a blinding disease that afflicts 5-11% of patients that undergo surgery to correct a rhegmatogenous retinal detachment (Han D: Proliferative vitreoretinopathy. Edited by Albert D, J W. M, DT. A, BA. B. Philadelphia, Elsevier Saunders, 2008, pp. 2315-2324). There are between 1,700-3,700 cases of PVR in the US annually (Wilkes S R et al., 1982 Am J Ophthalmol, 94:670-673; Haimann M H et al., 1982 Arch Ophthalmol, 100:289-292). The current treatment for PVR is repeat surgery to remove the epiretinal membrane that is causing retinal detachment, and to reattach the detached retina (Charteris D G, 1998 Br J Ophthalmol, 82:106), which is anatomically successful in only 60-80% of cases (Michels R G, Wilkinson C P, Rice T A: Retinal Detachment. Edited by St. Louis, Mosby, 1990, p. pp. 669-706; Mietz H and Heimann K, 1995 Br J Ophthalmol, 79:874-8775). Moreover, the procedure carries the risk of recurrence (Girard P et al., 1994 Retina, 14:417-424; Lleo Perez A et al., 2000 Arch Soc Esp Oftalmol, 75:741-750; Nagasaki H et al., 1991 Retina, 11:204-207; Rodriguez de la Rua E et al., 2005 Curr Eye Res, 30:147-153; Tseng W et al., 2004 Am J Ophthalmol, 137:1105-1115; Yoshino Y et al., 1989 Retina, 9:97-100). Prior to the invention described herein, efforts to identify non-surgical, i.e., pharmacological, approaches to treat PVR were not successful (Wiedemann P et al., 1998 Am J Ophthalmol, 126:550-559; Asaria R H et al., 2001 Ophthalmology, 108:1179-1183; Schiff W M et al., 2007 Arch Ophthalmol, 125:1161-1167).

(14) Nutlin-3a was first identified as a potent and selective small molecule inhibitor of the p53-MDM2 interaction. Subsequent studies showed that Nutlin-3a administration in vitro caused p53 stabilization and activation of the p53-pathway. Researchers have investigated the effects of Nutlin-3a administration to the eye, specifically in the context of a therapeutic strategy for treating retinoblastoma (Brennan et al., 2011 Cancer Res, 71(12): 4205-13). Retinoblastoma is a malignant tumor of the retina and it is estimated that up to 40% of retinoblastomas are hereditary. Retinoblastoma is a childhood cancer, and usually diagnosed in very young children between 12 months and 24 months of age. Because retinoblastomas retain wild-type p53 (instead, having a mutated RB1 gene that drives tumorigenesis), administration of Nutlin-3a may be useful as a cancer therapeutic by inducing effective p53-mediated apoptosis, senescence, or growth arrest in the tumor cells.

(15) The data described herein demonstrate the surprising results that Nutlin-3a administration inhibited or reduced proliferative vitreoretinopathy and retinal detachment. These results are particularly intriguing, even in light of Brennan et al., because PVR and retinoblastoma are distinct ocular conditions. First, the etiologies of PVR and retinoblastoma are completely different—retinoblastoma is the uncontrolled growth and division of cells driven most often by mutations in the Rb1 (retinoblastoma) gene, while PVR is caused by a spontaneous event occurring after injury, trauma, or surgical procedure. Second, Nutlin-3a was a known p53 activator, and thus, the anti-tumorigenic effects in a wild-type p53 retaining cancer, such as retinoblastoma, were well known in the cancer field. However, unlike in cancers, the role of p53 and/or MDM2 has never been identified or implicated, prior to the invention, in a non-cancer setting, e.g., the development or mechanisms of PVR or retinal detachment. And finally, the patient populations affected by retinoblastoma and PVR are also entirely distinct. Retinoblastoma can develop in utero, and is usually diagnosed between 12 and 24 months of age. Moreover, many retinoblastoma patients inherited the disease. In contrast, PVR is associated with retinal detachment, which is a spontaneous event occurring after injury, trauma, or surgical procedure. PVR and retinal detachment often occur in the elderly (e.g., greater than 65 years of age), very near-sighted individuals, or individuals with a family history of retinal detachment. Thus, the patients affected by PVR are typically older than 12 or 24 months, and/or have suffered from a previous injury, trauma or surgical procedure to or near the proximity of the eye.

(16) Proliferative Vitreoretinopathy

(17) PVR is a blinding disease associated with rhegmatogenous retinal detachment, for which there is currently no satisfactory treatment. The term “proliferation” in “PVR” refers to the proliferation of retinal pigment epithelial and glial cells, while the terms “vitreo” and “retinopathy” identify the tissues which are affected, namely the vitreous humor (or simply vitreous) and the retina. Specifically, PVR is a disease that develops as a complication, secondary to rhegmatogenous retinal detachment. PVR occurs in about 8-10% of patients undergoing primary retinal detachment surgery, and can prevent the successful surgical repair of rhegmatogenous retinal detachment. Prior to the invention described herein, there were no prophylactic/preventative options available to patients that were at risk of PVR, e.g., those patients that had undergone retinal surgery.

(18) The full-thickness retinal break (e.g., tears and holes) that is quintessential to rhegmatogenous retinal detachment results in exposure of cells to vitreous, a rich source of growth factors and cytokines (Oh K, Hartnett M, Landers I M: Pathogenic mechanisms of retinal detachment. Edited by Ryan S. Philadelphia, Elsevier Mosby, 2006). The accumulation of fluid in the sub-retinal space, along with the tractional force of the vitreous on the retina results in rhegmatogenous retinal detachment. Specifically, the RPE cells migrate into vitreous, proliferate, and synthesize extracellular matrix proteins (Han D: Proliferative vitreoretinopathy. Edited by Albert D, J W. M, DT. A, BA. B. Philadelphia, Elsevier Saunders, 2008, pp. 2315-2324). The cytokines present in the vitreous humor trigger the ability of the RPE to proliferate and migrate. This series of events culminates in the formation of a retina-associated membrane, which contracts and thereby causes retinal detachment and vision loss (Campochiaro P: The pathogenesis of proliferative vitreoretinopathy. Edited by Ryan S. Philadelphia, Elsevier Mosby, 2006).

(19) While cells (retinal pigment epithelial, glial, fibroblasts, etc. (Campochiaro P A, 1997 Arch Ophthalmol, 115:237-241; Baudouin C et al., 1990 Am J Ophthalmol, 110:593-598; Vinores S A et al., 1990 Invest Ophthalmol Vis Sci, 31:14-28) in PVR membranes express a plethora of cell surface receptors, the PDGF receptor α is essential for experimental PVR, and is associated with clinical PVR (Andrews A et al., 1999 Invest Ophthalmol Vis Sci, 40:2683-2689; Robbins S G et al., 1994 Invest Ophthalmol Vis Sci, 35 No 10:3649-3663; Cui J et al., 2009 Exp Eye Res, 88:438-444). The surprisingly prominent role of PDGFRα in PVR is related to the fact that it can be engaged by a wide spectrum of vitreal agents, which activate the receptor indirectly and thereby trigger a signature set of signaling events that includes suppression of p53 (Lei H et al., 2009 J Biol Chem, 284:6329-6336; Lei H et al., 2009 Invest Ophthalmol Vis Sci, 50:3394-3403; Lei H et al., 2011 Mol Cell Biol, 31:1788-1799).

(20) Predisposing factors for postoperative PVR are preoperative PVR, aphakia, high levels of vitreous proteins, duration of retinal detachment before corrective surgery, the size of the retinal hole or tear, intra-ocular inflammation, vitreous hemorrhage, vitreous liquidity, and trauma or injury to the eye. As described in Rodriguez de la Rua E et al., 2005 Curr Eye Res, 30:147-153, incorporated herein by reference, the risk for PVR was higher in patients >70 years, with intraocular pressure lower than 14 (OR: 3.84; CI 95%: 2.04-7.30), in retinal breaks larger than “1 clock hour” (OR: 2.54; CI: 1.28-5.05), extended retinal detachments (OR: 4.01; CI: 1.98-8.10), and reinterventions (OR: 1.55; CI: 1.14-9.22). Scleral surgery also was a risk factor for PVR (OR: 3.89; CI: 2.12-7.14) and aphakia/pseudophakia when scleral surgery is performed (OR: 3.33; CI: 1.54-7.22). In particular, some subjects that have undergone ocular surgeries, such as surgery to correct retinal detachments, are at increased risk for developing PVR.

(21) The Interaction of P53 and Nutlin-3a

(22) Various forms of cellular stress increase expression and activate p53, a tetrameric transcription factor, and thereby trigger the p53 pathway, which leads to cell cycle arrest, apoptosis and/or senescence (Levine A J et al., 2009 Nat Rev Cancer, 9:749-758). The finding that p53 and/or the p53 pathway is mutated in approximately 50% of solid tumors (Hainaut P and Hollstein M, 2000 Adv Cancer Res, 77:81-137) has lead to the development of pharmacological agents that stimulate the p53 pathway. For instance, the small molecule Nutlin-3a activates the p53 pathway by preventing p53 from interacting with Mdm2/Hdm2 (murine double min 2, also called Hdm2 in humans) (Vassilev L T et al., 2004 Science, 303:844-848), which reduces the level of p53 by a variety of mechanisms (Prives C, 1998 Cell, 95:5-8; Ofir-Rosenfeld Y et al., 2008 Mol Cell, 32:180-189; Sasaki M et al., 2011 Nat Med, 17:944-951).

(23) The amino acid sequence of human p53 (Genbank Accession No. AAD28535.1) is as follows (SEQ ID NO: 1):

(24) TABLE-US-00001 MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIE QWFTEDPGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWPLSSSVPSQKT YQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPP GTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVE YLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTII TLEDSSGNLLGRNSFEVRVCACPGRDRRTEKENLRKKGEPHHELPPGSTKR ALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGK EPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD
Orthologs for human p53 can be readily identified and are known in the art, for example, mouse (Genbank Accession No. AAC05704), rat (Genbank Accession No. AAH81788), cat (Genbank Accession No. P41685), dog (Genbank Accession No. AF060514) and horse (Genbank Accession No. X91793). Other isoforms and transcriptional variants of p53 are also known in the art.

(25) The amino acid sequence of human double min 2 (Hdm2) (Genbank Accession No. NP_002383) is as follows (SEQ ID NO: 2):

(26) TABLE-US-00002 MVRSRQMCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQK DTYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEH RKIYTMIYRNLVVVNQQESSDSGTSVSENRCHLEGGSDQKDLVQELQEEKP SSSHLVSRPSTSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESLA LCVIREICCERSSSSESTGTPSNPDLDAGVSEHSGDWLDQDSVSDQFSVEF EVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDSFEEDPEISLA DYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENST QAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSS IIYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHG KTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP
Orthologs for Hdm2 can be readily identified and are known in the art, for example, mouse (Genbank Accession No. AAB09030), rat (Genbank Accession No. NP_001101569.1), cat (Genbank Accession No. NP_001009346.1), dog (Genbank Accession No. AAG42840.0) and horse (Genbank Accession No. AAF28866.1).

(27) In 2004, Vassilev and co-workers (Hoffman-La Roche Inc., Nutley, N.J.) described a class of antagonists that inhibited the murine double min 2 (MDM2)-p53 complex. These antagonists are a group of cis-imidazoline analogues designated as the Nutlins. Through x-ray crystallography, the MDM2-p53 complex showed a well defined hydrophobic cleft which represented the binding site for p53. In addition, the structure revealed that this cleft was filled by only three side chains of the helical region of p53: Phe19, Leu26 and Trp23. This observation led to the possibility that a small molecular inhibitor could mimic these three amino acids and their orientation. The inhibitor could disrupt the MDM2-p53 interaction by binding specifically in this cleft, liberating functional p53. A class of small molecules, called Nutlins, were found to bind specifically into the p53-binding pocket of Mdm2/Hdm2, thereby preventing Mdm2 interaction with p53 and inhibiting Mdm2-dependent degradation of p53. Nutlin-3a (RG7112/RO5045337) is currently in clinical trials for certain tumors in which the Hdm2/p53 pathway is intact (Secchiero P et al., 2011 Curr Pharm Des, 17:569-577). Furthermore, an ophthalmic formulation of Nutlin-3a has been developed (Brennan R C et al., 2011 Cancer Res, 71:4205-4213). The results presented below demonstrate that the correlation between the PDGFRα-mediated decline in the level of p53 and development of PVR is causally related, and that Nutlin-3a-mediated stabilization of p53 prevents PVR.

(28) Nutlin-3a (RG7112/RO5045337) is a small molecule that is currently in clinical trials for certain types of cancer. Specifically, Nutlin-3 [(±)-4-[4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one] is a cis-imidazoline analog which inhibits the interaction between mdm2 and tumour suppressor p53. Nutlin-3 is arbitrarily referred to as enantiomer a because it appears as the first peak from chiral purification of racemic nutlin-3. It acts by preventing a decline in the level of p53, which is a transcription factor that is mutated in many, but not all tumors that occur in humans. By inhibiting the interaction between mdm2 and p53, Nutlin-3 stabilizes p53, and selectively induces a growth-inhibiting state called “senescence” in cancer cells. Specifically, in those tumors in which p53 remains intact (normal/wild type p53), Nutlin-3a prevents proliferation, and promotes the apoptosis/senescence of the tumor cells.

(29) Nutlins are cis-imidazoline analogs which inhibit the interaction between mdm2 and tumour suppressor p53. Nutlin-3 ((±)-[4-[4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one]) has the following chemical structure:

(30) ##STR00002##

(31) Cis-isomers of nutlin-3 are:

(32) ##STR00003##

(33) Trans-isomers of this compound are:

(34) ##STR00004##
Nutlin-3a is arbitrarily referred to as enantiomer “a” because it appears as the first peak from chiral purification of racemic nutlin-3. Nutlin-3a acts by preventing a decline in the level of p53, which is a transcription factor that is mutated in many, but not all tumors that occur in humans. By inhibiting the interaction between mdm2 and p53, Nutlin-3a stabilizes p53, and selectively induces a growth-inhibiting state called “senescence” in cancer cells. Specifically, in those tumors in which p53 remains intact (normal/wild type p53), Nutlin-3a prevents proliferation, and promotes the apoptosis/senescence of the tumor cells.

(35) Analogs of nutlin-3 may be a compound of formula (I):

(36) ##STR00005##
wherein
R.sup.1 is independently selected from F, Cl, Br, and I;
R.sup.2 is independently selected from F, Cl, Br, and I;
R.sup.3 is independently selected from hydrogen, C.sub.1-C.sub.6 alkyl, and C.sub.1-C.sub.6-alkoxy;
s is 0, 1, 2, 3, 4, or 5;
t is 0, 1, 2, 3, 4, or 5; and
u is 0, 1, 2, 3, 4, or 5;
or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

(37) Analogs of nutlin-3 may be a compound of formula (II):

(38) ##STR00006##
wherein
R.sup.1 is independently selected from F, Cl, Br, and I;
R.sup.2 is independently selected from F, Cl, Br, and I; and
R.sup.3 is independently selected from hydrogen, C.sub.1-C.sub.6 alkyl, and C.sub.1-C.sub.6-alkoxy;
or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

(39) In one aspect, R.sup.1 of formula (I) or (II) is Cl. In one aspect, R.sup.2 of formula (I) or (II) is Cl. In another aspect, R.sup.3 is methoxy or isopropyloxy.

(40) As used herein, “alkyl” or “C.sub.1-C.sub.6 alkyl” is intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 or C.sub.6 straight chain (linear) saturated aliphatic hydrocarbon groups and C.sub.3, C.sub.4, C.sub.5 or C.sub.6 branched saturated aliphatic hydrocarbon groups. For example, C.sub.1-C.sub.6 alkyl is intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 and C.sub.6 alkyl groups. Examples of alkyl include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.

(41) In certain embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g., C.sub.1-C.sub.6 for straight chain, C.sub.3-C.sub.6 for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms.

(42) The term “alkoxy” includes substituted and unsubstituted alkyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups.

(43) “Isomer” means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereoisomers”, and stereoisomers that are non-superimposable mirror images of each other are termed “enantiomers” or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a “racemic mixture”.

(44) A carbon atom bonded to four non-identical substituents is termed a “chiral center”. “Chiral isomer” means a compound with at least one chiral center. Compounds with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed “diastereomeric mixture”. When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center.

(45) Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).

(46) “Pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.

(47) “Solvate” means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H.sub.2O.

(48) As used herein, the term “analog” refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound. The analogs described herein also retain similar function to Nutlin-3a, in which the analog also reduces or inhibits the interaction or association between p53 and Hdm2, or increases intraocular p53 levels.

(49) As used herein, “blocking the interaction or association” or “inhibiting or reducing binding” refers to preventing or reducing the direct or indirect association of one or more molecules, peptides, or proteins; or preventing or reducing the normal activity of one or more molecules, peptides, or proteins. The interaction, association, or binding is covalent, non-covalent, or ionic.

(50) The present invention also provides functional derivatives of analogs of Nutlin-3a. As used herein, “functional analogs” of Nutlin-3a refers to small molecules, antibodies, polypeptides, or polynucleotides that inhibit or reduce the interaction or association between p53 and Hdm2. Preferably, the functional analog binds in the p53-binding pocket of Hdm2 and inhibits or reduces interaction, association or binding between p53 and Hdm2. Functional analogs of Nutlin-3a can be identified by screening methods known in the art. Suitable screening assays may utilize techniques known in the art such as two hybrid assay, fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), protein-fragment complementation (PCA), or co-immunoprecipitation assay which detect protein-protein interactions, to detect interaction of p53 and Hdm2, or inhibition or reduction of p53-Hdm2 binding.

(51) In particular, a screening assay to identify functional analogs was described in Vassilev et al. (2004, Science, 303:845-848), hereby incorporated by reference in its entirety. For example, the screening assay comprises assaying (i) stabilization and accumulation of p53 protein, (ii) activation of Hdm2 expression, or (iii) activation of other p53-regulated genes and the p53 pathway. For example, the skilled artisan, using the amino acid sequences disclosed herein for p53 and Hdm2 could use recombinant DNA methods well known in the art to construct expression vectors and in vitro translate the p53 and Hdm2 proteins. Incubation of the proteins with putative analogs of Nutlin or a library of agents to be screened can be performed with optimal conditions determined by the skilled artisan. Co-immunoprecipitation, immunoaffinity purification, western blotting or other methods well known in the art are then used to assess the binding between p53 and Hdm2, or the inhibition or reduction of binding by the introduction of the putative Nutlin analog. In other embodiments, stabilization and accumulation of p53 protein levels can be determined in vitro, through immunoblotting techniques utilizing p53-specific antibodies. In another embodiment, the activation of other p53-regulated genes and the p53 pathway, such as MDM2, apoptosis genes PUMA and NOXA, cell cycle regulators p21, and p53 itself can be assessed by determining or quantifying the expression levels by mRNA or protein.

(52) Pharmaceutical Compositions

(53) For administration to a subject such as a human or other mammal (e.g., companion, zoological or livestock animal), the Nutlin or analog thereof is desirably formulated into a pharmaceutical composition containing the active agent in admixture with one or more pharmaceutically acceptable diluents, excipients or carriers. Examples of such suitable excipients for can be found in U.S. Publication 2009/0298785 (incorporated by reference herein in its entirety), the Handbook of Pharmaceutical Excipients, 2nd Edition (1994), Wade and Weller, eds. Acceptable carriers or diluents for therapeutic use are well-known in the pharmaceutical art, and are described, for example, in Remington: the Science and Practice of Pharmacy, 20th Edition (2000) Alfonso R. Gennaro, ed., Lippincott Williams & Wilkins: Philadelphia, Pa. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.

(54) The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can contain as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s).

(55) Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.

(56) Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.

(57) Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition, Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can be also used.

(58) A person of ordinary skill in the art can easily determine an appropriate dosage to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage that will be most suitable for an individual subject based upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, general health, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. To determine a suitable dose, the physician or veterinarian could start doses levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. This is considered to be within the skill of the artisan and one can review the existing literature on a specific agent to determine optimal dosing.

(59) The compositions described herein comprising a Nutlin or an analog thereof can be administered to a subject via intravitreally of subconjunctivally. In some embodiments, the composition is administered in the form of a liquid (e.g., drop or spray) or gel suspension. Alternatively, the composition is applied to the eye via liposomes or infused into the tear film via a pump-catheter system. Further embodiments embrace a continuous or selective-release device, for example, membranes such as, but not limited to, those employed in the OCUSERT System (Alza Corp., Palo Alto, Calif.) In an alternative embodiment, the p53 activator is contained within, carried by, or attached to a contact lens, which is placed on the eye. Still other embodiments embrace the use of the composition within a swab or sponge, which is applied to the ocular surface.

(60) In some cases, the composition further comprises a pharmaceutically acceptable carrier, e.g., a pharmaceutically acceptable salt. Suitable ocular formulation excipients include FDA approved ophthalmic excipients, e.g., emulsions, solutions, solution drops, suspensions, and suspension drops, a list of which is provided in Table 1. Other suitable classifications include gels, ointments, and inserts/implants. Table 1 provides maximum percentages, when available, for the various formulation types.

(61) Exemplary excipients for use in optimizing ocular formulations include alcohol, castor oil, glycerin, polyoxyl 35 castor oil, Tyloxapol, polyethylene glycol 8000 (PEG-8000), ethanol, glycerin, cremaphor, propylene glycol (pG), polypropylene glycol (ppG), and polysorbate 80. In some cases, citrate buffer and sodium hydroxide are included to adjust pH. Preferably, the formulation for ocular delivery of nutlin-3a comprises 5% cremaphor, 10% pG, 15% pPG, and 70% phosphate buffered saline (PBS).

(62) TABLE-US-00003 TABLE 1 Exemplary Percentage According to the FDA Excipient Database Excipient/Co-solvent http://www.accessdata.fda.gov/scripts/cder/iig/index.cfm Acetic Acid 0.2 (solution), 0.9% (drops) Alcohol 0.5% (solution), 1.4% (solution, drops) Alcohol, dehydrated 0.5% (solution), 0.5% (solution, drops) Ammonium hydroxide N/A (solution; solution, drops) Anyhydrous trisodium citrate N/A (solution) Antipyrine 0.1% (solution) Benzalkonium chloride 2% (solution), 0.9% (drops) Benzododecinium 0.012% (solution) Boric acid 0.1% (emulsion), 37.2% (solution), 1.9% (solution, drops), 1% (suspension), 0.6% (suspension drops) Caffeine 2% (solution) Calcium chloride 0.02% (solution, drops) Carbomer 1342 0.05% (emulsion) Creatinine 0.5% (solution), 0.2% (solution, drops) Carbomer 934P 0.45% (suspension, drops) Carbomer Homopolymer Type B 0.5% (suspension), 0.45% (suspension, drops) Carboxymethylcellulose sodium 0.5% (solution, drops) Castor oil 5% (emulsion) Cetyl alcohol 0.5% (suspension) Chlorobutanol 0.5% (solution), 0.2% (solution, drops) Cholesterol N/A (powder, for suspension) Citric acid 0.2% (solution), 0.05% (solution, drops) Citric acid monohydrate 0.05% (solution; solution, drops) Diethanolamine N/A (solution) Divinylbenzene styrene 0.75% (suspension, drops) copolymer Edetate disodium 10% (solution), 0.1% (solution, drops), 0.13% (suspension), 0.101% (suspension, drops) Edetate sodium 0.02% (emulsion), 0.1% (solution), 0.02% (suspension) Gellan gum 0.6% (solution) Glycerin 2.2% (emulsion), 3% (solution), 2.6% (solution, drops), 2.5% (suspension; suspension, drops) Glyceryl stearate 0.5% (suspension) Hydrocarbon gel, plasticized N/A (suspension) Hydrochloric acid 1.06% (solution), 0.17% (solution, drops) Hydroxyethyl cellulose 0.5% (solution; solution, drops), 0.25% (suspension), 0.35% (suspension, drops) Hydroxyethyl methylcellulose 0.5% (solution) Hypromellose 2910 0.5% (solution; suspension; suspension, drops) Hypromelloses 50% (solution), 0.5% (solution, drops; suspension), 0.6% (suspension, drops) Lauralkonium chloride 0.005% (solution, drops) Lauroyl sarcosine 0.03% (suspension, drops) Light mineral oil N/A (suspension) Magnesium chloride 0.03% (powder, for solution), 0.0065% (solution, drops) Mannitol 23% (solution), 4.6% (solution, drops), 2.4% (suspension), 4% (suspension, drops) Methylcellulose 0.5% (solution) Methylparaben 0.05% (solution; solution, drops; suspension; suspension, drops) Mineral oil 0.1% (suspension) Nitric acid N/A (solution) Nitrogen N/A (solution) Nonoxynol-9 0.125% (solution) Nonoxynol-40 0.05% (solution), 0.01 (solution, drops) Octylphenol polymethylene N/A (solution) Phosphoric acid N/A (solution, drops) Polidronium chloride 0.0005% (solution, drops) Poloxamer 188 0.1% (solution; solution, drops) Poloxamer 407 0.2% (solution), 0.16% (solution, drops), 0.101% (suspension, drops) Polycarbophil 0.9% (solution), 0.859% (suspension, drops) Polyethylene glycol 300 N/A (solution) Polyethylene glycol 8000 2% (solution) Polyoxyl 35 castor oil 5% (solution; solution, drops) Polyoxyl 40 hydrogenated castor oil 0.5% (solution, drops) Polyoxyl 40 stearate 7% (solution), 0.5% (suspension) Polypropylene glycol 15% (solution) Polysorbate 20 0.05% (suspension) Polysorbate 80 4% (emulsion), 0.2% (solution), 1% (solution, drops), 0.1% (suspension; suspension, drops) Polyvinyl alcohol 1.4% (solution; solution, drops; suspension; suspension, drops) Potassium acetate 4% (powder, for solution) Potassium chloride 22.2% (solution), 0.14% (solution, drops), Potassium phosphate, monobasic 0.2% (solution), 0.065% (solution, drops), 0.44% (suspension) Potassium sorbate 0.47% (solution) Povidone K29/32 1.8% (solution) Povidone K30 2% (solution), 0.6% (suspension) Povidone 90 1.2% (solution) Propylene glycol 10% (solution), 0.75% (solution, drops), 5% (suspension), 1% (suspension, drops) Propylparaben 0.015% (solution; solution, drops), 0.01% (suspension; suspension, drops) Sodium acetate 0.05% (emulsion), 0.35% (solution), 1.279% (solution, drops) Sodium bisulfite 0.1% (solution; solution, drops); 0.06% (suspension) Sodium borate 0.543% (solution), 1.1% (solution, drops), 0.0285% (suspension, drops) Sodium borate decahydrate 0.15% (solution), 0.095% (solution, drops) Sodium carbonate 1% (solution) Sodium chloride 55% (solution), 0.9% (solution, drops), 0.85% (suspension), 0.68% (suspension, drops) Sodium citrate 2% (solution), 2.2% (solution, drops), 0.3% (suspension), 0.45% (suspension, drops) Sodium hydroxide 0.397% (emulsion), 0.1% (solution) Sodium metabisulfite 0.2% (solution), 0.25% (solution, drops), 0.1% (suspension, drops) Sodium nitrate 1.18% (solution) Sodium phosphate 0.81% (solution), 0.29% (solution, drops), 0.2% (suspension) Sodium phosphate dihydrate 0.03% (solution) Sodium phosphate, dibasic 0.29% (solution), 0.43% (suspension) Sodium phosphate, dibasic, 1.28% (solution), 1.4% (solution, drops), 0.25% (suspension) anhydrous Sodium phosphate, dibasic, 1.081% (solution), 1.201% (solution, drops) dihydrate Sodium phosphate, dibasic, 2.15% (solution), 2.5% (solution, drops), 0.866% (suspension), heptahydrate 0.431% (suspension, drops) Sodium phosphate, monobasic 0.19% (solution), 0.01% (solution, drops) Sodium phosphate, monobasic, 0.725% (solution), 0.78% (solution, drops), 0.65% (suspension), anhydrous 0.056% (suspension, drops) Sodium phosphate, monobasic, 1.158% (solution), 1.053% (solution, drops) dihydrate Sodium phosphate, monobasic, 0.54% (solution), 0.721% (solution, drops), 0.538% (suspension) monohydrate Sodium sulfate 0.226% (solution), 1.2% (suspension) Sodium sulfate anhydrous 0.152% (solution), 0.17% (solution, drops), 1.2% (suspension) Sodium sulfate decahydrate 0.09% (solution, drops) Sodium sulfite 0.2% (solution, drops) Sodium thiosulfate 5% (solution), 0.31% (solution, drops), 0.32% (suspension), 0.314% (suspension, drops) Sorbic acid 0.1% (emulsion), 0.2% (solution; solution, drops) Sorbitol 0.2% (solution), 0.25% (solution, drops) Stabilized oxychloro complex 0.005% (solution, drops) Sulfuric acid 0.02% (solution, drops) Thimerosal 0.01% (solution; solution, drops), 0.004% (suspension), 1% (suspension, drops) Tocophersolan 0.5% (solution, drops) Trisodium citrate dihydrate 0.3% (solution), 0.294% (solution, drops) Triton 720 N/A (solution) Tromethamine 0.936% (solution), 0.75% (solution, drops) Tyloxapol 0.1% (solution; solution, drops), 0.3% (suspension; suspension, drops) Xanthan gum 0.6% (solution) Zinc chloride 0.0025% (solution, drops)

(63) Preferably, the compositions are delivered by intravitreal injection or subconjunctival administration.

(64) As described in detail below, suppressing expression of p53 was a required event in two assays of PVR, namely, PDGFRα-mediated contraction of cells in a collagen gel and retinal detachment in an animal model of PVR. Furthermore, as described in detail below, preventing the decline in the level of p53 with agents such as Nutlin-3a protected from retinal detachment, which is the most vision-compromising component of PVR. Finally, as described herein, Nutlin-3a is effective in the clinical setting, as the small molecule prevented human PVR vitreous-induced contraction of cells isolated from a patient PVR membrane.

(65) As described below, Nutlin-3a was administered in a series of intravitreal injections. While this approach completely prevented retinal detachment, 50% of the rabbits developed vitreal traction (stage 2) (FIG. 2A). Since the last injection of Nutlin-3a was 23 days prior to the end of the experiment, the level of Nutlin-3a may have dropped below the therapeutic range. The recently-developed ocular formulation of Nutlin-3a, which can be administered as a subconjunctival injection (Brennan R C et al., 2011 Cancer Res, 71:4205-4213), is also suitable to achieve Nutlin-3a-mediated PVR prophylaxis.

(66) As described in detail below, Nutlin-3a treatment very effectively prevented retinal detachment, and also slowed formation of membranes (stage 1) (FIG. 2A), both of which are of clinical benefit. It was surprising that membranes were able to form in Nutlin-3a animals, because Nutlin-3 activates the p53 pathway (Vassilev L T et al., 2004 Science, 303:844-848), which counters cellular events that are intrinsic to membrane formation such as proliferation and survival (Lei H et al., 2010 Exp Eye Res, 90:376-381). However, this observation is consistent with reports that elevating p53 does not always cause apoptosis and/or cell cycle arrest (Giono L E et al., 2007 Mol Cell Biol, 27:4166-4178; Mendrysa S M et al., 2003 Mol Cell Biol, 23:462-472; Mendrysa S M et al., 2006 Genes Dev, 20:16-21). For instance, p53 is transiently elevated in mitotic cells of normal tissue in healthy animals, and fails to engage the p53 pathway because p53 is held in check by post-translational modifications (Loewer A et al., 2010 Cell, 142:89-100).

(67) The results described herein reveal that cellular responses associated with PVR do not have the same requirements. Contraction and proliferation require a decline in the level of p53 and a second PDGFRα-mediate event(s), whereas protection from apoptosis and senescence proceed when only p53 is suppressed (FIG. 6). Furthermore, while all 4 responses were completely dependent on expression of PDGFRα, contraction required the lowest level. This low threshold for PDGFRα expression and ability of contraction to proceed when other cellular response are impaired provide a plausible explanation for why therapies targeting other cellular responses have not been successful (Wiedemann P et al., 1998 Am J Ophthalmol, 126:550-559; Asaria R H et al., 2001 Ophthalmology, 108:1179-1183; Schiff W M et al., 2007 Arch Ophthalmol, 125:1161-1167).

(68) As described in detail below, epiretinal membranes formed in rabbits injected with cells that were unable to suppress p53 efficiently (sh PDGFRα), which was required for RV-mediated proliferation and viability, cellular events that are thought to be essential for membrane formation. Previous reports have shown that proliferation-incompetent cells induce PVR provided that they are injected at a sufficiently high level (Fastenberg D M et al., 1982 Am J Ophthalmol, 93:565-572). Thus, membranes may have formed in rabbits injected with sh PDGFRα cells because enough of them were injected.

(69) The examples below demonstrate that both molecular and pharmacological approaches indicate that reducing the level of p53 was permissive for retinal detachment, a process that involves contraction of the retina-associated membrane. A simple explanation for this phenomenon is that p53 suppresses the expression of genes that are required for retinal detachment. For instance, p53 may inhibit production of those extracellular matrix proteins that are required for contraction of the membrane (Iotsova V et al., 1996 Cell Growth Differ, 7:629-634). However, such an explanation appears inadequate for the in vitro contraction assays, which contained ample extracellular matrix proteins that are conducive for contraction. p53 may down regulate expression of integrins such as β1 (Qiu J et al., 2011 Mol Cell Biochem, 357:125-133), whose interaction with extracellular matrix proteins is essential for contraction. Alternatively, there may be a connection to EMP2 (epithelial cell membrane protein) and FAK (focal adhesion kinase), which are essential for contraction of collagen gels and strongly implicated in PVR (Morales S A et al., 2009 Invest Ophthalmol Vis Sci, 50:4949-4956; Morales S A et al., 2007 Exp Eye Res, 85:790-798; Morales S A et al., 2009 Invest Ophthalmol Vis Sci, 50:462-469; Morales S A et al., 2011 Invest Ophthalmol Vis Sci, 52:5465-5472; Telander D G et al., 2011 Curr Eye Res, 36:546-552).

(70) Finally, just as p53 suppresses cell cycle progression (Levine A J et al., 1991 Nature, 351:453-456), the results presented herein indicate that p53 is a checkpoint of retinal detachment. In contrast to genetic lesion of the p53 pathway that are present in approximately 50% of solid tumors (Hainaut P and Hollstein M, 2000 Adv Cancer Res, 77:81-137, epigenetic, environmental factors that result in non-canonical activation of PDGFRα drive p53-dependent blinding diseases such as PVR.

EXAMPLES

Example 1: Materials and Methods

(71) The materials and methods used in the examples described herein are set forth below.

(72) Major Reagents and Cell Culture

(73) The phospho-Y742 PDGFRα antibody was raised against the phospho-peptide [KQADTTQpYVPMLDMK (SEQ ID NO: 3), where the lower case “p” represents the phosphorylated Tyrosine residue] (Lei H et al., 2010 Am J Pathol, 177:132-140). The Ras GTPactivating protein (RasGAP) antibody was crude rabbit antiserum against a GST fusion protein including the SH2-SH3-SH2 region of the human RasGAP (Rosenkranz S et al., 1999 J Biol Chem, 274:28335-28343). Antibodies against PDGFRα, phospho-Akt (S473), Akt, and p53 were purchased from Cell Signaling Technology (Beverly, Mass.). Secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G, and goat anti-mouse immunoglobulin G) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Enhanced chemiluminescent substrate for detection of horseradish peroxidase was from Pierce Protein Research Products (Rockford, Ill.). ApoAlert annexin VFITC apoptosis kit and in situ β-galactosidase assay kit were purchased from Clontech Laboratories, Inc. (Mountain View, Calif.) and Agilent Technologies, (Santa Clara, Calif.), respectively. Nutlin-3a was purchased from Cayman Chemical (Ann Arbor, Mich.). Normal rabbit vitreous (RV) was prepared from frozen rabbit eyeballs as previously described (Lei H et al., 2009 J Biol Chem, 284:6329-6336). The level of PDGFs in RV is either very low, or below the level of detection (Lei H et al., 2007 Invest Ophthalmol Vis Sci, 48:2335-2342; Pennock S et al., 2011 Am J Pathol, 179:2931-2940).

(74) RPEM cells are RPE cells derived from a human epiretinal membrane, as previously described (Wong C A et al., 2002 Can J Ophthalmol, 37:211-220). Primary rabbit conjunctival fibroblasts (RCFs) were obtained and cultured as described previously (Ikuno Y et al., 2002 Invest Ophthalmol Vis Sci, 43:2406-2411). RCFs that stably expressed the shRNA targeting vector specific for GFP, PDGFRα, p53 or PDGFRα and p53 were designated sh GFP, sh PDGFRα sh p53 and sh PDGFRα/p53, respectively. F cells are immortalized mouse embryo fibroblasts derived from PDGFR knock-out mice that do not express either of the two PDGFR genes, Fα and Fβ cells are F cells in which PDGFRα or PDGFRβ has been re-expressed (Andrews A et al., 1999 Invest Ophthalmol Vis Sci, 40:2683-2689).

(75) Knockdown of PDGFRα and p53

(76) Oligos (GCCAGCTCTTATTACCCTCTA (SEQ ID NO: 4)) for PDGFRα, (CGGGCGTAAACGCTTCGAGAT (SEQ ID NO: 5)) for p53 and (ACAACAGCCACAACGTCTATA (SEQ ID NO: 6)) for GFP in a hairpin-pLKO.1 retroviral vector respectively, the packaging plasmid (pCMVdR8.91), the envelope plasmid (VSV-G/pMD2.G) and 293T packaging cells used. The shRNA lentiviruses were prepared as described previously (Lei H et al., 2011 Mol Cell Biol, 31:1788-1799). The viruses were used to infect RCF cells. Successfully infected cells were selected on the basis of their ability to proliferate in media containing puromycin (1 μg/ml). The resulting cells were characterized by western blot analysis using antibodies against PDGFRα, p53 and RasGAP (loading control).

(77) Western Blot

(78) Cells were grown to 90% confluence in serum-containing medium, and then incubated for 24 hr in medium without serum. Cells were stimulated (as detailed for each experiment), washed twice with ice-cold phosphate buffered saline (PBS), and lysed in extraction buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 20 μg/ml aprotinin, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride). Lysates were clarified by centrifugation at 13,000×g, 4° C. for 15 min. Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes, and then subjected to western blot analysis using indicated antibodies. Signal intensity was determined by densitometry and analyzed with the Quantity One (Bio-Rad) software.

(79) Collagen I Contraction Assay

(80) Cells were trypsinized, washed and resuspended in 1.5 mg/ml of neutralized collagen I (INAMED, Fremont, Calif.) (pH 7.2) at a density of 1×10.sup.6 cells/ml for RPEM or 5×10.sup.4 cells/ml for RCFs. The mixture was aliquoted into wells of a 24-well plate that had been preincubated overnight with 5 mg/ml bovine serum albumin in PBS. The collagen solution was solidified by incubating at 37° C. for 90 min, and overlaid with medium containing the desired agents. The media were replaced every day, and the gel diameter was measured on day 3. The gel area was calculated using the formula πr2, where r is the radius of the gel.

(81) Proliferation and Apoptosis Assays

(82) Proliferation and apoptosis was assayed as previously described (Lei H et al., 2009 J Biol Chem, 284:6329-6336). Briefly, RCFs were seeded into 24-well plates at a density of 50,000 cells/well in DMEM+10% FBS. After 6 hrs the cells had attached; the medium was aspirated, the cells were rinsed twice with PBS and the cells were cultured in serum-free DMEM with or without RV (1:3 dilution). The media were replaced every day. On Day 3, the cells were counted in a hemocytometer. At least three independent experiments were performed. To monitor apoptosis, RCFs were seeded into 6 cm-dishes at a density of 1×10.sup.5 cells per dish in DMEM+10% FBS. After the cells had attached the dishes, they were treated as described above in the proliferation Assay. On Day 3, the cells were harvested and stained with FITC-conjugated Annexin V and propidium iodide according to the instructions provided with the apoptosis kit (BD Biosciences, Palo Alto, Calif.). The cells were analyzed by flow cytometry in Coulter Beckman XL. At least three independent experiments were performed.

(83) Senescence Assay

(84) RCF cells were plated into a 12-well plate (10,000 cells/well) in DMEM (high glucose) supplemented with 10% FBS. After 6 hrs the medium was changed into DMEM with or without RV (1:3 dilution), and replenished every 24 hrs. On day 3, the β-galactosidase activity was assessed according to the manufacturer's instructions provide with the in situ β-galactosidase assay kit.

(85) Rabbit Model for PVR

(86) PVR was induced in Dutch Belted rabbits, purchased from Covance (Denver, Pa.), as previously described (Lei H et al., 2009 Invest Ophthalmol Vis Sci, 50:3394-3403). Briefly, a gas vitrectomy was performed by injecting 0.1 ml of perfluoropropane (C3F8) (Alcon, Fort Worth, Tex.) into vitreous. One week later, the right eye of rabbits was injected in one of two ways. For the experiment injecting RCFs expressing shRNAs, 0.1 ml of DMEM containing 1×10.sup.5 RCFs that were modified as outlined in the legend were injected along with 0.1 ml rabbit platelet-rich plasma. For the Nutlin-3a experiment, all rabbits were injected with 0.1 ml of DMEM containing 1×10.sup.5 unmodified RCFs, 0.1 ml rabbit platelet rich plasma and either not injected a third time, or injected with vehicle, or 0.1 ml of 200 μM Nutlin-3a. The vehicle or Nutlin-3a injection was repeated on day 3 and 5. The retinal status was evaluated with an indirect ophthalmoscope fitted with a +30 D fundus lens on day 1, 3, 5, 7, 14, 21 and 28. PVR was graded according to the Fastenberg scale of classification 41: stage 0, no disease; stage 1, epiretinal membrane; stage 2, vitreoretinal traction without retinal detachment; stage 3, localized retinal detachment (1-2 quadrants); stage 4, extensive retinal detachment (2-4 quadrants without complete detachment); stage 5, complete retinal detachment. On day 28, animals were sacrificed, and eyes were enucleated and frozen at −80° C.

(87) Immunohistochemistry

(88) Rabbit eyeballs were fixed in 10% formalin for 48 hr and embedded in paraffin after dehydration. Subsequently, 4 μm paraffin sections were prepared, dewaxed in xylene and rehydrated in ethanol, diluted ethanol and deionized water. Antigen retrieval that was performed by boiling the slides for 20 min in a citrate-based buffer (Vector Laboratories Inc., Burlingame, Calif.). The endogenous peroxidase activity was blocked by incubation with 1% H.sub.2O.sub.2 in methanol for 10 min and the endogenous avidin and biotin binding sites were blocked by incubation with avidin and biotin blocking buffers (Vector Laboratories). The resulting sections were first incubated in blocking buffer containing 3% goat serum, and then in primary antibody (diluted 1:200 in blocking buffer, anti-p53 from ABcam [Cambridge, Mass.]) overnight at 4° C. Incubation with secondary antibody (biotinylated goat anti-mouse; ABcam) was for one hour at room temperature. Finally, the ABC reagent (Vector Laboratories) was added for 45 min and the sections were stained with DAB (Thermo Scientific, Rockford, Ill.). The sections were observed and photographed under a microscope.

(89) Statistics

(90) The experimental data were analyzed using an unpaired t test and one way ANOVA and/or post tests. A p value of less than 0.05 was considered statistically significant.

Example 2: Suppressing p53 was Essential for RV-Induced Contraction, and Retinal Detachment

(91) RV contains a variety of non-PDGFs that indirectly activate PDGFRα and thereby chronically stimulate Akt (Lei H et al., 2011 Mol Cell Biol, 31:1788-1799), which phosphorylates and activates Mdm2 (Zhou B P et al., 2001 Nat Cell Biol, 3:973-982) that mediates a decline in the level of p53 (Ogawara Y et al., 2002 J Biol Chem, 277:21843-21850; Gottlieb T M, et al., 2002 Oncogene, 21:1299-1303; Haupt Y et al., 1997 Nature, 387:296-299). Nutlin-3a antagonizes the interaction of Mdm2 and p53, and thereby prevents Mdm2-mediated reduction of p53 (Vassilev L T et al., 2004 Science, 303:844-848). Because of these properties, it was determined whether Nutlin-3a would prevent RV-mediated reduction in the level of p53, contraction of cells in collagen gels and protect rabbits from developing PVR. Primary rabbit conjunctival cells (RCFs) were utilized in these experiments because they robustly contract collagen gels and induce PVR.

(92) Nutlin-3a effectively blocked the RV-mediated decline in the level of p53 and contraction of collagen gels (FIGS. 1A-1B). As expected, it had no effect on RV-induced phosphorylation of PDGFRα or activation of Akt (FIG. 1A), events that are upstream of the known action of Nutlin-3a. The minimum dose to prevent the RV-induced reduction of p53 was 2 micromolar (FIG. 8A), whereas the maximum tolerated dose was 30 micromolar (FIG. 8B). Moreover, multiple intravitreal injection of Nutlin-3a of up to 20 micromolar did not produce overt signs of retinal toxicity (FIGS. 9A-9B, and data not shown). These results set the stage to test if Nutlin-3a could prevent retinal detachment in an animal model of PVR.

(93) As shown in FIGS. 2A-2B, 100% of the animals in both control groups (uninjected, empty circles; or injected with vehicle, filled circles) developed complete retinal detachment (stage 5) by day 28. In contrast, 0% of the Nutlin-3a-treated animals (squares in FIG. 2A) succumbed to even partial retinal detachment (stage 3 or 4), although they did form membranes (stage 1 and 2 in FIG. 2A; FIGS. 9A-9B). Importantly, the p53 level was higher in epiretinal membranes isolated from Nutlin-3a-injected animals as compared with vehicle-injected controls (FIG. 2B). These observations indicate that Nutlin-3a treatment maintained a high level of p53 expression in cells of the epiretinal membrane and prevented retinal detachment.

(94) A molecular approach to assess the importance of reducing p53 for RV-mediated contraction and retinal detachment led to a similar conclusion. The overall strategy of this second approach was to reduce the PVR potential of RCFs by silencing expressing of PDGFRα, and then to test if it could be rescued by silencing expression of p53. Lentiviral-mediated delivery of shRNAs directed toward either PDGFRα, or p53 suppressed expression by at least 80% (FIG. 3A). As shown in FIG. 3B, RV-mediated suppression of p53 in sh PDGFRα cells was substantially reduced, although not completely eliminated. Similarly, RV-induced contraction of cells was largely, although not completely diminished in sh PDGFRα cells (FIG. 3C).

(95) To investigate whether the incomplete suppression of RV-mediated contraction was due to the residual expression of PDGFRα, the efficacy of Imatinib to interfere with RV-induced contraction was examined. Imatinib completely blocked RV-induced contraction in sh GFP cells (FIG. 10A), which indicated that one of the Imatinib targets (abl, c-kit PDGFRα and PDGFRβ (Druker B J et al., 2001 N Engl J Med, 344:1031-1037) was essential. The previously characterized panel of cells that do or do not express PDGFRs (Andrews A et al., 1999 Invest Ophthalmol Vis Sci, 40:2683-2689) provided the opportunity to identify the relevant Imatinib target. F cells, which are immortalized fibroblasts from mice lacking both PDGFR genes (but harboring all other Imatinib targets) contracted weakly to RV, and this responses was unaffected by Imatinib (FIG. 10B). Expressing PDGFRβ in these cells did not improve RV-induced contraction or generate sensitivity to Imatinib (FIG. 10B). Finally, expression of PDGFRα improved this RV-stimulated response, which was erased by Imatinib (FIG. 10B). These observations indicate that PDGFRα was the relevant target of Imatinib, and that the modest RV-induced contraction seen in sh PDGFRα cells (FIG. 3C) was due to residual expression of PDGFRα.

(96) As expected from previous studies assessing the importance of PDGFRα for experimental PVR (Andrews A et al., 1999 Invest Ophthalmol Vis Sci, 40:2683-2689; Lei H et al, 2009 Invest Ophthalmol Vis Sci, 50:3394-3403; Ikuno Y et al., 2000 Invest Ophthalmol Vis Sci, 41:3107-3116), there was a significantly statistic reduction in the PVR potential of sh PDGFRα cells (FIG. 4). The sh PDGFRα cells failed to induce retinal detachment, although they retained their ability to form membranes, which exerted traction of the retina (FIG. 4; compare empty and filled circles). Molecularly suppressing p53 in sh PDGFRα cells fully restored their ability to induce retinal detachment (FIG. 4; compare filled circles with squares). Thus, two different experimental approaches indicated that reducing the level of p53 was essential for RV-mediated contraction and retinal detachment in an animal model of PVR.

Example 3: PDGFRα Did More than Reduce p53 to Promote Contraction

(97) To assess if reducing p53 was the only PDGFRα-mediated event required for contraction in response to RV, this outcome was compared in sh GFP and sh p53 cells. If it was, then contraction of sh 53 cells would be RV-independent. As shown in FIGS. 11A-11C, this was not the case. While basal contraction of sh 53 cells was enhanced, they responded well to RV. These observations indicated that PDGFRα did more than reduce the level of p53 in order to mediate RV-dependent contraction.

(98) The same question was addressed for 3 additional cellular responses associated with PVR, proliferation and protection from apoptosis and senescence. Like contraction, proliferation of sh p53 cells was responsive to RV (FIG. 5A), whereas protection from apoptosis and senescence were fully engaged in unstimulated cells and RV did not enhance these responses further (FIGS. 5B and 5C). These findings indicate that reducing p53 was sufficient to trigger some of the RV-stimulated cellular responses that are associated with PVR (protection from apoptosis and senescence), whereas contraction and proliferation required an event(s) in addition to reducing the level of p53 (FIG. 6).

(99) Residual expression of PDGFRα in shPDGFRα cells provided an opportunity to compare cellular responses associated with PVR for their dependence on the level of expression of PDGFRα. RV was unable to promote proliferation of sh PDGFRα cells or protect them from apoptosis or senescence (FIGS. 5A-5C), which indicated that these responses required expression of PDGFRα in excess of 20% of the control level. In contrast, contraction remained responsive to RV (albeit weakly) in sh PDGFRα cells (FIG. 3C). Thus, contraction required less PDGFRα expression than did proliferation or protection from apoptosis and senescence.

Example 4: Relevance to Clinical PVR

(100) To begin to assess the clinical relevance of these findings the impact of Nutlin-3a on HV-mediated signaling events and contraction of RPE cells isolated from a human PVR membrane was considered. As shown in FIG. 7A, Nutlin-3a prevented the precipitous fall in the level of p53 observed in HV-treated control cells, without impacting upstream signaling events. Furthermore, Nutlin-3a inhibited HV-stimulated contraction of RPE-containing collagen gels (FIG. 7B). In addition, p53 was undetectable in epiretinal membranes from PVR patients (FIGS. 12A-12F). These findings indicate that Nutlin-3a had the potential to protect patients from developing PVR.

OTHER EMBODIMENTS

(101) While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

(102) The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

(103) While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.