WO2023122577A2 - Methods of treating cancer using liposomal particles comprising anticancer agents and pharmaceutical compositions related thereto - Google Patents

Abstract

This disclosure relates to methods of treating cancer using liposomal particles and pharmaceutical compositions related thereto. In certain embodiments, the anticancer agent is trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-1-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof optional in combination with another chemotherapy agent.

Description

METHODS OF TREATING CANCER USING LIPOSOMAL PARTICLES COMPRISING ANTICANCER AGENTS AND PHARMACEUTICAL COMPOSITIONS RELATED THERETO

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/291,548 filed December 20, 2021. The entirety of this application is hereby incorporated by reference for all purposes.

BACKGROUND

Leukemia is cancer of the bone marrow and the lymphatic system that results in the production of leukemia cells. As the number of leukemia cells increases, normal white blood cells, red blood cells, and platelets are reduced causing a number of issues such as spontaneous bleeding. Treating leukemia can be complex and sometimes lasts several years. For example, children with leukemia often receive a standard three drug therapy of L-asparaginase, vincristine, and dexamethasone for the first month of treatment. For children in high-risk groups, a fourth drug such as daunorubicin may be added. This initial regimen may be followed with other chemotherapy agents such as methotrexate and 6-mercaptopurine. Some of the agents are administered into the cerebrospinal fluid (CSF) in order to target and prevent leukemia cells from spreading to the brain and spinal cord. These treatments are not universally effective. Thus, there is a need for improved therapeutic options.

Alfayez et al. report a liposomal formulation of cytarabine and daunorubicin for the treatment of adults with newly diagnosed AML. Leukemia & Lymphoma, 2020, 61(2), 288-297. See also U.S. Pat. Nos. 10,927,418, 10,835,492, 8,022,279 and 8,431,806 and U.S. Pat. App. Pub. No. 2021/0169803.

Hu et al. report lipid-based dispersions useful for formulating therapeutic agents. U.S. Pat. App. Pub. No. 2009/0060998. See also U.S. Pat. App. Pub. No. 2017/0340624.

Wang et al. report the preparation of pyrrolopyrimidine compounds for the treatment of cancer. WO2013052417. See also U.S. Pat. Nos. 10,004,755, 9,273,056, and W02020205967

Ong et al. report evaluation of extrusion technique for nanosizing liposomes. Pharmaceutics, 2016, 8, 36, 1-12. Mukthavaram et al. report high-efficiency liposomal encapsulation of a tyrosine kinase inhibitor improves in vivo toxicity and tumor response profile. Int J Nanomedicine, 2013, 8: 3991- 4006.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to methods of treating cancer using liposomal particles and pharmaceutical compositions related thereto. In certain embodiments, the anticancer agent is trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H- pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof optional in combination with another chemotherapy agent.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof to a subject in need thereof. In certain embodiments the subject is a human subject. In certain embodiments, the liposomal particles further comprise another anticancer agent such as vincristine, L-asparaginase, dexamethasone, daunorubicin, methotrexate, 6-mercaptopurine, or combinations thereof.

In certain embodiments, the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 6-8: 2.5-1.5: 1.5-0.5 respectively, e.g., 7:2: 1 respectively. In certain embodiments, the liposomal particle has an average diameter of between 90 to 130 nm. In certain embodiments, the liposomal particle has an average diameter of between 70 to 180 nm. In certain embodiments, the liposomal particle has an average diameter of between 70 to 180 nm or between 70 to 250 nm for drug loaded nanoparticles. In certain embodiments, the liposomal particle has an average diameter of between 90 to 130 nm or between 70 to 250 nm for MRX-2843 loaded nanoparticles. In certain embodiments, the liposomal particle has an average diameter of between 90 to 130 nm or between 70 to 250 nm for vincristine loaded nanoparticles.

In certain embodiments, the amount of trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4- methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX- 2843) or salt that is administered is between 10 to 500 mg, 100 to 500 mg, or 50 to 400 mg, or 50 to 300 mg of MRX-2843 per dose. In certain embodiments, the liposomal particles further comprise another anticancer agent.

In certain embodiments, the cancer is a hematological cancer such as leukemia, lymphoma, or multiple myeloma.

In certain embodiments, trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt is administered once daily or once weekly.

In certain embodiments, trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt is administered once daily or once weekly in an amount of between 10 to 20 mg/kg.

In certain embodiments, trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt is administered once daily or once weekly in an amount of between 10 to 20 mg/kg in combination with another chemotherapy agent.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl) methyl] phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and vincristine or salt thereof to a subject in need thereof. In certain embodiments, the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 7:2: 1 respectively. In certain embodiments, the liposomal particles have an average diameter of between 70 to 180 nm. In certain embodiments, the cancer is a hematological cancer. In certain embodiments, the cancer is leukemia. In certain embodiments, the molecular ratio of MRX-2843 and vincristine is between 8 and 90. In certain embodiments, MRX-2843 and vincristine are contained in the same nanoparticle. In certain embodiments, MRX-2843 and vincristine are in separate nanoparticles.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d] pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and venetoclax to a subject in need thereof. In certain embodiments, the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 7:2: 1 respectively. In certain embodiments, the liposomal particles have an average diameter of between 70 to 180 nm. In certain embodiments, the cancer is a hematological cancer. In certain embodiments, the cancer is leukemia. In certain embodiments, the molecular ratio of MRX-2843 and venetoclax is between 0.2 and 20. In certain embodiments, the molecular ratio of MRX-2843 and venetoclax is between 0.05 and 20. In certain embodiments, MRX-2843 and venetoclax are contained in the same nanoparticle. In certain embodiments, MRX-2843 and venetoclax are in separate nanoparticles

In certain embodiments, this disclosure relates to pharmaceutical compositions comprising liposomal particles comprising trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof. In certain embodiments, the pharmaceutical composition is in the form of a pH buffered saline solution, e.g., a phosphate buffered saline solution.

In certain embodiments, this disclosure relates to pharmaceutical compositions comprising liposomal particles comprising trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d] pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and vincristine or salt thereof contained in the same nanoparticle.

In certain embodiments, this disclosure relates to pharmaceutical compositions comprising liposomal particles comprising trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d] pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and venetoclax or salt thereof contained in the same nanoparticle.

In certain embodiments, this disclosure relates to the production of liposomal particles using processes disclosed herein.

In certain embodiments, this disclosure relates to methods of preparing liposomal particles loaded with trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl] phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) comprising, mixing a dialkyl phosphatidylcholine, a dialkyl phosphatidyl alcohol, and cholesterol in an acidic aqueous buffer with sonification at greater than about 10 or 20 kHz providing an acidic lipid emulsion; extruding the lipid emulsion through a nanometer sized filter providing a solution of liposomal particles having an acid aqueous core; concentrating the liposomal particles by centrifuging and filtering the liposomal particles and/or tangential flow filtration, providing concentrated and purified liposomal particles having an acid aqueous core; contacting the concentrated and purified liposomal particles having an acidic aqueous core with a basic aqueous buffer providing a basic buffered solution of liposomal particles having an acid aqueous core; mixing the basic buffered solution of liposomal particles having an acid aqueous core with trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d] pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof, and optionally another chemotherapy agent, providing MRX-2843 loaded liposomal particles optionally loaded with another chemotherapy agent. In certain embodiments, dialkyl phosphatidylcholine is distearyl phosphatidylcholine and the dialkyl phosphatidyl alcohol is distearoyl phosphatidylglycerol.

In certain embodiments, this disclosure relates to the production of a medicament for uses disclosed herein comprising liposomal particles as disclosed herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figure 1A illustrates the preparation of MRX-2843 loaded liposomal particles using an acidic core and a basic exterior to drive absorption of agents into the lipid bilayer or core of the particles. Lipids distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol (7:2: 1 mol) were mixed, then dried by rotary evaporation and rehydrated with 250 mM ammonium sulfate buffer (about 500 - 600, or 250 - 600 for full range of all particles made) (pH 4.2-5.0) with high intensity ultrasonication. The lipid emulsion is extruded via high pressure nitrogen through 80 nm polycarbonate filters at 60 °C over 10 passes. The liposome solution is then concentrated and exchanged to phosphate buffer (pH 7.7-9.8) via centrifugal diafiltration of tangential flow filtration, then sterile- (0.45 micron-) filtered. Vincristine sulfate and/or MRX- 2843 is added to the liposome solution and mixed at 700 rpm for 60 min at 60 °C. Drug-loaded liposomes are then buffer exchanged to phosphate buffered saline (PBS) for injection. For MRX- 2843 particles or venetoclax particles, loaded at 4 °C with a magnetic stir bar mixing for 24 - 48 hrs.

Figure IB shows size data on liposomal particles prior to drug loading.

Figure 1C shows size data with MRX-2843 and vincristine loaded at a synergistic ratio, Avg. Diameter = 165.8 nm.

Figure 2A shows data where NSG mice were inoculated with a luciferase-expressing ETP- ALL cell line (LOUCY-luc) followed by treatment with MRX-2843 free drug or saline vehicle that were initiated 21 days later. Free drug and saline vehicle were administered once daily by oral gavage. Survival was monitored and differences were determined by log-rank analysis. MRX- 2843 free drug did not provide significant therapeutic benefit in this model.

Figure 2B shows data indicating MRX-2843 loaded nanoparticles mediate greater therapeutic efficacy than MRX-2843 free drug in an ETP- ALL xenograft model. NSG mice were inoculated with a luciferase-expressing ETP-ALL cell line (LOUCY-luc) and treatment with nanoparticles containing MRX-2843 or empty vehicle nanoparticles were initiated 21 days later. MRX2843 nanoparticles were administered once weekly for 4 weeks by intraperitoneal injection. Survival was monitored and differences were determined by log-rank analysis. MRX-2843 nanoparticles provided significant therapeutic benefit.

Figure 3A shows data indicating liposomal nanoparticles containing MRX-2843 and vincristine provide ratio-dependent therapeutic activity in an ETP-ALL xenograft model. NSG mice were inoculated with a luciferase-expressing ETP-ALL cell line (LOUCY-luc) and treatment with loaded nanoparticles containing MRX-2843 and vincristine (VINC) at ratios that provide antagonistic, additive, or synergistic anti-leukemia activity in cell culture assays according to the table or empty nanoparticle vehicle was initiated 21 days later. Nanoparticles were administered once weekly for 4 weeks by intraperitoneal injection. Survival was monitored and differences were determined by log-rank analysis. The additive effective formulation here is at a ratio of 103.5. Additive or Antagonistic particles may have synergistic efficacy in vivo extended to upper limit of about 240: 1 (MRX-2843 :vincristine).

Figure 3B shows data indicating that loaded nanoparticles containing MRX-2843 and vincristine provide therapeutic activity in an ETP-ALL xenograft model with more advanced disease. NSG mice were inoculated with a luciferase-expressing ETP-ALL cell line (LOUCY-luc) and treatment with nanoparticles containing a synergistic ratio of MRX-2843 and vincristine (SYN) was initiated 40 days later. Nanoparticles were administered once weekly for 8 weeks by intraperitoneal injection. Disease burden was monitored by bioluminescence imaging and was significantly decreased in mice treated with SYN nanoparticles relative to mice treated with vehicle. Disease burden was not significantly different between groups before initiation of treatment. Median survival was significantly prolonged from 25 days after initiation of treatment with vehicle to 49 days in mice treated with nanoparticles containing MRX-2843 and vincristine. Figure 4A shows results of principal component analyses of AML cell line dose responses done on combinations of MRX-2843 and venetoclax. Gene expression (Transcripts per million, TPM) of MRX-2843 molecular targets MERTK and FLT3, along with venetoclax target BCL-2, were modeled as descriptor variables with growth inhibition (GI), synergy, and drug combination metrics (total drug, drug ratio) to understanding relations among these variables. The first two principal components describe 30.19% and 24.97% of the variance, respectively, within the high- throughput screening dataset.

Figure 4B shows data using a combination of MRX and venetoclax nanoparticles incubated with OCI-AML5 cells. After 4 and 8 hours of incubation, cells were harvested, frozen, and analyzed via LC/MS for intracellular concentrations of drugs. Intracellular concentrations ranged from 0.1 - 0.01 (MRX: venetoclax) in the cytoplasmic space.

Figures 5A-5G show data indicating primary AML cells are effectively and synergistically growth inhibited by various ratios of MRX and venetoclax nanoparticles, despite FLT3 rescue. The cytokine components are listed at the top. Both medias are supported by 85% DMEM+ 15% FBS + lx pen/strep + 50 uM beta mercaptoethanol. The “cytokine media” has SCF (100 ng/mL), IL-6 (20 ng/mL), and TPO (10 ng/mL). The “FLT3 Rescue” media is the cytokine media plus FLT-3L (10 ng/mL) and IL-3 (10 ng/mL).

Figure 5 A illustrates a screen designed to determine the effectiveness of nanoparticles (NP) vs free drug (FD) in the presence of cytokine media (CK) and FLT-3 cytokines.

Figure 5B shows data on GI50s of MRX and venetoclax nanoparticles (NP) versus free drug (FD) for 4 patient samples in the presence of cytokine media (CK). GI50 refers to the dose of the drug that achieve 50% growth inhibition.

Figure 5C shows data on GI50s of MRX and venetoclax nanoparticles (NP) versus free drug (FD) for 4 patient samples in the presence of FLT-3 rescue cytokines (FLT-3L and IL-3).

Figure 5D shows overall drug combination responses comparing nanoparticle and free drug formulations of MRX-2843 and venetoclax in patient-derived samples of AML. Nanoparticles are more intensely and frequently synergistic than free drug, while overall achieving equivalent growth inhibition as free drug.

Figure 5E shows overall drug combination responses comparing nanoparticle and free drug formulations of MRX-2483 and venetoclax in patient-derived samples of AML after exposure to FLT3 rescue cytokines. Synergy in nanoparticle combinations is conserved and occurs more frequently than for free drug combinations.

Figure 5F shows ratio-dependent synergy responses occur most strong for a 5: 1 - 20: 1 (MRX-2843 :venetoclax) nanoparticle formulation, while synergy in free drug formulations is largely absent.

Figure 5G shows that after FLT3 rescue, while overall synergy in nanoparticles is dampened, it still occurs within ratios observed without FLT3 rescue, while free drug formulations demonstrate antagonistic responses.

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

"Embodiments" are examples of this disclosure which are not necessarily limited to these examples. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

"Subject" refers to any animal, preferably a human patient, livestock, rodent, monkey or domestic pet.

"Cancer" refers any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation of the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or others.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression.

As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.

The term "effective amount" refers to that amount of a compound or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, disease treatment, as illustrated below. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose will vary depending on, for example, the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

Liposomal Particles

Liposomes are microscopic vesicles made, in part, from phospholipids which form closed, fluid filled spheres when mixed with water. Phospholipid molecules are polar, having a hydrophilic ionizable head, and a hydrophobic tail consisting of long fatty acid chains. When sufficient phospholipid molecules are present in water, the tails spontaneously associate to exclude water. The result is a bilayer membrane in which fatty acid tails converge in the membrane's interior and polar heads point outward toward the aqueous medium. As the liposomes form, water soluble molecules can be incorporated into the aqueous interior, while lipophilic molecules tend to be incorporated into the lipid bilayer. Liposomes may be either multilamellar, onion-like structures, with liquid separating multiple lipid bilayers, or unilamellar liposome with a single bilayer surrounding an entirely liquid center.

Typically, the phosphatidyl choline provides the primary packing/entrapment/structural element of the liposome which provides a scaffold for therapeutic agents and the other lipid components. Typically, the phosphatidyl choline comprises mainly C16 or longer fatty-acid chains. Chain length provides for both liposomal structure and membrane width. Additionally, the fatty-acid chains may have at least one double bond, although this is not a requirement. Cholesterol typically provides a combination of stability and flexibility to liposomal particles. Phospholipids such as phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine are useful in the dispersions.

In certain embodiments, liposomal particles disclosed herein may be produce by a variety of techniques such as extrusion, ultrasonication, freeze-thaw sonication (FTS), sonication and homogenization.

In certain embodiments, anticancer agents or combinations thereof are incorporated into the lipid bilayer or aqueous internal compartment(s) of the liposomal particles either by passive or active loading procedures or some combination thereof. In passive loading, the anticancer agents or combinations thereof can be included in the preparation from which the liposomal particles are formed or, alternatively, the anticancer agents or combinations thereof can be added to the outside of preformed liposomal particles and loads passively down its concentration gradient into the liposomal particles. Optionally, unencapsulated material may be removed from the preparation by any suitable procedures. Alternatively, active loading procedures can be employed, such as ion gradients, ionophores, pH gradients and metal-based loading procedures based on metal complexation.

In certain embodiments, this disclosure relates to methods of preparing liposomal particles loaded with trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl] phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl] cyclohexanol (MRX-2843) comprising, mixing a dialkyl phosphatidylcholine, a dialkyl phosphatidyl alcohol, and cholesterol in an acid aqueous buffer with sonification at greater than about 10 or 20 kHz providing an acidic lipid emulsion; extruding the acidic lipid emulsion through a nanometer sized filter providing a solution of liposomal particles having an acid core; concentrating the liposomal particles by centrifuging and filtering the liposomal particles providing concentrated and purified liposomal particles having an acid core; contacting the concentrated and purified liposomal particles with a basic aqueous buffer providing a basic buffered solution of liposomal particles having an acid core; mixing the basic buffered solution of liposomal particles having an acid core with trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof, and optionally another chemotherapy agent, providing MRX-2843 loaded liposomal particles.

In certain embodiments, dialkyl phosphatidylcholine is distearyl phosphatidylcholine and the dialkyl phosphatidyl alcohol is distearoyl phosphatidylglycerol.

In certain embodiments, the acid aqueous buffer is an ammonium sulfate buffer at a pH of about 4.2-5.0.

In certain embodiments, extruding the lipid emulsion through a filter is a filter with a pore size diameter averaging between 60 and 150 nm and extruding is repeated 10 times.

In certain embodiments, filtering is through a syringe filter with a diameter averaging about 0.45 microns.

In certain embodiments, the basic aqueous buffer is a phosphate buffer at a pH of about

7.7-9.8. In certain embodiments, mixing the basic buffered solution of liposomal particles is at 700 rpm for 60 minutes

In certain embodiments, mixing the basic buffered solution of liposomal particles is with a magnetic stir bar for more than 24 or 48 hours.

In certain embodiments, said another chemotherapy agent is vincristine or salt thereof.

In certain embodiments, the molar ratio of vincristine to MRX-2843 is between 1 : 17 to 1 :72.

In certain embodiments, the molar ratio of vincristine to MRX-2843 is between 1 :34 to 1 :37.

In certain embodiments, said another chemotherapy agent is venetoclax or salt thereof.

In certain embodiments, the molar ratio of venetoclax to MRX-2843 is between 1 : 1000 to 13: 1 or 20:1.

In certain embodiments, the molar ratio of venetoclax to MRX-2843 is between 1 :9 to 1 : 11.

In certain embodiments, the ratios correspond to single agent MRX-2843 nanoparticles and single agent nanoparticles, e.g., vincristine/venetoclax.

In certain embodiments, the ratios correspond to simultaneously loading the agents into the same liposomal particles.

In certain embodiments, this disclosure relates to the production of a medicament for uses disclosed herein comprising liposomal particles as disclosed herein.

In certain embodiments, the liposomal particles are about 70-500 nm in diameter. In certain embodiments, the liposomal particles have a diameter of less than 300 nm, sometimes less than 200 nm. In certain embodiments, the average size of liposomal particles is approximately 70 to 180 nm in diameter. In certain embodiments, the average size of liposomal particles is approximately 70 to 180 nm in diameter. In certain embodiments, the liposome membrane is composed of distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG) and cholesterol (CHOL). In certain embodiments, the liposome membrane is composed of 50-80% DSPC, 1-20% DSPG and 1-20% CHOL. In certain embodiments, the liposome membrane is composed of 50-80% distearoyl phosphatidylcholine (DSPC) or dipalmitoyl phosphatidylcholine (DPPC), 1-20% DSPG or distearoyl phosphatidylinositol (DSPI), 1-20% CHOL. In certain embodiments, the liposomal particles contain a cryoprotectant in the intraliposomal medium. In certain embodiments, the cryoprotectant is a saccharide, either or both inside and outside of the liposomes. In certain embodiments, the cryoprotectant is trehalose outside the liposome.

In certain embodiments, the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 6-8: 2.5-1.5:1.5-0.5 respectively, e.g., about 7:2:1 respectively. In certain embodiments, the liposomal particle has an average diameter of between 70 to 180 nm.

In certain embodiments, the liposomal particles contain poly-(ethylene glycol) (PEG) on the surface of the liposomal particles to extend blood-circulation time and reduce mononuclear phagocyte system uptake. See Immordino et al., International Journal of Nanomedicine, 2006, 1(3), 297-315.

In certain embodiments, the liposome membrane is composed of DSPC, DSPG and CHOL in about a 7:2: 1 molar ratio and contain a cryoprotectant in the internal core liposomal medium. In one instance, the liposomal particles are prepared by methods disclosed herein wherein extruded liposomal particles or drug loaded liposomal particles are suspended in phosphate-buffered sucrose solution at pH of about 7.0. In another instance, the extruded liposomal particles are suspended in sucrose aqueous solution. In one embodiment, the extruded liposomal particles are suspended in 250-400 mM sucrose aqueous solution.

In certain embodiments, the liposomal particles comprise membrane-bound cryoprotectants to improve resistance to freezing and lyophilizing. In particular, sugars grafted onto phosphatidylcholine (PC) or PC: cholesterol liposomal membrane surfaces via oligo(ethylene oxide) linkers consisting of one to three repeating units. In certain embodiments, the particles comprise phosphatidylinositol.

In certain embodiments, a lyophilized composition comprising liposomal particles disclosed herein provide convenience in storage, preservation, and ease of shipping. These lyophilized compositions retain their characteristics over long periods of time.

Methods of use

In certain embodiments, this disclosure relates to methods of treating cancer using liposomal particles and pharmaceutical compositions related thereto containing an anticancer agent. In certain embodiments, the anticancer agent is trans-4-[2-[(2-cyclopropylethyl)amino]-5- [4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof optional in combination with another chemotherapy agent.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof to a subject in need thereof. In certain embodiments the subject is a human subject. In certain embodiments, the liposomal particles further comprise another anticancer agent such as vincristine, L-asparaginase, dexamethasone, daunorubicin, methotrexate, 6-mercaptopurine, or combinations thereof.

In certain embodiments, it is contemplated that a combination of chemotherapy agents may be in the same liposomal particles or prepared separately, e.g., wherein the individually loaded liposomal particles are mixed together in desirable ratios or administered individually in desirable rations. In certain embodiments, it is contemplated that first set of liposomal particles contain MRX-2843 alone and a second set of liposomal particles contain another chemotherapy agent alone wherein the first and second set of liposomal particles are administering in combination, or it is contemplated that individual liposomal particles contain both agents together.

In certain embodiments, the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 7:2: 1 respectively. In certain embodiments, the liposomal particle has an average diameter of between 70 to 180 nm. In certain embodiments, the cancer is a hematological cancer such as leukemia, lymphoma, or multiple myeloma.

In certain embodiments, trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt is administered once daily.

In certain embodiments, trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt is administered once daily in an amount of between 10 to 20 mg/kg.

In certain embodiments, trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt is administered once weekly in an amount of between 10 to 20 mg/kg optionally in combination with another chemotherapy agent. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof in combination with another anticancer agent to a subject in need thereof.

In certain embodiments the subject is a human subject. In certain embodiments, the liposomal particle further comprises another anticancer agent such as vincristine, L-asparaginase, dexamethasone, daunorubicin, methotrexate, 6-mercaptopurine, or combinations thereof. In certain embodiments, the cancer is a hematological cancer such as leukemia.

In certain embodiments, the subject is diagnosed with cancer or hematological malignancy. In certain embodiments, the hematological malignancy is acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia, acute monocytic leukemia (AMOL), chronic myeloid leukemia (CML), myeloproliferative neoplasms (MPNs), and lymphomas, Hodgkin's lymphomas, and non-Hodgkin's lymphomas such as Burkitt lymphoma, B-cell lymphoma.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising an anticancer agent disclosed herein which can be used alone in the treatment of each of the foregoing conditions or can be used to provide additive or potentially synergistic effects with certain existing chemotherapies, radiation, biological or immunotherapeutics (including monoclonal antibodies) and cancer vaccines.

In certain embodiments, pharmaceutical compositions comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof in enantiomeric excess as reported herein may be useful for restoring effectiveness of certain existing chemotherapies and radiation and or increasing sensitivity to certain existing chemotherapies and/or radiation.

In certain embodiments, this disclosure relates to assays, methods, systems, and kits for selecting a treatment regimen for a subject with a hematological cancer (e.g., leukemia) or a risk for a hematological cancer (e.g., leukemia), treating a subject with a hematological cancer (e.g., leukemia), and/or improving the effectiveness of a treatment regimen recommended for or administered to a subject with a hematological cancer (e.g., leukemia) or a risk for a hematological cancer (e.g., leukemia). A test sample for use in the assays, methods, systems, or kits described herein can be derived from a biological sample of the subject, e.g., a bone marrow, or blood sample or plasma or serum sample from the subject.

Depending upon selection of a biomarker, the test sample can be subjected to one or more analyses, e.g., including, but not limited to, genotyping assays, expression assays (e.g., protein and/or transcript levels), other assays capable of identifying a genotype or any combinations thereof. A plethora of such assays is known in the art, and many are commercially available such as microarrays and sequencing.

In certain embodiment, this disclosure relates to methods for the treatment of a subject at risk of, exhibiting symptoms of, suspected of, or diagnosed with a cancer or neoplasm selected from skin cancer, melanoma, breast cancer, cervical cancer, central nervous system tumors including primary CNS tumors such as glioma, glioblastomas, astrocytomas (including glioblastoma multiforme) and ependymomas, and secondary CNS tumors (i.e., metastases to the central nervous system of tumors originating outside of the central nervous system), colorectal cancer, including large intestinal colon carcinoma, gastric cancer, carcinoma of the head and neck including squamous cell carcinoma of the head and neck, hematologic cancers including leukemias and lymphomas such as acute lymphoblastic leukemia, acute myelogenous leukemia (AML), myelodysplastic syndromes, chronic myelogenous leukemia, Hodgkin's lymphoma, nonHodgkin's lymphoma, megakaryoblastic leukemia, multiple myeloma and erythroleukemia, hepatocellular carcinoma, lung cancer including small cell lung cancer and non-small cell lung cancer, ovarian cancer, endometrial cancer, pancreatic cancer, pituitary adenoma, prostate cancer, renal cancer, sarcoma, and thyroid cancers.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt in combination with vincristine.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt in combination with venetoclax. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt in combination with a MEK inhibitor such as pimasertib.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt in combination with daunorubicin and cytarabine.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt in combination with fludarabine and cytarabine.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt in combination with immune checkpoint inhibitors such as antibodies, e.g., anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e.g., nivolumab, pembrolizumab, cemiplimab) and anti-PD-Ll (e.g., atezolizumab, avelumab, durvalumab).

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and/or an anticancer agent disclosed herein such as a TAM family inhibitor (tyrosine kinases inhibitors of Tyro3, Axl, Flt3, or Mer) optionally in combination with anticancer agents such as vinca alkaloids, e.g.. vincristine, vinblastine, vinorelbine, vindesine, vinflunine.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and/or an anticancer agent disclosed herein such as a TAM family inhibitor (tyrosine kinases inhibitors of Tyro3, Axl, Flt3 , or Mer) optionally in combination with anticancer agents such as vinca alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, vinflunine and a dihydrofolate reductase inhibitor, e.g., methotrexate, raltitrexed, pemetrexed, or pralatrexate.

In certain embodiments, the Mer inhibitor is 4-(2-(butylamino)-5-(4-((4-methylpiperazin- l-yl)methyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-l-ol (UNC2025), N-(4-(lH- imidazol-l-yl)benzyl)-2-(butylamino)-4-((4-hydroxycyclohexyl)amino)pyrimidine-5- carboxamide (UNC2881), 2,6-dimethylpyridin-4-yl)(4-(7-((lr,4S)-4-hydroxycyclohexyl)-2-(((S)- pentan-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)piperidin-l-yl)methanone (UNC5293) or salts thereof.

In certain embodiments, the Axl inhibitor is cabozantinib (BMS-907351), N-(4-((2-amino- 3-chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-ethoxy-l-(4-fluorophenyl)-2-oxo-l,2- dihydropyridine-3 -carboxamide (BMS-777607), bemcentinib (R428), cabozantinib malate (XL184), ningetinib, N-(5-((6,7-dimethoxyquinolin-4-yl)oxy)pyridin-2-yl)-2,5-dioxo-l-phenyl- l,2,5,6,7,8-hexahydroquinoline-3-carboxamide (ONO-7475), 2-(((3-methylisoxazol-5- yl)methyl)thio)-N-(2-((2-nitro-4-(trifluoromethyl)phenyl)amino)ethyl)benzamide (RU-301), glesatinib (MGCD265), N-(3-fluoro-4-((3-phenyl-lH-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2- (4-fluorophenyl)-l,5-dimethyl-3-oxo-2,3-dihydro-lH-pyrazole-4-carboxamide (NPS-1034), or salts thereof.

In certain embodiments, the Flt3 inhibitor is sunitinib, N-(4-((2-amino-3-chloropyridin-4- yl)oxy)-3-fluorophenyl)-4-ethoxy-l-(4-fluorophenyl)-2-oxo-l,2-dihydropyridine-3 -carboxamide (BMS777607), quizartinib (AC220), dovitinib (TKI-258), fedratinib (TG101348), amuvatinib (MP-470), tandutinib (MLN518), (E)-(4-(2-(lH-indazol-3-yl)vinyl)phenyl)(piperazin-l- yl)methanone (KW-2449), (E)-N-(5-methyl-lH-pyrazol-3-yl)-6-(4-methylpiperazin-l-yl)-2- styrylpyrimidin-4-amine (ENMD-2076), certinib (LDK378), pacritinib (SB1518), 2-(3,4- dimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (TCS 359), (E)-N-(l- ((5-(2-((4-cyanophenyl)amino)-4-(propylamino)pyrimidin-5-yl)pent-4-yn-l-yl)amino)-l- oxopropan-2-yl)-4-(dimethylamino)-N-methylbut-2-enamide (FF-10101), isoguanosine, 8- bromo-2-((l-methylpiperidin-4-yl)amino)-4-((4-phenoxyphenyl)amino)pyrido[4,3-d]pyrimidin- 5(6H)-one (G-749), l-(5-((7-(3-morpholinopropoxy)quinazolin-4-yl)thio)-l,3,4-thiadiazol-2-yl)- 3-(p-tolyl)urea (SKLB4771), gilteritinib (ASP2215), 2-hydroxy-l-(2-((9-(4-methylcyclohexyl)- 9H-pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidin-2-yl)amino)-7,8-dihydro-l,6-naphthyridin-6(5H)- yl)ethan-l-one (AMG 925) or salts thereof.

In certain embodiments, the Tyro3 inhibitor is N-(4-((2-amino-3-chloropyridin-4-yl)oxy)- 3 -fluorophenyl)-4-ethoxy-l-(4-fluorophenyl)-2-oxo-l,2-dihydropyridine-3 -carboxamide (BMS777607), N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-3-(4-fluorophenyl)-l- isopropyl-2,4-dioxo-l,2,3,4-tetrahydropyrimidine-5-carboxamide (RXDX-106), N-(4-((6,7- dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-4-ethoxy-l-(4-fluoro-2-methylphenyl)-lH- pyrazole-3 -carboxamide (LDC1267) or salts thereof.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof or an anticancer agent disclosed herein such as a TAM family inhibitor (tyrosine kinases inhibitors of Tyro3, Axl, Flt3, or Mer) optionally in combination with anticancer agents such as BCL2 inhibitor, e.g., venetoclax, obatoclax, or gossypol.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof or an anticancer agent disclosed herein such as a TAM family inhibitor (tyrosine kinases inhibitors of Tyro3, Axl, Flt3, or Mer) optionally in combination with anticancer agents such as a Mek inhibitor, e.g., pimasertib.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof or an anticancer agent disclosed herein such as a TAM family inhibitor (tyrosine kinases inhibitors of Tyro3, Axl, Flt3, or Mer) optionally in combination with vyxeos (CPX-35, liposomal formulation of cytarabine and daunorubicin at a fixed 5:1 molar ratio). In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and/or an anticancer agent disclosed herein such as a TAM family inhibitor (tyrosine kinases inhibitors of Tyro3, Axl, Flt3 , or Mer) optionally in combination with a purine analog such as fludarabine.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and/or an anticancer agent disclosed herein such as a TAM family inhibitor (tyrosine kinases inhibitors of Tyro3, Axl, Flt3, or Mer) optionally in combination with a purine analog such as fludarabine and a cytosine antimetabolite such as cytarabine.

In certain embodiments, the methods of administration reported herein is in a subject with a lymphodepleted environment due to prior or concurrent administration of lymphodepleting agents. In certain embodiments, lymphodepleting agents are cyclophosphamide and fludarabine.

Additional chemotherapy agents include molecules or derivatives such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado- trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa- 2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5 -fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MV AC).

Pharmaceutical compositions

In certain embodiments, this disclosure relates to pharmaceutical compositions comprising liposomal particles comprising trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof. In certain embodiments, the pharmaceutical composition is in the form of a pH buffered saline solution, e.g., a phosphate buffered saline solution.

In certain embodiments, the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 7:2: 1 respectively. In certain embodiments, the liposomal particle has an average diameter of between 70 to 150 nm. In certain embodiments, trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l- piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt is between 100 to 500 mg of MRX-2843 per dose. In certain embodiments, the liposomal particle further comprises another anticancer agent.

In certain embodiments, this disclosure relates to the production of a medicament for uses disclosed herein comprising liposomal particles as disclosed herein.

Pharmaceutical compositions typically comprise an effective amount of compounds and a suitable pharmaceutical acceptable carrier. The preparations can be prepared in a manner known per se, which usually involves mixing the compounds according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

Pharmaceutical compositions comprising compound(s) of the present disclosure can be administered to a subject either alone or as a part of a single pharmaceutical composition. In certain embodiments, the pharmaceutical composition is in the form of a sterilized pH buffered aqueous salt solution or a saline phosphate buffer between a pH of 6 to 8, optionally comprising a saccharide or polysaccharide.

The pharmaceutical compositions of the present disclosure can be administered to subjects either topically to the skin, orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), intraci sternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments, or drops), or as a buccal or nasal spray. Pharmaceutically acceptable salts, solvates, and hydrates of the compounds listed are also useful in the method of the disclosure and in pharmaceutical compositions of the disclosure.

Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions for reconstitution into sterile injectable solutions or dispersions.

Prevention of the action of microorganisms may be controlled by addition of any of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like.

Dosage forms for topical administration include ointments, powders, sprays, and inhalants wherein the active agent(s) are admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this disclosure.

The pharmaceutical preparations of the disclosure are preferably in a unit dosage form, and can be suitably packaged, for example in a syringe, vial, bottle, sachet, ampoule, or box in any other suitable single-dose or multi-dose holder or container (which can be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure e.g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

EXAMPLES

Liposomal particles

Experiments were performed to determine the combined anticancer activity of small molecules: the cytotoxic chemotherapies, vincristine, venetoclax, and methotrexate, as well as MRX-2843, a small inhibitor of molecule Mer tyrosine kinase and FLT3 inhibitor. A method was devised by which MRX-2843 may be encapsulated within lipid vesicles (i.e., liposomes) both alone and in combination with vincristine or venetoclax at drug ratios selected to maximize anticancer activity based upon performance in the prior drug screens. Drug formulations were tested in mouse models of T cell acute lymphoblastic leukemia (T-ALL).

Striking and unexpectedly high activity from single-agent formulations of MRX-2843 were observed. Specifically, MRX-2843 dosed orally, once daily at its maximum tolerable dose (MTD, 65 mg/kg) very modestly slows leukemia progression, whereas liposomal MRX-2843 achieves near complete leukemia control when dosed systemically, well below its respective MTD, i.e., once weekly dose of 16 mg/kg. These results were unexpected given that the oral bioavailability of MRX-2843 is high (78%). Therapeutic performance of the combination drug formulation was likewise highly impressive with near complete leukemia control at a near identical dose of MRX-2843 (17.4 mg/kg total drug, once weekly). The composition and methods used to prepare formulations of MRX and/or vincristine or venetoclax are provided below.

Preparation of ratiometric drug-loaded liposomes

A mixture of lipids comprising DSPC (l,2-Distearoyl-sn-glycero-3-phosphocholine; Coatsome™ MC-8080 (DSPC)) in chloroform (25 mg/mL), DSPG (l,2-Distearoyl-sn-glycero-3- phosphoglycerol, sodium salt; Coatsome™ MG-8080LS (DSPG-Na)) in chlorofomrmethanol (5: 1 v/v; 5 mg/mL), and Cholesterol (PHR1533-500mg) in chloroform (25 mg/mL) is prepared at molar ratio 7:2: 1 (DSPC:DSPG:Chol). Organic solvent is evaporated by rotary evaporation under vacuum at 30° C (Rotavapor™ R-100). The resulting lipid foam is left in vacuum overnight to evaporate residual organic solvent.

Lipid foams are rehydrated with 250 - 600 mM Ammonium Sulfate (AS) buffer (pH = 4.2) 567 mM typically, and are subsequently sonicated at 60° C, at high power and under sterile conditions, with cup horn sonication (Q700™ Sonicator). Lipid vesicles in ammonium sulfate buffer are then size homogenized under high pressure extrusion with nitrogen gas (Liposofast™ LF-50). Liposomes undergo 10 extrusion passes with a single polycarbonate filter membrane of 0.08 pm pore size (Nuclepore™ Track-Etched Membrane), sandwiched between polyester drain discs at 60° C.

Extraliposomal AS buffer is then exchanged for phosphate buffer (Potassium Phosphate Monobasic Anhydrous (KH2PO4) at 2.52 g/L; Sodium Phosphate Dibasic Anhydrous (Na2HPO4) at 25.71 g/L; pH = 7.70) via centrifugal spin diafiltration (Amicon Ultracel™ 15, MWCO lOOkDa, regenerated cellulose membranes) at a minimum dilution of 1 :256 (AS Phosphate buffer). Liposomes are syringe filtered with cellulose acetate (0.45 pm) to ensure sterility. Method for loading MRX-2843 alone: 4 °C temperature, magnetic stir bar, 2 days of stirring results in MRX- 2843 liposomal diameter at an average of 133.2 nm, or 60 °C for 60 mins 700 rpm. There are two methods to load MRX-2843 nanoparticles.

To prepare drug-loaded liposomes, MRX-2843 (10 mg/mL in water) and vincristine sulfate are then mixed at various molar ratios with liposomes (see table below). Mixtures are loaded into 1.5 mL tubes, heated to 60 °C on a heat block, and mixed at 700 rpm for 1 hr. The drug-loaded liposomes are then allowed to cool to room temperature. They are then loaded into 100 kDa MWCO dialysis bags and twice dialyzed in 3L of phosphate buffered saline for a minimum of 6 hours.

Alternatively, one can add magnetic stir bar technique for MRX and venetoclax single agent particles. MRX-2843 is prepared at 13.12% (wt/wt) in a liposome formulation. The mixture is set to stir at 4 °C for 48 hours, and then dialyzed. Single agent magnetic stir bar loading provides MRX-2843: 2970.466 uM and Lipids: 9.680 mg/mL. Magnetic stir bar loading provides venetoclax: 31.222 uM and Lipids: 9.055 mg/mL.

The liposome sizes can vary based on which drug(s) are being loaded, e.g., about between 70 and 120 nm range for unloaded particles; about between 130 and 200 nm for MRX-2843; and about between 140 and 200 nm for MRX-2843 and vincristine, 90 and 130 nm for venetoclax nanoparticles, or other agent in the same particle.

Table 1 shows the molar concentrations of agents loaded into liposomal particles

Table 2 Drug Formulation Data

Some experiments indicate that the synergistic lower bound is 11.5 not 22.4. Table 3 shows favorable drug combinations from in vitro screening results (non-formulated drugs) by HTS

Some experiments indicate MRX-2843 and vincristine combinations: Synergistic lower ratio for

T-ALL, ETP-ALL, and T-ALL + ETP-ALL is 8.5, upper bound may extend to 90; optimal is still 35. Additivity extends from 90 - 160, optimal is 120. Re-evaluation of MRX-2843 and venetoclax synergy in AML: upper bound at 20. Optimal at 0.2 or 5.

Therapeutic approach for pediatric leukemia

Overall survival rates in pediatric leukemia have been improved through the use of intensive, high dose, multi-agent combination chemotherapy; however, outcomes remain poor in certain high-risk subsets, including early T-precursor acute lymphoblastic leukemia (ETP-ALL), infant ALL and acute myeloid leukemia (AML). Additionally, patients in high-risk subsets are exposed to more toxic therapy and have a high prevalence of late effects. Therefore, improved therapeutic approaches are needed for these patients.

A high-throughput drug screening was utilized and formulated to identify synergy between small molecule inhibitors of the receptor tyrosine kinases MER (MERTK) and FLT3 and the anti- apoptotic protein BCL-2. Lipid particles containing these agents were tested in AML, infant ALL and ETP-ALL models in order to identify a therapeutic that improves the therapeutic index via synergistic drug encapsulation. Co-delivery of molecularly targeted therapies utilizing a dual MERTK/FLT3 inhibitor and BCL-2 inhibitors can be used to improve the growth inhibition (GI) of pediatric AML, ETP-ALL, and infant ALL cells in vitro.

A high-throughput screening strategy was utilized to identify optimal ratiometric dosing for pairwise drug combinations targeting MERTK/FLT3 and BCL-2 across twelve different leukemia cell lines providing the comprehensive concentration- and ratiometric-dependent resolution needed to nominate synergistic drug combinations. Methods were developed to encapsulate nominated synergistic drug combinations in nanoparticle liposomes and optimize drug loading conditions to enable co-delivery of synergistic ratios. Liposomes were engineered using sonication of rehydrated lipid foam filled by high pressure extrusion to make unilamellar vesicles. Synergistic combinations of drug can be loaded by passive rehydration and a chemical buffer gradient allowing for the maintenance of drug ratios of nanoparticles in storage. Effective (high GI) and synergistic combinations can be used in methods including co-delivery by encapsulation in liposomal nanocarriers.

In in vitro contexts, synergistic ratios are desirable. Additive/antagonistic ratios may also be desirable in treatment contexts. A high-throughput drug screening of MRX-2843 and vincristine ratios (MRX-2843 / vincristine) were performed and mean synergy scores (bliss independence model) were evaluated across T-ALL and ETP-ALL cell lines: Synergistic ratios range from 8 - 90 (tested ratios: 8.93 between 71.4). Additive ratios range from between 90-200 or additive ratios range from 90 - 160 (tested ratios: 142.9), and antagonistic ratios range are greater than 160 or 200 (tested ratios: 285.7 plus). Nanoparticles (MRX-2843 and vincristine in the same nanoparticle) were tested in vitro and in vivo. Synergistic ratio ranges tested were between 11.54 and 30.42. Additive ratio ranges tested were between 91.75 and 127.59, and antagonistic ratio ranges tested were between 167.63 and 246.06. Molar ratios are in terms of the molar amounts of MRX-2843 to vincristine drug. For example, a molar ratio of 30 means that there are 30 moles of MRX-2843 to every 1 mole of vincristine contained in the same nanoparticle.

High-throughput screen of MRX-2843 and three BCL-2 inhibitors, including venetoclax, gossypol and obatoclax, tested on AML cell lines

Ratiometric screens of variant concentration of MRX-2843 and three BCL-2 inhibitors individually, including venetoclax, gossypol and obatoclax, were tested on multiple AML cell lines and infant ALL cell lines. Results were compared to identify optimal combinations. Surface plots were generated depicting combination responses within AML cell lines. Dose responses curves include as four-parameter logistic curves. Levels of synergy were identified for combinations of MRX-2843 and venetoclax.

Principal component analyses were performed on AML cell lines to identify dose responses to combinations of MRX-2843 and venetoclax. Gene expression of MRX-2843 molecular targets MERTK and FLT3, along with venetoclax target BCL-2, were modeled as descriptor variables with growth inhibition (GI), synergy, and drug combination metrics (total drug, drug ratio). The first two principal components describe 30.19% and 24.97% of the variance, respectively, within the high-throughput screening dataset.

Primary AML cells are effectively and synergistically growth inhibited by various ratios of MRX and venetoclax nanoparticles, despite FLT3 rescue

Experiments were performed to determine combination dose responses in 5 primary AML cell samples across four ratios of MRXvenetocalx (1 :20, 1 :5, 5:1, 20: 1) for nanoparticle and free drug formulations. Nanoparticles demonstrate the majority of synergy responses regardless of cytokine media. Ratios 5: 1 and 20: 1 (MRX: venetoclax) are most synergistic across 5 patients when summing synergy scores across all drug concentrations tested within a ratio. Nanoparticles are more synergistic (Bliss Independence model) than free drug combinations. Synergy is dampened overall, but despite the challenge of FLT3 rescue, 5: 1 and 20: 1 ratios are most synergistic in nanoparticles. When talking about molar ratios for the MRX-2843 :venetoclax drug combination, a molar ratio of 30 would be 30 moles of MRX-2843 for every 1 mole of venetoclax.

MRX-2843 is contained in one nanoparticle, and venetoclax is contained in the other. Thus, the molar ratio refers to the mixture of these drugs in separate nanoparticles. Nanoparticle tests (MRX- 2843 in one particle, venetoclax in a different particle) in primary AML patient samples. Synergistic ratios tested were 0.2 - 20. Additive ratios tested were 0.05 - 0.2.

Claims

1. A method of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl) methyl] phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and vincristine or salt thereof to a subject in need thereof.

2. The method of claim 1, wherein the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 7:2: 1 respectively.

3. The method of claim 2, wherein the liposomal particles have an average diameter of between 70 to 250 nm.

4. The method of claim 1, wherein the cancer is a hematological cancer.

5. The method of claim 1, wherein the cancer is leukemia.

6. The method of claim 1, wherein the molecular ratio of MRX-2843 and vincristine is between 8 and 90.

7. The method of claim 1, wherein MRX-2843 and vincristine are contained in the same nanoparticle.

8. The method of claim 1, wherein MRX-2843 and vincristine are in separate nanoparticles

9. A pharmaceutical composition comprising liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d] pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and vincristine or salt thereof contained in the same nanoparticle.

10. The pharmaceutical composition of claim 9, wherein the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 7:2: 1 respectively.

11. A method of treating cancer comprising administering an effective amount of liposomal particles comprising trans-4-[2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl) methyl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and venetoclax to a subject in need thereof.

12. The method of claim 10, wherein the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 7:2: 1 respectively.

13. The method of claim 12, wherein the liposomal particles have an average diameter of between 70 to 250 nm.

14. The method of claim 10, wherein the cancer is a hematological cancer.

15. The method of claim 10, wherein the cancer is leukemia.

16. The method of claim 10, wherein the molecular ratio of MRX-2843 and venetoclax is between 5 : 1 and 20: 1.

17. The method of claim 10, wherein MRX-2843 and venetoclax are contained in the same nanoparticle.

18. The method of claim 10, wherein MRX-2843 and venetoclax are in separate nanoparticles

19. A pharmaceutical composition comprising liposomal particles comprising trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d] pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof and venetoclax or salt thereof contained in the same nanoparticle.

20. The pharmaceutical composition of claim 8, wherein the liposomal particles contain distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol in a molar ration of about 7:2: 1 respectively.

21. A method of preparing liposomal particles loaded with trans-4-[2-[(2- cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3-d] pyrimidin-7-yl]cyclohexanol (MRX-2843) comprising, mixing a dialkyl phosphatidylcholine, a dialkyl phosphatidyl alcohol, and cholesterol in an acid aqueous buffer with sonification at greater than 10 kHz providing an acidic lipid emulsion; extruding the acidic lipid emulsion through a nanometer sized filter providing a solution of liposomal particles having an acidic core; concentrating the liposomal particles having an acid core by centrifuging and filtering the liposomal particles having an acid core providing concentrated and purified liposomal particles having an acid core; contacting the concentrated and purified liposomal particles having an acidic core with a basic aqueous buffer providing a basic buffered solution of liposomal particles having an acid core; mixing the basic buffered solution of liposomal particles having an acid core with trans-4- [2-[(2-cyclopropylethyl)amino]-5-[4-[(4-methyl-l-piperazinyl)methyl]phenyl]-7H-pyrrolo[2,3- d]pyrimidin-7-yl]cyclohexanol (MRX-2843) or salt thereof, and optionally another chemotherapy agent, providing MRX-2843 loaded liposomal particles.

22. The method of claim 21 wherein dialkyl phosphatidylcholine is distearyl phosphatidylcholine and the dialkyl phosphatidyl alcohol is distearoyl phosphatidylglycerol.

23. The method of claim 21 wherein the acid aqueous buffer is an ammonium sulfate buffer at a pH of about 4.2.

24. The method of claim 21 wherein extruding the lipid emulsion through a filter is a filter with a pore size diameter averaging between 60 and 150 nm.

25. The method of claim 21 wherein filtering is through a syringe filter with a diameter averaging about 0.45 microns.

26. The method of claim 21 the basic aqueous buffer is a phosphate buffer at a pH of about 7.7.

27. The method of claim 21 wherein mixing the basic buffered solution of liposomal particles is at 700 rpm for 60 minutes or for more than 24 hours.

28. The method of claim 21 wherein said another chemotherapy agent is vincristine or salt thereof.

29. The method of claim 21 wherein said another chemotherapy agent is venetoclax or salt thereof.