WO2023034605A1

WO2023034605A1 - Polymeric nanoparticles comprising a histone deacetylase 6 / phosphoinositide 3-kinase-8 dual inhibitor and related methods

Info

Publication number: WO2023034605A1
Application number: PCT/US2022/042521
Authority: WO
Inventors: Surender Kharbanda; Sachchidanand TIWARI; Ashish THAKUR; Gregory Tawa; Gurmit Grewal; Harpal Singh
Original assignee: Hillstream Biopharma Inc; US Department of Health and Human Services
Current assignee: Tharimmune Inc; US Department of Health and Human Services
Priority date: 2021-09-03
Filing date: 2022-09-02
Publication date: 2023-03-09
Anticipated expiration: 2024-03-03

Abstract

The present disclosure relates to a composition comprising: polymeric nanoparticles comprising hybrid block copolymers comprising a poly(lactic acid)-poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PPG-PEG-PLA) penta-block copolymer with a methoxy poly(ethylene glycol)-poly(lactic acid) (m-PEG-PLA) di-block copolymer; and an HDAC6/PI3K-δ dual inhibitor; and/or BCL-2 inhibitor. Methods related to the use and manufacture of the polymeric nanoparticles are also disclosed.

Description

POLYMERIC NANOPARTICLES COMPRISING A HISTONE DEACETYLASE 6 / PHOSPHOINOSITIDE 3-KINASE-8 DUAL INHIBITOR AND RELATED METHODS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/260,904, filed September 3, 2021. The entire contents of this application are incorporated herein by reference.

FIELD

[0002] This disclosure relates to polymeric nanoparticles comprising a histone deacetylase 6 (HDAC6) / phosphoinositide 3-kinase-6 (PI3K-6) dual inhibitor and related methods of using the polymeric nanoparticles, treating of cancer, and making the polymeric nanoparticles.

BACKGROUND

[0003] Conventional chemotherapeutic agents used in the treatment of cancer can suffer from resistance of the cancer cells to the chemotherapeutic agents or from toxicity induced in healthy cells/tissues. Delivery of anticancer drugs would be more effective if the delivery system were able to effectuate treatment with smaller amounts of drugs and/or new combinations of drugs to mitigate resistance. There is a pressing need for such delivery systems.

SUMMARY

[0004] In one aspect, this disclosure provides a composition comprising polymeric nanoparticles comprising hybrid block copolymers comprising a poly(lactic acid)- poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol)-poly(lactic acid) (PLA- PEG-PPG-PEG-PLA) penta-block copolymer with a methoxy poly(ethylene glycol)- poly(lactic acid) (m-PEG-PLA) di-block copolymer; and a histone deacetylase 6 (HDAC6) / phosphoinositide 3-kinase-6 (PI3K-6) dual inhibitor. The HDAC6/PI3K-6 dual inhibitor is associated with the polymeric nanoparticles. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound III. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound XL.

[0005] In another aspect, the present disclosure provides a composition comprising polymeric nanoparticles comprising block copolymers comprising a PLA-PEG-PPG-PEG- PLA penta-block copolymer with an m-PEG-PLA di-block copolymer; and a B cell lymphoma-2 (BCL-2) inhibitor. The BCL-2 inhibitor is associated with the polymeric nanoparticles. In some embodiments, the BCL-2 inhibitor is venetoclax. In some embodiments, the BCL-2 inhibitor is navitoclax.

[0006] In another aspect, the present disclosure provides a composition comprising an HDAC6/PI3K-6 dual inhibitor; and a BCL-2 inhibitor.

[0007] In another aspect, the present disclosure provides a composition comprising polymeric nanoparticles comprising block copolymers comprising a PLA-PEG-PPG-PEG- PLA penta-block copolymer with an m-PEG-PLA di-block copolymer; an HDAC6/PI3K-6 dual inhibitor; and a BCL-2 inhibitor. The HDAC6/PI3K-6 dual inhibitor and the BCL-2 are associated with the polymeric nanoparticles. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound III. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound XL. In some embodiments, the BCL-2 inhibitor is venetoclax. In some embodiments, the BCL-2 inhibitor is navitoclax. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4, or 1 :5. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio from 5: 1 to 1 :5. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 1 : 1. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 1 : 1. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 2: 1. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 2: 1. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 3: 1. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 3: 1. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 1 :2. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 1 :2. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 1 :3. In some embodiments, the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 1 :3. In some embodiments, less PLA-PEG-PPG-PEG-PLA penta-block copolymer is present, by mass, than m-PEG-PLA di-block copolymer. In some embodiments, a mass ratio of PLA-PEG- PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer is from 1 :20 to 1 : 10. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer is from 1 : 15 to 1 : 5. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer is from 1 :8 to 3:8. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG- PLA penta-block copolymer to m-PEG-PLA di-block copolymer is from 1 :3 to 1 :2. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG- PLA di-block copolymer is from 1 :2 to 1 : 1. In some embodiments, the average diameter of the polymeric nanoparticles is between 50 and 170 nm. In some embodiments, the average diameter of the polymeric nanoparticles is between 60 and 130 nm. In some embodiments, the average diameter of the polymeric nanoparticles is between 60 and 100 nm. In some embodiments, the average diameter of the polymeric nanoparticles is between 80 and 110 nm. In some embodiments, the average diameter of the polymeric nanoparticles is between 100 and 170 nm. In some embodiments, a poly dispersity index (PDI) of the polymeric nanoparticles is not more than 0.5. In some embodiments, a PDI of the polymeric nanoparticles is not more than 0.3. In some embodiments, a zeta potential of the polymeric nanoparticles is from -5 mV to -40 mV. In some embodiments, the composition further comprises a PEG-PPG-PEG tri-block copolymer.

[0008] In another aspect, a pharmaceutical composition comprising the composition disclosed herein is provided. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0009] In another aspect, the present disclosure provides a method of reducing proliferation, survival, migration, or colony formation ability of a rapidly proliferating cell in a subject in need thereof, comprising contacting the cell with a therapeutically effective amount of a composition disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cell is a cancer cell.

[0010] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising a composition or pharmaceutical composition disclosed herein. In some embodiments, the cancer comprises a solid tumor cancer or a cancer of the blood. In some embodiments, the cancer is selected from the group consisting of breast cancer, leukemia, lymphoma, colon cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, and combinations thereof. In some embodiments, the cancer or breast cancer comprises triple negative breast cancer (TNBC). In some embodiments, the cancer or breast cancer comprises ER⁺ breast cancer. In some embodiments, the cancer comprises acute myeloid leukemia. In some embodiments, the cancer is metastatic. In some embodiments, the method further comprises administering an additional anti-cancer therapy to the subject. In some embodiments, the additional anti-cancer therapy comprises surgery, chemotherapy, radiation, hormone therapy, immunotherapy, or a combination thereof. In some embodiments, the cancer is resistant or refractory to a chemotherapeutic agent. In some embodiments, the subject is a human. In some embodiments, the composition or pharmaceutical composition is administered intravenously, intratumorally, or subcutaneously.

[0011] In another aspect, the present disclosure provides a composition or pharmaceutical composition disclosed herein for use in the treatment of cancer.

[0012] In another aspect, the present disclosure provides the use of a composition or pharmaceutical composition disclosed herein for the manufacture of a medicament for the treatment of cancer.

[0013] In some embodiments, the therapeutically effective amount comprises an HDAC6/PI3K-6 dual inhibitor and a BCL-2 inhibitor. In some embodiments, the therapeutically effective amount comprises an HDAC6/PI3K-6 dual inhibitor and a BCL-2 inhibitor is synergistic in comparison to a therapeutically effective amount of the HDAC6/PI3K-6 dual inhibitor or the BCL-2 inhibitor administered alone. In some embodiments, the BCL-2 inhibitor is venetoclax. In some embodiments, the BCL-2 inhibitor is navitoclax. In some embodiments, the therapeutically effective amount is used in the reduction of proliferation, survival, migration, or colony formation ability of a rapidly proliferating cell in a subject. In some embodiments, the rapidly proliferating cell is a cancer cell. In some embodiments, the cancer is breast cancer. In some embodiments, the breast cancer is ER⁺ breast cancer.

[0014] In another aspect the present disclosure provides a method of manufacturing a composition disclosed herein comprising mixing penta-block PLA-PEG-PPG-PEG-PLA and di-block m-PEG-PLA block copolymers dissolved in acetonitrile with the HDAC6/PI3K-6 dual inhibitor and/or the BCL-2 inhibitor to form a first mixture; mixing the first mixture with a PEG-PPG-PEG tri-block copolymer dissolved in water to form a second mixture; stirring the second mixture and evaporating the acetonitrile; and filtering the stirred and evaporated second mixture, thereby manufacturing the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Aspects, features, benefits, and advantages of the embodiments described herein will be apparent with regard to the following description, appended claims, and accompanying drawings. The drawings depict exemplary embodiments of the disclosure and are not intended to be limiting.

[0016] FIG. 1 illustrates the reaction scheme of ring opening polymerization of L-lactide.

[0017] FIG. 2 shows an HPLC chromatogram of the 7679 (Compound XL) (right-hand peak) and 1925 (Compound III) (left-hand peak) molecules.

[0018] FIG. 3 contains line graphs showing percent release per day (FIG. 3A) and percent cumulative release (FIG. 3B) of 7679 (Compound XL) (filled black squares) and 1925 (Compound III) (filled red circles) from polymeric nanoparticles.

[0019] FIG. 4 presents a series of confocal laser scanning micrographs showing DAPI (FIG. 4A) (blue), Rhodamine B (FIG. 4B) (red), and merged staining (FIG. 4C) of MDA-MB-468 breast cancer cells exposed to Rhodamine B loaded polymeric nanoparticles.

[0020] FIG. 5 contains line graphs showing percent cell viability in ZR-75-1 human breast cancer cell line treated with increasing concentrations of 7679 (Compound XL) (red circles) or 7679 NPs (black squares) (FIG. 5A) and 1925 (Compound III) (red circles) or 1925 NPs (black squares) (FIG. 5B).

[0021] FIG. 6 contains line graphs showing percent cell viability in SUM 149 breast cancer cells treated with increasing concentrations of 7679 (Compound XL) (red circles) or 7679 NPs (black squares) (FIG. 6A) and 1925 (Compound III) (red circles) or 1925 NPs (black squares) (FIG. 6B).

[0022] FIG. 7 contains line graphs showing percent cell viability in MDA-MB-468 cells (FIG. 7 A) and E0771 murine breast cancer cells (FIG. 7B) treated with increasing concentrations of 7679 (Compound XL) (red circles) or 7679 NPs (black squares). [0023] FIG. 8 contains a line graph showing percent cell viability in MCF-7 mammary cancer cells treated with increasing concentrations of 7679 (Compound XL) (red circles) or 7679 NPs (black squares).

[0024] FIG. 9 contains line graphs showing percent cell viability in SW620 colon cancer cells treated with increasing concentrations of 7679 (Compound XL) (red circles) or 17679 NPs (black squares) (FIG. 9A) and 1925 (Compound III) (red circles) or 1925 NPs (black squares) (FIG. 9B).

[0025] FIG. 10 contains line graphs showing percent cell viability in HCT116 colon cancer cells (FIG. 10A) and MC38 murine colon adenocarcinoma cells (FIG. 10B) treated with increasing concentrations of 7679 (red circles) or 7679 NPs (black squares).

[0026] FIG. 11 contains line graphs showing percent cell viability in U266B 1 human myeloma cells treated with increasing concentrations of 7679 (Compound XL) (red circles) or 7679 NPs (black triangles) (FIG. 11 A) and 1925 (Compound III) (red circles) or 1925 NPs (black squares) (FIG. 11B).

[0027] FIG. 12 contains a line graph showing percent cell viability in U937 lymphoma cells treated with increasing concentrations of 7679 (Compound XL) (red circles) or 7679 NPs (black triangles).

[0028] FIG. 13 contains a line graph showing tumor growth in a breast cancer syngeneic mouse model in control (circles), 12.5 mg/kg nanoparticle encapsulated Compound XL (7679) (squares) and 25 mg/kg nanoparticle encapsulated 7679 (Compound XL) (triangles). Treatments were administered once per week for three weeks.

[0029] FIG. 14 contains a line graph showing percent survival of mice in a breast cancer syngeneic mouse model in control (red), 12.5 mg/kg nanoparticle encapsulated 7679 (Compound XL) (blue) and 25 mg/kg nanoparticle encapsulated 7679 (Compound XL) (green).

[0030] FIG. 15 contains a line graph showing tumor growth in a breast cancer syngeneic mouse model in vehicle (blue circles), 25 mg/kg nanoparticle encapsulated idelalisib (orange squares) and 25 mg/kg nanoparticle encapsulated 7679 (Compound XL) (black triangles).

[0031] FIG. 16A contains fluorescence images showing biodistribution of nanoparticles encapsulating Indocyanine Green (ICG) in a breast cancer syngeneic mouse model. FIG. 16B contains a bar graph showing tumor uptake of ICG from nanoparticles and free ICG at three hours in a breast cancer syngeneic mouse model. FIG. 16C contains a bar graph showing tumor uptake of ICG from nanoparticles (right bar) and free ICG (left bar) at 3, 24 and 48 hours in a breast cancer syngeneic mouse model.

[0032] FIG. 17 contains line graphs showing percent cell viability in SUM149 cells (FIG. 17A) and HCT116 cells (FIG. 17B) treated with increasing concentrations of Compound XL (7679) (black circles), navitoclax (ABT-263) (blue squares) and a 1 : 1 (red triangles pointed up) and 1 :2 (green triangles pointed down) mass ratio of 7679 (Compound XL) to navitoclax (ABT-263).

[0033] FIG. 18 contains line graphs showing percent cell viability in THP1 monocyte-like leukemia cells treated with increasing concentrations of 7679 (Compound XL) (black circles), ABT-199 (venetoclax) (blue squares) and a 1 : 1 (red triangles pointed up) and 1 :2 (green triangles pointed down) mass ratio of 7679 (Compound XL) to ABT-199 (venetoclax).

[0034] FIG. 19 contains line graphs showing percent cell viability in THP1 cells treated with increasing concentrations of 7679 (Compound XL) (black circles), ABT-263 (navitoclax) (blue squares) and a 1 : 1 (red triangles pointed up) and 1 :2 (green triangles pointed down) mass ratio of 7679 (Compound XL) to ABT-263 (navitoclax).

[0035] FIG. 20 contains line graphs showing percent cell viability in HL60 myeloid leukemia cells treated with increasing concentrations of a 2: 1 mass ratio of 7679 (Compound XL) to ABT-199 (venetoclax) (red, filled circles), a 2: 1 mass ratio of 7679 (Compound XL) to ABT-263 (navitoclax) (green, filled squares), 7679 (Compound XL) (black diamonds), ABT-199 (venetoclax) (blue empty circles) and ABT-263 (navitoclax) (pink empty squares).

[0036] FIG. 21 contains line graphs showing percent cell viability in Mv411 myelomonocytic leukemia cells treated with increasing concentrations of a 2: 1 mass ratio of 7679 (Compound XL) to ABT-199 (venetoclax) (red filled circles), a 2: 1 mass ratio of 7679 (Compound XL) to ABT-263 (navitoclax) (green filled squares), 7679 (Compound XL) (black diamonds), ABT- 199 (venetoclax) (empty circles) and ABT-263 (navitoclax) (empty squares).

[0037] FIG. 22 contains line graphs showing percent cell viability in Mv411 cells treated with increasing concentrations of a 2: 1 mass ratio of 7679 (Compound XL) to ABT-199 (venetoclax) (red filled circles), a 2: 1 mass ratio of 7679 (Compound XL) to ABT-263 (navitoclax) (green filled squares), 7679 (Compound XL) (black diamonds), ABT- 199 (venetoclax) (blue empty circles) and ABT-263 (navitoclax) (pink empty squares).

[0038] FIG. 23 shows an

NMR spectra of nanoparticles (NPs) comprising hybrid block copolymers comprising a PLA-PEG-PPG-PEG-PLA penta-block copolymer with an m-PEG- PLA di-block copolymer described herein.

[0039] FIG. 24 shows an FT-IR spectra of nanoparticles comprising hybrid block copolymers comprising a PLA-PEG-PPG-PEG-PLA penta-block copolymer with an m-PEG- PLA di-block copolymer described herein.

[0040] FIG. 25A is a line graph showing comparative hydrodynamic size distribution profiles of single and dual drug encapsulated NPs. FIG. 25B shows a TEM image of PI3K- 6/HDAC6 dual inhibitor / navitoclax (NAV)-loaded nanoparticles with the PI3K-6/HDAC6 dual inhibitor present with the navitoclax at a 1 :3 mass ratio. Scale bar: 200 nm. FIG. 25C shows another TEM image of the same nanoparticles. Scale bar: 20 nm.

[0041] FIG. 26A is a line graph showing release kinetics of PI3K-6/HDAC6 NPs. Empty squares show percent release per day, and filled squares show percent cumulative release. FIG. 26B is a line graph showing release kinetics of Nav-NPs. Empty circles show percent release per day, and filled circles show percent cumulative release. FIG. 26C is a line graph showing in vitro release profile of PI3K-6/HDAC6-NAV NPs. Empty triangles (pointed up) show percent release of Nav per day. Filled triangles (pointed up) show cumulative release of navitoclax. Empty triangles (pointed down) show percent release of PI3K-6/HDAC6 per day. Filled triangles (pointed down) show cumulative release of PI3K-6/HDAC6.

[0042] FIGs. 27A, B, and C are line graphs showing comparative % cell inhibition plots of PI3K-6/HDAC6-NPs (empty squares), NAV-NPs (filled circles), and PI3K-6/HDAC6-NAV- NPs with a 1 :3 (filled triangles, pointed down), 3: 1 (filled triangles, pointed up), and 1 : 1 mass ratio of PI3K-6/HDAC6 dual inhibitor to navitoclax (filled diamonds) in MCF7 cells (FIG. 27A), ZR-75-1 cells (FIG. 27B), and EAC cells (FIG. 27C).

[0043] FIGs. 28A, B, and C are bar graphs showing comparative % cell viability bar graphs of NAV-NPs, PI3K-6/HDAC6-NPs, and PI3K-8/HDAC6-NAV-NPs with a 1 : 1, 3:1, or 1 :3 ratios of PI3K-6/HDAC6 dual inhibitor to navitoclax at lOpM concentration in MCF7 cells (FIG. 28A), ZR-75-1 cells (FIG. 28B), and EAC cells (FIG. 28C). [0044] FIGs. 29A, B, and C are line graphs showing combination index value vs Fraction affected plots of PI3K-6/HDAC6-NAV-NPs with a 1 :3 (filled triangles, pointed down), 3: 1 (filled triangles, pointed up), and 1 : 1 mass ratio of PI3K-6/HDAC6 dual inhibitor to navitoclax (filled diamonds) in MCF7 cells (FIG. 29A), ZR-75-1 cells (FIG. 29B), and EAC cells (FIG. 29C).

[0045] FIG. 30A is a line graph showing relative tumor volume in an ER⁺ EAC syngeneic mice breast cancer model. Relative tumor volume is shown for an untreated group (circles), PI3K-6/HDAC6-NPs treated group (squares), NAV-NPs treated group (triangles, pointed up) and PI3K-6/HDAC6-NAV-NPs treated group (triangles, pointed down). FIG. 30B is a graph showing relative tumor volume % of independent mice from each group shown in FIG. 30A, with untreated (circles), PI3K-6/HDAC6-NPs (squares), NAV-NPs (triangles, pointed up) and PI3K-6/HDAC6-NAV-NPS group (triangles, pointed down) shown.

[0046] FIG. 31A is a line graph showing average body weight of ER⁺ EAC syngeneic mice breast cancer model mice treated with PI3K-6/HDAC6-NPs (squares), NAV-NPs (triangles, pointed up) and PI3K-6/HDAC6-NAV-NPs (triangles, pointed down). FIG. 31B is a line graph showing Kaplan Meier survival curves of EAC cell line-derived syngeneic breast cancer tumor bearing mice upon intravenous administration of PBS, PI3K-6/HDAC6-NPs, NAV-NPs, and PI3K-6/HDAC6-NAV-NPs. The lines showed lowest probability of survival in the order of PBS, PI3K-8/HDAC6-NPs, Nav-NPs, and PI3K-8/HDAC6-NAV-NPs. Mice were treated twice a week for three weeks.

[0047] FIGs. 32A and B are bar graphs showing nephrotoxicity evaluation of PI3K- 6/HDAC6-NPS on EAC tumor bearing mice. Shown are mg/dl creatine (FIG. 32A) and mg/dl blood urea (FIG. 32B) in healthy mice, untreated mice, and PI3K-6/HDAC6-NPs treated mice.

[0048] FIGs. 33A, B, and C are bar graphs showing hepatotoxicity evaluation of PI3K- 6/HDAC6-NPS on EAC tumor bearing mice. Shown are U/liter aspartate aminotransferase (AST; FIG. 33A), U/liter alanine aminotransferase (ALT; FIG. 33B), and mg/dl bilirubin total (FIG. 33C) in healthy mice, untreated mice, and PI3K-6/HDAC6-NPs treated mice.

[0049] FIG. 34 contains H&E stained images of heart, lung, liver, spleen, and kidney of untreated (PBS) and PI3K-6/HDAC6-NPs treated mice. DETAILED DESCRIPTION

[0050] It will be appreciated that for clarity, the following disclosure will describe various aspects of embodiments. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment,” “an embodiment,” “an example embodiment,” “some embodiments,” or the like means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” “an example embodiment,” or “some embodiments” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the disclosure. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0051] In one aspect, the present disclosure provides nanoparticles encapsulating dual inhibitor (PI3K-6-HDAC6) that is useful for treating different cancer indications. Also provided are nanoparticles encapsulating PI3K-6/HDAC6 dual inhibitors and BCL-2 inhibitors. Also provided are pharmaceutical compositions comprising nanoparticles described herein, along with associated methods.

Definitions

[0052] For convenience, before further description of the present disclosure, certain terms used in the specification, examples, and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. [0053] The articles “a,” “an,” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0054] The terms “comprise,” “comprising,” “including,” “containing,” “characterized by” and grammatical equivalents thereof are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only.”

[0055] As used herein, “consisting of’ and grammatical equivalents thereof exclude any element, step or ingredient not specified in the claim.

[0056] As used herein, the terms “about” or “approximately” mean within 5% of a given value or range.

[0057] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values.

[0058] As used herein, the term “nanoparticle” refers to particles in the range between 10 nm to 1000 nm in diameter, wherein diameter refers to the diameter of a perfect sphere having the same volume as the particle. The term “nanoparticle” is used interchangeably as “nanoparticle(s).” In some cases, the diameter of the particle is in the range of about 1-1000 nm, 10-500 nm, 20-300 nm, or 100-300 nm. In various embodiments, the diameter is about 30-170 nm. In certain embodiments, the diameter of the nanoparticle is about 1, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 nm. In other embodiments, the diameter of the nanoparticle is 1, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 nm.

[0059] As used herein, the terms “polymer” or “polymeric” are given their ordinary meanings as used in the art, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units may all be identical, or in some cases, there may be more than one type of repeat unit present within the polymer. When more than one type of repeat unit is present in a single polymer, the polymer is termed a “copolymer” or “co-polymer.”

[0060] As used herein, the term “polymeric nanoparticle” refers to a nanoparticle made up of a polymer.

[0061] As used herein, a “block copolymer” is a copolymer formed when repeat units cluster together and form groups (“blocks”) of repeating units. As used herein, a “hybrid” block copolymer comprises a mixture of different block copolymers, such as the m-PEG-PLA diblock and PLA-PEG-PPG-PEG-PLA penta-block described herein.

[0062] As used herein, the term “associated substantially with” in the context of a nanoparticle means a substance is encapsulated by the nanoparticle, adsorbed to the nanoparticle, or conjugated to a surface of the nanoparticle. In some embodiments, when a substance is associated substantially with a nanoparticle, at least 20%, at least 40%, at least 60%, at least 80%, at least 90%, at least 95%, or at least 99% of the mass of the substance is encapsulated by the nanoparticle, adsorbed to the nanoparticle, or conjugated to the surface of the nanoparticle. In some embodiments, the nanoparticle is a polymeric nanoparticle. In some embodiments, the mass of the substance associated substantially with the nanoparticle is in reference to a pharmaceutical composition comprising the substance and the nanoparticle ready for administration to a subject. In some embodiments, the mass of the substance associated substantially with the nanoparticle is in reference to the amount of a substance that is associated substantially with a nanoparticle when the substance/nanoparticle composition is being formed.

[0063] As used herein, an “emulsifier” and “emulsion” are given their ordinary meaning as used in the art. That is, an emulsion is a chemical mixture comprising a dispersed phase and a continuous phase, wherein the phases are normally immiscible. An emulsifier can stabilize the components of an emulsion such that the kinetic stability of the emulsion is increased. Examples of emulsifiers that may optionally be included in a composition with the polymeric nanoparticles of the present disclosure include PEG-PPG-PEG of different molecular weights from 1,000 Daltons to 13,000 Daltons such as, for example, from 4,000 Daltons to 13,000 Daltons or from 1,000 Daltons to 6,000 Daltons and sodium lauryl sulphate. An emulsifier may or may not be added to the polymeric nanoparticles of the present disclosure (e.g., may or may not be added during preparation thereof). The emulsifier may be a polymeric emulsifier (e.g., the PEG-PPG-PEG tri -block copolymer). The emulsifier may be a non- polymeric emulsifier (e.g., sodium lauryl sulfate). Polymeric and non-polymeric emulsifiers may be used alone, in combination, or not at all. In embodiments where the emulsifier is a polymeric emulsifier it is external to the nanoparticle itself. For example, in some embodiments PEG-PPG-PEG is a component of the nanoparticle and can also be used as an emulsifier. The PEG-PPG-PEG used as an emulsifier is external and distinct from the PEG- PPG-PEG that is making up the nanoparticles. The term external emulsifier can also refer to non-polymeric emulsifiers as these are also not part of the nanoparticles. An external polymeric emulsifier refers to a polymeric emulsifier that does not make up a portion of the nanoparticles.

[0064] The terms “combination,” “therapeutic combination,” or “pharmaceutical combination” as used herein refer to the combined administration of two or more therapeutic agents (e.g., co-delivery). Components of a combination therapy may be administered simultaneously or sequentially, i.e., at least one component of the combination is administered at a time temporally distinct from the other component(s). In embodiments, a component(s) is administered within one month, one week, 1-6 days, 18, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour, or 30, 20, 15, 10, or 5 minutes of the other component(s).

[0065] In some embodiments, polymeric nanoparticles comprising a pharmaceutical combination or a pharmaceutical composition comprising polymeric nanoparticles comprising a pharmaceutical combination, or both, as provided herein display a synergistic effect. The term “synergistic effect” as used herein, refers to action of two agents such as, for example, navitoclax and a PI3K-6/HDAC6 dual inhibitor to produce an effect, for example, slowing the symptomatic progression of cancer or symptoms thereof, which is greater than the simple addition of the effects of each drug administered by themselves (either administered by themselves using the polymeric nanoparticle delivery system, or delivered by themselves wherein the agent is delivered by conventional means). In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is ER⁺ breast cancer. In some embodiments, the PI3K-6/HDAC6 dual inhibitor and the navitoclax are present in the nanoparticle in a 1 :3 mass ratio. A synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the pharmaceutical combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

[0066] In a further embodiment, the provided herein is a polymeric nanoparticle comprising a synergistic pharmaceutical combination for administration to a subject, wherein the dose range of each component corresponds to the synergistic ranges suggested in a suitable tumor model or clinical study.

[0067] The term “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions and/or dosage forms, which are, within the scope of sound medical judgment, suitable for contact with the tissues a warm-blooded animal, e.g., a mammal or human, without excessive toxicity, irritation allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.

[0068] A “therapeutically effective amount” of a polymeric nanoparticle comprising one or more therapeutic agents is an amount sufficient to provide an observable or clinically significant improvement over the baseline clinically observable signs and symptoms of the disorders treated with the combination.

[0069] The terms “subject” or “patient” as used herein are intended to include animals, which are capable of suffering from or afflicted with a cancer or any disorder involving, directly or indirectly, a cancer. Examples of subjects include mammals, e.g., humans, apes, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In an embodiment, the subject is a human, e.g., a human suffering from cancer.

[0070] The terms “treating” or “treatment” as used herein comprise a treatment relieving, reducing or alleviating at least one symptom in a subject or producing a delay in the progression of a disease. For example, treatment can be the diminishment of one or several symptoms of a disorder or complete eradication of a disorder, such as cancer. Within the meaning of the present disclosure, the term “treat” also denotes to arrest and/or reduce the risk of worsening a disease. The terms “prevent,” “preventing,” or “prevention” as used herein comprise the prevention of at least one symptom associated with or caused by the state, disease or disorder being prevented. [0071] As used herein, the term “human equivalent dose” refers to a dose of a composition to be administered to a human that is calculated from a specific dose used in an animal study.

[0072] As used herein, a “stabilizer” reduces or eliminates changes in diameter and/or PDI of polymeric nanoparticles during storage or lyophilization. Examples of stabilizers that can be used with the polymeric nanoparticles of the present disclosure include mannose, betalactose, trehalose, sodium cholate, and glucose. When a stabilizer is employed, it may be present in a weight of about 5% to about 50% of the total weight of the polymer such as, for example, 10% to 50%, 30% to 50%, or 40% to 50% of the total weight of the polymer. In some embodiments, the stabilizer can comprise glucose.

[0073] As used herein, the term “rapidly proliferating cells” refers to cells having the capacity for autonomous growth (e.g., cancer cells).

Histone Deacetylase 6 (HDAC6) / Phosphoinositide 3-Kinase-b (PI3K-3) Dual Inhibitors

[0074] According to certain embodiments, HDAC6/PI3K-6 dual inhibitors are associated with the nanoparticles described herein. In certain embodiments, the HDAC6/PI3K-6 dual inhibitors described herein are as described in U.S. Patent Publication No. 2020/0165257, incorporated by reference herein, in its entirety. In some embodiments, compounds of Formulae 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, shown below, and which may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g., asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms, comprise HDAC6/PI3K-6 dual inhibitors. These compounds can be, for example, racemates or optically active forms. For compounds with two or more asymmetric elements, these compounds can additionally be mixtures of diastereomers. For compounds having asymmetric centers, all optical isomers in pure form and mixtures thereof are encompassed. In these situations, the single enantiomers, z.e., optically active forms, can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column. All forms are contemplated herein regardless of the methods used to obtain them. [0075] All forms (for example solvates, optical isomers, enantiomeric forms, polymorphs, free compound, and salts) of an active agent may be employed either alone or in combination.

[0076] All compounds are understood to include all possible isotopes of atoms occurring in the compounds. Isotopes include those atoms having the same atomic number but different mass numbers and encompass heavy isotopes and radioactive isotopes. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include ⁿC, ¹³C, and ¹⁴C. Accordingly, the compounds disclosed herein may include heavy or radioactive isotopes in the structure of the compounds or as substituents attached thereto. Examples of useful heavy or radioactive isotopes include ¹⁸F, 15_N, 18Q 76_Br, 125_{I? and} 13 lj

[0077] Formulae 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 include all pharmaceutically acceptable salts thereof.

[0078] The term “substituted” means that any one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom’s normal valence is not exceeded. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation into an effective therapeutic agent.

[0079] A dash

that is not between two letters or symbols is used to indicate a point of attachment for a substituent.

[0080] The term “alkyl” includes both branched and straight chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms, generally from 1 to about 8 carbon atoms. The term C1-C5 alkyl as used herein indicates an alkyl group having from 1, 2, 3, 4, or 5 carbon atoms.

[0081] The terms “halo” or “halogen” mean fluoro, chloro, bromo, or iodo, and are defined herein to include all isotopes of same, including heavy isotopes and radioactive isotopes. Examples of useful halo isotopes include ¹⁸F, ⁷⁶Br, and ¹³¹I. Additional isotopes will be readily appreciated by one of skill in the art. [0082] The term “chiral” refers to molecules, which have the property of non- superimposability of the mirror image partner.

[0083] The term “stereoisomer” refers to compounds, which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0084] The term “diastereomer” refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis, crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column.

[0085] The term “enantiomer” refers to two stereoisomers of a compound, which are non- superimposable mirror images of one another. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.

[0086] Stereochemical definitions and conventions used herein generally follow Parker, S. P., ed., 1984, McGraw-Hill, “Dictionary of Chemical Terms,” McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds” (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, ie., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory.

[0087] The terms “racemic mixture” or “racemate” refer to an equimolar (or 50:50) mixture of two enantiomeric species, devoid of optical activity. A racemic mixture may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.

[0088] The term “pharmaceutically acceptable salts” includes derivatives of the disclosed compounds in which the parent compound is modified by making inorganic and organic, nontoxic, acid, or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, iso-propanol, or acetonitrile are used, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts.

[0089] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)_n-COOH where n is 0-4, and the like. Lists of additional suitable salts may be found, e.g., in G. Steffen Paulekuhn, et al., Journal of Medicinal Chemistry 2007, 50, 6665 and Handbook of Pharmaceutically Acceptable Salts: Properties, Selection and Use, P. Heinrich Stahl and Camille G. Wermuth, Editors, Wiley- VCH, 2002.

[0090] In an embodiment, a dual inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HD AC), a pharmaceutically acceptable salt thereof, a prodrug thereof, or solvate thereof are provided. The dual inhibitor may include a core containing a quinazoline moiety or a quinazolin-4(3H)-one moiety, a kinase hinge binding moiety, and a histone deacetylase pharmacophore. [0091] In an embodiment, the histone deacetylase pharmacophore may include:

but is not limited thereto.

[0092] In the above formulae, at least one non-adjacent -CH2- group may be optionally replaced with -O-; n may be 1, 2, 3, 4, and 5; J may be CH or N; M may be CH or N; W may be N, O, or S; X may be CH or N; T may be CH or N; Q may be -(CH2)_P-, -(CH2)_pNH(CH2)r- , -NH(CH2)_P-, or -(CH2)_PNH-, wherein p and r may each independently be 0, 1, 2, 3, or 5; Y may be CH or N; R³ may be:

wherein R⁴ and R⁵ may each independently be H or a C1-C5 alkyl group; R⁶ is H or a C1-C4 alkyl group.

[0093] The kinase hinge binding moiety may include, but is not limited thereto:

wherein R1 may be a C1-C5 alkyl group;

R⁷ may be H, a C1-C5 alkyl group, a C1-C5 alkyl containing 1-5 fluorine atoms, a C1-C5 alkyl containing 1-5 deuterium atoms, or NH2;

R⁸ may be H, a C1-C5 alkyl group, Cl, CONH2, or CN;

R⁹ may be H, a C1-C5 alkyl group, a C1-C5 alkyl containing 1-5 fluorine atoms, a C1-C5 alkyl containing 1-5 deuterium atoms, or NH2; and

X may be CH or N. [0094] In an embodiment, the core of the dual inhibitor may be represented by Formula 1 : I'ormuh I

wherein Ar is an aryl or heteroaryl group unsubstituted or substituted with 1-3 C1-C6 alkyl groups, indicates a binding site to the histone deacetylase pharmacophore, and “**” indicates a binding site to the kinase hinge binding moiety.

[0095] For example, the histone deacetylase pharmacophore may be:

[0096] For example, the kinase hinge binding moiety may be:

wherein R¹ may be a C1-C5 alkyl group;

R⁷ may be H, a C1-C5 alkyl group, a C1-C5 alkyl containing 1-5 fluorine atoms, a C1-C5 alkyl containing 1-5 deuterium atoms, or NFF;

R⁸ may be H, a C1-C5 alkyl group, Cl, CONH2, or CN;

R⁹ may be H, a C1-C5 alkyl group, a C1-C5 alkyl containing 1-5 fluorine atoms, a C1-C5 alkyl containing 1-5 deuterium atoms, or NFF; and

X may be CH or N.

[0097] In another embodiment, the core of the dual inhibitor may be represented by Formula

2, but is not limited thereto:

wherein

R² may be hydrogen, a halogen, or a C1-C5 alkyl group. indicates a binding site to the histone deacetylase pharmacophore, and “**” indicates a binding site to the kinase hinge binding moiety.

[0098] For example, the histone deacetylase pharmacophore may be:

[0099] For example, the kinase hinge binding moiety may be:

wherein R¹ may be a C1-C5 alkyl group;

R⁸ may be H, a C1-C5 alkyl group, Cl, CONH2, or CN;

X may be CH or N.

[0100] In an embodiment, the dual inhibitor may be represented by Formula 3:

Formula 3

[0101] In Formula 3, R¹ may be a C1-C5 alkyl group, X may be CH or N, and Z may be:

but is not limited thereto, wherein in the above formulae, at least one non-adjacent -CH2- group may be optionally replaced with -O-; n may be 1, 2, 3, 4, and 5; J may be CH or N; M may be CH or N; W may be N, O, or S; X may be CH or N; T may be CH or N; Q may be - (CH2)_P-, -(CH₂)_PNH(CH₂)r-, -NH(CH2)_P-, or -(CH2)_PNH-, wherein p and r may each independently be 0, 1, 2, 3, or 5; Y may be CH or N; R³ may be:

wherein R⁴ and R⁵ may each independently be H or a C1-C5 alkyl group;

R⁶ is H or a C1-C4 alkyl group. [0102] In an embodiment, the dual inhibitor may be represented by Formula 4:

Forwuhs 4

In Formula 4,

R¹ may be a C1-C5 alkyl group;

R⁷ may be H, a C1-C5 alkyl group, a C1-C5 alkyl containing 1-5 fluorine atoms, a Ci- C5 alkyl containing 1-5 deuterium atoms, or NFF;

R⁸ may be H, a C1-C5 alkyl group, Cl, CONH2, or CN;

R⁹ may be H, a C1-C5 alkyl group, a C1-C5 alkyl containing 1-5 fluorine atoms, a Ci- C5 alkyl containing 1-5 deuterium atoms, or NFF; and

X may be CH or N; and

Z may be:

but is not limited thereto, wherein in the above formulae, at least one non-adjacent -CH2- group may be optionally replaced with -O-; n may be 1, 2, 3, 4, and 5; J may be CH or N; M may be CH or N; W may be N, O, or S; X may be CH or N; T may be CH or N; Q may be -(CH2)_P-, -(CH2)_pNH(CH2)r- , -NH(CH2)_P-, or -(CH2)_PNH-, wherein p and r may each independently be 0, 1, 2, 3, or 5; Y may be CH or N; R³ may be:

[0103] In another embodiment, the dual inhibitor may be represented by Formula 5:

F nwla 5

[0104] In Formula 5,

R¹ may be a C1-C5 alkyl group;

R² may be hydrogen, a halogen, or a C1-C5 alkyl group;

X may be CH or N; and

Z may be:

but is not limited thereto, wherein in the above formulae, at least one non-adjacent -CH2- group may be optionally replaced with -O-; n may be 1, 2, 3, 4, and 5; J may be CH or N; M may be CH or N; W may be N, O, or S; X may be CH or N; T may be CH or N; Q may be - (CH2)-, -(CH₂)_PNH(CH₂)r-, -NH(CH2)_P-, or -(CH2)_PNH-, wherein p and r may each independently be 0, 1, 2, 3, or 5; Y may be CH or N; R³ may be:

wherein R⁴ and R⁵ may each independently be a C1-C5 alkyl group; R⁶ may be H or a C1-C4 alkyl group.

[0105] In another embodiment, the dual inhibitor may be represented by Formula 6:

In Formula 6,

R¹ may be a C1-C5 alkyl group;

R² may be hydrogen, a halogen, or a C1-C5 alkyl group;

R⁷ may be H, a C1-C5 alkyl group, a C1-C5 alkyl containing 1-5 fluorine atoms, a Ci- C5 alkyl containing 1-5 deuterium atoms, or NH2;

R⁸ may be H, a C1-C5 alkyl group, Cl, CONH2, or CN;

R⁹ may be H, a C1-C5 alkyl group, a C1-C5 alkyl containing 1-5 fluorine atoms, a Ci-

Cs alkyl containing 1-5 deuterium atoms, or NH2; and X may be CH or N; and Z may be:

but is not limited thereto, wherein in the above formulae, at least one non-adjacent -CH2- group may be optionally replaced with -O-; n may be 1, 2, 3, 4, and 5; J may be CH or N; M may be CH or N; W may be N, O, or S; X may be CH or N; T may be CH or N; Q may be - (CH2)-, -(CH₂)_PNH(CH₂)r-, -NH(CH₂)_P-, or -(CH₂)_PNH-, wherein p and r may each independently be 0, 1, 2, 3, or 5; Y may be CH or N; R³ may be:

wherein R⁴ and R⁵ may each independently be H or a C1-C5 alkyl group; R⁶ is H or a C1-C4 alkyl group;

[0106] The HDAC6/PI3K-6 dual inhibitors may be represented by one of the following compounds:

[0107] The kinase may be a phosphoinositide 3-kinase (PI3K).

[0108] In an embodiment, a dual inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HD AC) represented by Formula 7 or Formula 8 is provided:

[0109] In Formulae 7 and 8, Ar is an aryl or heteroaryl group unsubstituted or substituted with 1-3 Ci-Ce alkyl groups, R² is hydrogen, a halogen, or a C1-C5 alkyl group, A is histone deacetylase pharmacophore, and B is a kinase hinge binding moiety described in detail above.

[0110] In another embodiment, a pharmaceutically acceptable salt, a prodrug, or solvate of the dual inhibitor represented by Formulae 7 and 8 is provided.

[OHl] In another embodiment, a method for treating or diagnosing cancer in a mammal is provided. The method includes administering to the mammal a pharmaceutical composition, including an effective amount of an active agent, wherein the active agent is the dual inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HDAC), a pharmaceutically acceptable salt thereof, a prodrug thereof, or solvate thereof.

[0112] In another embodiment, an inhibitor of histone deacetylase (HDAC), a pharmaceutically acceptable salt thereof, a prodrug thereof, or solvate thereof is provided. The HDAC inhibitor may include a core containing a quinazolin-4(3H)-one moiety and a histone deacetylase pharmacophore.

[0113] The HDAC inhibitor may be represented by Formula 9, but is not limited thereto:

wherein Ar may be an aryl or heteroaryl group unsubstituted or substituted with 1-3 Ci-Ce alkyl groups, may be:

wherein in the above formulae, at least one non-adjacent — CH2— group may be optionally replaced with — O— ; n may be 1, 2, 3, 4, and 5; J may be CH or N; M may be CH or N; W may be N, O, or S; X may be CH or N; T may be CH or N; Q may be -(CH2)-, - (CH₂)_PNH(CH₂)r-, -NH(CH2)_P-, or -(CH2)_PNH-, wherein p and r may each independently be 0, 1, 2, 3, or 5; Y may be CH or N; R³ may be:

wherein R⁴ and R⁵ may each independently be H or a C1-C5 alkyl group; and R⁶ may be H or a C1-C4 alkyl group, and “**” may be H, Ci-Ce alkyl, C3-C6 cycloalkyl, or aryl.

[0114] The HDAC inhibitor may be represented by one of the following compounds:

[0115] In an embodiment, an inhibitor of histone deacetylase (HDAC) represented by Formula 10 is provided:

Fomw’h? 10

In Formula 10,

Ar is an aryl or heteroaryl group unsubstituted or substituted with 1-3 Ci-Ce alkyl group,

E is histone deacetylase pharmacophore, and

G is H, Ci-Ce alkyl, C3-C6 cycloalkyl or aryl.

In another embodiment, a pharmaceutically acceptable salt, a prodrug, or solvate of the HDAC inhibitor represented by Formula 10 is provided. B Cell Lymphoma-2 (BCL-2) Inhibitors

[0116] According to certain embodiments, BCL-2 inhibitors are associated with the nanoparticles described herein. In some embodiments, BCL-2 inhibitors are combined with HDAC6/PI3K-6 dual inhibitors on the same nanoparticles. In some embodiments, BCL-2 inhibitors associated with nanoparticles are combined with HDAC6/PI3K-6 dual inhibitors associated with distinct nanoparticles. BCL-2 inhibitors include venetoclax (ABT-199), navitoclax (also referred to herein as Nav; NAV; and ABT-263), ABT-737, gossypol (AT- 101), apogossypolone (ApoG2), and obatoclax; selective inhibitors of nuclear export (SINEs), e.g., selinexor (KPT-330).

Polymeric Nanoparticles

[0117] Nanoparticles (also referred to herein as “NPs”) can be produced as nanocapsules or nanospheres. Drug loading in the nanoparticle can be performed by either an adsorption process or an encapsulation process (Spada et al., 2011, Protein delivery of polymeric nanoparticles, World Academy of Science, Engineering and Technology: 76, incorporated herein, by reference, in its entirety). Nanoparticles, by using both passive and active targeting strategies, can enhance the intracellular concentration of drugs in cancer cells while avoiding toxicity in normal cells. When nanoparticles bind to specific receptors and enter the cell, they are usually enveloped by endosomes via receptor-mediated endocytosis, thereby bypassing the recognition of P-glycoprotein, one of the main drug resistance mechanisms (Cho et al., 2008, Therapeutic Nanoparticles for Drug Delivery in Cancer, Clin. Cancer Res., 2008, 14: 1310-1316, incorporated herein, by reference, in its entirely).

[0118] Nanoparticles are removed from the body by opsonization and phagocytosis (Sosnik et al., 2008, Polymeric Nanocarriers: New Endeavors for the Optimization of the Technological Aspects of Drugs, Recent Patents on Biomedical Engineering, 1 : 43-59, incorporated herein, by reference, in its entirety). Nanocarrier based systems can be used for effective drug delivery with the advantages of improved intracellular penetration, localized delivery, protection of drugs against premature degradation, controlled pharmacokinetic and drug tissue distribution profile, lower dose requirement, and cost effectiveness (Farokhzad OC, et al., Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. Proc. Natl. Acad. Sci. USA 2006,103 (16): 6315-20; Fonseca C, et al., Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti -turn oral activity. J. Controlled Release 2002; 83 (2): 273-86; Hood et al., Nanomedicine, 2011, 6(7): 1257-1272, incorporated herein, by reference, in their entireties).

[0119] The uptake of nanoparticles is inversely proportional to their size. Due to their small size, the polymeric nanoparticles have been found to evade recognition and uptake by the reticulo-endothelial system (RES) and can thus circulate in the blood for an extended period (Borchard et al., 1996, Pharm. Res. 7: 1055-1058, incorporated herein, by reference, in its entirety). Nanoparticles are also able to extravasate at the pathological site like the leaky vasculature of a solid tumor, providing a passive targeting mechanism. Due to the higher surface area leading to faster solubilization rates, nano-sized structures usually show higher plasma concentrations and area under the curve (AUC) values. Lower particle size helps in evading the host defense mechanisms and increases the blood circulation time. Nanoparticle size affects drug release. Larger particles exhibit slower diffusion of drugs into the system. Smaller particles offer larger surface area but lead to faster drug release. Smaller particles tend to aggregate during storage and transportation of nanoparticle dispersions. Hence, a compromise between a small size and maximum stability of nanoparticles is desired. The size of nanoparticles used in a drug delivery system should be large enough to prevent their rapid leakage into blood capillaries but small enough to escape capture by fixed macrophages that are lodged in the reticuloendothelial system, such as the liver and spleen.

[0120] In addition to their size, the surface characteristics of nanoparticles are also an important factor in determining the life span during circulation. Nanoparticles should ideally have a hydrophilic surface to escape macrophage capture. Nanoparticles formed from block copolymers with hydrophilic and hydrophobic domains meet these criteria. Controlled polymer degradation also allows for increased levels of agent delivery to a diseased state. Polymer degradation can also be affected by the particle size. Degradation rates increase with increase in particle size in vitro (Biopolymeric nanoparticles; Sundar et al., 2010, Science and Technology of Advanced Materials; doi: 10.1088/1468-6996/11/1/014104, incorporated herein, by reference, in its entirety).

[0121] Poly(lactic acid) (PLA) has been approved by the US FDA for applications in tissue engineering, medical materials, and drug carriers. US2006/0165987A1, incorporated herein, by reference, in its entirety, describes a stealthy polymeric biodegradable nanosphere comprising poly(ester)-poly(ethylene) multiblock copolymers and optional components for imparting rigidity to the nanospheres and incorporating pharmaceutical compounds. US2008/0081075A1, incorporated herein, by reference, in its entirety, discloses a novel mixed micelle structure with a functional inner core and hydrophilic outer shells, selfassembled from a graft macromolecule and one or more block copolymer.

[0122] US2010/0004398A1, incorporated herein, by reference, in its entirety, describes a polymeric nanoparticle of shell/core configuration with an interphase region and a process for producing the same.

[0123] Provided herein are polymeric nanoparticles for the delivery of chemotherapeutic compounds. The inventors of the present disclosure have developed polymeric nanoparticles comprising formulations of chemotherapeutic compounds. The polymeric nanoparticles are useful for the delivery of drugs. For example, the nanoparticles can find use in treatment of diseases exhibiting rapid cell division such as various cancers by delivering appropriate chemotherapeutic agents.

[0124] Accordingly, provided herein is a composition comprising: a) polymeric nanoparticles comprising hybrid block copolymers comprising a poly(lactic acid)-poly(ethylene glycol)- poly(propylene glycol)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PPG-PEG-PLA) penta-block with a methoxy -poly(ethylene glycol)-poly(lactic acid) (m-PEG-PLA) di-block; b) PI3K-6-HDAC6 dual inhibitors; and c) a BCL-2 or BCL-xL/BCL-2 inhibitor. In some embodiments, both the dual inhibitor and the BCL-2/BCL-xL inhibitor are associated with the polymeric nanoparticles.

[0125] For example, the PI3K-6-HDAC6 dual inhibitors and the BCL-2/BCL-xL inhibitors can be associated with the polymeric nanoparticles by being contained within an enclosed region of a shell of polymer. Alternatively, or additionally, the drugs can be interspersed within the polymer that forms the shell, or the drugs can adhere to an outside surface of the shell. The drugs can be associated with the polymeric nanoparticle in any manner suitable to carry and deliver the drugs to locations of disease in need of treatment.

[0126] In certain embodiments, the PI3K-6-HDAC6 dual inhibitors and the BCL-2/BCL-xL inhibitors can both be associated substantially with the same polymeric nanoparticles. In some embodiments, the PI3K-6-HDAC6 dual inhibitors and BCL-2/BCL-xL inhibitors are encapsulated by the nanoparticle. [0127] In certain embodiments, the polymeric nanoparticles can comprise both hydrophobic and hydrophilic block copolymers. In an embodiment, the polymeric nanoparticles provided herein comprise hybrid block copolymers comprising a poly(lactic acid)-poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PPG-PEG- PLA) penta-block with a methoxy -poly(ethylene glycol) (m-PEG-PLA) di-block. The PLA- PEG-PPG-PEG-PLA penta-block copolymer can be formed from PEG-PPG-PEG tri-block copolymer and PLA via ring opening polymerization of the lactide. In certain embodiments, the molecular weight of the penta-block copolymer can range from 5,000 g/mol to 40,000 g/mol. In certain embodiments, the molecular weight range of di-block copolymer can be from 2,000 g/mol to 40,000 g/mol.

[0128] Poly(lactic acid) (PLA) is a hydrophobic polymer and can be a component of the polymeric nanoparticles. As alternative to PLA or in addition to PLA, poly(glycolic acid) (PGA) and block copolymer of poly lactic acid-co-glycolic acid (PLGA) may also be used. The hydrophobic polymer can also comprise a biologically derived polymer or a biopolymer. The molecular weight of the PLA used is generally in the range of about 2,000 g/mol to 80,000 g/mol. Thus, in an embodiment, the PLA used is in the range of about 10,000 g/mol to 80,000 g/mol. The average molecular weight of PLA may also be about 70,000 g/mol. As used herein, one g/mol is equivalent to one “Dalton” (z.e., Dalton and g/mol are interchangeable when referring to the molecular weight of a polymer). “Kilodalton” (or “kDa”) as used herein refers to 1,000 Daltons.

[0129] Polyethylene glycol) (PEG) is another suitable component of the polymer used to form the polymeric nanoparticles. PEG can impart hydrophilicity, reduce phagocytosis by macrophages, and/or reduce immunological recognition. Block copolymers like poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (PEG-PPG-PEG) are hydrophilic or hydrophilic-hydrophobic copolymers that can be components of the polymeric nanoparticles of the present disclosure. For example, the PLA-PEG-PPG-PEG-PLA penta- block copolymer can be formed from ring opening polymerization using lactide and also by using m-PEG for the di-block. In general, block copolymers of the present disclosure may have two, three, four, five, or more distinct blocks.

[0130] In a further embodiment, the polymeric nanoparticles provided herein comprise a methoxy-poly(ethylene glycol)-poly(lactic acid) (m-PEG-PLA) di-block copolymer. [0131] In some embodiments, a first block copolymer of the instant disclosure consists essentially of two segments of poly(lactic acid) (PLA), separated by a segment of poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (PEG-PPG-PEG), to form the PLA-PEG-PPG-PEG-PLA penta-block.

[0132] In some embodiments, a second block of the instant disclosure consists essentially of an m-PEG-PLA di-block copolymer.

[0133] In some embodiments, the first and second block copolymers of the instant disclosure can be combined to form the polymeric nanoparticles of the instant disclosure. In some embodiments, the process described in Example 1 of the present disclosure can be used to accomplish the combination.

[0134] In some embodiments, the polymeric nanoparticles of the instant disclosure can be biodegradable.

[0135] In various embodiments, the nanoparticles comprise QUATRAMER™ reagent, which comprises a PLA-PEG-PPG-PEG-PLA penta-block with a methoxy-poly(ethylene glycol)- polylactic acid (m-PEG-PLA) di-block copolymer. QUATRAMER™ reagent is available from Hillstream Biopharma; Bridgewater, NJ, USA.

[0136] The PLA-PEG-PPG-PEG-PLA penta-block copolymer and the m-PEG-PLA di-block copolymer may optionally be combined in specific ratios. As used herein, such ratios are expressed in the form of Mass_penta-biock:Massdi-biock, unless stated otherwise. In some embodiments, less PLA-PEG-PPG-PEG-PLA penta-block copolymer is present, by mass, than the m-PEG-PLA di-block. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG- PLA penta-block copolymer to m-PEG-PLA di-block copolymer can be from 1 :20 to 1 : 1. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m- PEG-PLA di-block copolymer can be from 1 : 15 to 1 :2. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer can be from 1 : 10 to 1 :2. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta- block copolymer to m-PEG-PLA di-block copolymer can be from 1 :8 to 1 : 10 or can be about 1 :9. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer can be from 1 :3 to 1 :5 or can be about 1 :4. In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG- PLA di-block copolymer can be from 1 :2 to 3 :8 or can be about 3:7. [0137] In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer can be at least 1 :20 such as, for example, at least 1 : 19, at least 1 : 18, at least 1 : 17, at least 1 : 16, at least 1 : 15, at least 1 : 14, at least 1 : 13, at least 1 : 12, at least 1 : 11, at least 1 : 10, at least 1 :9, at least 1 :8, at least 1 :7, at least 1 :6, at least 1 :5, at least 1 :4, at least 1 :3, or at least 1 :2.

[0138] In some embodiments, a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer can be not more than 1 : 1 such as, for example, not more than 1 :2, not more than 1 :3, not more than 1 :4, not more than 1 :5, not more than 1 :6, not more than 1 :7, not more than 1 :8, not more than 1 :9, not more than 1 : 10, not more than 1 : 11, not more than 1 : 12, not more than 1 : 13, not more than 1 : 14, not more than 1 : 15, not more than 1 : 16, not more than 1 : 17, not more than 1 : 18, or not more than 1 : 19.

[0139] The polymeric nanoparticles of the instant disclosure have, in various embodiments, a diameter that is an average of a distribution of nanoparticles in a particular population. The polymeric nanoparticles have dimensions that can be measured using a transmission electron microscope, or another suitable technique that can allow for measurements of the diameters of a sample of a population of polymeric nanoparticles.

[0140] In some embodiments, the diameter of the nanoparticles can be at least 50 nm such as, for example, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, at least 130 nm, at least 140 nm, at least 150 nm, or at least 160 nm.

[0141] In some embodiments, the diameter of the nanoparticles can be not more than 170 nm such as, for example, not more than 160 nm, not more than 150 nm, not more than 140 nm, not more than 130 nm, not more than 120 nm, not more than 110 nm, not more than 100 nm, not more than 90 nm, not more than 80 nm, not more than 70 nm, or not more than 60 nm.

[0142] In some embodiments, the diameter of the nanoparticles can range from 50 nm to 170 nm such as, for example, from 60 nm to 130 nm, from 60 nm to 100 nm, from 80 nm to 110 nm, from 90 to 130 nm, from 100 to 170 nm, or any other suitable range, based on the properties of the polymeric nanoparticles (e.g., the precise drugs associated therewith).

[0143] In some embodiments, a poly dispersity index (PDI) of the polymeric nanoparticles is not more than 0.50 such as, for example, not more than 0.45, not more than 0.40, not more than 0.35, not more than 0.30, not more than 0.25, not more than 0.20, not more than 0.15, not more than 0.10, or not more than 0.05. In some embodiments, the PDI is from 0.05 to 0.2. As used herein, the PDI is a ratio of the mass average molar mass of the penta- and di-block copolymers in the polymeric nanoparticles to the number average molar mass of the penta- and di-block copolymers in the polymeric nanoparticles. PDI may also be referred to simply as, “dispersity.”

[0144] The number average molar mass is defined as below:

where Ni is the number of molecules of molecular mass M.

[0145] The mass average molar mass is defined as below:

where Ni is the number of molecules of molecular mass M.

[0146] In some embodiments, mass average molar mass and number average molar mass can be measured by any suitable process such as, for example, gel permeation chromatography, viscometry via the Mark-Houwink equation, or colligative methods (for number average molar mass); or static light scattering, small angle neutron scattering, X-ray scattering, or sedimentation velocity (for number average molar mass).

[0147] In some embodiments, a zeta potential of the polymeric nanoparticles is from -5 mV to -40 mV such as, for example, -5 mV to -30 mV, -5 to -25 mV, or -5 to -15 mV. As used herein, zeta potential is a measure of the electrical potential difference at the slipping plane. The slipping plane is the interface of mobile fluid around a particle (e.g., a polymeric nanoparticle of the present disclosure) with fluid components that remain attached to the particle surface (e.g., via adsorption and/or electrostatic interaction).

[0148] The zeta potential and PDI (Poly dispersity Index) of the nanoparticles may be calculated (see U.S. Patent No. 9,149,426, incorporated herein by reference, in its entirety).

[0149] In addition to the polymer components described herein, the compositions provided herein can comprise one or more HDAC6/PI3K-6 dual inhibitors. The polymeric nanoparticles can associate with the HDAC6/PI3K-6 dual inhibitors. For example, in some embodiments, the polymeric nanoparticles can encapsulate the HDAC6/PI3K-6 dual inhibitors and/or adsorb to the HDAC6/PI3K-6 dual inhibitors. The polymeric nanoparticles can associate with the HDAC6/PI3K-6 dual inhibitors in any manner suitable to carry the HDAC6/PI3K-6 dual inhibitors throughout a subject’s body and deliver the chemotherapeutic compounds to a diseased cell (e.g., a rapidly dividing cell such as a cancer cell).

[0150] In addition to the polymer components described herein, the compositions provided herein can comprise one or more HDAC6/PI3K-6 dual inhibitors and one or more BCL-2 inhibitors. The polymeric nanoparticles can associate with the HDAC6/PI3K-6 dual inhibitors and the BCL-2 inhibitors. For example, in some embodiments, the polymeric nanoparticles can encapsulate the HDAC6/PI3K-6 dual inhibitors and the BCL-2 inhibitors and/or adsorb to the BCL-2 inhibitors. The polymeric nanoparticles can associate with the HDAC6/PI3K-6 dual inhibitors and the BCL-2 inhibitors in any manner suitable to carry the HDAC6/PI3K-6 dual inhibitors and the BCL-2 inhibitors throughout a subject’s body and deliver the chemotherapeutic compounds to a diseased cell (e.g., a rapidly dividing cell such as a cancer cell).

[0151] The inventors of the present disclosure have determined that when one or more HDAC6/PI3K-6 dual inhibitors and one or more BCL-2 inhibitors are both included in a composition of the present disclosure (e.g., associated with polymeric nanoparticles of the present disclosure), improved performance (e.g., improved growth inhibition of diseased cells) can be obtained, compared to the activity of the individual drugs. In certain embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors are associated with the same polymeric nanoparticles. In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors are associated with a first set of polymeric nanoparticles and the one or more BCL-2 inhibitors are associated with a second set of polymeric nanoparticles. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is selected from one or more of Compounds I-LXIII, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is selected from one or more of Compounds III and XL, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound III, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound XL, described above. In some embodiments, the BCL-2 inhibitor is selected from one or more of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, gossypol (AT-101), apogossypolone (ApoG2), and obatoclax; selective inhibitors of nuclear export (SINEs), e.g., selinexor (KPT-330). In some embodiments, the BCL-2 inhibitor is selected from one or more of venetoclax (ABT-199) and navitoclax (ABT-263). In some embodiments, the BCL-2 inhibitor is venetoclax. In some embodiments, the BCL-2 inhibitor is navitoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound III and venetoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound III and navitoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound XL and venetoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound XL and navitoclax.

[0152] Additionally, it has been determined that the ratio of the one or more HDAC6/PI3K-6 dual inhibitors to the one or more BCL-2 inhibitors can impact the performance of the chemotherapeutic compounds. Unless stated otherwise, all ratios disclosed herein for the one or more HDAC6/PI3K-6 dual inhibitors compared to the one or more BCL-2 inhibitors are written with the number referring to the one or more HDAC6/PI3K-6 dual inhibitors first and the number referring to the relative amount of the one or more BCL-2 inhibitors second. However, it is noted that the opposite order is used occasionally and explicitly in the Examples. It is understood that, for example, a 1 :3 ratio of one or more HDAC6/PI3K-6 dual inhibitors to one or more BCL-2 inhibitors is equivalent to, and inherently discloses, a 3: 1 ratio of one or more BCL-2 inhibitors to one or more HDAC6/PI3K-6 dual inhibitors.

Additionally, unless stated otherwise, the ratios herein are based on measurements of the masses of the respective chemical species.

[0153] In some embodiments, the total mass of the one or more HDAC6/PI3K-6 dual inhibitors can be greater than or equal to the total mass of the one or more BCL-2 inhibitors. In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of at least 1.1 : 1, at least 1.2: 1, at least 1.3: 1, at least 1.4: 1, at least 1.5: 1, at least 2: 1, at least 2.5: 1, or at least 3: 1. In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of 1 : 1 to 3.5: 1, or a mass ratio of 1.5: 1 to 2.5: 1. In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of about 2:1. In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of about 3: 1 (e.g., 3: 1). In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of not more than 3: 1, not more than 4: 1, or not more than 5: 1.

[0154] In some embodiments, the total mass of the one or more HDAC6/PI3K-6 dual inhibitors can be less than or equal to the total mass of the one or more BCL-2 inhibitors. In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of not more than 1 : 1.5, not more than 1 : 1.6, not more than 1 : 1.7, not more than 1 : 1.8, not more than 1 : 1.9, not more than 1 :2, not more than 1 :2.5, or not more than 1 :3. In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of 1 : 1 to 1 :3.5, or a mass ratio of 1 : 1.5 to 1 :2.5. In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of about 1 :2. In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of about 1 :3 (e.g., 1 :3). In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of at least 1 :3, at least 1 :4, or at least 1 :5.

[0155] In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of about 2: 1. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is selected from one or more of Compounds I-LXIII, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is selected from one or more of Compounds III and XL, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound III, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound XL, described above. In some embodiments, the BCL-2 inhibitor is selected from one or more of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, gossypol (AT-101), apogossypolone (ApoG2), and obatoclax; selective inhibitors of nuclear export (SINEs), e.g., selinexor (KPT-330). In some embodiments, the BCL-2 inhibitor is selected from one or more of venetoclax (ABT-199) and navitoclax (ABT-263). In some embodiments, the BCL-2 inhibitor is venetoclax. In some embodiments, the BCL-2 inhibitor is navitoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound III and venetoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound III and navitoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound XL and venetoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound XL and navitoclax.

[0156] In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors can be present in a mass ratio of about 1 :2. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is selected from one or more of Compounds I-LXIII, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is selected from one or more of Compounds III and XL, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound III, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound XL, described above. In some embodiments, the BCL-2 inhibitor is selected from one or more of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, gossypol (AT-101), apogossypolone (ApoG2), and obatoclax; selective inhibitors of nuclear export (SINEs), e.g., selinexor (KPT-330). In some embodiments, the BCL-2 inhibitor is selected from one or more of venetoclax (ABT-199) and navitoclax (ABT-263). In some embodiments, the BCL-2 inhibitor is venetoclax. In some embodiments, the BCL-2 inhibitor is navitoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound III and venetoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound III and navitoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound XL and venetoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound XL and navitoclax.

[0157] As described below, preparation of polymeric nanoparticles can optionally involve addition of a PEG-PPG-PEG tri-block copolymer. The inventors of the present disclosure have found that including such a tri-block copolymer can improve the stability of the polymeric nanoparticles and/or can serve as an emulsifier for other components. In some embodiments, the tri-block copolymer may be associated with or associated substantially with the polymeric nanoparticles. In some embodiments, the tri-block copolymer may not be associated with the polymeric nanoparticles. In some embodiments, the tri-block copolymer comprises a central polypropylene glycol) block flanked by two poly(ethylene glycol) blocks. In some embodiments, the tri-block copolymer comprises poloxamer 407, PLURONIC® F127, or PLURONIC® L61, or poloxamer 181. PLURONIC® L61 can comprise a tri-block copolymer comprising a central polypropylene glycol) block flanked by two poly(ethylene glycol) blocks. PLURONIC® L61 is available from BASF SE, Ludwigshafen, Germany.

Methods for preparing polymeric nanoparticles

[0158] The present disclosure provides a method of manufacturing nanoparticles comprising: a) mixing penta-block PLA-PEG-PPG-PEG-PLA and di-block m-PLA-PEG hybrid block copolymers dissolved in acetonitrile with one or more HDAC6/PI3K-6 dual inhibitors and at least one BCL-2 inhibitor dissolved in DMSO to form a first mixture; b) mixing the first mixture with a PEG-PPG-PEG tri-block copolymer dissolved in water and Triethylamine (TEA) to form a second mixture; c) stirring the second mixture and evaporating the acetonitrile and TEA; and d) filtering the stirred and evaporated second mixture, thereby manufacturing the nanoparticles.

[0159] In some embodiments, the penta-block and di-block copolymers are present in the acetonitrile at 10 mg to 40 mg per mL of acetonitrile. In some embodiments, the penta-block and di-block copolymers are present in the acetonitrile at 15 mg to 30 mg per mL of acetonitrile. In some embodiments, the penta-block and di-block copolymers are present in the acetonitrile at about 20 mg per mL of acetonitrile.

[0160] In some embodiments, the one or more HDAC6/PI3K-6 dual inhibitors and one or more BCL-2 inhibitors are dissolved in DMSO such that the total mass of drugs is dissolved at 0.05 mg per pL of DMSO to 0.2 mg per pL of DMSO.

[0161] In some embodiments, the PEG-PPG-PEG tri-block copolymer is dissolved in the water at 2.5 mg of PEG-PPG-PEG tri-block copolymer per mL of water to 10 mg of PEG- PPG-PEG tri-block copolymer per mL of water. In some embodiments, the PEG-PPG-PEG tri-block copolymer is dissolved in the water at 3.5 mg of PEG-PPG-PEG tri-block copolymer per mL of water to 7.5 mg of PEG-PPG-PEG tri-block copolymer per mL of water. In some embodiments, the PEG-PPG-PEG tri-block copolymer is dissolved in the water at about 5 mg per mL of water.

[0162] In some embodiments, the PEG-PPG-PEG tri-block copolymer comprises poloxamer 407. In some embodiments, the PEG-PPG-PEG tri-block copolymer comprises PLURONIC® F127. PLURONIC® F 127 and poloxamer 407 both comprise a tri-block copolymer comprising a central polypropylene glycol) block flanked by two poly(ethylene glycol) blocks. The approximate lengths of the two PEG blocks can be 101 repeat units, while the approximate length of the propylene glycol block can be 56 repeat units.

PLURONIC® F127 is available from BASF SE, Ludwigshafen, Germany.

[0163] It is understood that both PLURONIC® F127 and poloxamer 407 comprise the same tri-block copolymer, but they may vary from each other based on their respective molecular weights and/or the number of monomers in each of their blocks. In some embodiments, the PLURONIC® F127 and/or poloxamer 407 can comprise a molecular weight of from 10,500 g/mol to 14,500 g/mol such as, for example a molecular weight of about 12,600 g/mol.

[0164] In some embodiments, the TEA is added in an amount of 0.5 pL to 2 pL for every 1 mL of water. In some embodiments, the TEA is added in an amount of about 0.5 pL for every 1 mL of water. In some embodiments, the TEA is added in an amount of about 1 pL for every 1 mL of water. In some embodiments, the TEA is added in an amount of about 2 pL for every 1 mL of water.

[0165] Before the preparation of polymeric nanoparticles as described above, the penta-block and di-block copolymers may be prepared and used as reagents for preparation of the polymeric nanoparticles. These can be prepared as described in Example 1 from a poloxamer copolymer such as poloxamer 181 (for the penta-block copolymer), methoxy -poly(ethylene glycol) (m-PEG) (for the di-block copolymer), initiator, and PLA. The m-PEG can comprise a molecular weight from 2kDa to lOkDa. A ring-opening polymerization of lactide in the presence of a Sn-catalyst can be employed, or any other suitable technique as determined by the skilled artisan. In some embodiments, the poloxamer 181 can comprise a molecular weight of 1,000 g/mol to 3,000 g/mol. In some embodiments, the poloxamer 181 can comprise a molecular weight of about 2,000 g/mol. The appropriate molecular weight can be selected in order to, for example, improve the properties (e.g., stability) of the polymeric nanoparticles.

Pharmaceutical Compositions

[0166] Also provided herein is a pharmaceutical composition comprising the polymeric nanoparticle compositions described herein for use in medicine and in other fields that use a carrier system or a reservoir or depot of nanoparticles. The polymeric nanoparticles can be used in prognostic, therapeutic, diagnostic, and/or theranostic compositions. Suitably, the nanoparticles of the present disclosure are used for drug and agent delivery (e.g., within a tumor cell), as well as for disease diagnosis and medical imaging in human and animals. Thus, the instant disclosure provides a method for the treatment of disease using the nanoparticles, further comprising a chemotherapeutic agent, as described herein. The nanoparticles of the present disclosure can also be used in other applications such as chemical or biological reactions where a reservoir or depot is required, as biosensors, as agents for immobilized enzymes and the like.

[0167] Thus, in an aspect, provided herein is a pharmaceutical composition comprising a) polymeric nanoparticles comprising hybrid block copolymers comprising a PLA-PEG-PPG- PEG-PLA penta-block copolymer with an m-PEG-PLA di-block copolymer; and b) one or more HDAC6/PI3K-6 dual inhibitors. The one or more HDAC6/PI3K-6 dual inhibitors are associated with the polymeric nanoparticles.

[0168] Thus, in an aspect, provided herein is a pharmaceutical composition comprising a) polymeric nanoparticles comprising hybrid block copolymers comprising a PLA-PEG-PPG- PEG-PLA penta-block copolymer with an m-PEG-PLA di-block copolymer; b) one or more HDAC6/PI3K-6 dual inhibitors; and c) one or more BCL-2 inhibitors. Both the one or more HDAC6/PI3K-6 dual inhibitors and the one or more BCL-2 inhibitors are associated with the polymeric nanoparticles. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is selected from one or more of Compounds I-LXIII, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is selected from one or more of Compounds III and XL, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound III, described above. In some embodiments, the HDAC6/PI3K-6 dual inhibitor is Compound XL, described above. In some embodiments, the BCL-2 inhibitor is selected from one or more of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, gossypol (AT-101), apogossypolone (ApoG2), and obatoclax; selective inhibitors of nuclear export (SINEs), e.g., selinexor (KPT-330). In some embodiments, the BCL-2 inhibitor is selected from one or more of venetoclax (ABT-199) and navitoclax (ABT-263). In some embodiments, the BCL-2 inhibitor is venetoclax. In some embodiments, the BCL-2 inhibitor is navitoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound III and venetoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound III and navitoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound XL and venetoclax. In some embodiments, the combination of HDAC6/PI3K-6 dual inhibitor and BCL-2 inhibitor is Compound XL and navitoclax. [0169] Suitable pharmaceutical compositions or formulations can contain, for example, from about 0.1% to about 99.9%, preferably from about 1% to about 60%, of the active ingredient(s). Pharmaceutical formulations for enteral or parenteral administration are, for example, those in unit dosage forms, such as sugar-coated tablets, tablets, capsules or suppositories, or ampoules. If not indicated otherwise, these are prepared in a manner known per se, for example by means of conventional mixing, granulating, sugar-coating, dissolving, or lyophilizing processes. It will be appreciated that the unit content of a combination partner contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount may be reached by administration of a plurality of dosage units.

[0170] The pharmaceutical compositions can contain, as the active ingredient, one or more of nanoparticles in combination with one or more pharmaceutically acceptable carriers (excipients). In making the compositions of the disclosure, the active ingredient is typically mixed with an excipient, diluted by an excipient, or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0171] Some examples of suitable excipients include lactose (e.g., lactose monohydrate), dextrose, sucrose, sorbitol, mannitol, starches (e.g., sodium starch glycolate), gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, colloidal silicon dioxide, microcrystalline cellulose, polyvinylpyrrolidone (e.g., povidone), cellulose, water, syrup, methyl cellulose, and hydroxypropyl cellulose. The formulations can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; and flavoring agents.

[0172] The liquid forms in which the compounds and compositions of the present disclosure can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Methods comprising use of polymeric nanoparticles

[0173] The polymeric nanoparticles and pharmaceutical compositions disclosed herein can be used to treat or prevent any condition or disorder which is known to or suspected of benefitting from treatment with PI3K-6-HDAC6 dual inhibitor with or without BCL-2/BCL- xL inhibitor.

[0174] In one aspect, the polymeric nanoparticles and/or pharmaceutical compositions disclosed herein can be used to reduce proliferation, survival, migration, or colony formation ability of a rapidly proliferating cell in a subject in need thereof. This can be accomplished by contacting the cell with a therapeutically effective amount of the polymeric nanoparticles and/or pharmaceutical compositions. Such a method can be conducted in vivo (e.g., in a cancer patient), in vitro, or ex vivo.

[0175] In some embodiments, the cell can be a cancer cell or a cancer stem cell.

[0176] In another aspect, the polymeric nanoparticles and/or pharmaceutical compositions disclosed herein can be used to treat or prevent cancer or a precancerous condition. In some embodiments, the cancer can be, a cancer cell or a cancer stem cell. In some embodiments, the cancer can be a solid tumor cancer or a cancer of the blood. In some embodiments, the cancer can be selected from the group consisting of breast cancer, ovarian cancer, pancreatic cancer, leukemia, lymphoma, osteosarcoma, gastric cancer, prostate cancer, colon cancer, non-small cell lung cancer and small cell lung cancer, liver cancer, kidney cancer, head and neck cancer, cervical cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, multiple myeloma, and combinations thereof. In some embodiments, the cancer can be selected from the group consisting of breast cancer, leukemia, lymphoma, colon cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, and combinations thereof.

[0177] In some embodiments, the cancer can comprise triple negative breast cancer (TNBC). In some embodiments, the cancer can comprise ER⁺ breast cancer.

[0178] In some embodiments, the cancer can be acute myeloid leukemia. [0179] In some embodiments, the cancer may be an affliction of a subject. In some embodiments, the subject may be a human.

[0180] In some embodiments, the treatment using the polymeric nanoparticles or pharmaceutical composition comprising them can comprise administration of an additional anti-cancer therapy. The additional anti-cancer therapy can comprise any medically suitable therapy that could be combined with the polymeric nanoparticles disclosed herein. Such combinations of therapies can increase the overall effectiveness of cancer treatments.

[0181] In some embodiments, the additional anti-cancer therapy can comprise surgery; chemotherapy, radiation, hormone therapy, immunotherapy, or a combination thereof.

[0182] Additional anti-cancer therapies that may be combined with the polymeric- nanoparticle-based therapies disclosed herein include: lenalidomide and crizotinib. Additional anti-cancer therapies that may be combined with the polymeric-nanoparticle- based therapies disclosed herein include: gleevec, herceptin, avastin, PD-1 checkpoint inhibitors, PDL-1 checkpoint inhibitors, CTLA-4 checkpoint inhibitors, tamoxifen, trastuzamab, raloxifene, fluorouracil/5-fu, pamidronate disodium, anastrozole, exemestane, cyclophos-phamide, letrozole, toremifene, fulvestrant, fluoxymester-one, trastuzumab, methotrexate, megestrol acetate, docetaxel, paclitaxel, testolactone, aziridine, vinblastine, capecitabine, goserelin acetate, zoledronic acid, taxol, vinblastine, vincristine, and/or anthracyclines such as Doxorubicin and Pirarubicin.

[0183] In some embodiments, the cancer can be resistant to certain chemotherapeutic agents. Administration of the of the polymeric nanoparticles of the present disclosure can be an alternative therapy when a different therapy, vulnerable to resistance, has been attempted unsuccessfully. Alternatively, or additionally, the therapies of the instant disclosure can offer alternative forms or administration of chemotherapeutic drugs that can reduce the effect of resistance to the drugs.

[0184] In some embodiments, the composition or pharmaceutical composition comprising the polymeric nanoparticles can be administered to the subject via an administration route. For example, the composition or pharmaceutical composition can be administered intravenously, intratumorally, or subcutaneously.

[0185] In some embodiments of the methods, the composition can be administered at least once per day, once every other day, once per week, twice per week, once per month, or twice per month. In an embodiment of the methods, the composition is administered at least once per day. In an embodiment of the methods, the composition is administered at least once every other day. In an embodiment of the methods, the composition is administered at least once per week. In an embodiment of the methods, the composition is administered at least twice per week. In an embodiment of the methods, the composition is administered at least once per month. In an embodiment of the methods, the composition is administered at least twice per month. In another embodiment, the composition is administered more than once per day.

[0186] In some embodiments of the methods, the composition is administered over a period of three weeks. In other embodiments of the methods, the composition is administered over a period of 30 days. In other embodiments of the methods, the composition is administered over a period of 60 days. In other embodiments of the methods, the composition is administered over a period of 90 days. In other embodiments of the methods, the composition is administered over a period of 120 days. In other embodiments of the methods, the composition is administered over a period of 150 days. In other embodiments of the methods, the composition is administered over a period of 6 months. In other embodiments of the methods, the composition is administered over a period of about 6 months to about 1 year. In other embodiments of the methods, the composition is administered over a period of about 1 year to about 2 years.

[0187] The effective dosage of the polymeric nanoparticles provided herein may vary depending on the chemotherapeutic agent(s) used, the mode of administration, the condition being treated, and the severity of the condition being treated. The dosage regimen of the polymeric nanoparticle can be selected in accordance with a variety of factors, including the route of administration and the renal and hepatic function of the patient. In certain embodiments, the therapeutically effective amount can be a human equivalent dose that is determined from an animal experiment.

EXAMPLES

[0188] The disclosure will now be illustrated with working examples, which are intended to illustrate the various aspects of the disclosure and are not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions.

Example 1. Ring opening polymerization of PLA-PEG-PPG-PEG-PLA penta-block and hybrid block co-polymeric nanoparticles

[0189] The m-PEG-PLA + PLA-PEG-PPG-PEG-PLA (hybrid copolymer) and PLA-PEG- PPG-PEG-PLA (penta-block copolymer) were prepared by ring opening polymerization using stannous octoate. The scheme of the ring opening polymerization reaction is shown in FIG. 1.

[0190] Generally, the ring opening polymerization comprised the following steps. First, a polymer comprising m-PEG (with a molecular weight that can be from 2kDa to 10 kDa), L- lactide, and a block co-polymer comprising PEG-PPG-PEG were dissolved in an organic solvent to obtain a solution. About 0.005% weight percent of lactide, stannous octoate, and a base were added to the solution to obtain a reaction mixture. The reaction mixture was stirred in presence of nitrogen and at 170°C for 3 hours, to obtain a hybrid block copolymer of PLA, chemically modified with a block copolymer or polymer. The block polymer was dissolved in an organic solvent and made into a homogenized mixture. The homogenized mixture was added to an aqueous phase to obtain an emulsion. Finally, the emulsion was stirred to obtain biodegradable polymeric nanoparticles, to promote L-lactide to undergo ring opening polymerization.

[0191] One method included a mixture of initiators i.e., PLURONIC® L61 (0.0625 mM) and mPEG (0.1875 mM) were added in to a vacuum dried two neck round bottom flask. The flask was heated to 110°C using a magnetic stirrer hot plate to get well mixed molten phase as well as to remove any moisture content from both the initiators. L-lactide (50 mM) was added into the flask at 50°C under nitrogen blanket. After melting of L-lactide, stannous octoate (0.005 wt % of L-lactide) was added into the flask. Then the flask was heated to 170°C for three hours with continuous stirring under a nitrogen environment. After three-hours reaction, the flask was cooled to room temperature, and DCM was added to dissolve the solid block copolymer. Methanol (~8X volume of DCM) was used to precipitate the block copolymer from DCM soluble unreacted initiators, monomer, and catalyst. The precipitated block copolymer was filtered using Whatman filter paper and dried overnight under a vacuum. [0192] The synthesized block copolymer was characterized using gel permeation chromatography (GPC, Viscoteck GPCmax system), to evaluate its molecular weight and poly dispersity index (Mw/Mn). The chemical composition of the block copolymer was confirmed by

NMR in CDCh at 400 MHz (Bruker) and Fourier-transform infrared spectroscopy (Spectrum Two, FT-IR Spectrometer, Perkin Elmer, USA).

[0193] The advantages of adopting the ring opening polymerization over the condensation polymerization are summarized in Table 1.

Example 2, Condensation polymerization reaction for the production of PLA-PEG-PPG- PEG-PLA

[0194] A condensation polymerization reaction for the production of PLA-PEG-PPG-PEG- PLA comprised the following steps. 5 g of poly (lactic acid) (PLA) with an average molecular weight of 60,000 g/mol was dissolved in 100 ml CH2Q2 (dichloromethane) in a 250 ml round bottom flask. To this solution, 0.7 g of PEG-PPG-PEG polymer (molecular weight range of 1100-8400 g/mol) was added. The solution was stirred for 10-12 hours at 0°C. To this reaction mixture, 5 ml of 1% N,N-di cyclohexylcarbodiimide (DCC) solution was added followed by slow addition of 5 ml of 0.1% 4-Dimethylaminopyridine (DMAP) at - 4°C to 0°C/sub-zero temperatures. The reaction mixture was stirred for the next 24 hours followed by precipitation of the PLA-PEG-PPG-PEG block copolymer with diethyl ether and filtration using Whatman filter paper No. 1. The PLA-PEG-PPG-PEG block copolymer precipitates so obtained were dried under low vacuum and stored at 2°C to 8°C until further use.

Table 1 — Advantages of ring opening polymerization

Example 3, Selection and characterization of HDAC6/PI3K-5 dual inhibitor

[0195] Histone deacetylase 6 (HDAC6) / phosphoinositide 3-kinase-6 (PI3K-6) dual inhibitors Compound III (1925) and Compound XL (7679), described above, were selected out of about ten candidates for nanoparticle generation. A standard curve for each dual inhibitor was made using an HPLC method using a WATERS XBridge RP C18, 4.6 x 250 mm column, with a flow rate of 1 mL/min, absorbance at 254 nm, column temperature of - 50°C at a total run time of 15 minutes. Table 2 shows the parameters of the run.

Table 2 — Run parameters

[0196] FIG. 2 shows the HPLC chromatogram with 1925 showing a retention time of 6.37 min (blue) and Compound XL (7679) showing a retention time of 6.84 min (green). There was a single peak for each molecule.

Example 4, Characterization of PLA-PEG-PPG-PEG-PLA penta-block and hybrid block co- polymeric nanoparticles

[0197] Blank hybrid m-PEG-PLA + PLA-PEG-PPG-PEG-PLA nanoparticles, synthesized by ring opening polymerization, were compared to penta-block PLA-PEG-PPG-PEG-PLA polymer nanoparticles based on yield, monomer to initiator mole ratio, nanoparticle size, and stability. The results are shown in Table 3. It was observed that the hybrid blank m-PEG- PLA + PLA-PEG-PPG-PEG-PLA at [M]/[I] ratio of 133 and blank PLA-PEG-PPG-PEG- PLA at [M]/[I] ratio of 126 were the most stable. Table 3 — Characterization of blank PLA based block co-polymeric nanoparticles

a. gel permeation chromatography b. nanoparticle

[0198] Compound XL (7679) and Compound III (1925) were incorporated into the hybrid nanoparticles. The properties of these nanoparticles are shown in Table 4, below. Table 4 — Characterization of nanoparticles encapsulating HDAC6/PI3K-6 dual inhibitor

[0199] Lyophilized drug loaded Nanoparticles (10 mg-200 mg) were dispersed in 1-5 ml PBS at pH 7.4. Following incubation at 37°C, filtrates were collected at different time intervals indicated in FIG. 3 and lyophilized before HPLC measurement. FIG. 3 A shows release of drug per day from drug encapsulated in nanoparticles and FIG. 3B shows cumulative release.

Example 5, HDAC6/PI3K-5 dual inhibitor associated with nanoparticles decreases viability of cancer cell lines

[0200] The nanoparticles used in Example 4 were loaded with dye to test cellular uptake into an MDA-MB-468 breast cancer cell line. As shown in FIGs. 4A, B, and C, nanoparticles loaded with rhodamine were taken up into MDA-MB-468 cells. FIG. 5 shows decrease in percent cell viability in ZR-75-1 cells, a hormone dependent breast cancer cell line, with increasing concentrations of 7679 (FIG. 5A) and 1925 (FIG. 5B) both in solution and encapsulated in nanoparticles. FIG. 6 shows decrease in percent cell viability in SUM 149 cells, a triple negative breast cancer cell line, with increasing concentrations of Compound XL (7679) (FIG. 6 A) and Compound III (1925) (FIG. 6B) both in solution and encapsulated in nanoparticles. FIG. 7 shows decrease in percent cell viability in MDA MB 468 cells, a breast cancer cell line, (FIG. 7A) and in E0771 cells, a breast cancer cell line, Compound XL (7679) (FIG. 7B) with increasing concentrations of Compound XL (7679). FIG. 8 shows decrease in percent cell viability in MCF-7 cells, a hormone dependent breast cancer cell line, with increasing concentrations of Compound XL (7679) both in solution and encapsulated in nanoparticles. FIG. 9 shows decrease in percent cell viability in SW620 cells, a colon cancer cell line, with increasing concentrations of Compound XL (7679) (FIG. 9A) and Compound III (1925) (FIG. 9B) both in solution and encapsulated in nanoparticles. FIG. 10 shows decrease in percent cell viability in HCT116 cells, a human colon cancer cell line, (FIG. 10A) and MC38 cells, a mouse colon cancer cell line (FIG. 10B) with increasing concentrations of Compound XL (7679) both in solution and encapsulated in nanoparticles. FIG. 11 shows decrease in percent cell viability in U266B1 cells, a multiple myeloma cell line, with increasing concentrations of Compound XL (7679) (FIG. 11 A) and Compound III (1925) (FIG. 1 IB) both in solution and encapsulated in nanoparticles. FIG. 12 shows decrease in percent cell viability in U937 cells, a leukemia cell line, with increasing concentrations of Compound XL (7679) both in solution and encapsulated in nanoparticles.

[0201] IC50 values for these results are shown in Table 5, below. Table 5 — IC50 Values for 7679, 7679 NPs, 1925, and 1925 NPs

Example 6, Use of nanoparticles encapsulating HDAC6/PI3K-5 dual inhibitors in an in vivo mouse cancer model

[0202] The nanoparticles from Example 4 loaded with Compound XL (7679) were injected into a breast cancer syngeneic mouse model once a week intravenously for three weeks. Ehrlich ascites Cells were injected into mice and Ehrlich ascites Tumor (EAT) growth and percent increase in tumor growth compared to control is shown in FIG. 13. 12.5 and 25 mg of nanoparticles per kg of mouse weight were compared to control. Survival of mice was monitored for 50 days, and the results shown in FIG. 14. Nanoparticles encapsulating Compound XL (7679) were more effective than control in reducing tumor growth in mice and promoting mouse survival.

[0203] Nanoparticles loaded with Compound XL (7679) and nanoparticles loaded with idelalisib were administered to the same syngeneic mouse model. Both sets of nanoparticles were administered at 25 mg/kg twice a week for three weeks. Tumor growth was monitored for 36 days as shown in FIG. 15. Nanoparticles encapsulating Compound XL (7679) were more effective than nanoparticles encapsulating idelalisib in reducing tumor growth in mice. [0204] Nanoparticles loaded with indocyanine green dye were used to show biodistribution of nanoparticles in a syngeneic mouse model. Tumors took up more indocyanine over 3, 24, and 48 hour time periods when indocyanine was delivered with nanoparticles as opposed to indocyanine alone, as shown in FIG. 16. The nanoparticles described in Example 4 are able to concentrate concentration of substances encapsulated within the nanoparticles in tumor cells.

Example 7, HDAC6/PI3K-5 dual inhibitor with venetoclax or navitoclax decreases viability of cancer cell lines

[0205] The free Compound XL (7679) and free navitoclax and/or venetoclax were used to treat cell lines. FIG. 17 shows decrease in percent cell viability in SUM 149 cells, a triple negative breast cancer cell line, (FIG. 17A) and HCT116 cells, a colon cancer cell line, (FIG. 17B) with increasing concentrations of 7679 and navitoclax alone and in combination at two different mass ratios. FIG. 18 shows decrease in percent cell viability in THP1 cells, an acute monocytic leukemia cell line, with increasing concentrations of Compound XL (7679) and venetoclax (199) alone and in combination at two different ratios. FIG. 19 shows decrease in percent cell viability in THP1 cells with increasing concentrations of Compound XL (7679) and navitoclax alone and in combination at two different ratios. FIG. 20 shows decrease in percent cell viability in HL60 cells, an acute monocytic leukemia cell line, with increasing concentrations of Compound XL (7679) and venetoclax (199) or navitoclax (263) alone and in combination. A summary of the IC50 of these combinations in AML cell lines is shown in Tables 6 and 7, below.

Table 6 — Single Drug

Table 7 — Combination 2:1

[0206] FIG. 21 shows decrease in percent cell viability in Mv411 cells, an acute monocytic leukemia cell line, with increasing concentrations of Compound XL (7679), navitoclax (ABT263) and venetoclax (ABT199) alone and in combination at a 2: 1 7679 to venetoclax or navitoclax ratio. FIG. 22 shows decrease in percent cell viability in Mv411 cells with increasing concentrations of Compound XL (7679), navitoclax (ABT263) and venetoclax (ABT199) alone and in combination at a 2: 1 Compound XL (7679) to venetoclax or navitoclax ratio. A summary of the IC50 of these combinations in AML cell lines is shown in Tables 8, 9 and 10, below.

Table 8 — Single Drug

Table 9 — Combination 2:1

Table 10 — Combination 1:2

Example 8, Preparation and characterization of drug encapsulating nanoparticles

[0207] To evaluate the suitable PLA based hybrid block copolymer for in vitro and in vivo studies, blank NPs from block copolymers were prepared using the nanoprecipitation method. In brief, PLA block copolymer (100 mg) was dissolved in 5 ml acetonitrile (20 mg/ml) at ~50-60°C for 10-25 minutes in hot water bath. PLURONIC® F-127 emulsifier solution (5 mg/ml) was prepared in double-distilled water in a beaker with the help of a stirrer (-700 rpm). After complete dissolution, PLA block copolymer solution was injected in an aqueous F127 emulsifier solution (10 mg/2 ml) during continuous stirring (600-700 rpm) using a 26- gauge needle. NPs solution was kept on stirring overnight at room temperature to evaporate acetonitrile and stabilize nanoparticles. Suitable block copolymer was evaluated through NPs stability study, which was done for 15 days’ time at 4°C. NP stability was defined according to their change in size in comparison to the initial size measured with DLS instrument (Anton Paar Litesizer 500). For this study, 10 mg/ml NPs in double distilled water were kept at 4°C and size measurements were done on daily basis.

[0208] To prepare PI3K/HDAC (PI3K/HDAC-NPs) and navitoclax encapsulated NPs (Nav- NPs), drug was dissolved in DMSO (1 mg/30pl, sonication for 5 min) and mixed with dissolved PLA block-copolymer solution at 1 : 10 weight by mass ratio of drug and polymer, then this solution was injected into F-127 emulsifier solution to prepare Drug-NPs. To prepare dual drug encapsulated NPs, three drug mass ratios were used, /.< ., 1 : 1, 3 : 1 and 1 :3 (Table 11), while the drug to polymer mass ratio was kept constant, z.e., 1 : 10. Drug encapsulated NPs were filtered using Amicon® 3kDa ultrafilter (Millipore) through centrifugation at 4000 rpm for 60 minutes at ~10°C to remove unencapsulated drug. Glucose at concentration of 25 wt% of PLA block copolymer was added into the Drug-NPs then the solution was lyophilized to obtain drug encapsulated NPs. These lyophilized Drug-NPs were stored at -20°C until use.

[0209] The collected filtrate was analyzed for unencapsulated PI3K/IC and navitoclax using HPLC. Drug encapsulation efficiency (%) was calculated using the following formula: 100

[0210] The hydrodynamic diameter of prepared nanoparticles was assessed using DLS instrument (Anton Paar Litesizer 500). Prepared Drug-NPs were also characterized using transmission electron microscopy (TEM) for their shape, size, and morphology.

[0211] The shape of the nanoparticles obtained by the process mentioned above is essentially spherical. The particle size range was about 30 to 120 nm. The hydrodynamic radius of the nanoparticle was measured using a dynamic light scattering (DLS) instrument and is in the range of 50-140 nm.

[0212] The above method was also used to prepare SAHA, IDL, Rho-B, and ICG encapsulated NPs. Example 9, Characterization of polymeric nanoparticles

[0213] Molecular weight, Mw, and Mn obtained for nanoparticles comprising hybrid block copolymers comprising a PLA-PEG-PPG-PEG-PLA penta-block copolymer with an m-PEG- PLA di-block copolymer was ~32 kDa and ~28 kDa respectively as calculated using GPC Omnisec software. The synthesized polymer showed poly dispersity index (Mw/Mn) of —1.1, confirming the narrow molecular weight distribution. The structural characterization of PLA block copolymer was done using ^TH NMR (FIG. 23).

[0214] The peak at -3.4 ppm (a), ~3.5 ppm (b), and -1.1 ppm (c) confirmed the presence of CH, CH2, and CH3 protons of PPG unit available in PLURONIC® L61 (Wang et al., 2012, Poly (caprolactone)-modified PLURONIC® Pl 05 micelles for reversal of paclitaxel - resistance in SKOV-3 tumors, Biomaterials 33(18):4741-4751). The presence of PEG in PLURONIC® and mPEG, was confirmed by the singlet peak at ~3.6 ppm (d) of CH2CH2 protons. Peak at -1.58 ppm (e) represented CH3 proton and peak at -5.19 ppm (f) represents tertiary proton of PLA available in penta-block (PLA-PEG-PPG-PEG-PLA) and di-block (m- PEG-PLA) copolymers (Gupta et al., 2018, Concomitant delivery of paclitaxel and NuBCP-9 peptide for synergistic enhancement of cancer therapy, Nanomedicine: Nanotechnology, Biology and Medicine 14(4): 1301-1313).

[0215] Further confirmation of the hybrid block copolymer was done using FT-IR spectroscopic study, shown in FIG. 24. The PLA block copolymer showed characteristic peaks of C=O at 1753 cm'¹. FT-IR spectra exhibited a broad peak -3750 cm'¹ and a sharp peak at 1084 cm'¹ due to terminal hydroxy group -OH and C-0 stretching in PLURONIC® and PLURONIC® modified PLA block copolymer (Su et al., 2002, FTIR spectroscopic study on effects of temperature and polymer composition on the structural properties of PEO- PPO- PEO block copolymer micelles, Langmuir 18(14):5370-5374). Additionally, aliphatic CH stretching band of PLA at 2994 cm'¹ and of PLURONIC® at 2880 cm'¹ was also observed in FIG. 24 (Li et al., 2015, In situ gel-forming AP-57 peptide delivery system for cutaneous wound healing, International Journal of Pharmaceutics 495(l):560-571). Thus, these data confirmed the successful synthesis of PLA block copolymer comprising mPEG and PLURONIC®. Example 10. Characterization of drug encapsulating polymeric nanoparticles

[0216] The synthesized hybrid block copolymers comprising a PLA-PEG-PPG-PEG-PLA penta-block copolymer with an m-PEG-PLA di-block copolymer was used to prepare single and dual drug encapsulating nanoparticles (NPs), i.e., PI3K-6/HDAC6-NPs, NAV-NPs, and PI3K-6/HDAC6-NAV-NPs through a nanoprecipitation method. These drug encapsulated NPs were characterized for hydrodynamic diameter using DLS. Table 11 shows the hydrodynamic diameter of single and dual drug encapsulating NPs. The blank NPs showed lowest hydrodynamic diameter of 79 nm as compared to PI3K-6/HDAC6-NPs (109 nm) and NAV-NPs (169 nm). The larger size of drug encapsulating NPs could be due to the drug encapsulation in the hydrophobic core of the NPs (Gupta et al., 2018, Concomitant delivery of paclitaxel and NuBCP-9 peptide for synergistic enhancement of cancer therapy, Nanomedicine: Nanotechnology, Biology and Medicine 14(4): 1301-1313). Navitoclax is a relatively large molecule (molecular weight of 974.6 g/mol) and more hydrophobic as compared to the PI3K-6/HDAC6 dual inhibitor (molecular weight of 576 g/mol), which could result in comparatively larger NP size. The PI3K6/HADC6-NAV-NPs (1 : 1, 3: 1, and 1 :3 mass ratios of PI3K6/HADC6 to NAV) have shown the hydrodynamic diameter higher than the PI3K-6/HDAC6-NPs and lower than the NAV-NPs (FIG. 25 A). As the concentration of PI3K-6/HDAC6 dual inhibitor increases in the NPs the hydrodynamic size of NPs shifted towards the lower size. The PDI of all prepared NPs was in the range of 0.09-0.2 (data not shown), which is suitable for drug delivery applications (Masarudin et al., 2015, Factors determining the stability, size distribution, and cellular accumulation of small, monodisperse chitosan nanoparticles as candidate vectors for anticancer drug delivery: application to the passive encapsulation of [14C]-doxorubicin, Nanotechnology, Science and Applications 8:67). The TEM images (FIGs. 25B and FIG. 25C) of PI3K-6/HDAC6-NAV-NPs (1 :3 mass ratio of PI3K6/HADC6 to NAV NPs, Table 11) showed a size of 140±7 nm with uniform spherical core-shell morphology (FIG. 25C), which is lower than the hydrodynamic size obtained with DLS (Ghorbani et al., 2018, A novel polymeric micelle-decorated Fe 3 O 4/ Au core-shell nanoparticle for pH and reduction-responsive intracellular co-delivery of doxorubicin and 6-mercaptopurine, New Journal of Chemistry 42(22): 18038-18049).

[0217] The drug encapsulation efficiency (EE) of single and dual drug-NPs calculated for PI3K-6/HDAC6 dual inhibitor and navitoclax are in the range of 90-98% (Table 11). Both the drug molecules are hydrophobic in nature, so they were readily encapsulated into the hydrophobic core of the polymeric NPs and resulted in higher encapsulation efficiency.

Table 11 — Characterization of nanoparticles

Example 1 1. /// vitro release assessment from drug encapsulating NPs

[0218] In vitro release study of PI3K-6/HDAC6-NPs, NAV-NPs and PI3K-6/HDAC6-NAV- NPs was done in phosphate buffer saline (PBS, pH-7.4). Lyophilized drug-NPs (50 mg) were dispersed in PBS (10 ml) and the NPs solution was kept in an incubator shaker at 37°C, 120 rpm. The NPs solution was filtered at predetermined points (every 24 hours for 10 days) using Amicon® 3kDa ultrafilter at 4000 rpm for 60 min. The collected filtrates were lyophilized and analyzed using HPLC.

[0219] PI3K-6/HADC6-NPs and NAV-NPs showed a cumulative release of 29 ± 4% and 24 ± 4%, respectively. The PI3K-6/HDAC6-NAV-NPs showed cumulative release of 24 ± 2% for both the drug molecules, z.e., navitoclax and PI3K-6/HADC6 dual inhibitor. Both the single and dual drug NPs showed a burst release of up to 9% in the initial days followed by sustained 2-4% release up to 10 days. Initial higher percent release of the drug from the drug- NPs could be due to the adsorption of drug molecules on the NP surface, resulting in faster dissociation than entrapped drug molecules in the NP’s hydrophobic core. These data confirmed the slow and sustained release of PI3K-6/HDAC6 dual inhibitor and navitoclax from PI3K-6/HDAC6-NAV-NPs for a longer duration (FIGs. 26A-26C). Example V In vitro cytotoxicity study

[0220] The proliferation inhibition study of PI3K-6/HDAC6-NPs, NAV-NPs, and PI3K- 6/HDAC6-NAV-NPs (weight ratios of 1 : 1, 3: 1, and 1 :3 PI3K6/HADC6 to NAV) was determined on ER⁺ breast cancer cell lines including MCF7, ZR-75-1, and EAC (Ehrlich ascites cancer; mouse breast cancer cell line). MCF7 cells were grown in DMEM medium containing 10% FBS and 100 U/ml pen/strep solution. ZR-75-1, and EAC cells were grown in RPMI medium containing 10% FBS and 100 U/ml pen/strep solution. Cells were plated at density of 2500-4000 cells per well in flat bottom 96 well plates (Nest Biotechnology, China). Cells were treated for 72h with PI3K-6/HDAC6-NPs, NAV-NPs, and PI3K- 6/HDAC6-NAV-NPS (mass ratios of 1 : 1, 3 : 1, and 1 :3 PI3K6/HADC6 to NAV) at final drug molar concentration of 0.001, 0.01, 0.1, 1, 10, and 100 pM. Molar concentration of PI3K- 6/HDAC6 and navitoclax in encapsulating NPs was calculated based on their encapsulation efficiency. Cancer cell growth inhibition was evaluated using MTT assay (Wang et al., 2012, Poly (caprolactone)-modified Pluronic Pl 05 micelles for reversal of paclitaxel-resistance in SKOV-3 tumors, Biomaterials 33(18):4741-4751). The half maximal inhibitory drug concentration (IC50) was determined using GraphPad Prism 9 software. All the experiments were done in triplicates.

[0221] In vitro anti-cancer activity of PI3K-6/HADC6-NPs and NAV-NPs and PI3K- 6/HDAC6-NAV-NPS were evaluated, and results are given in Table 12 and FIGs. 27A-27C. All the three combinations (NPs with 1 : 1, 1 :3, and 3 : 1 ratios of PI3K6/HADC6 to NAV) have shown lower IC50 values as compared to PI3K-6/HADC6-NPs and NAV-NPs in all three cancer cell lines (FIGs. 27A-27C). When the cells were treated with 10 pM of all three PI3K- 6/HDAC6-NAV-NPS, the cell viability was significantly decreased to 10.11 ± 2.97 as compared to treatment with PI3K-6/HADC6-NPs and NAV-NPs (42.98 ± 18.23) (FIGs. 28A- 28C). The lower IC50 values of PI3K-HADC-Nav-NPs on cancer cells could be due to the simultaneous inhibition of multiple pathways which resulted in higher killing of cancer cells (Anderson et al., 2016, PIK3CA mutations enable targeting of a breast tumor dependency through mTOR-mediated MCL-1 translation, Science Translational Medicine 8(369):369ral75-369ral75; Merino et al., 2016, Targeting BCL-2 to enhance vulnerability to therapy in estrogen receptor-positive breast cancer, Oncogene 35(15): 1877-1887). Taken together, these findings confirmed the synergistic effect of PI3K-6/HDAC6 dual inhibitor and navitoclax combination for ER⁺ breast cancer therapy. Table 12 — ICso values of Drug-NPs on ER⁺ breast cancer cell lines

Example 13, Combination index (CI) analysis

[0222] Combination index (CI) analysis was performed on Compusyn Software to determine the synergistic (CI=<1), additive (CI=I) and antagonistic (CI=>1) cytotoxic effects ofPI3K- 6/HDAC6-NAV-NPs at constant drug molar ratios.

[0223] The CI values of NPs with 1 : 1, 1 : 3, and 3 : 1 ratios of PI3K6/HADC6 to NAV on MCF7, ZR-75 and EAC cells are given in Table 13. The CI value vs fraction affected plots for MCF7, ZR-75, and EAC cells are shown in FIGs. 29A-29C. The PI3K-6/HDAC6-NAV- NPs of 1 :3 weight ratio has shown lowest CI value of 0.17, 0.44 and 0.49 for ZR-75, MCF7 and EAC, respectively, as compared to 1 : 1 and 3: 1 NPs. Therefore, PI3K-6/HDAC6-NAV- NPs of 1 :3 has been used for in vivo anti-cancer therapeutic efficacy studies.

Table 13 — CI value of PI3K-6/HDAC6-NAV-NPs on ER+ breast cancer cell lines

Example 14, In vivo anti-cancer therapeutic efficacy, survival, and toxicity studies

[0224] In vivo experiments were done on Balb/c mice (weight 24-27 gram) under ethical clearance (122/IAEC/2019) obtained from CPCSCA committee, All India Institute of Medical Sciences (AIIMS) New Delhi, India. EAC cells (10⁸ cells/100pl PBS) were subcutaneously (s.c.) injected into the right hind limb of the Balb/c mice to generate subcutaneous tumor. Tumor volume was measured using a digital Vernier calliper and calculated using the following equation:

Tumor volume (mm³) = (W² x L) -H 2 where W and L are width and length of the tumor, respectively.

[0225] Five days after EAC inoculation, mice bearing tumors (tumor volume 290-400 mm³) were divided into four groups (5 mice/group) of control (PBS), 4 mg/kg PI3K-6/HDAC6- NPs, 4 mg/kg NAV-NPs, and 4 mg/kg PI3K-6/HDAC6-NAV-NPs. All NPs were injected intravenously (IV) in the lateral tail vein of mice. Doses were given as twice a week for three weeks, on day 6, 10, 13, 17, 20, and 24 days after EAC inoculation. The changes in mice body weight and tumor volume were recorded every week.

[0226] Relative tumor regression % (RTR%) was calculated using the following equation:

RTR % = ^Cavfl~^Tavg _x 100%

Cavg where, Cavg = mean relative tumor volume of the control group on study day of interest and Tavg = mean relative tumor volume of the drug treated group on study day of interest.

[0227] In vivo tumor growth inhibition by PI3K-6/HADC6-NPs, NAV-NPs and PI3K- 6/HDAC6-NAV-NPS (3: 1 mass ratio of PI3K6/HADC6 to NAV) was evaluated in syngeneic breast cancer model (FIG. 30 A). All the NP formulations were given at a dose of 4 mg/kg via intravenous injection twice a week for three weeks. Treatment with PI3K-6/HADC6-NPs (4 mg/kg) and NAV-NPs (4 mg/kg) showed relative tumor regression percent (RTR%) of ~19 and -67 as compared to control group (PBS). While the treatment with PI3K-6/HDAC6- NAV-NPs (4 mg/kg) has shown -99% of RTR. These data confirm the synergistic effect of PI3K-6/HADC6 dual inhibitor and navitoclax combinatorial treatment. A significant change in relative tumor volume of treated vs untreated groups can be seen in FIG. 30B.

[0228] The lack of related toxicity of treated and untreated mice groups was observed through lack of change in mice body weight. All treated groups have not shown any significant body weight change of mice (FIG. 31 A). The comparative survival of treated and untreated groups was also observed up to 60 days from the day of EAC inoculation (FIG.

3 IB). The untreated and PI3K-6/HDAC6-NPs treated groups showed the median survival of 31 and 35 days, respectively. While the NAV-NP treated group showed median survival of 45 days, which could be due to better tumor regression capability of BCL-2 inhibitor as compared to PI3K-6/HDAC6 inhibitor (Kumar et al., 2014, Novel polymeric nanoparticles for intracellular delivery of peptide cargos: antitumor efficacy of the BCL-2 conversion peptide NuBCP-9, Cancer Research 74(12):3271-3281). The PI3K-8/HDAC6-NAV-NPs treated group has showed death of only one mouse out of five on day 49. From the survival study, it can be concluded that PI3K-6/HDAC6-NAV-NPs are more efficient for ER⁺ breast cancer tumor therapy.

[0229] Nephrotoxicity, hepatotoxicity, and histopathological studies were performed to get more insight into the therapeutic efficacy of PI3K-6/HDAC6-NAV-NPs. The creatinine (FIG. 32, top panel) and blood urea (FIG. 32, bottom panel) levels of control (PBS) and PI3K- 6/HDAC6-NAV-NPS did not show any change as compared to healthy mice, confirming no toxicity with PI3K-6/HDAC6-NAV-NP treatment. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) showed (FIG. 33) higher value in case of untreated mice, which could be due to EAC tumor growth related liver necrosis and inflammation (Gowda et al., 2022, Ehrlich ascites carcinoma mice model for studying liver inflammation and fibrosis, Advances in Cancer Biology-Metastasis 100029). The liver tissue section of untreated mice showed (FIG. 34) deformities in hepatocytes, alteration in lobular structure, vacuolization, swelling, and fibrosis further confirming the EAC related toxicities (Kapoor et al., 2014, Anticancer effect of dl-glyceraldehyde and 2-deoxyglucose in Ehrlich ascites carcinoma bearing mice and their effect on liver, kidney and haematological parameters, Indian Journal of Clinical Biochemistry 29(2):213-220). The ALT, AST, and bilirubin levels (FIG. 33) in mice treated with PI3K-6/HDAC6-NAV-NPs did not show any significant change as compared to healthy mice. In FIG. 34, kidney tissue section of untreated mice showed congested vascular spaces with extensive haemorrhage into glomeruli. Normal renal cortex and glomerular tufts can be seen in PI3K-6/HDAC6-NAV-NP treated kidney tissue section. All these changes suggested that PI3K-6/HDAC6-NAV-NP treatment is effective for ER⁺ breast cancer therapy without toxicity.

Example 15, Materials and statistical analysis

[0230] Materials. Navitoclax (Nav or NAV) was purchased from MedChem Express (USA). PI3K/HDAC dual inhibitor was provided by NCATS, NUT USA, as a gift (Thakur, et al., 2020, Design, synthesis, and biological evaluation of quinazolin-4-one-based hydroxamic acids as dual PI3K/HDAC inhibitors, Journal of Medicinal Chemistry 63(8):4256-4292). L- Lactide was procured from Purac (The Netherlands). Methoxy polyethylene glycol (mPEG, Mn-5000), PLURONIC® L-61(PEG-PPG-PEG; Mn-2000), F-127, trifluoro acetic acid (TFA), chloroform-d (CDCh), and stannous octoate were procured from Sigma Aldrich. Cell culture media (DMEM and RPMI), fetal bovine serum (FBS), and pen/strep solution were purchased from Gibco. Cancer cell lines, including MCF7 and ZR-75-1 were received as a gift from DFCI, Harvard Medical School, Boston, USA. Ehrlich ascites carcinoma (EAC) cells were procured from National Centre for Cell Science (NCCS), Pune, India. MTT was purchased from Hi-Media Laboratories (India). Dichloromethane (DCM), methanol, and acetonitrile (ACN) were procured from Merck India.

[0231] Statistical analysis. All data are represented as mean ± standard deviation (SD). Graph pad prism 9 software was used for all statistical analysis. The statistical significance was determined using ANOVA followed by Bonferroni post hoc correction with p<0.05 as the minimal significance level.

Claims

76 CLAIMS

1. A composition comprising: a) polymeric nanoparticles comprising hybrid block copolymers comprising a poly(lactic acid)-poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol)- poly(lactic acid) (PLA-PEG-PPG-PEG-PLA) penta-block copolymer with a methoxy poly(ethylene glycol)-poly(lactic acid) (m-PEG-PLA) di-block copolymer; and b) a histone deacetylase 6 (HDAC6) / phosphoinositide 3-kinase-6 (PI3K-6) dual inhibitor, wherein the HDAC6/PI3K-6 dual inhibitor is associated with the polymeric nanoparticles.

2. The composition of claim 1, wherein the HDAC6/PI3K-6 dual inhibitor is Compound III

77

3. The composition of claim 1, wherein the HDAC6/PI3K-6 dual inhibitor is Compound XL

4. A composition comprising: a) polymeric nanoparticles comprising block copolymers comprising a PLA-PEG- PPG-PEG-PLA penta-block copolymer with an m-PEG-PLA di-block copolymer; and b) a B cell lymphoma-2 (BCL-2) inhibitor, wherein the BCL-2 inhibitor is associated with the polymeric nanoparticles.

5. The composition of claim 4, wherein the BCL-2 inhibitor is venetoclax.

6. The composition of claim 4, wherein the BCL-2 inhibitor is navitoclax.

7. A composition comprising: a) an HDAC6/PI3K-6 dual inhibitor; and b) a BCL-2 inhibitor.

8. A composition comprising: a) polymeric nanoparticles comprising block copolymers comprising a PLA-PEG- PPG-PEG-PLA penta-block copolymer with an m-PEG-PLA di-block copolymer; b) an HDAC6/PI3K-6 dual inhibitor; and c) a BCL-2 inhibitor, 78 wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 are associated with the polymeric nanoparticles.

9. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor is Compound III

10. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor is Compound XL

11. The composition of claim 7 or 8, wherein the BCL-2 inhibitor is venetoclax.

12. The composition of claim 7 or 8, wherein the BCL-2 inhibitor is navitoclax. 79

13. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4, or 1 :5.

14. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio from 5: 1 to 1 :5.

15. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 1 : 1.

16. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 1 : 1.

17. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 2: 1.

18. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 2: 1.

19. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 3: 1.

20. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 3 : 1.

21. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 1 :2.

22. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 1 :2.

23. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of about 1 :3. 80

24. The composition of claim 7 or 8, wherein the HDAC6/PI3K-6 dual inhibitor and the BCL-2 inhibitor are present in a mass ratio of 1 :3.

25. The composition of any one of claims 1-6 or 8, wherein less PLA-PEG-PPG-PEG- PLA penta-block copolymer is present, by mass, than m-PEG-PLA di-block copolymer.

26. The composition of claim 25, wherein a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer is from 1 :20 to 1 : 10.

27. The composition of claim 25, wherein a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer is from 1 : 15 to 1 :5.

28. The composition of claim 25, wherein a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer is from 1 :8 to 3 :8.

29. The composition of claim 25, wherein a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer is from 1 :3 to 1 :2.

30. The composition of claim 25, wherein a mass ratio of PLA-PEG-PPG-PEG-PLA penta-block copolymer to m-PEG-PLA di-block copolymer is from 1 :2 to 1 : 1.

31. The composition of any one of claims 1-6 or 8, wherein the average diameter of the polymeric nanoparticles is between 50 and 170 nm.

32. The composition of any one of claims 1-6 or 8, wherein the average diameter of the polymeric nanoparticles is between 60 and 130 nm.

33. The composition of any one of claims 1-6 or 8, wherein the average diameter of the polymeric nanoparticles is between 60 and 100 nm.

34. The composition of any one of claims 1-6 or 8, wherein the average diameter of the polymeric nanoparticles is between 80 and 110 nm. 81

35. The composition of any one of claims 1-6 or 8, wherein the average diameter of the polymeric nanoparticles is between 100 and 170 nm.

36. The composition of any one of claims 1-6 or 8, wherein a poly dispersity index (PDI) of the polymeric nanoparticles is not more than 0.5.

37. The composition of any one of claims 1-6 or 8, wherein a PDI of the polymeric nanoparticles is not more than 0.3.

38. The composition of any one of claims 1-6 or 8, wherein a zeta potential of the polymeric nanoparticles is from -5 mV to -40 mV.

39. The composition of any one of claims 1-6 or 8, further comprising a PEG-PPG-PEG tri-block copolymer.

40. A pharmaceutical composition comprising the composition of any one of claims 1-8, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

41. A method of reducing proliferation, survival, migration, or colony formation ability of a rapidly proliferating cell in a subject in need thereof, comprising contacting the cell with a therapeutically effective amount of a composition comprising: the composition of any one of claims 1-8.

42. The method of claim 41, wherein the cell is a cancer cell.

43. A method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising: the composition of any one of claims 1-8.

44. The method of claim 43, wherein the cancer comprises a solid tumor cancer or a cancer of the blood. 82

45. The method of claim 43, wherein the cancer is selected from the group consisting of breast cancer, leukemia, lymphoma, colon cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, and combinations thereof.

46. The method of claim 43, wherein the cancer or breast cancer comprises triple negative breast cancer (TNBC).

47. The method of claim 43, wherein the cancer or breast cancer comprises ER⁺ breast cancer.

48. The method of claim 43, wherein the cancer comprises acute myeloid leukemia.

49. The method of claim 43, wherein the cancer is metastatic.

50. The method of claim 43, further comprising administering an additional anti-cancer therapy to the subj ect.

51. The method of claim 50, wherein the additional anti-cancer therapy comprises surgery, chemotherapy, radiation, hormone therapy, immunotherapy, or a combination thereof.

52. The method of claim 43, wherein the cancer is resistant or refractory to a chemotherapeutic agent.

53. The method of claim 43, wherein the subject is a human.

54. The method of claim 43, wherein the composition or pharmaceutical composition is administered intravenously, intratumorally, or subcutaneously.

55. The composition of any one of claims 1-39 or the pharmaceutical composition of claim 40 for use in the treatment of cancer.

56. Use of the composition of any one of claims 1-39 or the pharmaceutical composition of claim 40 for the manufacture of a medicament for the treatment of cancer.

57. A method of manufacturing the composition of any one of claims 1-6 or 8 comprising: a) mixing penta-block PLA-PEG-PPG-PEG-PLA and di-block m-PEG-PLA block copolymers dissolved in acetonitrile with the HDAC6/PI3K-6 dual inhibitor and/or the BCL-2 inhibitor to form a first mixture; b) mixing the first mixture with a PEG-PPG-PEG tri-block copolymer dissolved in water to form a second mixture; c) stirring the second mixture and evaporating the acetonitrile; and d) filtering the stirred and evaporated second mixture, thereby manufacturing the composition.