COMBINATION FORMULATIONS OF TAXANES AND MTOR INHIBITORS

Cross-Reference to Related Applications

[0001] This application claims priority of U.S. Provisional Application No. 62/777,704, filed 10 December 2018, the disclosure of which is herein incorporated by reference in its entirety.

Technical Field

[0002] The invention relates to compositions and methods for improved delivery of combinations of therapeutic agents. More particularly, the invention concerns delivery systems which provide combinations of taxanes and mTOR inhibitors.

Background Art

[0003] The progression of many life-threatening diseases such as cancer, AIDS, infectious diseases, immune disorders and cardiovascular disorders are influenced by multiple molecular mechanisms. Due to this complexity, achieving cures with a single agent has been met with limited success. Thus, combinations of agents have often been used to combat disease, particularly in the treatment of cancers. It appears that there is a strong correlation between the number of agents administered and cure rates for both solid and blood cancers (Frei, et al, Clin. Cancer Res. (1998) 4:2027-2037; Fisher, M. D.; Clin Colorectal Cancer (2001) Aug; l(2):85-6).

[0004] Taxanes are known antineoplastic agents that are typically used in combination with other agents to treat a variety of cancers. Generally taxanes, such as paclitaxel, refer to a family of products extracted from the leaves and bark of the European yew tree ( Taxus Baccata and other species of the Taxus family), as well as semi-synthetic products, such as docetaxel, obtained from baccatin III or from 10-deacetylbaccatin III which are also extracted from yew. Docetaxel acts by disrupting microtubule disassembly, resulting in inhibition of mitosis, and ultimately leading to apoptosis. Examples of cancer which can be treated with docetaxel are: locally advanced or metastatic breast cancer, non-small cell lung cancer, hormone-refractory prostate cancer and ovarian cancer, and gastric cancer.

[0005] mTOR inhibitors are a class of agents which act to inhibit the mammalian target of rapamycin (mTOR) which is a serine/threonine-specific protein kinase that belongs to the family of phosphatidylinositol-3 kinase related kinases. Everolimus is the 40-0(2-hydroxyethyl) derivative of rapamycin (sirolimus) and works similarly to sirolimus as an mTOR inhibitor. Cellular effects of everolimus include enhancement of apoptosis in some tumour cell lines as well as inhibition of cell proliferation, migration, and angiogenesis in some human cancers. Oral administration of everolimus has been approved or shown effective in the treatment of a number of cancers including advanced kidney cancer (US FDA approved in March 2009), subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) in patients who are not suitable for surgical intervention (US FDA October 2010), progressive or metastatic pancreatic neuroendocrine tumors not surgically removable (May 2011), breast cancer in post-menopausal women with advanced hormone-receptor positive, HER2-negative type cancer, in conjunction with exemestane (US FDA July 2012) and progressive, well-differentiated non-functional, neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin with unresectable, locally advanced or metastatic disease (US FDA February 2016); as well as non cancer indications such as prevention of organ rejection after liver transplant (Feb 2013), prevention of organ rejection after renal transplant (US FDA April 2010), and, tuberous sclerosis complex-associated partial-onset seizures for adult and pediatric patients aged 2 years and older (US FDA April 2018).

[0006] Free drug combination therapies using oral everolimus and intravenously (IV) administered docetaxel have been tested in some cancers but have yet to show a significant improvement in efficacy. Ramalingam et al, conducted a phase II study to evaluate the efficacy of this combination in advanced non-small cell lung cancer (NSCLC) and reported that although the combination of everolimus and docetaxel was tolerated, there was no clear signal of enhanced efficacy in an unselected population of NSCLC patients (Ramalingam et al., J Thorac Oncol. 2013 Mar;8(3):369-72.).

[0007] Docetaxel is a front-line therapy for metastatic castration-resistant prostate cancer (mCRPC); however, resistance to docetaxel is a key problem in current prostate cancer management seeing as half of patients do not respond to therapy, and those who initially respond ultimately relapse. The mTOR pathway has been implicated in prostate cancer chemoresistance and several studies indicate that mTOR inhibition is a valid strategy for docetaxel sensitization in prostate cancer cells. One study reports on patients with CRCP that were treated with a multi-drug combination of intravenous docetaxel and bevacizumab (a known inhibitor of angiogenesis) given on day 1 along with oral everolimus for 21 days and showed that the combination can be safely administered however their results“do not support the hypothesis that this combination of agents improves upon the results obtained with docetaxel monotherapy in an unselected population of chemotherapy-naive patients with CRPC” (Gross et ah, Clin Genitourin Cancer. 2018 Feb; 16(l):el l-e21). Another phase I study in which 10 mg/d oral Everolimus was combined with 60 mg/m ² IV docetaxel reported that the combination“was reasonably well tolerated”; however, they concluded“that the response rate to combination therapy would be unlikely to exceed that of docetaxel alone administered at 75 mg/m ² .” (Courtney et al., Clin Genitourin Cancer. 2015 Apr; 13(2): 113-123.)

[0008] It is understood that combinations of drugs are most effective when the ratio experienced by the subject is non-antagonistic or synergistic; that the value of such a ratio may be predetermined in vitro in a model system and that controlling the pharmacokinetics of the administered ratio permits the effect of the non-antagonism or synergy to be exhibited in treatment. See, for example, US patents 7,850,990 and 9,271,931, wherein pharmacokinetics are controlled by associating the drug combination with lipid based particles, such as liposomes. Alternative delivery systems include nanoparticles, such as those described in EP patent 1,786,443 and Canadian patent 2,574,767. Particularly useful nanoparticulate formulations are disclosed in US 10,285,951. In many instances, however, the enhanced efficacy exhibited by particular delivery systems for particular drug combinations exceeds expectations. This is the case in the present instance.

[0009] Investigators of the present invention have identified particular delivery vehicle formulations required to accommodate a combination of a taxane or taxane derivative (such as docetaxel) and an mTOR inhibitor or derivative thereof (such as everolimus), which result in superior drug retention and sustained drug release of each agent. Furthermore, they have surprisingly demonstrated that certain ratios of these drugs, when encapsulated in delivery vehicles and parenterally administered, can be successfully maintained in the blood compartment over time resulting in enhanced efficacy compared to the free drug cocktail. In some embodiments the ratio may be maintained for at least 1-6 hours or at least 1-4 hours. Formulating these drugs in nanoparticulate carriers provides a novel approach to co-deliver such anticancer drug combinations with widely differing physicochemical properties, mechanisms of action and/or routes of administration while maintaining optimal drug:drug ratios in vivo thus resulting in enhanced efficacy. Disclosure of the Invention

[0010] The invention relates to compositions and methods for administering effective amounts of mTOR inhibitor and taxane drug combinations using nanoparticles that are stably associated therewith at least one taxane agent and one mTOR inhibitor. These compositions allow the two or more agents to be delivered to the disease site in a coordinated fashion, thereby assuring that the agents will be present at the disease site at a desired ratio. Thus, by“stably associated” is meant that the pharmacokinetics of the delivery of each drug is controlled by its association with the nanoparticle so that the administered ratio is maintained after administration for sufficient time to permit a non-antagonistic ratio administered to exert its effect. Thus the administered ratio will be maintained in the blood for 1-6 hours or at least 1-4 hours. This result will be achieved whether the agents are co-encapsulated in a nanoparticulate delivery vehicle, or are encapsulated in a separate nanoparticles administered such that desired ratios are maintained at the disease site. The pharmacokinetics (PK) of the composition are controlled by the nanoparticles themselves such that coordinated delivery is achieved (provided that the PK of the delivery systems are comparable).

[0011] Thus, in one aspect, the invention provides a nanoparticle composition for parenteral administration comprising at least one taxane and one mTOR inhibitor associated with the nanoparticles at therapeutically effective ratios especially those that are non-antagonistic. The therapeutically effective non-antagonistic ratio of the agents can be determined by assessing the biological activity or effects of the agents on relevant cell culture or cell-free systems, in addition to tumor homogenates from individual patient biopsies, over a range of concentrations, as well as in vivo. Frequent combinations are docetaxel with everolimus, among other taxane derivatives together with everolimus. Also, a combination is provided comprising a taxane derivative and an mTOR inhibitor such as everolimus or sirolimus, among other known mTOR inhibitors. Any method which results in determination of a ratio of agents which maintains a desired therapeutic effect may be used, including in vivo analysis.

[0012] The composition comprises at least one taxane and one mTOR inhibitor in a ratio of the taxane to mTOR inhibitor which exhibits a desired biologic effect to relevant cells in culture, cell-free systems or tumor homogenates. Preferably, the ratio is a mole ratio at which the agents are non-antagonistic. By“relevant” cells, applicants refer to at least one cell culture or cell line which is appropriate for testing the desired biological effect. As these agents are used as antineoplastic agents,“relevant” cells are those of cell lines identified by the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI)/National Institutes of Health (NIH) as useful in their anticancer drug discovery program. Currently the DTP screen utilizes 60 different human tumor cell lines. The desired activity on at least one of such cell lines would need to be demonstrated. By“tumor homogenate,” the applicant refers to cells generated from the homogenization of patient biopsies or tumors. Extraction of whole tumors or tumor biopsies can be achieved through standard medical techniques by a qualified physician and homogenization of the tissue into single cells can be carried out in the laboratory using a number of methods well-known in the art.

[0013] In another aspect, the invention is directed to a method to deliver a therapeutically effective amount of an mTOR inhibitontaxane derivative combination (e.g., everolimus:docetaxel) to a desired target by administering the compositions of the invention.

[0014] The invention is also directed to a method to deliver a therapeutically effective amount of an mTOR inhibitontaxane derivative combination by administering a taxane stably associated with a first delivery vehicle and an mTOR inhibitor stably associated with a second delivery vehicle. The first and second delivery vehicles may be contained in separate vials, the contents of the vials being administered to a patient simultaneously or sequentially. In one embodiment, the ratio of the mTOR inhibitor and taxane derivative is non-antagonistic. In some embodiments, the taxane and/or mTOR inhibitor are provided as a drug conjugate.

[0015] In another aspect, the invention is directed to a method to prepare a therapeutic composition comprising nanoparticles containing a ratio of at least one taxane and one mTOR inhibitor which provides a desired therapeutic effect which method comprises providing a panel of at least one taxane and one mTOR inhibitor wherein the panel comprises at least one, but preferably a multiplicity of ratios of said drugs, testing the ability of the members of the panel to exert a biological effect on a relevant cell culture, cell-free system or tumor homogenate over a range of concentrations, selecting a member of the panel wherein the ratio provides a desired therapeutic effect on said cell culture, cell-free system or tumor homogenate over a suitable range of concentrations; and stably associating the ratio of drugs represented by the successful member of the panel into nanoparticulate drug delivery vehicles. In preferred embodiments, the abovementioned desired therapeutic effect is non-antagonistic (i.e. synergistic or additive). [0016] As further described below, in a preferred embodiment, in designing an appropriate combination in accordance with the method described above, the non-antagonistic ratios are selected as those that have a combination index (Cl) of < 1.1. In further embodiments, suitable nanoparticle formulations are designed such that they stably incorporate an effective amount of a taxane:mTOR inhibitor combination (e.g., docetaxeheverolimus) and allow for the sustained release of both drugs in vivo. Preferred formulations contain drug conjugates of at least one of the taxane or mTOR inhibitor. More preferred formulations contain drug conjugates of both a taxane and an mTOR inhibitor, such as for example, a drug conjugate of docetaxel and a drug conjugate of everolimus. Most preferred formulations contain drug conjugates of both docetaxel and everolimus wherein each is separately linked to a hydrophobic moiety such as cholesterol.

Brief Description of the Drawings

[0017] Figure 1 is a diagram outlining the method of the invention for determining an appropriate ratio of therapeutic agents to include in formulations.

[0018] Figure 2 shows the concentration of cells affected in C-33 A cells of everolimus and docetaxel alone and at 1: 1, 2:1 and 1:2 ratios. Also shown is an isobologram generated at 30% effective dose (ED) values.

[0019] Figure 3A is a graph showing the concentration of docetaxel-cholesterol conjugate (Procet) remaining in the plasma (ug/mL) at various times after intravenous administration when the drug conjugate was formulated in nanoparticles.

[0020] Figure 3B is a graph showing % injected dose over time of docetaxel-cholesterol conjugate remaining in the plasma at various times after intravenous administration when the drug conjugate was formulated in nanoparticles.

[0021] Figure 3C is a graph showing the concentration of everolimus-cholesterol conjugate (ProEver) remaining in the plasma (ug/mL) at various times after intravenous administration when the drug conjugate was formulated in nanoparticles.

[0022] Figure 3D is graph showing % injected dose over time of everolimus-cholesterol conjugate remaining in the plasma at various times after intravenous administration when the drug conjugate was formulated in nanoparticles.

[0023] Figure 4A is a graph showing the % of injected dose over time of both docetaxel- cholesterol and everolimus-cholesterol conjugates remaining in the plasma at various times after intravenous administration when the drug conjugates were formulated in PLA-PEG containing nanoparticles at a 1: 1: 12 drug:drug:polymer ratio.

[0024] Figure 4B is a graph showing the % of injected dose over time of both docetaxel- cholesterol and everolimus-cholesterol conjugates remaining in the plasma at various times after intravenous administration when the drug conjugates were formulated in PLA-PEG containing nanoparticles at a 1: 1:9 drug:drug:polymer ratio.

[0025] Figure 4C is a graph showing the % of injected dose over time of both docetaxel- cholesterol and everolimus-cholesterol conjugates remaining in the plasma at various times after intravenous administration when the drug conjugates were formulated in PLA-PEG containing nanoparticles at a 1: 1:6 drug:drug:polymer ratio.

[0026] Figure 5A is a graph showing the efficacy of co-loaded nanoparticles with docetaxeheverolimus drug conjugates at various ratios compared to the individual free drugs and the free drug cocktail when administered via i.v. against the PC3 (PTEN ^mut ) human prostate cancer cell model in nude mice. Mice were organized into appropriate groups consisting of a control (“Untreated”) and treatment groups including empty nanoparticles (“Vehicle”), free docetaxel (lOmg/kg), free everolimus (lOmg/kg), free cocktail of docetaxeheverolimus (10: 10 mg/kg) and docetaxeheverolimus drug conjugates co-loaded in nanoparticles at either a 1: 1, 1:5, 1: 10 or 5:1 ratios at various doses as indicated.

[0027] Figure 5B is a graph showing the efficacy of empty nanoparticles (“Vehicle”), free docetaxel (5 mg/kg), free everolimus (20 mg/kg), free drug cocktail (docetaxeheverolimus, 5 mg/kg:20mg/kg), docetaxel/everolimus co-loaded into nanoparticles (“DENP”) resulting in a final docetaxeheverolimus ratio of 1: 1 (5:5 docetaxeheverolimus mg/kg), or 5: 1 (45:9 docetaxeheverolimus mg/kg), docetaxel and /everolimus separately loaded into nanoparticles and co-administered (“NPD+NPE”) resulting in a final docetaxeheverolimus ratio of 5: 1 (15:3 docetaxeheverolimus mg/kg), and docetaxel (15 mg/kg) and everolimus ( 3 mg/kg) each separately encapsulated in nanoparticles and administered alone (“NPD” and “NPE”, respectively).

[0028] Figure 5C is a graph showing the efficacy of empty nanoparticles (“Vehicle”), and docetaxel/everolimus co-loaded into nanoparticles (“DENP”) resulting in a final docetaxeheverolimus ratio of 1: 1 (10: 10 docetaxeheverolimus mg/kg), high dose 5: 1 (60: 12 docetaxel:everolimus mg/kg), 2: 1 (12.5:6 docetaxel :everoli mus mg/kg), and 5: 1 (15:3 docetaxekeverolimus mg/kg).

[0029] Figure 6 are a group of graphs showing the percent change in body weight over time for the study groups shown in Figure 5A.

[0030] Figure 7 is a graph showing the percent change in mean body weight for study groups given either free or encapsulated docetaxel or everolimus.

[0031] Figure 8 is a graph showing the efficacy of co-loaded nanoparticles with docetaxekeverolimus drug conjugates at various ratios compared to the free drug cocktail when administered via i.v. against the MDA-MB-231 (PTEN ^wt ) human breast adenocarcinoma cell model in nude mice. Mice were organized into appropriate treatment groups consisting of empty nanoparticles (“Vehicle”), the free drug cocktail of docetaxekeverolimus (5:5 mg/kg) and docetaxekeverolimus drug conjugates co-loaded in nanoparticles (“NP D:E”) at either a 1: 1, 1:5, 1: 10 or 5: 1 ratios at various doses as indicated.

[0032] Figure 9 show the individual mice tumor volumes over time for the formulations shown in Figure 8 as well as for free docetaxel as indicated.

[0033] Figure 10A is a graph showing the efficacy of co-loaded nanoparticles with docetaxekeverolimus drug conjugates at various ratios compared to the free drug cocktail when administered via i.v. against the MDA-MB-468 (PTEN ^del ) human breast adenocarcinoma cell model in SCID Beige mice. Mice were organized into appropriate treatment groups consisting of a control (“Untreated”) and treatment groups including empty nanoparticles (“Vehicle”), free docetaxel, free everolimus, the free drug cocktail of docetaxekeverolimus (5:5mg/kg) and docetaxekeverolimus drug conjugates co-loaded in nanoparticles (“NP D:E”) at either a 1: 1, 1:5, 1: 10 or 5: 1 ratios at various doses as indicated.

[0034] Figure 10B is a graph showing the efficacy of co-loaded nanoparticles with docetaxekeverolimus drug conjugates at various ratios compared to the free drug cocktail when administered via i.v. against the MDA-MB-468 (PTEN ^del ) human breast adenocarcinoma cell model in SCID Beige mice on an accelerated dosing schedule as compared to that shown in Figure 8A. Mice were organized into appropriate treatment groups consisting of empty nanoparticles (“Vehicle”), free docetaxel, the free drug cocktail of docetaxekeverolimus (5:5 mg/kg) and docetaxekeverolimus drug conjugates co-loaded in nanoparticles (“NP D:E”) at either a 1: 1, 1:5, 1: 10 or 5: 1 ratios at various doses as indicated. [0035] Figure 11 shows a group of graphs of

[0036] individual mice tumor volumes over time for some of the study groups shown in Figures 10A and 10B.

[0037] Figure 12 is a graph showing the mean percent body weight change for the study groups shown in Figure 10A.

[0038] Figure 13 A is graph showing the concentration over time of docetaxel-cholesterol drug conjugate (“ProCet”) formulated in nanoparticles and stored at 4°C (“4C”) or room temperature (“RT”) for up to 3 months.

[0039] Figure 13B is graph showing the concentration over time of everolimus-cholesterol drug conjugate (“ProEver”) formulated in nanoparticles and stored at 4°C or room temperature for up to 3 months.

Modes of Carrying Out the Invention

[0040] The invention provides compositions comprising nanoparticles stably associated therewith at least one taxane and one mTOR inhibitor, wherein the taxane and mTOR inhibitor are present at non-antagonistic taxane/mTOR inhibitor ratios.

[0041] Preferably, nanoparticle compositions provided herein will include nanoparticles stably associated therewith at least one taxane and one mTOR inhibitor in a ratio of the taxane / mTOR inhibitor which exhibits a non-antagonistic effect to relevant cells or tumor cell homogenates.

[0042] Preferably, nanoparticle compositions of the invention will include nanoparticles stably associated therewith docetaxel and everolimus. More preferably, docetaxel and everolimus will be present in compositions of the invention at a docetaxel: everolimus ratio of between 100:1 and 1:100 or of between 40:1 and 1:40, even more preferably the ratio of docetaxel to everolimus will be in the range of 40:1 and 1:1, 30:1 and 1:1, 20:1 and 1:1 or 10:1 and 1:1. In some embodiments, nanoparticle compositions of the invention will include nanoparticles stably associated therewith docetaxel and everolimus, wherein the ratio of docetaxel to everolimus will be in the range of 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1,

32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1. In some

embodiments, the ratio of docetaxel to everolimus will be in the range of 10:1 and 4:1. In some embodiments, the ratio is a mole or molar ratio. In some embodiments, the ratio is a weight ratio.

[0043] In further embodiments of the invention, the above described nanoparticles are administered with a third or fourth agent. Any therapeutic, diagnostic or cosmetic agent may be included.

[0044] In another aspect of the invention, at least one of the taxane or mTOR inhibitor is provided as a drug conjugate. Preferably the taxane and/or mTOR inhibitor are conjugated to a hydrophobic moiety. More preferably, the hydrophobic moiety is a hydrophobic polymer or a natural product such as, but not limited to, cholesterol or a C22 fatty acid.

[0045] The nanoparticles of the present invention may be used not only in parenteral administration but also in topical, nasal, subcutaneous, intraperitoneal, intramuscular, aerosol or oral delivery or by the application of the delivery vehicle onto or into a natural or synthetic implantable device at or near the target site for therapeutic purposes or medical imaging and the like. Preferably, the nanoparticles of the invention are used in parenteral administration, most preferably, intravenous administration.

[0046] The preferred embodiments herein described are not intended to be exhaustive or to limit the scope of the invention to the precise forms disclosed. They are chosen and described to best explain the principles of the invention and its application and practical use to allow others skilled in the art to comprehend its teachings.

[0047] Unless defined otherwise, all terms of art, notations and other scientific terms or terminology used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[0048] As used herein,“a” or“an” means“at least one” or“one or more.”

Abbreviations and/or Definitions:

[0049] The term“therapeutic agent” or“drug” as used herein refers to chemical moieties used in a variety of therapeutic, including pharmaceutical applications. The solubility range of the drugs range from“insoluble” in water or buffer, to those that are“sparingly soluble” or “soluble.”

[0050] As used herein,“insoluble in aqueous medium” means that the substance can be dissolved in an aqueous solution at physiological ionic strength only to the extent of 0.05 mg/ml or less. It is recognized that almost no substances are completely insoluble in aqueous medium, and that the salt concentration or osmolality of the medium may also influence solubility.

“Insoluble in aqueous medium,” according to the present definition, assumes the osmolality, ionic strength, and pH of physiologically compatible solutions. Alternatively,“insolubility in pure water” may be used as the standard if so specified. “Insolubility in water” is defined as < 0.05 mg/ml of pure water.

[0051] Similarly,“sparingly soluble” and“soluble” may be described in terms of reference to either“aqueous medium” as defined above or in“pure water.” Substances that are“soluble” in aqueous medium dissolve at least to the extent of being equal to or greater than 1.0 mg/ml of the physiological solution; substances that are“sparingly soluble” in aqueous medium dissolve only to the extent of less than 1.0 mg/ml but more than 0.05 mg/ml of the physiological solution.

[0052] “Hydrophobic moiety” is defined as a moiety which is insoluble in aqueous solution as defined above. The hydrophobic moiety may be a hydrophobic polymer such as

polycaprolactone (PCL) or may be a hydrophobic small molecule such as a vitamin or a steroid. It may be monovalent - i.e., have a suitable functional group for coupling only to a single active through a linker - or may be multivalent - i.e., able to couple to multiple actives through a linker.

[0053] An“amphiphilic stabilizer” or“amphiphilic moiety” is a compound having a molecular weight greater than about 500 that has a hydrophilic region and a hydrophobic region. Preferably the molecular weight is greater than about 1,000, or greater than about 1,500, or greater than about 2,000. Higher molecular weight moieties, e.g., 25,000 g/mole or 50,000 g/mole, may be used. “Hydrophobic” is defined as above. “Hydrophilic” in the context of the present invention refers to moieties that have a solubility in aqueous solution ( i.e ., a physiological solution as defined above) of at least 1.0 mg/ml. Typical amphiphilic stabilizers are copolymers of hydrophilic regions and hydrophobic regions. Thus, in the amphiphilic stabilizer, the hydrophobic region, if taken alone, would exhibit a solubility in aqueous medium of less than 0.05 mg/ml and the hydrophilic region, if taken alone, would exhibit a solubility in aqueous medium of more than 1 mg/ml. Examples include copolymers of polyethylene glycol and polylactic acid (PLA), including poly-L-lactide (PLLA), poly-D, L-lactide (a PDLA or PLLDA); or copolymers of polyethylene glycol and poly(lactic-co-glycolic acid) (PLGA or PLG) or copolymer of polyethylene glycol and polycaprolactone (PCL). In some embodiments, the weight ratio of the hydrophobic portion to the hydrophilic portion is between 8.5 and 12.5 and/or the hydrophobic portion has a molecular weight of 8- 25 kD. In other embodiments, the ratio of the hydrophobic portion to the hydrophilic portion is 4:1, 3:1, 2:1 or 1:1 and / or the hydrophobic portion has a molecular weight of 10 or 11 or 20 or 25 kD.

[0054] A“linker” refers to any covalent bond, to a divalent residue of a molecule, or to a chelator (in the case where the active (drug) is a metal ion or organic metallic compound, e.g., cisplatin) that allows the hydrophobic moiety to be attached to the active agent. The linker may be selectively cleavable upon exposure to a predefined stimulus, thus releasing the active agent from the hydrophobic moiety. The site of cleavage, in the case of the divalent residue of a molecule may be at a site within the residue, or may occur at either of the bonds that couple the divalent residue to the agent or to the hydrophobic moiety. The predefined stimuli include, for example, pH changes, enzymatic degradation, chemical modification or light exposure.

Convenient conjugates are often based on hydrolyzable or enzymatically cleavable bonds such as esters, carbonates, carbamates, disulfides and hydrazones.

[0055] In some instances, the conditions under which the drug performs its function are not such that the linker is cleaved, but the drug is able to perform this function while still attached to the particle. In this case, the linker is described as“non-cleavable,” although virtually any linker could be cleaved under some conditions; therefore,“non-cleavable” refers to those linkers that do not necessarily need to release the drug from the particle as the active performs its function. Taxanes

[0056] Taxanes are a class of widely used anticancer drugs. They are diterpenes which are naturally produced by plants belonging to the Taxus genus ( e.g ., Yews). “Taxanes” as used herein includes paclitaxel (Taxol™), docetaxel (Taxotere™), cabazitaxel and other taxane analogs or derivatives thereof. Paclitaxel is a used for treating a range of carcinomas. Another taxane, cabazitaxel, has been approved by the FDA to treat hormone-refractory prostate cancer.

[0057] The anti-tubulin agent docetaxel is one of the most broadly used chemotherapies, with approvals in locally advanced or metastatic breast cancer, head and neck cancer, gastric cancer, hormone -refractory prostate cancer and non- small-cell lung cancer. It is approximately twice as potent as paclitaxel (essentially due to docetaxel’ s effect on the centrosome of the mitotic spindle), however it has similar efficacy as paclitaxel which may be due to the fact that docetaxel is prone to cellular drug resistance via a number of different mechanisms.

[0058] An exemplified embodiment of a taxane drug conjugate for use of the invention is a docetaxel-cholesterol conjugate (or“docetaxel-linker-hydrophobic moiety”) shown below:

mTQR Inhibitors

[0059] “mTOR inhibitor” as used herein is meant to include any inhibitor with modulates activity at any point along the mTOR pathway, including the PI3K/AKT/mTOR or“PAM” pathway (note that PI3K is Phosphoinositide 3-Kinase and AKT is Protein Kinase B). Such inhibitors include, for example, rapamycin (sirolimus), temsirolimus, everolimus or other rapamycin derivatives (“rapalogs”). Some act through ATP competitive inhibition while others are non- ATP inhibitors, all of which are included herein. [0060] Activation of the PAM pathway is thought to be an important oncogenic driver in as many as 20% of all solid tumors, but no precision therapies are currently approved for use in these populations. Although evidence suggests that everolimus is active against tumors with PAM pathway activation, it is currently only used in unselected populations of a few tumor types. Everolimus has a relatively narrow therapeutic window, and efficacious doses are associated with significant rates of Grade 3 and 4 adverse events. Thus there exists a need to develop improved everolimus, and other mTOR inhibitor, formulations with increased efficacy and/or reduced toxicity.

[0061] mTOR inhibitors of the invention are preferably present as a drug conjugate. More preferably the drug conjugate is provided in a nanoparticle. Exemplified embodiments of mTOR inhibitor drug conjugates for use of the invention are everolimus-cholesterol drug conjugates (or “everolimus -linker-hydrophobic moiety”) linked through either a glycolate or succinate linkage such as the example shown below:

Drug Conjugates

[0062] The preferred drug delivery approach applied here was to combine two well-known concepts, namely the use of drug conjugates and the utilization of micellar or nanoparticle delivery vehicles. The goal of most prodrug or drug conjugate technologies is typically to make hydrophobic drugs more hydrophilic for increased solubility in an aqueous environment.

However, by making them more hydrophobic and consequently more compatible with polymer based delivery systems it is possible to adjust the properties of two disparate drugs such that their effective release rates are matched. Here, drugs are made more hydrophobic by linking them to a hydrophobic anchor (or“hydrophobic moiety”). Micelles or lipophilic nanoparticle carriers are used to maintain these drug conjugates in aqueous environment since the individual drugs themselves are otherwise insoluble.

[0063] In some embodiments, nanoparticle delivery systems employed in this work require the synthesis of taxane drug conjugates, docetaxel for example, to increase the hydrophobicity of the drug. While docetaxel itself is nearly insoluble in water, it possesses sufficient aqueous solubility that it can rapidly partition out of some delivery systems, typically with half-lives on the order of a few minutes. The objective of this work was to control the pharmacokinetics of the formulated drug in vivo while maximizing efficacy. A variety of drug conjugates were synthesized, characterized and formulated into nanoparticles to characterize the effects of iterative changes to the dmg-linker-hydrophobic moiety structural motif on partition kinetics and efficacy. A spectrum of in vitro and in vivo anti-tumor activity was observed, often dictated by the physical and chemical properties of the drug conjugate rather than the delivery vehicle itself.

[0064] Parameters that are likely to affect the in vivo availability of a drug when optimizing the design of formulations of the invention include: (1) the plasma elimination of the delivery vehicle; (2) the partitioning rate of the drug out of the particle; and (3) the hydrolysis rate of the drug conjugate In an ideal system the nanoparticles remain intact upon i.v. administration, they are cleared relatively slowly from the central blood compartment, and drug conjugate hydrolysis is relatively rapid (preferably through enzymatic means rather than pH to avoid stability issues in the formulation). Though not wishing to be bound by any one particular theory, the rate limiting process affecting drug availability in the compositions of the present invention may be the partitioning rate of the drug conjugate from the particle to the plasma. A series of drug conjugates based on docetaxel and everolimus were investigated in order to achieve control of the pharmacokinetic behavior of a taxane/mTOR inhibitor drug combination in vivo.

[0065] PCT/US 2016/042330 and US Patent No. 8486924, describe the design of

nanoparticles with prolonged circulation half-lives where the release of one or two agents is modulated by manipulating the composition of the nanoparticle amphiphilic stabilizer and/or the degree of hydrophobicity or they hydrophobic moiety and/or the lability of the linkers, both are considered for use in this invention and are incorporated herein by reference in their entireties.

[0066] Preferred hydrophobic moieties include polymers or natural products. Examples of suitable hydrophobic polymeric moieties include but are not limited to polymers of the following: acrylates including methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate (BA), isobutyl acrylate, 2-ethyl acrylate, and t-butyl acrylate; methacrylates including ethyl methacrylate, n-butyl methacrylate, and isobutyl methacrylate; acrylonitriles; methacrylonitrile; vinyls including vinyl acetate, vinylversatate, vinylpropionate, vinylformamide, vinylacetamide, vinylpyridines, and vinylimidazole; aminoalkyls including aminoalkylacrylates,

aminoalkylmethacrylates, and aminoalkyl(meth)acrylamides; styrenes; cellulose acetate phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose phthalate, and the polymers poly(D,L lactide), poly(D,L-lactide-co-glycolide), poly(glycolide), poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters), polyesters, poly(hydroxyvaleric acid),

polydioxanone, poly(ethylene terephthalate), poly(malic acid), poly(tartronic acid),

polyanhydrides, polyphosphazenes, poly(amino acids) and their copolymers (see generally, Ilium, L., Davids, S. S. (eds.) Polymers in Controlled Drug Delivery, Wright, Bristol, 1987; Arshady, J. Controlled Release (1991) 17:1-22; Pitt, Int. J. Phar. (1990) 59:173-196; Holland, et al, J. Controlled Release (1986) 4:155-180); hydrophobic peptide-based polymers and copolymers based on poly(L-amino acids) (Lavasanifar, A., et al, Advanced Drug Delivery Reviews (2002) 54:169-190), poly(ethylene- vinyl acetate) ("EVA") copolymers, silicone rubber, polyethylene, polypropylene, polydienes (polybutadiene, polyisoprene and hydrogenated forms of these polymers), maleic anhydride copolymers of vinyl-methylether and other vinyl ethers, polyamides (nylon 6,6), polyurethane, poly(ester urethanes), poly(ether urethanes), poly(ester- urea). Particularly preferred polymeric hydrophobes include poly(ethylenevinyl acetate), poly (D,L-lactic acid) oligomers and polymers, poly (L-lactic acid) oligomers and polymers, poly (glycolic acid), copolymers of lactic acid and glycolic acid, poly (caprolactone), poly

(valerolactone), polyanhydrides, copolymers of poly (caprolactone) or poly (lactic acid). Natural products as“hydrophobic moieties” should have functional groups or groups that can be converted to functional groups for conjugation including: hydrophobic vitamins (for example vitamin E, vitamins K and A), fatty acids (preferably long-chain or C18-C24 fatty acids), carotenoids and retinols (for example beta carotene, astaxanthin, trans and cis retinal, retinoic acid, folic acid, dihydrofolate, retinyl acetate, retinyl palmitate), cholecalciferol, calcitriol, hydroxycholecalciferol, ergocalciferol, i -tocopherol, (^-tocopherol acetate, (^-tocopherol nicotinate, cholesterol and estradiol. Preferred natural products for use as hydrophobic moieties of the invention are cholesterol or a C18-C24 fatty acid. Depending on the nature of the hydrophobic moiety, it may be able to accommodate more than one, including substantially more than one drug through a multiplicity of linking sites. Polymeric moieties may have as many as 100 sites whereby drugs could be linked. Simpler hydrophobic moieties, such as vitamin E, may provide only one such site. Thus, the number of drugs coupled to a single hydrophobic moiety may be only 1, or may be 2, 5, 10, 25, 100 and more, and all integers in between. For instance, the polymers set forth above can readily be provided with a multiplicity of functional groups for coupling to the drug. Difunctional hydrophobic moieties would include the hydrophobic polymer chains listed above that have two terminal OH, COOH, or NH2 groups. Multifunctional hydrophobic moieties include all of those listed above that have multiple OH, COOH, or NH2 groups on some or all of the monomer units on the polymer backbone. These functional groups are merely illustrative; other moieties which could form functional groups for linking include phenyl substituents, halo groups, and the like. Typically, when the hydrophobic moiety is a hydrophobic polymer, it may have multiple sites for linkage. When the hydrophobic moiety is a relatively small molecule, it will accommodate only the number of linkers for which it has available functional groups. Linkers or cross-linkers for use in the invention include succinate and diglycolate as well as other linkers such as those described in

PCT/US2005/025549 which is incorporated herein by reference.

[0067] Amphiphilic stabilizers and hydrophobic moieties for use in the invention also include those described in PCT/US2005/025549 or US Patent No. 8486924.

Determining Non-Antagonistic Taxane:mTQR Inhibitor Ratios

[0068] In one aspect of the invention taxane agents and mTOR inhibitors will be encapsulated into nanoparticles at synergistic or additive ( i.e . non-antagonistic) ratios. Determination of ratios of agents that display synergistic or additive combination effects may be carried out using various algorithms, based on the types of experimental data described below. These methods include isobologram methods (Loewe, et al., Arzneim-Forsch (1953) 3:285-290; Steel, et al., Int. J. Radiol. Oncol. Biol. Phys. (1979) 5:27-55), the fractional product method (Webb, Enzyme and Metabolic Inhibitors (1963) Vol. 1, pp. 1-5. New York: Academic Press), the Monte Carlo simulation method, CombiTool, ComboStat and the Chou-Talalay median-effect method based on an equation described in Chou, J. Theor. Biol. (1976) 39:253-276; and Chou, Mol. Pharmacol. (1974) 10:235-247). Alternatives include surviving fraction (Zoli, et al, Int. J. Cancer (1999) 80:413-416), percentage response to granulocyte/macrophage-colony forming unit compared with controls (Pannacciulli, et al, Anticancer Res. (1999) 19:409-412) and others (Berenbaum, Pharmacol. Rev. (1989) 41:93-141; Greco, et al., Pharmacol Rev. (1995) 47:331-385).

[0069] The Chou-Talalay median-effect method is preferred and is described in US Patent No.7,850,990 which is incorporated herein by reference.

[0070] Non-antagonistic ratios of two or more agents can also be determined using in vivo screening. Non-antagonistic ratios of two or more agents can be determined for disease indications other than cancer and this information can be used to prepare therapeutic formulations of two or more drugs for the treatment of these diseases. With respect to in vitro or in vivo assays, many measurable endpoints can be selected from which to define drug synergy, provided those endpoints are therapeutically relevant for the specific disease.

[0071] As set forth above, the in vitro studies on cell cultures will be conducted with “relevant” cells. The choice of cells will depend on the intended therapeutic use of the agent. In vitro studies on individual patient biopsies or whole tumors can be conducted with“tumor homogenate,” generated from homogenization of the tumor sample(s) into single cells. In vivo studies will be conducted using animal studies, preferably with mice of various strains that bear a cancer cell or xenograft model.

Nanoparticles

[0072] Nanoparticle formulations of the invention are preferably less than 100 nm, less than 80 nm, or more preferably 20-80 nm, or even more preferably 40-80 nm in average diameter. In some embodiments, the average diameter of nanoparticle formulations of the invention are preferably less than 70 nm. In one embodiment, nanoparticles are formed by assembling into a particle at least one taxane or mTOR inhibitor drug conjugate wherein the taxane or mTOR inhibitor is coupled through a linker to a hydrophobic moiety; and at least one amphiphilic stabilizer comprising a hydrophobic portion and a hydrophilic portion. In some embodiments, the amphiphilic stabilizer is copolymer which includes polyethylene glycol (PEG). In certain embodiments, the weight ratio of hydrophobic portion to the hydrophilic portion of the amphiphilic stabilizer is in the range of 8:5 to 12:5, preferably 10:5 (or 2: 1) and wherein the hydrophobic portion has a molecular weight of 8 kD to 25 kD, or 8 kD-20 kD or 8 kD- 15 kD. This permits ready partition from the particles of the drug conjugate(s) in intact form whereupon release of the therapeutic agent itself when the drug conjugate is liberated to the bloodstream is relatively rapid.

[0073] As noted above, preferred examples employ drug conjugates of docetaxel and everolimus and nanoparticle delivery vehicles to facilitate pharmacokinetic control. By making these drugs more hydrophobic and consequently more compatible with polymer based delivery systems, the pharmacokinetics of the drug combination compositions can be controlled. It is also possible to adjust the properties of formulations containing additional antineoplastic agents such that their effective release rates in vivo are matched to that of the two-drug combinations. Nanoparticle carriers can be used to suspend these drug conjugates and other agents in an aqueous environment.

[0074] As shown below, long circulating drug conjugate nanoparticles provide significantly enhanced therapeutic activity over the non-encapsulated drug combinations or free drug cocktails; these types of formulations of the invention are therefore advantageous per se.

[0075] Thus, exemplary pharmaceutical compositions are those that comprise nanoparticles formed from a drug conjugate of a taxane and a drug conjugate of an mTOR inhibitor, which drug conjugates are conjugates of said agents each coupled to a hydrophobic moiety through a linker wherein said nanoparticles also comprise a lipid and/or an amphiphilic stabilizer. In some embodiments, no lipid is required. In certain embodiments, a targeting agent is included in the nanoparticles. In some embodiments, a targeting agent is attached to the amphiphilic stabilizer. Preferably the amphiphilic stabilizer is a copolymer comprising PEG.

[0076] The invention also includes methods to administer the above combinations using the compositions of the invention, to combine the compositions of the invention with formulations of additional antineoplastic agents and administer these and to methods of preparing these compositions and formulations.

[0077] The nanoparticle delivery vehicles of the present invention may be used not only in parenteral administration but also in topical, nasal, subcutaneous, intraperitoneal, intramuscular, aerosol or oral delivery or by the application of the delivery vehicle onto or into a natural or synthetic implantable device at or near the target site for therapeutic purposes or medical imaging and the like. Preferably, the nanoparticle delivery vehicles of the invention are used in parenteral administration, most preferably, intravenous administration. [0078] The nanoparticle delivery vehicles of the present invention may be used to treat cancer or other hematological disorders including, but not limited to, breast cancer (such as locally advanced or metastatic breast cancer), lung cancer (such as non-small cell lung cancer), prostate cancer (such as hormone-refractory prostate cancer or castration-resistant prostate cance), ovarian cancer, Head and Neck Squamous Cell Carcinoma (HNSCC) or gastric/GEJ cancers (e.g. tumors of the gastroesophageal junction).

[0079] In some embodiments, the cancer or hematological disorder is associated with a PTEN (Phosphatase and Tensin Homolog) mutation. PTEN is a protein that, in humans, is encoded by the PTENgene. Mutations of this gene are a step in the development of many cancers. Genes corresponding to PTEN (orthologs) have been identified in most mammals for which complete genome data are available. PTEN acts as a tumor suppressor gene through the action of its phosphatase protein product. This phosphatase is involved in the regulation of the cell cycle, preventing cells from growing and dividing too rapidly. It is a target of many cancer drugs.

[0080] The protein encoded by this gene is a phosphatidylinositol-3,4,5-trisphosphate 3- phosphatase. It contains a tensin-like domain as well as a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating Akt/PKB signaling or the PAM pathway. Thus in some embodiments, cell used in the present invention include both PTEN wild-type (PTEN ^wt ) and thus no PAM pathway activation, as well as PTEN deleted (PTEN ^del ) which have PAM activated. mTOR inhibitors as provided in formulations of the invention may be more active in these PTEN deleted cell lines or cancers.

[0081] The delivery vehicle compositions of the present invention may be administered to warm-blooded animals, including humans as well as to domestic avian species. For treatment of human ailments, a qualified physician will determine how the compositions of the present invention should be utilized with respect to dose, schedule and route of administration using established protocols. Such applications may also utilize dose escalation should agents encapsulated in delivery vehicle compositions of the present invention exhibit reduced toxicity to healthy tissues of the subject. Preferably, the pharmaceutical compositions of the present invention are administered parenterally, i.e., intraarterially, intravenously, intraperitoneally, subcutaneously, or intramuscularly. More preferably, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus or infusional injection. For example, see Rahman, et al, U.S. patent No. 3,993,754; Sears, U.S. patent No. 4,145,410; Papahadjopoulos, et al., U.S. patent No. 4,235,871; Schneider, U.S. patent No. 4,224,179; Lenk, et al, U.S. patent No. 4,522,803; and Fountain, et al, U.S. patent No. 4,588,578, incorporated by reference.

[0082] Pharmaceutical compositions comprising delivery vehicles of the invention are prepared according to standard techniques and may comprise water, buffered water, 0.9% saline, 0.3% glycine, 5% dextrose, iso-osmotic sucrose solutions and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like. Additionally, the delivery vehicle suspension may include polymer or lipid-protective agents which protect the nanopoarticles against free-radical and peroxidative damages on storage. Free -radical quenchers, such as alpha-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, may be used.

[0083] In some embodiments of the invention, at least 85%, 90%, or 95% of each drug is retained in the nanoparticles after storage for at least 3 months, 4 months, 5 months, 6 months, or greater than 6 months when stored at room temperature or when stored at 4 deg C.

[0084] The concentration of delivery vehicles in the pharmaceutical formulations can vary widely, such as from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, and the like, in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment

[0085] Preferably, the pharmaceutical compositions of the present invention are administered intravenously. Dosage for the delivery vehicle formulations will depend on the ratio of drug to polymer and the administrating physician’s opinion based on age, weight, and condition of the patient.

[0086] In addition to pharmaceutical compositions, suitable formulations for veterinary use may be prepared and administered in a manner suitable to the subject. Preferred veterinary subjects include mammalian species, for example, non-human primates, dogs, cats, cattle, horses, sheep, and domesticated fowl. Subjects may also include laboratory animals, for example, in particular, rats, rabbits, mice, and guinea pigs.

Kits

[0087] The therapeutic agents in the invention compositions may be formulated separately in individual compositions wherein each of the taxane and mTOR inhibitor are stably associated with appropriate nanoparticles. These compositions can be administered separately to subjects as long as the pharmacokinetics of the delivery vehicles are coordinated so that the ratio of taxane:mTOR inhibitor administered is maintained at the target for treatment. Thus, it is useful to construct kits which include, in separate containers, a first composition comprising nanoparticles stably associated with at least one taxane (or taxane derivative) and, in a second container, a second composition comprising an mTOR inhibitor (preferably nanoparticles stably associated with at least one mTOR inhibitor). The containers can then be packaged into the kit.

[0088] The kit will also include instructions as to the mode of administration of the compositions to a subject, at least including a description of the ratio of amounts of each composition to be administered. Alternatively, or in addition, the kit is constructed so that the amounts of compositions in each container is pre-measured so that the contents of one container in combination with the contents of the other represent the correct ratio. Alternatively, or in addition, the containers may be marked with a measuring scale permitting dispensation of appropriate amounts according to the scales visible.

[0089] The present invention is further described by the following examples. The examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplifications, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.

EXAMPLES Example 1

Docetaxel:Everolimus Synergy In Vitro is Drug Ratio Dependent

[0090] Many combinations of two or more drugs have the ability to exhibit synergistic effects. Similarly, combinations of the same two or more drugs may also show additive or antagonistic interactions. In order to identify ratios of a taxane and an mTOR inhibitor that are non- antagonistic, various combinations of docetaxel and everolimus were tested for their cytotoxic effects in vitro. More specifically, drug ratios that demonstrate synergy or additivity over a broad range of drug concentrations were investigated.

[0091] Measuring additive, synergistic or antagonistic effects was performed using everolimus: docetaxel at 10: 1, 5: 1, 2: 1, 1: 1, 1:2, 1:5 and 1: 10 ratios in a number of cancer cell lines all of which come from the American Type Culture Collection (ATCC).

[0092] Compound preparation. Solid powders of both everolimus and docetaxel free drug were weighed on a calibrated balance and dissolved in 100 % DMSO. DMSO samples were stored at room temperature. On the day of the experiment, the compound stocks were diluted in DMSO to concentrations of 3160 their IC50. Both solutions were subsequently mixed in the seven ratios indicated above.

[0093] Cell proliferation assay. A cell assay stock was thawed and diluted in appropriate medium and dispensed in a 384- well plate, at a concentration of 100 - 6400 cells per well in 45 mΐ medium. The margins of the plate were filled with phosphate-buffered saline. Plated cells were incubated in a humidified atmosphere of 5% C02 at 37 °C. After 24 hours, 5 mΐ of the appropriate compound combination or single agent was added and plates were further incubated for another 72 hours. After 72 hours, plates were cooled in 30 minutes to room temperature and 24 mΐ of ATPlite IStep™ (PerkinElmer) solution was added to each well, and subsequently shaken for 2 minutes. After 10 minutes of incubation in the dark at room temperature, the luminescence was recorded on an Envision multimode reader (PerkinElmer).

[0094] Controls: t = 0 signal. On a parallel plate, 45 mΐ cells were dispensed in quadruplicate and incubated in a humidified atmosphere of 5 % CO2 at 37 °C. After 24 hours, plates were cooled to room temperature in 30 minutes. 5 m i DMSO-containing Hepes buffer and 24 mΐ ATPlite IStep™ solution were added and subsequently mixed for 2 minutes. Luminescence was measured after 10 minutes incubation (= luminescence _t =o) in the dark. [§§95] Cell growth control. The cellular doubling times of all cell lines were calculated from the t = 0 hours and t = end growth signals of the untreated cells. If the doubling time was out of specification (0.5 - 2.0 times deviating from historic average) the assay was invalidated.

[0096] Maximum signals. On each 384-well plate, the maximum luminescence was recorded after incubation for 72 hours without compound in the presence of 0.4% DMSO. All equivalent wells (usually 14) were averaged. This average is defined as: luminescence _{a ntreated,t} =72 _h .

[0097] Dose response curves. Accurate single agent ICsos were used for combination analysis. For each single agent its dose-response signal was fitted by a 4-parameter logistics curve using XL-fit 5 (IDBS software):

luminescence = bottom + (top-bottom) / ( 1 + 10 ^{(log IC50 log |tpd| l hl111} )

[cpd] is the compound concentration tested hill is the Hill-coefficient. Bottom and top are the asymptotic minimum and maximum of the curve.

[0098] Combination Index (Cl) determination. A combination index (Cl) is determined for each everolimus:docetaxel dose using Calcusyn which is based on Chou and Talalay's theory of dose-effect analysis, in which a "median-effect equation" has been used to calculate a number of biochemical equations that are extensively used in the art. Derivations of this equation have given rise to higher order equations such as those used to calculate Combination Index (Cl). As mentioned previously, Cl can be used to determine if combinations of more than one drug and various ratios of each combination are antagonistic (CI>1.1), additive (0.9<CI>1.1) or synergistic (CKO.9). Cl plots are typically illustrated with Cl representing the y-axis versus the proportion of cells affected, or fraction affected (f _a ), on the x-axis (see Figure 1). In addition, Cl is defined for a certain percentage cell viability (V), which is the signal related to a non-exposed control: V = 100 % x luminescencereated,t=72h / luminescence _{Ua reated,t} =72h· The concentrations of the two compounds cpdl and cpd2 needed to reach a certain percentage cell viability V in combination are then compared to the concentrations needed as single agents:

Cl ₍ 100-19 = [cpdl]vl IC _(100-Vj,cpdl + [cpd2]y / IC _(100-Vj,cpd2

[0099] For example [cpdl] 50 signifies the concentration of cpdl in a mixture that gives 50% viability. ICso. _cpdi would signify the IC50 of cpdl alone. The Cl is labelled by %-effect, to follow conventions, so CI75 signifies the Cl at 25 % viability. The data from these studies show the Cl of each cell line as a function of drug:drug ratio and illustrate that particular combinations of docetaxel and everolimus are antagonistic while others are synergistic or additive. [0100] Curve shift analysis. This analysis provides a visual confirmation of synergy. The concentrations of the mixtures of compounds 1 and 2 ( cpdl and cpd2), and the single agents, were expressed in terms of IC50 equivalents (in‘units’ of IC50):

[mix] = [cpdl] / ICso.cpdi + [ cpd2 ] / ICso,cpd2

[0101] The dose-response signal was fitted by a 4-parameter logistics curve using XL-fit 5 (IDBS software)

luminescence = bottom + (top-bottom) / (1 + 10 ^{(log X log l} "" ^{x | 1 1} " ¹¹¹ )

[0102] Here hill is the Hill-coefficient and X the inflection point of the curve. Bottom and top are the asymptotic minimum and maximum of the curve. Because [mix ] is expressed in terms of IC50 equivalents, the curves of the single agents will overlap and their inflection point will lie at a value of 1. The IC50 values that are used in the calculations, are those determined in parallel for the single agents.

[0103] In mixtures where synergy is absent, curves will overlap those of the single agents. In mixtures where there is synergy, curves will shift leftward towards lower IC50 equivalents: the mixture appears more potent than expected on basis of the individual constituents. This is a good indicator of synergy (Zhao et al. Clin. Cancer Res. 10: 7994-8004).

[0104] Isobolograms. An isobologram is a dose-oriented plot which reveals whether drug combinations are synergistic. It is defined at a certain effect level, which is usually 75 %. If the single agent curves do not achieve this efficacy level, the isobologram level is set at 50 % or 30 %. If single agents do not reach the 30 % effect, no isobologram is drawn. On the axis, the calculated doses of the single compounds are plotted that give the pre-set growth effect. Both points are connected with a straight line (additivity line). For the drug combinations, it is calculated which dilutions give the pre-set growth effect and the concentrations of the individual components at this point are plotted in the isobologram. In case of an additive drug effect, the drug combination will lie close to the additivity line. In case of synergy or antagonism, the points will lie under or above the line, respectively.

[0105] As indicated above, the data from these studies show the Cl of each cell line as a function of drug:drug ratio and illustrate that particular combinations of docetaxel and everolimus are antagonistic while others are synergistic or additive. In addition, Figure 2 shows the cell viability of C-33 A cells as a function of drug concentration for free docetaxel, free everolimus and 3 combinations of docetaxel/everolimus as both overlays and as an Isobologram with 30% effect level.

Example 2

In Vivo Pharmacokinetic Analysis of Docetaxel and Everolimus Drug Conjugates

[0106] For pharmaceutical formulations in particular it is important for the drugs to exhibit prolonged circulation in vivo. In order to determine whether docetaxel and everolimus drug conjugates formulated in nanoparticles showed extended circulation lifetimes, ProCet (docetaxel- cholesterol) and ProEver (everolimus-cholesterol) single agent nanoparticles were formed and plasma levels in mice were measured over time.

[0107] Docetaxel and everolimus drug conjugates were generated by separately linking each drug to cholesterol through a diglycolate linker.

[0108] Synthesis of drug conjugates: diglycolic anhydride was added slowly to a solution of cholesterol in pyridine. The solvent was then removed and the crude product was washed with water and 0.1M HC1. The crude product was recovered by filtration, then dissolved in methylene chloride and purified by silica gel chromatography by gradient elution with methanol. The purified cholesteryl diglycolate was then conjugated to docetaxel or everolimus through standard esterification conditions, and purified using silica gel chromatography.

[0109] Each drug conjugate is combined with one or more co-polymers (e.g. PLA-PEG or PLGA-PEG) to generate nanoparticles. Preferred copolymers are PEG copolymers that were used at the following molecular weights (“K” refers to thousands): PLA(10K)-PEG(5K);

PLA(8K)-PEG(5K); PLA(13K)-PEG(2K); PLA(14K)-PEG(4K); PLA(11K)-PEG(5K);

PLGA( 10K)-PEG(5K) ; PLGA(10K)-PEG(2K); and PLGA(15K)-PEG(5K).

[0110] Nanoparticles comprising PLA-PEG showed optimal size and stability at a 2: 1 PLA:PEG ratio, and PLA(10K)-PEG(5K) was used in the studies below. Different

dmg:polymer ratios were evaluated for the ability to coordinate release rates of the two drug conjugates (see Figures 4A-4C as discussed below). Nanoparticles were generated by rapidly mixing water and a miscible solvent containing the formulation components in a confined space using carefully controlled flow rates. Under conditions where the rate of mixing exceeds the rate of precipitation, these devices have been shown to achieve homogenous particle formation kinetics, resulting in minimum sized particles for a particular formulation composition. [0111] Female BDF-1 mice (7-10 days old; 18-26 grams) were administered the nanoparticle-containing drug conjugates at a drug dose of 10 mg/kg and at the indicated time points (3 mice per time point), blood was collected by cardiac puncture and placed into EDTA coated microtainers. The samples were centrifuged and plasma was carefully transferred to another tube. Plasma docetaxel-cholesterol and everolimus-cholesterol levels were determined by HPLC-UV methods. The total drug (counts) per mouse in the blood compartment was calculated assuming a plasma volume of 0.04125 mL per gram of body weight.

[0112] As shown in Figures 3A and 3C, the concentration of ProCet and ProEver, respectively, were maintained in the plasma over time and at about the same rate.

[0113] As shown in Figures 3B and 3D, the percent injected dose of each drug conjugate, reached the level of approximately 50% of the injected dose for 12 and 16 hours for ProEver and ProCet, respectively.

[0114] The results in Figures 4A-5C show the percent injected dose of each drug conjugate when formulated in nanoparticles with PLA-PEG at either a 1: 1: 12 drug:drug:polymer ratio (Figure 4A), a 1: 1:9 dmg:dmg:polymer ratio (Figure 4B), or a 1: 1:6 dmg:drug:polymer ratio (Figure 4C). These results show that the 1: 1: 12 drug to polymer ratio is optimal for extended circulation half-life of both ProCet and ProEver.

Example 3

Docetaxel and Everolimus Co-Formulated in Nanoparticles Demonstrate Ratio-Dependent

Efficacy

[0115] To maximize the therapeutic activity of drug combinations and to capture the non- antagonistic benefits observed in vitro , the drug combination needs to be delivered to the disease site at the optimal drug to drug ratio(s). Nanoparticle formulations containing the two drug conjugates at fixed ratios shown to be non-antagonistic in tissue culture were developed allowing extended and coordinate in vivo drug release (see Figures 3A-3D). The antitumor activity of these formulations was then evaluated in PC3 human prostate cancer mouse models (Figure 5).

[0116] Taxane and everolimus drug conjugates were generated by separately linking each to cholesterol through a diglycolate linker as described in Example 2. Nanoparticles comprising PLA(10K)-PEG(5K) were formed with docetaxel-cholesterol and everolimus-cholesterol drug conjugates at either a 1: 1, 1:5, 1: 10 or 5: 1 ratio. [0117] In order to perform tumor studies in mice, animals are inoculated subq. with about lxlO ⁶ PC3 tumors cells which were then allowed to grow for 14 days prior to initiation of treatment. Using a 28g needle, mice are inoculated subcutaneously with ~ 1 x 10 ⁶ tumor cells on day 0 (one inoculum/mouse) in a volume of 50 pL. When tumors reach a defined size of approximately 150-to-250 mm ³ , either one-day prior to treatment or on the day of treatment (-day 10-14), all tumors are measured. After selecting the appropriate tumor sizes, excluding tumors too small or large, the tumors are randomly distributed (n=10) and the mean tumor volume of the groups are determined.

[0118] Mice are organized into appropriate groups and consist of control and treatment groups such as, saline control, vehicle control, positive control and various dilutions of test articles. Mice are injected intravenously or via oral gavage for the everolimus control agent with the required volume of sample to administer the prescribed dose to the animals based on individual mouse weights as outlined in Table 1 below:

Table 1: Treatment Regimens

[0119] Tumor growth measurements were monitored using vernier calipers beginning on the day of treatment. Tumor length measurements (mm) were made from the longest axis and width measurements (mm) will be perpendicular to this axis. From the length and width

measurements tumor volumes (cm ³ ) were calculated according to the equation (L X W ² /2). Animal weights were collected at the time of tumor measurement. [0120] Individual mouse body weights were recorded at various days (generally two days apart such as Monday, Wednesday and Friday) during the efficacy study and for a period of 14-days after the last dosing.

[0121] All animals were observed at least once a day, more if deemed necessary, during the pre-treatment and treatment periods for mortality and morbidity. In particular, signs of ill health are based on body weight loss, change in appetite, rough coat, lack of grooming, behavioral changes such as altered gait, lethargy and gross manifestations of stress. If signs of severe toxicity or tumor-related illness were, the animals were euthanized (CO2 asphyxiation) and a necropsy performed to assess other signs of toxicity. Moribund animals must be terminated for humane reasons and the decision to terminate was at the discretion of the Animal Care

Technician and the Study Director/Manager. Any and all of these findings were recorded as raw data and the time of death will be logged as the following day.

[0122] Data are presented in either tabular or figure form as follows:

Plot of individual mouse tumor volumes with respect to each group, prior to treatment start and after grouping.

Mean body weights for each group as a function of time.

Mean tumor volumes for each group as a function of time.

Raw data including figures and tables are generated and include tumor growth vs. time, tumor growth inhibition, and tumor growth delay.

Summary of abnormal or remarkable observations.

[0123] Any individual animal with a single observation of > than 30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized. Any group with a mean body weight loss of >20 % or >10% mortality was to have dosing stopped. The group would not be euthanized and recovery would be allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint would be euthanized. If the group treatment related body weight loss was recovered to within 10% of the original weights, dosing could resume at a lower dose or less frequent dosing schedule. Exceptions to non treatment body weight % recovery could be allowed on a case-by-case basis. [0124] Endpoint: Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1000 mm ³ or 60 days, whichever came first. Responders could be followed longer.

[0125] As shown in Figure 5A, mice were organized into control (“Untreated”) and treatment groups including empty nanoparticles (“Vehicle”), free docetaxel, free everolimus, free drug cocktail and docetaxel/everolimus co-loaded in PLA-PEG nanoparticles (“NPD:E”) resulting in a final docetaxel: everolimus ratio of 1: 1 (5:5 docetaxel: everolimus mg/kg), 1:5 (5:25 docetaxel: everolimus mg/kg), 1: 10 (5:50 docetaxel: everolimus mg/kg), or 5: 1 (25:5 docetaxel: everolimus mg/kg). Female NCr nu/nu mice were dosed post tumor cell inoculations as per Table 1 for the nanoparticle formulations (free drugs were given using standard dosing schedules as listed in Table 1 and shown in Figure 65A). Animals were weighed and monitored for survival and in-life observations are collected at the time of weight measurement. Figure 5A illustrates the results of these experiments.

[0126] As Figure 5A indicates, a significantly enhanced antitumor activity for the nanoparticle formulation containing docetaxel: everolimus drug conjugates co-loaded at about a 5: 1 ratio was observed compared to each single agent, as well as free drug cocktail. These results demonstrate that fixing non-antagonistic docetaxel: everolimus ratios by encapsulating them inside appropriately designed nanoparticles can dramatically improve antitumor activity.

[0127] The graphs in Figure 6 show the percent change in body weight over time for these treatment groups. As shown, there was improved body weight gain in the nanoparticle-treated groups compared to the free drugs.

[0128] As shown in Figures 5B and 5C, mice were dosed with increasing amounts of docetaxel and/or everolimus. Treatment groups included empty nanoparticles (“Vehicle”), free docetaxel (5 mg/kg), free everolimus (20 mg/kg), free drug cocktail (docetaxekeverolimus, 5 mg/kg:20mg/kg), docetaxel/everolimus co-loaded into nanoparticles (“DENP”) resulting in a final docetaxekeverolimus ratio of 1: 1 (5:5 docetaxekeverolimus mg/kg), or 5: 1 (45:9 docetaxekeverolimus mg/kg), docetaxel and /everolimus separately loaded into nanoparticles and co-administered (“NPD+NPE”) resulting in a final docetaxekeverolimus ratio of 5: 1 (15:3 docetaxekeverolimus mg/kg), and docetaxel (15 mg/kg) and everolimus ( 3 mg/kg) each separately encapsulated in nanoparticles and administered alone (“NPD” and “NPE”, respectively). As shown in Figure 5B, increasing the amount of docetaxel and thus increasing the ratio of docetaxel to everolimus above 1: 1 results in enhanced efficacy.

[0129] Mice were also organized into treatment groups for various DENP formulations at increasing drug doses. As shown in Figure 5C, treatment groups included empty nanoparticles (“Vehicle”), and docetaxel/everolimus co-loaded into nanoparticles (“DENP”) resulting in a final docetaxel: everolimus ratio of 1: 1 (10: 10 docetaxel: everolimus mg/kg), high dose 5: 1 (60: 12 docetaxel: everolimus mg/kg), 2: 1 (12.5:6 docetaxel: everolimus mg/kg), and 5: 1 (15:3 docetaxel: everolimus mg/kg). Similar to Figure 5B, the results show that increasing the dosage amount of docetaxel at the 5: 1 ratio results in enhanced efficacy compared to a 1: 1 ratio.

[0130] Figure 7 is a graph showing the percent change in mean body weight for treatment groups that were given either free or nanoparticle-encapsulated docetaxel or everolimus (see Table 2 below). The groups were dosed using 30 mg/kg, 45 mg/kg, 60 mg/kg or 90 mg/kg of docetaxel in a nanoparticle (“DNP”) and shows that there was no impact on body weight at doses up to 60 mg/kg. Other groups were dosed with 15 mg/kg, 30 mg/kg, 45 mg/kg or 60 mg/kg everolimus in a nanoparticle (“ENP”) and the graph in Figure 7 shows that there was a reduction in body weight at amounts above 15 mg/kg encapsulated everolimus.

Table 2: Treatment Groups

Example 4

Efficacy of Docetaxel and Everolimus Nanoparticles in Breast Cancer Tumor Models

[0131] The antitumor activity of nanoparticle formulations containing the two drugs at various fixed drug ratios as described above were evaluated in the human breast adenocarcinoma xenograft mouse model with the human breast cancer cell line MDA-MB-231. The MDA-MB- 231 cell line is a human breast cancer cell line that was established from a pleural effusion of a 51 -year-old Caucasian female with a metastatic mammary adenocarcinoma and is one of the most commonly used breast cancer cell lines in medical research laboratories. MDA-MB-231 is a "basal" type and triple negative (ER, PR and HER2 negative) breast cancer cell line and is wild type for PTEN (Phosphatase and Tensin Homolog). It is generally considered as a positive control for the MDA-MB-468 breast cancer cell line which has a PTEN deletion (discussed in Example 5).

[0132] The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORCl, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORCl/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORCl and mTORC2; and GDC-0941 is a pan- PI3K inhibitor. The level of potentiation of everolimus inhibitory activity (measured by IC ₅ o values) has been found to be cell line-specific and it is known that these breast cancer cell lines are sensitive to everolimus.

[0133] Tumors were grown and measured as described above and female NCr nu/nu mice were similarly treated and monitored. Mice were dose as outlined in Table 3:

Table 3: Treatment Regimens

[0134] As shown in Figure 10A, mice were organized into treatment groups including empty nanoparticles (“Vehicle”), free drug cocktail (both drugs at 5 mg/kg or “5mpk”) and docetaxel/everolimus co-loaded in PLA-PEG nanoparticles (“NPD:E”) resulting in a final docetaxel: everolimus drug conjugate ratio of 1: 1 (5:5 docetaxel: everolimus mg/kg), 1:5 (5:25 docetaxel: everolimus mg/kg), 1: 10 (5:50 docetaxel: everolimus mg/kg), or 5: 1 (25:5 docetaxel: everolimus mg/kg). Mice were dosed post tumor cell inoculations as per Table 2 for the nanoparticle formulations (free drugs were given using standard dosing schedules as shown in Table 2 and Figure 8). Animals were weighed, monitored for survival, tumors were measured and in-life observations are collected at the time of measurements. Figure 9 illustrates the results of these individual experiments and shows significant tumor regression (9/9 mice) in the nanoparticle formulation containing docetaxel: everolimus drug conjugates co-loaded at about a 5: 1 ratio (“NP D:E 5: 1”).

[0135] As Figure 8 summarizes, a significantly enhanced antitumor activity for NP D:E 5: 1 (25mg:5mg) was observed compared to the free drug cocktail (p<0.0001) as well as the other nanoparticle formulations. These results demonstrate that fixing docetaxekeverolimus at a 5: 1 ratio by encapsulating them inside appropriately designed nanoparticles can dramatically improve antitumor activity in vivo.

Example 5

Efficacy of Docetaxel and Everolimus Nanoparticles in Tumor Models with PTEN Mutations

[0136] The antitumor activity of nanoparticle formulations containing the two drugs at various fixed drug ratios as described in above were evaluated in a PTEN mutated human breast adenocarcinoma xenograft mouse model with the human tumor cell line MDA-MB-468. [0137] Tumors were grown and measured as described above with the exception that female SCID-beige mice were inoculated with 5xl0 ⁶ MDA-MB-468 tumor cells in 50% Matrigel subq. in flank. Mice were similarly treated and monitored as before and dosed as outlined in Table 4:

Table 4. Treatment Regimens

[0138] As shown in Figure 8, mice were organized into control (“Untreated”) and treatment groups including empty nanoparticles (“Vehicle”), free docetaxel (5 mg/kg), free everolimus (5 mg/kg), free drug cocktail (both 5 mg/kg) and docetaxel/everolimus drug conjugates co-loaded in PLA-PEG nanoparticles (“NPD:E” or“NP DOC:EV”) resulting in a final docetaxel: everolimus ratio of 1:1 (5:5 docetaxel: everolimus mg/kg), 1:5 (5:25 docetaxel: everolimus mg/kg), 1:10 (5:50 docetaxel: everolimus mg/kg), or 5:1 (25:5 docetaxekeverolimus mg/kg). Mice were dosed post tumor cell inoculations as outlined in Table 4 for the nanoparticle formulations (free drugs were given using standard dosing schedules as shown in Table 4 and Figure 10A). For the 1:1 nanoparticle formulation, two different dosing schedules were tested; one on days 1, 8 and 15 as indicated above and the other on a q4d dosing schedule (days 1, 5, and 9). The nanoparticle formulations were dosed on days 1, 8, and 15 (free drugs were given using standard dosing schedules as shown in Figure 10B). Animals were weighed and monitored for survival and in-life observations at the time of weight measurement. Figure 11 illustrates the results of these experiments for individual mice in each treatment group. Furthermore, Figure 12 shows the mean percent body weight for each of the treatment groups. Tolerability, expressed as the percent change in body weight of the mice, from the studies presented in Examples 3-5 are summarized in Table 5 below:

Table 5. Summary of Percent Change in Body Weight with Nanoparticle (“NP”) or Free Drugs

[0139] The results in Figure 10A and 10B show that all of the nanoparticle formulations exhibited enhanced efficacy in comparison to the free drugs and the free drug cocktail as well as the empty nanoparticle control.

Example 6

Efficacy of Docetaxel and Everolimus Nanoparticles in Ovarian Cancer Tumor Models

[0140] The antitumor activity of nanoparticle formulations containing the two drugs at various fixed drug ratios as described above are evaluated in the human ovarian carcinoma xenograft mouse model with the human ovarian cancer cell line A2780. The A2780 cell line is a human ovarian cancer cell line that was established from an ovarian endometroid adenocarcinoma tumor in an untreated patient so the cell line has not been exposed to any anticancer drugs or chemicals. The cell line has an epithelial morphology and it is commonly used as a model to observe the effects of, and test the potency of various chemicals, methods of delivery and treatments for ovarian cancer [0141] Tumors are grown and measured as described above and female NCr nu/nu mice are similarly treated and monitored. Mice are dosed as outlined in Table 6:

Table 6: Treatment Regimens

#-Control Group:

Group 1: Dose Vehicle 1 30 minutes prior to Vehicle 2

Groups 4, and 8: Dose Everolimus 30 minutes prior to Docetaxel

[0142] Vehicle 1 = 0.5% Methylcellulose : 0.2% Tween 80 in DI Water; 0.5%

Methylcellulose : 0.2% Tween 80 in DI Water. Vehicle 2 = 7.5% Ethanol : 7.5% Tween 80 in D5W; 7.5% Ethanol : 7.5% Tween 80 in D5W. Dosing volume = 10 mL/kg (0.200 mL/20 g mouse). Adjust volume accordingly for body weight.

[0143] Mice are organized into treatment groups including empty nanoparticles (“Vehicle 1” and“Vehicle 2”), free drug cocktail (everolimus at lmg/kg or 0.25 mg/kg and docetaxel at lOmg/kg) and docetaxel/everolimus co-loaded in PLA-PEG nanoparticles (“NP DOC:EV”) resulting in a final docetaxel: everolimus drug conjugate ratio of 10:1 (10:1 docetaxel: everolimus mg/kg), 20:1 (10:0.5 docetaxel: everolimus mg/kg), or 40:1 (10:0.25 docetaxel: everolimus mg/kg). Mice are dosed post tumor cell inoculations as per Table 6 for the nanoparticle formulations (free drugs are given using standard dosing schedules as shown in Table 6). Animals are weighed, monitored for survival, tumors are measured and in-life observations are collected at the time of measurements.

[0144] These studies can demonstrate that fixing docetaxeheverolimus at a synergistic ratio by encapsulating them inside appropriately designed nanoparticles may dramatically improve antitumor activity in vivo.

Example 7

Stability of Docetaxel and Everolimus Drug Conjugates

[0145] The practical application of nanoparticle preparations as drug delivery vehicles is limited by the chemical and physical stability of the preparation. Commercialization requires long term stability at both the chemical and physical levels.

[0146] The stability of nanoparticle formulations comprising docetaxel and everolimus cholesterol conjugates (at a 1: 1 ratio) was monitored by HPLC after long-term storage at 4°C and room temperature (“RT”).

[0147] Nanoparticle formulations stored in 300 mM sucrose solution at 4°C or RT were found to be physically stable for three months by both visual inspection and DLS size measurements. As shown in Figure 13A, the concentration of docetaxel-cholesterol conjugate (“ProCet”) was maintained for up to 3 months. Similarly, Figure 13B shows the same results for the everolimus drug conjugate, ProEver. The concentration of the drug conjugates is maintained in the dosing solution at about 95% for up to 3 months demonstrating significant retention of the drugs upon storage. In addition, no particle size change was observed in either 4°C or room temperature storage conditions.

[0148] The above examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Many variations to those described above are possible. Since modifications and variations to the examples described above will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.