Pharmaceutical Compositions, Formulations And Methods For The Treatment Of Retinoblastoma

Abstract

The present invention provides a method for the treatment of retinoblastoma comprising administering a composition comprising a therapeutically active agent to a subject in need thereof by injection of the composition into the vitreous cavity, suprachoroidal space, supraciliary space or sub-Tenon's space of the eye adjacent to a retinoblastoma tumour. The invention also provides a composition comprising at least one therapeutically active agent selected from the group consisting of a Bcl-2 inhibitor or a topoisomerase inhibitor for use in the treatment of retinoblastoma, wherein the composition is for administration into the vitreous cavity, suprachoroidal space, sub-Tenon's space, or supraciliary space adjacent to a retinoblastoma tumour in an eye. Also provided is a kit comprising a Bcl-2 inhibitor and a topoisomerase inhibitor for use in the treatment of retinoblastoma, wherein the Bcl-2 inhibitor and the topoisomerase inhibitor are for separate, simultaneous or sequential administration. The invention also provides a kit comprising a composition comprising at least one therapeutically active agent and a cannulation or catheterization device for use in the treatment of retinoblastoma.

Claims

1. A method for the treatment of retinoblastoma comprising administering a composition comprising a therapeutically active agent to a subject in need thereof by injection of the composition into the vitreous cavity, suprachoroidal space, supraciliary space or sub-Tenon's space of the eye adjacent to a retinoblastoma tumour.

2. The method of claim 1, wherein the therapeutically active agent is selected from the group consisting of a Bcl-2 inhibitor, a HDAC inhibitor or a topoisomerase inhibitor.

3. The method of claim 1 or claim 2, wherein the method comprises a further step of administering a composition comprising a therapeutically active agent, wherein the therapeutically active agent is selected from the group consisting of a Bcl-2 inhibitor or a topoisomerase inhibitor.

4. The method of any one of claims 1 to 3 wherein the Bcl-2 inhibitor is selected from the group consisting of TW-37, venetoclax, navitoclax, ABT-737, sabutoclax, obatoclax, ABT-263, oblimersen, AT101, SS5746, APG-1252, APG-2575, S55746 or UBX1967/1325.

5. The method of any one of claims 1 to 3 wherein the topoisomerase inhibitor is selected from the group consisting of topotecan, irinotecan, doxorubicin, irinotecan, daunorubicin, SN-38, voreloxin, belotecan or semisynthetic derivatives of podophyllotoxin (etoposide).

6. The method of any one of claims 1 to 3 wherein the HDAC inhibitor is selected from the group consisting of vorinostat, belinostat, panobinostat, romidepsin, entinostat, mocetinostat, CUDC-101, tacedinaline or nicotinamide.

7. The method of any one of claims 1 to 6, wherein the method comprises a further step of administering a composition comprising a DNA damaging agent.

8. The method of claim 7 wherein the DNA-damaging agent is selected from the group consisting of altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, dactinomycin, ilfosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, procarbazine, streptozocin, temozolomide, thiotepa or trabectedin.

9. A composition comprising at least one therapeutically active agent selected from the group consisting of a Bcl-2 inhibitor, a HDAC inhibitor or a topoisomerase inhibitor for use in the treatment of retinoblastoma, wherein the composition is for administration into the vitreous cavity, suprachoroidal space, sub-Tenon's space, or supraciliary space adjacent to a retinoblastoma tumour in an eye.

10. The composition for use of claim 9, wherein the Bcl-2 inhibitor is selected from the group consisting of TW-37, venetoclax, navitoclax, ABT-737, sabutoclax, obatoclax, ABT-263, oblimersen, AT101, SS5746, APG-1252, APG-2575, S55746 or UBX1967/1325.

11. The composition for use of claim 9, wherein the topoisomerase inhibitor is selected from the group consisting of topotecan, irinotecan, doxorubicin, irinotecan, daunorubicin, SN-38, voreloxin, belotecan or semisynthetic derivatives of podophyllotoxin (etoposide).

12. The composition for use of claim 9, wherein the HDAC inhibitor is selected from the group consisting of vorinostat, belinostat, panobinostat, romidepsin, entinostat, mocetinostat, CUDC-101, tacedinaline or nicotinamide.

13. The composition for use of claim 9, wherein the composition comprises a Bcl-2 inhibitor, an excipient comprising an amphiphilic polymer, and an aqueous solution, wherein the Bcl-2 inhibitor is associated with the excipient in the form of micelles suspended in the aqueous solution.

14. The composition for use of claim 13, wherein the amphiphilic polymer comprises a polyethylene glycol conjugated lipid.

15. The composition for use of claim 14, wherein the polyethylene glycol conjugated lipid is selected from the group consisting of polyethylene glycol conjugated 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), conjugated 1,2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), conjugated 1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) or conjugated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

16. The composition for use of claim 15, wherein the polyethylene glycol in the polyethylene glycol conjugated lipid has a molecular weight range of 100 to 5000 Daltons.

17. The composition for use of any one of claims 13 to 16, where the Bcl-2 inhibitor is TW-37.

18. The composition for use of claim 17, wherein the concentration of the Bcl-2 inhibitor is in the range of 1.5 μM to 50 μM.

19. The composition for use of any one of claims 14 to 18, wherein the conjugated lipid in the polyethylene glycol conjugated lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000].

20. The composition for use of claim 19 wherein the concentration of the conjugated lipid is in the range of 2.5 μM to 50 μM.

21. The composition for use of any one of claims 13 to 20, wherein the molar ratio of the Bcl-2 inhibitor to amphiphilic polymer is in the range of 1:2 to 2:1.

22. The composition for use of any one of claims 13 to 21, wherein the composition further comprises a topoisomerase inhibitor.

23. A kit comprising a Bcl-2 inhibitor, a HDAC inhibitor and/or a topoisomerase inhibitor for use in the treatment of retinoblastoma, wherein the Bcl-2 inhibitor and the topoisomerase inhibitor are for separate, simultaneous or sequential administration.

24. The kit of claim 23, wherein the Bcl-2 inhibitor is selected from the group consisting of TW-37, venetoclax, navitoclax, ABT-737, sabutoclax, obatoclax, ABT-263, oblimersen, AT101, SS5746, APG-1252, APG-2575, S55746 or UBX1967/1325.

25. The kit of claim 23, wherein the topoisomerase inhibitor is selected from the group consisting of topotecan, irinotecan, doxorubicin, irinotecan, daunorubicin, SN-38, voreloxin, belotecan or semisynthetic derivatives of podophyllotoxin (etoposide).

26. The kit of claim 23, wherein the HDAC inhibitor is selected from the group consisting of vorinostat, belinostat, panobinostat, romidepsin, entinostat, mocetinostat, CUDC-101, tacedinaline or nicotinamide.

27. The use of a Bcl-2 inhibitor, a HDAC inhibitor or a topoisomerase inhibitor in the manufacture of a medicament for the treatment of retinoblastoma by administration into the vitreous cavity, suprachoroidal space or sub-Tenon's space adjacent to a retinoblastoma tumour in an eye.

28. The use of claim 27, wherein the Bcl-2 inhibitor is selected from the group consisting of TW-37, venetoclax, navitoclax, ABT-737, sabutoclax, obatoclax, ABT-263, oblimersen, AT101, SS5746, APG-1252, APG-2575, S55746 or UBX1967/1325.

29. The use of claim 27, wherein the topoisomerase inhibitor is selected from the group consisting of topotecan, irinotecan, doxorubicin, irinotecan, daunorubicin, SN-38, voreloxin, belotecan or semisynthetic derivatives of podophyllotoxin (etoposide).

30. The use of claim 27, wherein the HDAC inhibitor is selected from the group consisting of vorinostat, belinostat, panobinostat, romidepsin, entinostat, mocetinostat, CUDC-101, tacedinaline or nicotinamide.

31. A kit comprising a composition comprising at least one therapeutically active agent and a cannulation or catheterization device for use in the treatment of retinoblastoma in an eye, wherein the at least one therapeutically active agent is selected from the group consisting of a Bcl-2 inhibitor, a HDAC inhibitor or a topoisomerase inhibitor and wherein the cannulation or catheterization device is configured for delivery of the composition to the suprachoroidal space or supraciliary space.

32. The kit of claim 31, further comprising a pharmaceutically acceptable diluent.

33. The kit of claim 31, wherein the cannulation or catheterization device is configured to deliver injection volume in a range from 10 to 100 microliters.

34. A method for preparing the composition for use of claims 13 to 22 comprising: mixing the Bcl-2 inhibitor with an organic solvent to dissolve the Bcl-2 inhibitor; filtering sterilely the mixture; adding the organic solvent mixture to a volume of sterile filtered aqueous solution containing the amphiphilic polymer excipient; and mixing the formulated composition to produce Bcl-2 inhibitor containing micelles in an aqueous solution.

35. The method of claim 34, wherein the Bcl-2 inhibitor is TW-37.

36. The method of claim 34 or claim 35, wherein the amphiphilic polymer excipient is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000].

37. The method of claims 34 to 36, wherein the organic solvent is DMSO.

Description

[0060] In the Examples, reference is made to the following Figures in which:

[0061] FIG. 1 shows the results of retinoblastoma cell proliferation inhibition assays with a topoisomerase inhibitor (topotecan). FIG. 1a shows results for Y79 cell line. FIG. 1b shows results for WERI cell line. FIG. 1c shows results for BJ cell line.

[0062] FIG. 2 shows the results of retinoblastoma cell proliferation inhibition assays with a Bcl-2 inhibitor (TW-37). FIG. 2a shows results for Y79 cell line. FIG. 2b shows results for WERI cell line. FIG. 2c shows results for BJ cell line.

[0063] FIG. 3 shows the retinoblastoma cell areas of eyes from in-vivo study of retinoblastoma treatment. FIG. 3a shows cell areas from histological sections after H&E staining. FIG. 3b shows cell areas from histological section after staining with anti-human antibody.

[0064] FIG. 4 shows histology images of the retinoblastoma cells on the retina from in-vivo study of retinoblastoma treatment. Arrows indicate evidence of retinoblastoma cell proliferation on the lining of retina.

[0065] FIG. 5 shows the results of retinoblastoma cell proliferation inhibition assays with combination of a topoisomerase inhibitor (topotecan) and a Bcl-2 inhibitor (TW-37). FIG. 5a shows results for Y79 cell line. FIG. 5b shows results for WERI cell line. FIG. 5c shows results for BJ cell line.

[0066] FIG. 6 shows the ocular pharmacokinetic results of suprachoroidal administration of a micelle formulation of TW-37 in rabbit eyes.

EXAMPLE 1: RETINOBLASTOMA CELL PROLIFERATION INHIBITION ASSAYS WITH BCL-2 INHIBITOR AND TOPOISOMERASE Inhibitor

[0067] Testing of candidate compounds in a cell-based assay to determine the extent of cell inhibition with two human retinoblastoma cell lines (Y79 and WERI-Rb1) was performed. A normal fibroblast cell line (BJ) was used to identify active agents that selectively affect retinoblastoma. The cells were tested for mycoplasma contamination prior to use. Exponentially growing cells were plated in 384-well white, flat bottom, low flange, tissue culture treated assay plates and incubated overnight at 37° C. in a humidified 5% CO.sub.2 incubator. DMSO inhibitor stock solutions were added the following day by manual pin transfer with 50SS pins to a top final concentration of 50 μM and 3 μM in 0.25% DMSO and then diluted 1/3 for a total of ten testing concentrations for each dilution scheme. Combined these two dilution schemes captured twenty data points from 50 μM to 0.2 nM. For Y79, the cells were plated to 1,000/cells per well in 25 microliters of complete media. For WERI-RB-1, the cells were plated to 2,000 cells/well in 25 microliters of complete media. For BJ, the cells were plated to 1,000/cells per well in 30 microliters of complete media. After addition of the candidate compounds, the number of cells was determined following a 72 hour incubation period using the Cell Titer Glo Reagent (Promega, Madison, Wis.). Luminescence was measured on a Clariostar plate reader (BMG Labtech). Assay endpoints were normalized from 0% (DMSO only) to 100% inhibition and fit to a semi-log plot using n=3 technical replicates and the four parameter variable slope algorithm in GraphPad Prism. The experiments were replicated a second time by a different operator to ensure reproducibility of the data.

[0068] Over 25 inhibitors of cellular pathways involved in cell growth and apoptosis were screened including Chetomin, Daprodustat, MK-8617, BAY-85-3934 (Molidustat), BAY-87-2243, 2-Methoxyestradiol, Vincristine-Sulfate, Calcitriol, Carboplatin, Melphalan, Etoposide, Lificiguat, Nutlin-3, Nutlin-3A, Idasanutlin, IOX2, RV1162, PTC-209, Cerdulatinib, Idarubicin, Cabatzitaxel, Romidepsin, TW-37, Flavopirodol, Obatoclax, BAY-61-3606, Topotecan, Doxorubicin. Evaluation of the cell inhibition and death curves provided an estimation of the active agent concentration for 50% cell inhibition (EC50). The results identified compounds with promising effectiveness against retinoblastoma with less toxicity to normal cells to provide a therapeutic range of treatment. The greatest effectiveness was found with Bcl-2 inhibitors (TW-37, sabutoclax), a topoisomerase inhibitor (topotecan) and a HDAC inhibitor (vorinostat).

[0069] Topotecan inhibits topoisomerase I activity by stabilising the topoisomerase I-DNA covalent complexes during S phase of cell cycle, thereby inhibiting re-ligation of topoisomerase I-mediated single-strand DNA breaks and producing potentially lethal double-strand DNA breaks when encountered by the DNA replication machinery. Topotecan demonstrated significant growth inhibition of retinoblastoma cells at low μM concentrations with very low toxicity to normal cells (p<0.001 at 1 μM). Topotecan demonstrated an EC50 of 0.069 μM for Y79 cell line, 0.039 μM for WERI cell line and >2.57 μM for BJ cell line. Two replicate assays were performed, confirming the results. (See FIGS. 1a, 1b, 1c).

[0070] TW-37 binds to the BH3 (Bcl-2 homology domain 3) binding groove of Bcl-2 and competes with pro-apoptotic proteins (such as Bid, Bim and Bad) preventing their heterodimerisation with Bcl-2, and therefore allowing these proteins to induce apoptosis. TW-37 demonstrated significant retinoblastoma cell kill at very low μM active agent concentration with very low toxicity to normal cells. (p<0.001 at 1 μM). TW-37 demonstrated an EC50 of 0.335 μM for Y79 cell line, 0.278 μM for WERI cell line and >8.76 μM for BJ cell line. Two replicate assays were performed, confirming the results (See FIG. 2a, 2b, 2c).

[0071] Vorinostat inhibits HDAC activity and inhibits class I and class II HDAC enzymes. The resulting accumulation of acetylated histones and acetylated proteins induces cell cycle arrest and apoptosis of some transformed cells. Vorinostat demonstrated an EC50 of 2.84 μM for Y79 cell line, 1.37 μM for WERI cell line and >54.4 μM for BJ cell line.

[0072] Sabutoclax is a pan-Bcl-2 family inhibitor that may activate caspase-3/7 and caspase 9, and may modulate Bax, Bim, PUMA and survivin expression. The agent provides reactivation of apoptosis mediated by several anti-apoptotic Bcl-2 family proteins. Sabutoclax demonstrated an EC50 of 0.316 μM for Y79 cell line, 0.211 μM for WERI cell line and >3.65 μM for BJ cell line.

EXAMPLE 2: RETINOBLASTOMA IN VIVO MODEL TREATED WITH BCL-2 INHIBITOR AND TOPOISOMERASE INHIBITOR

[0073] Human retinoblastoma tumour cells, Y79 (ATCC@ HTB-18), were grown in media consisting of RPMI 1640 containing 20% FBS, L-glutamine 200 mM (100×), penicillin/streptomycin 5,000 U/ml and amphotericin B 250 μg/ml, to a target suspension density of 3×10.sup.5 cells/flask in 20 ml of media in a T75 flask. Thirty-two eyes in 16 immunosuppressed rabbits were inoculated with 200,000 cells in 30 μl of serum free media to the posterior retinal surface by intravitreal injection. The animals were studied in 4 groups of 8 eight eyes. Two groups were administered topotecan prepared in 30 μl of sterile saline injected intravitreally through a 29 gauge needle in the posterior region of the vitreous cavity near the tumour cells. One topotecan group was administered a 10 μg dose and a second group administered a 50 μg dose. The topotecan groups were administered the active agent formulation at 2, 3 and 4 weeks after tumour cell inoculation. One group was treated with 10 μg of TW-37 in 30 ul of DMSO injected in the vitreous cavity through a 29 gauge needle near the posterior retina adjacent to the tumour cells. The TW-37 group was administered the active agent formulation 3 and 4 weeks post tumour cell inoculation. A fourth group was treated with a sham injection of 30 μl sterile saline through a 29 gauge needle in the posterior region of the vitreous near the tumour cells at 2, 3 and 4 weeks after tumour cell inoculation. All animals were culled at 5 weeks to allow processing of the retina and retinoblastoma tumour cells on the retina for histological examination. Macro photography of the flat mounted retinae recorded tumour cell survival on the retinae and then representative sections were removed for fixation and subsequent processing for histology. All samples were cut at 5 μm and stained by H&E and replicate slides stained with human mitochondrial marker antibody to positively identify the human retinoblastoma cells. Slides were then scanned using a slide scanner (Histech or Hamamatsu S360) and retinoblastoma cell area on the retina quantified using CaseViewer software. The retinoblastoma cell areas from both H&E staining and antibody staining were lower compared to sham with both doses of topotecan treatment and the TW-37 treatment. The high dose topotecan treatment demonstrated statistically significant reduction in retinoblastoma cells in the eyes as compared to sham with p<0.01. The TW-37 treatment demonstrated statistically significant reduction in retinoblastoma cells in the eyes as compared to sham with p<0.01.

[0074] The cell area results are shown in FIGS. 3a and 3b. Representative histology images are shown in FIG. 4 with arrows indicating the retinoblastoma cells on the retina.

EXAMPLE 3: RETINOBLASTOMA CELL PROLIFERATION INHIBITION ASSAYS WITH A COMBINATION OF BCL-2 INHIBITOR AND TOPOISOMERASE INHIBITOR

[0075] Using the cell assay method of Example 1, cell inhibition with combinations of TW-37 and topotecan was examined. Assays were performed with Topotecan titrated into the assay with TW-37 at constant concentration set to 0.662 μM. Topotecan concentrations of 0 μM, 0.0033 μM, 0.0264 μM and 0.1037 μM in DMSO were studied. The combination of TW-37 and Topotecan demonstrated additive inhibition of human retinoblastoma cell lines WERI and Y-79 with only slight toxicity to normal (BJ) cells. The results are shown in FIGS. 5a, 5b, 5c.

EXAMPLE 4: MICELLULAR FORMULATION OF BCL-2 INHIBITOR WITH PEG-PHOSPHOLIPIDS

[0076] Micellular formulations of TW-37 were prepared. TW-37 solutions were prepared in DMSO at concentrations of 10 to 90 mM. PEG-phospholipid solutions were prepared with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] (18:0 PEG550 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (18:0 PEG1000 PE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (14:0 PEG1000 PE) dissolved in deionized water at concentrations of 5 to 45 mM. Equal volumes (20 μL) of MPEG solution and TW-37 solutions were combined in Eppendorf tubes and vortex mixed briefly at a molar stoichiometric ratio of TW-37 to PEG-phospholipid of 2:1. The solutions were examined for the presence of micelles by brightfield microscopy to identify the spherical micelles and changes over time such as loss of micelles, non-spherical particles, and aggregation. Formulations prepared with 18:0 PEG550 PE solutions at 5 and 15 mM (resulting in final formulation concentrations of 2.5 and 7.5 mM) with TW-37 solutions at 10 and 30 mM (resulting in final formulation concentrations of 5 and 15 mM) respectively demonstrated poor micelle formation. Formulations prepared with 18:0 PEG1000 PE solutions at 5, 15 and 45 mM (resulting in final formulation concentrations of 2.5, 7.5, and 22.5 mM) with TW-37 solutions at 10, 30, and 90 mM (resulting in final formulation concentrations of 5, 15, and 45 mM) respectively demonstrated micelle formation, with a greater number of micelles at the highest concentrations. However, the micelles showed limited stability with active agent escaping from the micelles to form crystals in the aqueous phase after 6 days at room temperature. Formulations prepared with 14:0 PEG1000 PE solutions at 5, 15 and 45 mM (resulting in final formulation concentrations of 2.5, 7.5, and 22.5 mM) with TW-37 solutions at 10, 30, and 90 mM (resulting in final formulation concentrations of 5, 15, and 45 mM) respectively demonstrated good micelle formulation with numerous micelles and no active agent crystals observed.

[0077] In similar study, equal volumes (20 μL) of MPEG solution in deionized water and TW-37 solutions in DMSO were combined in Eppendorf tubes and vortex mixed briefly at a molar stoichiometric ratio of TW-37 to PEG-phospholipid of 1:2. The solutions were examined for the presence of micelles by brightfield microscopy to identify the spherical micelles and changes over time such as loss of micelles, non-spherical particles, and aggregation. Formulations prepared with 18:0 PEG1000 PE solutions at 5, 15 and 45 mM (resulting in final formulation concentrations of 2.5, 7.5 and 22.5 mM) with TW-37 solutions at 2.5, 7.5, and 22.5 mM (resulting in final formulation concentrations 1.25, 3.75, 11.25 mM) respectively demonstrated micelle formulation, with a greater number of micelles at the highest concentrations. Formulations prepared with 14:0 PEG1000 PE solutions at 5, 15 and 45 mM (resulting in final formulation concentrations of 2.5, 7.5, 22.5 mM) with TW-37 at 2.5, 7.5, and 22.5 mM (resulting in final formulation concentrations of 1.25, 3.75, 11.25 mM) respectively demonstrated good micelle formulation with numerous micelles and no TW-37 crystals observed.

[0078] In a separate study, 18:0 PEG550 PE, 18:0 PEG1000 PE, 14:0 PEG1000 PE were prepared in deionized water at 10 and 15 mM. Solutions of TW-37 in DMSO were prepared at 3, 5, 7.5, and 10 mM. Equal 20 μL volumes of the solutions were mixed in an Eppendorf tube and vortex mixed to promote micelle formation. Brightfield microscopy showed formulations with 14:0 PEG1000 PE and TW-37 near 1:1 molar stoichiometry demonstrated the best micelle formation with greater number of micelles and no crystal formation indicating high association of the TW-37 with the micelles.

EXAMPLE 5: MICELLULAR FORMULATION OF BCL-2 INHIBITOR WITH PEG-PHOSPHOLIPID 14:0 PEG1000 PE

[0079] Micellular formulations of TW-37 were prepared with PEG-phospholipid 14:0 PEG1000 PE as the micelle formulation excipient. TW-37 solutions were prepared in DMSO at concentrations of 7.5, 10, 15 and 20 mM. The PEG-phospholipid was prepared in deionized water at concentrations of 10, 15, 20, and 30 mM. Equal 20 μL volumes of the solutions were mixed in an Eppendorf tube and vortex mixed to promote micelle formation. The mixed formulations were examined for the presence of micelles by brightfield microscopy to identify the spherical micelles and changes over time such as loss of micelles, non-spherical particles, aggregation and active agent crystal formation in the aqueous phase, indicating escape of active agent from the micelles. The micellular formulations were stored in the dark at room temperature and examined by microscopy over a 3 week period. The following table characterized the formulations at 3 weeks.

[0080] The two most stable formulations were prepared with PEG-phospholipid solutions at 10 mM and TW-37 solutions at 7.5 mM (final formulation concentration of 5 mM and 3.75 mM from dilution) and PEG-phospholipid solutions at 30 mM and TW-37 at 20 mM (final formulation concentrations of 15 mM and 10 mM from dilution). In general, the formulations with greatest stability were observed with molar stoichiometries of approximately 1:1 PEG-phospholipid to TW-37, or slightly greater than 1:1 to provide some excess of PEG-phospholipid to TW-37.

TABLE-US-00001 PEG-phospholipid TW-37 7.5 mM TW-37 15 mM TW-37 20 mM TW-37 30 mM [mM] crystals micelles crystals micelles crystals micelles crystals micelles 10 no good yes good yes no Yes Full of suspension amount of micelles crystals micelles 15 yes good yes good yes good Yes Full of suspension suspension suspension, crystals full of micelles 20 no few no good no full of Yes crystals micelles suspension, micelles and full of micelles micelles 30 no few yes few no less No full of micelles micelles micelles micelles than normal

EXAMPLE 6: STABILITY OF MICELLULAR FORMULATIONS OF BCL-2 INHIBITOR

[0081] A formulation with equal volumes of PEG-phospholipid 14:0 PEG1000 PE at 30 mM and TW-37 at 15 mM was prepared to produce a final formulation of 15 mM PEG-phospholipid and 7.5 mM TW-37. The formulation was protected from light and stored at −80° C., −20° C., 4° C. and room temperature. The formulation demonstrated approximately full recovery at when stored at −80, −20 and 4° C. after 4 weeks, indicating formulation stability. The room temperature sample showed TW-37 content of 80.3% at four weeks.

EXAMPLE 7: PHARMACOKINETIC STUDY OF BCL-2 INHIBITOR

[0082] A micellular formulation of TW-37 was prepared and administered into the suprachoroidal space of New Zealand White rabbits. A micellular formulation was prepared with an equal volumes of 4.8 mM TW-37 in DMSO added to 9.6 mM PEG-phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (14:0 PEG1000 PE) in deionized water for a final formulation of 2.9 mM TW-37 and 4.8 mM PEG-phospholipid. Both solutions were filter sterilized by passage through a sterile 0.2 micron nylon syringe filter into a sterile vial to produce a sterile formulation. The mixture was vortex mixed to produce a micelle suspension. A 25 μg dose of TW-37 in approximately 15 μL volume of the formulation was administered into the suprachoroidal space of twenty-four eyes in 12 rabbits. Eight eyes in 4 rabbits were administered 15 μL of a vehicle control into the suprachoroidal space, prepared identically to the active agent containing formulation but without TW-37. A flexible catheter with 250 micron OD and 140 micron ID was surgically introduced into the suprachoroidal space in the anterior region of the eye at the pars plana. The catheter was advanced posteriorly toward the posterior region of the suprachoroidal space. The catheter was configured to conduct light and provided illumination of the catheter tip and shaft to determine the catheter position and configuration by trans-scleral visualization. The illuminated tip of the catheter was used to position the catheter in the posterior region of the suprachoroidal space. The illuminated shaft of the catheter was used to manipulate and position the catheter to direct the injection toward the posterior region of the space. The study consisted of four groups, with each group consisting of six eyes administered the TW-37 formulation and two eyes administered the vehicle control. The eyes were examined by slit lamp in the anterior segment and by indirect ophthalmoscopy in the posterior segment prior to euthanasia for each time point at 1, 3, 7, 14 days post administration. The eyes were dissected and the vitreous, retina and choroidal separated and processed for TW-37 tissue concentration by LCMS. The tissue concentrations of TW-37 in the retina, choroid and vitreous are shown in FIG. 6.

[0083] The choroid demonstrated the highest level of TW-37 with the retina demonstrating a lower level of TW-37, generally following the pharmacokinetics of the choroid levels. The results indicate that the suprachoroidal space and choroid acted as a reservoir for the TW-37 and TW-37 passed into the retina to reach therapeutic levels. TW-37 had a peak concentration at 3 days in the choroid decreasing to near baseline at 14 days. TW-37 had a peak concentration in the retina at 7 days, decreasing to near baseline at 14 days. A single administration of TW-37 in the micellular formulation provided 14 days of tissue exposure of TW-37 to the target retina. The vitreous showed relatively low levels of TW-37 indicating low exposure to the anterior tissues of the eye and systemically.