WO2014031856A1 - Combination therapy using pi3 kinase and braf inhibitors - Google Patents

Description

COMBINATION THERAPY USING PI3 KINASE AND BRAF INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No. 61/692,625, filed August 23, 2012, which is incorporated in its entirety herein by reference.

BACKGROUND OF THE INVENTION [002] There is a need for new therapeutic regimens for treatment of cancer.

SUMMARY OF THE INVENTION

[003] Provided herein is a method for treating cancer in an individual in need thereof comprising administration of a B-raf inhibitor and a PI3 kinase inhibitor to the individual.

[004] In some embodiments, the PI3 kinase inhibitor is a wortmannin analog.

[005] In some embodiments, the PI3 kinase inhibitor is a compound selected from

Formula IIA Formula IIB

wherein Y is a heteroatom selected from nitrogen and sulfur and R 1 and R 2 are

independently selected from an unsaturated alkyl, cyclic alkyl, or R 1 and R 2 together with Y form ajieterocycle.

[006] In some embodiments, the PI3 kinase inhibitor is an irreversible PI3 kinase inhibitor.

[007] In some embodiments, the PI3 kinase inhibitor is

[008] In some embodiments, the B-raf inhibitor is selected from

[009] In some embodiments, the B-raf inhibitor is vemurafenib.

[010] In some embodiments, the B-raf inhibitor is PLX 4720.

[011] In some embodiments, the cancer is a BRAF mutant cancer. [012] In some embodiments, the cancer is selected from the group consisting of head and neck cancer, lung cancer, ovarian cancer, colon cancer, breast cancer, pancreatic cancer, cervical cancer, prostate cancer, and melanoma.

[013] In some embodiments, the cancer is melanoma.

[014] In some embodiments, the cancer is advanced-BRAF mutant melanoma.

[015] In some embodiments, the individual has not been treated with a selective BRAF inhibitor.

[016] In some embodiments, the individual does not have any active central nervous system metastases.

[017] In some embodiments, the individual does not have poorly controlled diabetes.

[018] In some embodiments, the individual is not HIV-positive.

[019] In some embodiments, PX-866 is administered orally at doses of 6 mg or 8 mg once per day on days 1-28 of the first 28-day cycle of therapy.

[020] In some embodiments, vemurafenib is administered orally at doses of 720 mg or 960 mg twice per day on days 9-28 of the first 28-day cycle of therapy.

[021] In some embodiments, both PX-866 and vemurafenib are administered on days 1-28 of subsequent cycles of therapy.

[022] In some embodiments, PX-866 is administered orally at doses of 6 mg or 8 mg once per day and vemurafenib is administered orally at doses of 720 mg or 960 mg twice per day.

[023] In some embodiments, 1-6 cycles of therapy are administered to the individual.

[024] In some embodiments, more than 6 cycles of therapy are administered to the individual.

[025] Further proided herein is a method for treating a BRAF mutant cancer in an individual in need thereof comprising administration of PX-866 and vemurafenib to the individual in need thereof.

INCORPORATION BY REFERENCE

[026] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

[027] Provided herein, in certain embodiments, are methods for treating cancer in a subject with a combination therapy of a BRAF inhibitor and a PI3 kinase inhibitor. In some embodiments, a PI3 kinase inhibitor is a wortmannin analog. Also provided herein are compounds, pharmaceutical compositions and medicaments comprising such compounds for a combination therapy of a cancer. Phosphatidylinositol-3-kinase (PI-3K)

[028] Phosphatidylinositol-3 -kinases (PI-3K) are a family of intracellular lipid kinases that play a critical role in transmitting signals from cell surface receptors on the plasma membrane to downstream signaling intermediates. There are three classes of PI-3K, classified based upon their structure and substrate specificity. Class I PI-3K are heterodimers formed by a regulatory subunit and a catalytic pi 10 subunit that phosphorylate membrane-associated phosphatidylinositol 4,5- bisphosphate (PIP2) to form phosphatidylinositol 3, 4, 5-trisphosphate (PIP3). PIP3 binds to the serine/threonine protein kinase AKT, which is the primary effector of PI-3K, triggering activation of downstream signaling intermediates including mTOR, with subsequent effects on cell growth and metabolism, survival, proliferation, and angiogenesis. The tumor suppressor gene phosphatase and tensin homolog (PTEN) counteracts the activity of Class I PI-3K by dephosphorylating PIP3 back to PIP2. PI-3K activation affects other AKT-independent pathways including Bruton tyrosine kinase and Tec family kinases, serum and glucocorticoid regulated kinases, and regulators of GTPases, although the role of these pathways is less well-defined.

[029] Class I PI-3K is further divided into Class IA and Class IB subfamilies. Class IA PI-3K are formed by a regulatory p85 subunit (PIK3R1) and a catalytic pi 10 subunit that are primarily activated by receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), IGF, PDGF, and Her2/neu. Several isoforms exist for each subunit, including α, β and δ isoforms of pi 10. The a and β isoforms are expressed ubiquitously, whereas expression of the δ isoform is restricted to leukocytes. Class IB PI-3Ks are composed of a pi 10 subunit and a plOl regulatory subunit.

Phosphatidylinositol-3-kinase (PI-3K) in Cancer

[030] Increased signaling through Class IA PI-3Ks has been implicated in many different forms of cancer, making it a rational and attractive target for drug development. Cancers in which PI-3K pathway abnormalities have been identified include NSCLC, breast carcinoma, ovarian carcinoma, endometrial carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck

(SCCHN), cervical cancer, glioblastoma, melanoma, and colorectal carcinoma (CRC), among others. Mechanisms that lead to increased signaling through the PI-3K pathway include increased RTK activity (e.g., EGFR in CRC, SCCHN, and NSCLC), activating mutations in the pi 10a isoform, mutations in the p85 subunit, and mutations and deletions in PTEN. Amplification of the PI-3K catalytic subunit alpha (PIK3CA) gene has also been observed in a number of tumors, including over 30% of SCCHN. In melanoma, reports of somatic mutations and deletions in PTEN have ranged from 29% to 43%. [031] Increased signaling through Class IA PI-3Ks has been implicated in many different forms of cancer. Cancers in which PI-3K pathway abnormalities have been identified include non-small cell lung cancer (NSCLC), breast carcinoma, ovarian carcinoma, endometrial carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck (SCCFiN), cervical cancer, glioblastoma, melanoma, and colorectal carcinoma. PI-3Ks are also contemplated in other cancers. Reported mechanisms which lead to increased signaling through the PI-3K pathway include increased receptor tyrosine kinase (RTK) activity, activating mutations in the pi 10 a isoform, mutations in the p85 subunit, and mutations and deletions in PTEN. Amplification of the PIK3CA gene has also been observed in a number of tumors, including squamous cell carcinomas of the lung and head and neck, although this observation has not yet been linked directly to increased PI-3K activity. BRAF

[032] BRAF is a human gene coding for the protein B-Raf. The gene is also referred to as proto- oncogene B-Raf and v-Raf murine sarcoma viral oncogene homo log Bl, while the protein is more formally known as serine/threonine -protein kinase B-Raf (BRAF). The B-Raf protein is involved in sending signals inside cells, which are involved in directing cell growth. BRAF inhibition causes programmed cell death in cancer cells (e.g., melanoma cell lines) by inhibition of the BRAF/MEK/ERK pathway.

[033] Melanoma cases in the US are increasing, with an estimated incidence of > 65,000 cases in 2010, with approximately 9,000 deaths. A key discovery is the finding that approximately 50% of melanomas possess an activating mutation in the BRAF gene (primarily T1799A: V600E) (that is, at amino acid position number 600 on the B-Raf protein, the normal valine is replaced by glutamic acid). This discovery, in turn, has allowed for the development of drugs selectively targeting mutant BRAF. Vemurafenib (also known as PLX4032, RG7204 or R05185426) and GSK2118436 are potent and selective inhibitors of the most common mutant forms of BRAF. Phase 1, 2, and 3 clinical studies of vemurafenib, a selective BRAF inhibitor, have been completed. Melanoma cells without this mutation are not inhibited by vemurafenib; the drug paradoxically stimulates normal BRAF and may promote tumor growth in such cases.

[034] Vemurafenib has been approved by the FDA for treatment of melanoma. Relapses after treatment with vemurafenib are unfortunately inevitable. Median PFS was approximately 7 months for the initial vemurafenib Phase 1 extension study. The experience with targeted therapies in other diseases such as chronic myelogenous leukemia (CML) and non-small cell lung cancer (NSCLC) suggests that understanding the molecular origins of resistance will lead to improved patient outcomes and the development of effective second-line targeted therapies. Mechanisms of Resistance to Selective BRAF Inhibition

[035] Published clinical data with the first generation selective BRAF inhibitors have shown antitumor activity in advanced incurable melanoma. Responses have been limited to those patients with V600E BRAF mutant melanoma. Despite these recent advances, disease progression following treatment with selective BRAF inhibitors appears inevitable for most patients. Median PFS from published reports is approximately 7 months, although the database is small and therefore the confidence intervals around this point estimate are still broad.

[036] Preclinical insights regarding the mechanisms of resistance to BRAF inhibitors and analysis of on-treatment tumor biopsies in patients receiving selective BRAF inhibitors suggest that resistance is mechanistically strikingly different from patterns of resistance to other tyrosine kinase inhibitors. In particular, mutations in the target kinase that prevent the drug from binding have not been detected. Instead, a variety of Oncogene by-pass' events emerge that allow the cell to evade the effects of BRAF pathway blockade by the drug. To date, observations of resistance include the following

• BRAF pathway by-pass through activation of RAF 1 and COT/TP L2, thereby restoring MEK activation

• Activating mutations in NRAS that restore MEK activation

• Increased activation of the receptor tyrosine kinase PDGFRp, which acts via an

unspecified MEK-independent mechanism

• RAF kinase switching such that cells use the CRAF and ARAF isoforms to activate the MAPK pathway

• Enhanced IGF-1R and PI-3K/AKT activity in melanoma cell lines resistant to BRAF inhibitors. Preclinical studies combining IGF-1R/PI-3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells

• In a tumor sample obtained at the time of progression, homozygous deletion of PTEN was observed, a mutation that was not present in the pre -treatment biopsy.

• MEK1 mutation also has been reported as a mechanism of resistance.

• Novel BRAF(V600E) splicing variants lacking the RAS-binding domain resulting in a novel form of BRAF(V600E) that facilitates enhanced BRAF dimerization with low RAS activation and ERK signaling in the presence of vemurafenib.

[037] A variety of clinical study strategies are underway or planned to prevent and/or overcome resistance to BRAF inhibitors, including combining selective BRAF and MEK inhibitors or studies designed to overcome the induced oncogene by-pass events. However, Applicants took a different approach. However, combination therapy using targeted agents presents numerous challenges. 1. In particular, it is ultimately likely that combination therapy targeting multiple pathways will be required in order to optimize patient therapy and prolong survival

2. Combining targeted agents is not always straightforward. The drugs do have significant toxicities alone and with sometimes unpredictable and severe side effects in combination. Careful Phase 1 studies with a limited number of experimental agents being given at the same time with PK and pharmacodynamic endpoints are mandatory before more elaborate regimens can be contemplated and evaluated in the clinic

3. Strategies to prospectively identify patients that will benefit from novel combinations as a first line strategy, or at the time of progression on vemurafenib therapy, are currently unavailable.

[038] Given the unpredictable nature of combination therapy, and unpredictable reasons for development of resistance to BRAF inhibitors in the clinic, Applicants explored combination therapy for treatment of cancer using vemurafenib in combination with a PI3 kinase inhibitor (e.g., PX-866).

Treatment of Cancer

[039] Cancers treatable by combination therapies described herein include, but are not limited to, breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, parapharyngeal cancer, gastrointestinal cancer, glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, renal cancer, pancreatic cancer, retinoblastoma, cervical cancer, uterine cancer, Wilm's tumor, multiple myeloma, skin cancer, lymphoma, leukemia, blood cancer, anaplastic thyroid tumor, sarcoma of the skin, melanoma, adenocystic tumor, hepatoid tumor, non-small cell lung cancer, chondrosarcoma, pancreatic islet cell tumor, prostate cancer including castration resistant forms, ovarian cancer, and/or carcinomas including but not limited to squamous cell carcinoma of the head and neck, colorectal carcinoma, glioblastoma, cervical carcinoma, endometrial carcinoma, gastric carcinoma, pancreatic carcinoma, leiomyosarcoma and breast carcinoma. In some embodiments, the combination therapies described herein treat a lung cancer such as non-small cell lung cancer (NSCLC). In other embodiments, the combination therapies described herein treat a head and neck cancer such as squamous cell carcinoma of the head and neck (SCCHN). In other embodiments, the combination therapies described herein treat a melanoma. In other embodiments, the combination therapies described herein treat a BRAF inhibitor-resistant melanoma. In other embodiments, the combination therapies described herein treat a treatment naive melanoma.

[040] The combination therapies described herein treat various stages of cancer including stages which are locally advanced, metastatic and/or recurrent. In cancer staging, locally advanced is generally defined as cancer that has spread from a localized area to nearby tissues and/or lymph nodes. In the Roman numeral staging system, locally advanced usually is classified in Stage II or III. Cancer which is metastatic is a stage where the cancer spreads throughout the body to distant tissues and organs (stage IV). Cancer designated as recurrent generally is defined as the cancer has recurred, usually after a period of time, after being in remission or after a tumor has visibly been eliminated. Recurrence can either be local, i.e., appearing in the same location as the original, or distant, i.e., appearing in a different part of the body. In certain instances, a cancer treatable by combination therapies described herein is unresectable, or unable to be removed by surgery. In further instances, a cancer treatable by the combination therapies described herein is incurable, i.e., not treatable by current treatment methods.

[041] In some embodiments, the combination therapies described herein are administered as a first-line or primary therapy, i.e. subjects are treatment naive. Other subjects suitable for treatment by the combination therapies described herein include those that have completed first-line anticancer therapy. First-line anti-cancer therapies include chemotherapy, radiotherapy,

immunotherapy, gene therapy, hormone therapy, surgery or other therapies that are capable of negatively affecting cancer in a patient, such as for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[042] In additional emboidiments, subjects suitable for treatment by the combination therapies described herein include those that are administered a combination of a PI3 kinase inhibitor and a BRAF inhibitor, in combination with one or more than one additional therapy selected from chemotherapy, radiotherapy, immunotherapy, gene therapy, hormone therapy, surgery and/or other therapies that are capable of negatively affecting cancer in a patient, such as for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[043] Chemotherapies for first-line and subsequent therapy include, but are not limited to, hormone modulators, androgen receptor binding agents (e.g., anti-androgens, bicalutamide, flutamide, nilutamide, MDV3100), gonadotropin-releasing hormone agonists and antagonists (e.g., leuprolide, buserelin, histrelin, goserelin, deslorelin, nafarelin, abarelix, cetrorelix, ganirelix degarelix), androgen synthesis inhibitors (abiraterone, TOK-001), temozolomide, mitozolomide, dacarbazine, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, cabazitaxel, paclitaxel, gemcitabine, navelbine, farnesyl -protein transferase inhibitors, transplatinum, 5- fluorouracil, capecitabine, vincristin, vinblastin and methotrexate, topoisomerase inhibitors (e.g., irinotecan, topotecan, camptothecin, etoposide) or any derivative related agent of the foregoing. Many of the above agents are also referred to as hormone therapy agents such as, for example, androgen receptor binding agents, gonadotropin-releasing hormone agonists and antagonists, androgen synthesis inhibitors, estrogen receptor binding agents as well as aromatase inhibitors.

[044] Radiotherapies for first-line and subsequent therapy include factors that cause DNA damage and include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors include microwaves and UV- irradiation. It is likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays may range from daily doses of 50 to 200 roentgens for prolonged periods of time (e.g., 3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[045] Immunotherapies generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, a tumor antigen or an antibody specific for some marker on the surface of a tumor cell. The tumor antigen or antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. An antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. Alternatively, an tumor antigen may stimulate a subject's immune system to target the specific tumor cells using cytotoxic T cells and NK cells. Immunotherapies include Sipuleucel-T (Provenge®), bevacizumab and the like.

[046] A gene therapy includes a therapeutic polynucleotide is administered before, after, or at the same time as a combination therapy. Therapeutic genes may include an antisense version of an inducer of cellular proliferation (oncogene), an inhibitor of cellular proliferation (tumor

suppressor), or an inducer of programmed cell death (pro-apoptotic gene). [047] Surgery of some type is performed for resectable cancers. Surgery types include preventative, diagnostic or staging, curative and palliative surgery and can be performed as a first- line and subsequent therapy.

[048] In some embodiments, the combination therapies described herein are administered as a second-line therapy after a first-line therapy becomes ineffective or the cancer is recurrent. In other embodiments, the combination therapies described herein administered as a third-line therapy after the first- and second-line therapy fails. In further embodiments, individuals are preselected for having completed a first- or second-line therapy. In some instances, the combination therapies described herein are administered to patients for whom prior platinum-based therapy has failed. In other instances, the combination therapies described herein are administered to patients for whom prior irinotecan therapy has failed.

[049] Subjects, in some embodiments, can also be prescreened or preselected for sensitivity and/or effectiveness of the combination therapies described herein. A subject can be examined for certain biomarkers that allow the subject to be amenable to a combination therapy. For example, biomarkers such as phosphatase and tensin homolog (PTEN) mutations and activating mutations of PI-3K catalytic subunits may increase sensitivity to the combination therapies described herein whereas other mutations such as Ras pathway mutations may decrease sensitivity. In some embodiments, a subject is preselected based on, for example, PTEN mutational status, PTEN copy number, PI3K gene amplification, PI3K catalytic subunit alpha (PIK3CA) mutational status, K-ras mutational status, and/or B-raf mutational status. Additional biomarker candidates are

contemplated and described in the below section.

BRAF Inhibitors

[050] In some embodiments, a BRAF inhibitor for combination therapies described herein is vemurafenib. In additional embodiments a BRAF inhibitor is selected from

PI3 kinase inhibitors

[051] In some embodiments, the PI-3 kinase inhibitor is selected from PX-866, XL 147, GDC- 0941 (Genentech/Roche), CAL-101 (Calistoga), NVP-BKM120 (Novartis), ZSTK474 (Zenyaku Kogyo), NVP-BYL179 (Novartis), AMG319 (Amgen), GDC0032 (Genentech/Roche), A66, AS- 252424, AS-604850, AS-605240, AZD6482, CAY10505, CH5132799, D-106669, GSK1059615, PIK-293, PIK-90, PIK-93, GNE-490, CNX-1351 (Celgene/Avila), INK117 (Intellikine), PIK-39, BAY806946 (Bayer), XL 765 (Exelixis), GDC-0980 (Genentech), GSK 2126458

(GlaxoSmithKline), NVP-BEZ235 (Novartis), NVP-BGT226 (Novartis), PF04691503 (Pfizer), PKI587 (Pfizer), SF1126 (Semaphore), D-87503, GSK2126458, IC-87114, PI-103, PIK-294, PIK- 75, PKI-402, PKI-587 (PF-05212384), Quercetin (Sophoretin), TG100-115, TGX-221, A-769662, phenformin hydrochloride, and PP121. [052] In some embodiments, PI3 kinase inhibitors include one or more of the following: Exelixis),

Genentech/Roche),

-13-

(ZSTK474, Zenyaku Kogyo),

605240),

(CAY10505),

(GSK2126458), ¾N (IC-87114),

-16-

(Quercetin (Sophoretin))

BAY806946 (Bayer), GDC0032 (Genentech/Roche), BYL719 (Novartis), AMG319 (Amgen), CNX-1351 (Celgene/Avila), INK117 (Intellikine) and the like.

[053] Wortmannin is a naturally occurring compound isolated from culture broths of fungal strains, Penicillium wortmannin, Talaromyces wortmannin, Penicillium Funiculosum and related micro-organisms. Wortmannin irreversibly inhibits PI-3K through covalent interaction with a

802

specific lysine on the kinase: Lys of the ATP binding pocket of the catalytic site of the pi 10a

883

isoform or Lys of the p 110γ isoform. Most isoforms of PI-3K, such as p 110a, p 110β, p 1105 and pi 10γ for example, are inhibited equally by wortmannin. Wortmannin demonstrates liver and hematologic toxicity, however, and is a biologically unstable molecule. Samples stored as aqueous solutions at either 37°C or 0°C at neutral pH are subject to decomposition by hydrolytic opening of the furan ring. It has been shown that the electrophilicity of the furan ring is central to the inhibitory activity of wortmannin. The irreversible inhibition of PI-3K occurs by formation of an enamine following the attack of the active lysine of the kinase on the furan ring at position C(20) of wortmannin. Decomposition of wortmannin interferes with its inhibitory activity on PI-3Ks.

Although wortmannin is a nanomolar inhibitor of PI-3K, its instability and toxicity to the liver results in variable activity in animal models. Wortmannin analogs that improve toxicity and stability of the base wortmannin compound are described herein.

[054] In some embodiments, wortmannin analogs suitable for combination therapies described herein include compounds of Formula IA or IB:

Formula IA Formula IB

wherein:

— is an optional bond;

n is 1-6;

Y is a heteroatom;

R 1 and R 2 are independently selected from an unsaturated alkyl, non-linear alkyl, cyclic

1 2

alkyl, and substituted alkyl or R and R together with the atom to which they are attached form a heterocycloalkyl group;

R is absent, H, or Ci-C₆ substituted or unsubstituted alkyl;

R⁴ is (C=0)R⁵, (C=0)OR⁵, (S=0)R⁵, (S0₂)R⁵, (P0₃)R⁵, (C=0)NR⁵R⁶;

R⁵ is substituted or unsubstituted Ci-C₆ alkyl; and

R⁶ is substituted or unsubstituted Ci-C₆ alkyl.

[055] In some embodiments, wortmannin analogs suitable for combination therapies described herein include compounds of Formula IIA or IIB:

Formula IIA Formula IIB

wherein Y is a heteroatom and R 1 and R 2 are independently selected from an unsaturated alkyl, non-linear alkyl, cyclic alkyl, and substituted alkyl or R 1 and R 2 together with Y form a

heterocycle.

[056] In certain embodiments of compounds of formula IIA or IIB, Y is a heteroatom selected from nitrogen and sulfur and R 1 and R 2 are independently selected from an unsaturated alkyl, cyclic alkyl, or Ri and R₂ together with Y form a heterocycle.

[057] In further embodiments, a wortmannin analog is Acetic acid 4-diallylaminomethylene-6- hydroxy-l-a-methoxymethyl-10 ,13 -dimethyl-3,7,17-trioxo-l,3,4,7,10,l 1β,12,13,14α,15, 16,17- dodecahydro-2-oxa-cyclopenta[a] henanthren-l 1-yl ester (PX-866) having the structure,

[058] In yet further embodiments, a wortmannin analog is Acetic acid 6-hydroxy-la- methoxymethyl- 10β , 13 -dimethyl-3 ,7, 17-trioxo-4-pyrrolidin- 1 -methylene- 1,3,4,7,10,

1 i ,12,13,14a,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-l 1-yl (PX-867) having the structure,

[059] In additional embodiments, wortmannin analogs suitable for combination therapies described herein include compounds selected from, but not limited to, PX-868, PX-870, PX-871, PX-880, PX-881, PX-882, PX-889, PX-890, DJM2-170, DJM2-171, DJM2-177, DJM2-181 and combinations thereof. In some embodiments, wortmannin analogs suitable for combination therapies described herein include compounds described in GB Pat. No. 2302021, which compounds are incorporated herein by reference.

Further forms of Wortmannin analogs

[060] In the scope of the embodiments, wortmannin analogs include further forms of the compounds described herein such as pharmaceutically acceptable salts, solvates (including hydrates), amorphous phases, partially crystalline and crystalline forms (including all polymorphs), prodrugs, metabolites, N-oxides, isotopically-labeled and stereo-isomers. Wortmannin analogs can be prepared as a pharmaceutically acceptable salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, for example an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. In addition, the salt forms of the disclosed compounds can be prepared using salts of the starting materials or intermediates.

[061] In some of the embodiments described herein, wortmannin analogs can be prepared as a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically acceptable salt) by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid, including, but not limited to, inorganic acids such as hydrochloric acid,

hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid metaphosphoric acid, and the like; and organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, Q-toluenesulfonic acid, tartaric acid, trifluoroacetic acid, citric acid, benzoic acid, 3-(4- hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, arylsulfonic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-l-carboxylic acid,

glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-l -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid.

[062] Alternatively, in some of the embodiments described herein, wortmannin analogs can be prepared as a pharmaceutically acceptable base addition salts (which is a type of a pharmaceutically acceptable salt) by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base, including, but not limited to organic bases such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like and inorganic bases such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[063] It should be understood that a reference to a pharmaceutically acceptable salt includes the solvent addition forms or crystal forms thereof, particularly solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of wortmannin analogs can be conveniently prepared or formed during the processes described herein. By way of example only, hydrates of wortmannin analogs can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents including, but not limited to, dioxane, toluene, alkyl acetate, anisole, tetrahydrofuran or methanol. In addition, the compounds provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds and methods provided herein.

[064] In some of the embodiments described herein, wortmannin analogs include crystalline forms, also known as polymorphs. Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Various factors such as the recrystallization solvent, rate of crystallization, and storage temperature may cause a single crystal form to dominate.

Certain crystalline forms of PX-866 are described in WO 2012/092288 and are incorporated herein by reference.

[065] In some of the embodiments described herein, wortmannin analogs in unoxidized form can be prepared from N-oxides of compounds of Formula (IA or IB) by treating with a reducing agent, such as, but not limited to, sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, or the like in a suitable inert organic solvent, such as, but not limited to, acetonitrile, ethanol, aqueous dioxane, or the like at 0 to 80°C. [066] In some embodiments, wortmannin analogs are isotopically-labeled, which are identical to those recited in the various formulae and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. In some embodiments, one or more hydrogen atoms are replaced with deuterium. In some embodiments, metabolic sites on the compounds described herein are deuterated. In some embodiments, substitution with deuterium affords certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements.

[067] In some of the embodiments described herein, wortmannin analogs can be prepared as prodrugs. Prodrugs are generally drug precursors that, following administration to a subject and subsequent absorption, are converted to an active, or a more active species via some process, such as conversion by a metabolic pathway. Some prodrugs have a chemical group present on the prodrug that renders it less active and/or confers solubility or some other property to the drug. Once the chemical group has been cleaved and/or modified from the prodrug the active drug is generated. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. An example, without limitation, of a prodrug would be a wortmannin analog which is administered as an ester (the "prodrug") to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial. A further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group where the peptide is metabolized to reveal the active moiety.

[068] In some of the embodiments described herein, wortmannin analogs are metabolites. A "metabolite" of a wortmannin analog disclosed herein is a derivative of that wortmannin analog that is formed when the wortmannin analog is metabolized. The term "active metabolite" refers to a biologically active derivative of a wortmannin analog that is formed when the wortmannin analog is metabolized (biotransformed). The term "metabolized," as used herein, refers to the sum of the processes (including, but not limited to, hydrolysis reactions and reactions catalyzed by enzymes) by which a particular substance is changed by an organism. Thus, enzymes may produce specific structural alterations to a wortmannin analog. For example, cytochrome P450 catalyzes a variety of oxidative and reductive reactions while uridine diphosphate glucuronyltransferases (UGT) catalyze the transfer of an activated glucuronic-acid molecule to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines and free sulphydryl groups (e.g. conjugation reactions). Further information on metabolism is available in The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). In one embodiment, metabolites of the compounds disclosed herein are identified either by administration of compounds to a host and analysis of tissue samples from the host, or by incubation of compounds with hepatic cells in vitro and analysis of the resulting compounds.

[069] Metabolites of wortmannin analogs, in some embodiments described herein, include, but are not limited to, metabolites resulting from first pass metabolism. In some embodiments, the metabolite is a 17-hydroxy (17-OH) derivative of a wortmannin analog. In some embodiments, the metabolite is a derivative of PX-866. In other embodiments, the metabolite is a derivative of PX- 867.

[070] In some instances a metabolite of PX-866 has the following structural formula:

In other instances a metabolite of PX-867 has the following structural formula:

[072] In further embodiments, a metabolite of a wortmannin analog is a 11,17-hydroxy (11,17- OH) derivative of a wortmannin analog. [073] In some instances a metabolite of PX-866 has the following structural formula:

[074] In other instances a metabolite of PX-867 has the following structural formula:

PX-866

[075] PX-866 (acetic acid 4-diallylaminomethylene-6-hydroxy-l-a-methoxymethyl-10p, 13 β- dimethyl-3,7,17-trioxo-l,3,4,7,10,l ip,12,13,14a,15,16,17-dodecahydro-2-oxa- cyclopenta[a]phenanthren-l 1-yl ester), a derivative of wortmannin, is a novel agent that

irreversibly inhibits PI-3K, a lipid kinase that mediates signal transduction cascades essential for cell proliferation, survival, and differentiation.

[076] PX-866 is an pan-isoform inhibitor of Class I P1-3K that covalently binds to ATP binding site of the pi 10 catalytic subunit. Described herein are studies that illustrate rapid metabolism of PX-866 to a 17-hydroxy PX-866 derivative. The 17-hydroxy PX-866 metabolite has a 2-5 fold increase in potency in cell proliferation assays versus pi 10a and ρΐ ΐθβ isoforms. For example, in cell based assays, potency of the 17-hydroxy metabolite is pi 10a IC50 14nM vs 39nM for the parent compound (PX-866), potency of the 17-hydroxy metabolite is pi 10β IC50 57nM vs. 88nM for the parent compound (PX-866). [077] Table 1 illustrates the potency of 17-hydroxy PX-866 metabolite in in vitro kinase assays:

Pharmacology

[078] PX-866 is a potent, irreversible inhibitor of PI-3K. In vitro, PX-866 is a potent inhibitor of Class I PI-3K, with IC₅₀ values that range from 39 to 183 nM. Inhibition of the PI-3K pathway in tissue culture by PX-866 has been observed with IC₅₀ values ranging from 50 nM to 500 nM using tumor-derived cell lines, and PX-866 inhibits tumor cell proliferation in vitro with IC₅₀ values ranging from 360 nM to 8.2 μΜ. 17-OH PX-866, a metabolite of PX-866 identified in rodents and humans, is also a potent inhibitor of PI-3K and demonstrates an equal or greater affinity for Class I PI-3K and equal or better IC₅₀ values in cell proliferation assays, when compared with PX-866.

[079] PX-866 inhibits tumor growth in multiple xenograft models as a single agent and in combination with cytotoxic agents, targeted therapies, and radiation. Antitumor efficacy with PX-866 is achieved in vivo in xenograft models with intravenous (IV) doses of 8 mg/kg to

12 mg/kg or oral (PO) doses of 2 mg/kg to 4 mg/kg and is associated with substantial PI-3K pathway inhibition using the phosphorylation of AKT as a pharmacodynamic endpoint.

ADME and Pharmacokinetics

[080] Preclinical absorption, distribution, metabolism, and excretion (ADME) and PK studies have been performed in mice, rats, and dogs. PX-866 is absorbed rapidly within the first 60 minutes after oral administration and eliminated at a rapid rate (t ₂ ~ 0.25 - 2 hours) in all species tested. In mice and rats, drug volumes of distribution were large and the clearance of drug from plasma was very rapid, exceeding that of hepatic blood flow, suggesting that non-hepatic clearance mechanisms may play a major role in the elimination of the drug. In dogs, the drug clearance was less rapid and the volume of distribution was much lower compared to the rodent species. Oral bioavailability of PX-866 was generally < 5%.

[081] Studies performed using radio-labeled PX-866 in mice suggested that first pass drug metabolism, rather than drug absorption issues, may account for the low oral bioavailability measurements. The observation that oral delivery of PX-866 results in greater potency in tumor efficacy studies as well as superior pharmacodynamic effects compared with IV dosing supports the idea that first pass metabolism of the drug may result in the production of metabolites that contribute to the efficacy of the drug. Consistent with this, an active metabolite of PX-866 with increased potency relative to the parent molecule, 17-OH PX-866, has been identified in rodents and humans. In rodents, PX-866 is rapidly metabolized to 17-OH PX-866, resulting in plasma concentrations that are equal to or exceed the parent drug levels in rodent oral PK studies.

[082] The PK profile of PX-866 in humans has been consistent with preclinical studies, with evidence of rapid conversion of PX-866 to 17-OH PX-866. With rare exceptions, the parent drug has been below the limits of detection in patients. Production of 17-OH PX-866 is rapid, with a time to maximum concentration (T_max) of 0.66 hours and a mean residence time (MRT) of 3.7 to 5.6 hours. Based on mean maximum concentration (C_max) and area under the concentration time curve (area under the curve [AUC]) values, 17-OH PX-866 PK appears to be dose-proportional, although significant inter- and intra-patient variability has been observed. The C_max of the 17-OH PX-866 metabolite is equal to or exceeds peak levels observed in mice treated at an efficacious dose of PX-866 (2 mg/kg). In addition, the AUC/kg for the 17-OH PX-866 metabolite in humans substantially exceeds the AUC/kg in mice due to an increase in MRT in humans. No evidence of drug accumulation or reduction has been seen to date.

PX-866 Tumor Efficacy Studies

[083] PX-866 has activity in a number of tumor-derived cell lines, including NSCLC, prostate, breast, ovarian, and colorectal cancers, and glioblastoma. In mouse tumor xenograft models, PX- 866 was efficacious as a single agent and in combination with chemotherapy, radiation, and targeted signal transduction inhibitors. As a single agent, in these models, it was also demonstrated

473

that the phosphorylation of Akt at Ser prepared from excised xenografted tumors was effectively inhibited as well. The activity of PX-866 in these tumor cell lines correlated with the mutational status of the PI-3K and Ras pathways. There was a trend for greater tumor control in models harboring mutations that are associated with increased PI-3K pathway activity, such as pi 10a mutations or PTEN deletion. Consistent with published preclinical data indicating activation of the Ras pathway can drive resistance to PI-3K inhibition, cells with mutations in the Ras oncogene, in the absence of concomittent PI-3K pathway mutation, demonstrated the lowest response to PX-866.

Preclinical studies of PX-866 and Vemurafenib

[084] In vitro studies were conducted to evaluate the effects of combining PX-866 with vemurafenib using melanoma cell lines harboring BRAF V600E mutations. The combination of PX-866 with vemurafenib produced a synergistic inhibition of cell proliferation in all melanoma cell lines tested, including A101D, A375, A2058, Colo829, SK-MEL-3 and SK-MEL-5. The mean combination indices (CIs), based on the combination of PX-866 and vemurafenib at EC50, EC75, EC90, and EC95 are shown in Table 2. ICs for these cell lines indicate PX-866 and vemurafenib produce synergistic to strongly synergistic effects on cell proliferation in vitro and support the hypothesis that co-inhibition of PI-3K and BRAF signaling may provide enhanced therapeutic benefit in patients with melanoma.

Table 2 Combination Indices for PX-866 and Vemurafenib

* Results from two separate experiments

Table 1 Planned Dose Escalation Cohorts

Preclinical studies of PX-866 and PLX-4720

[085] PX-866 and PLX-4720 was tested in a melanoma tumor xenograft animal model to evaluate the effects of the combination on tumor volume and weight. 1205Lu human melanoma cells which harbor BRAFV600E mutation were implanted into mice. After tumor volume reached about 300 mm , mice (n=10/group) were treated either with (1) vehicle, (2) PX-866, 2 mg/kg q2d, (3) PLX- 4720, 200 mg/kg diet, or (4) PX-866/PLX-4720 combined. Tumor volume and weight were assessed for 14 days from treatment.

[086] The tumor volume measurements show that vehicle treated mice had an average tumor volume of greater than 1000 mm at the end of 14 days. PX-866 treated mice had an average tumor volume of about 900 mm . PLX-4720 treated mice had an average tumor volume of about 700 mm . Finally, mice treated with the PX-866/PLX-4720 combination showed an average tumor volume of about 250 mm indicating that the combination was effective at suppressing tumor growth in the xenograft model and perhaps was effective at decreasing tumor volume.

[087] The tumor weight results were similar to the volume measurements. Vehicle treated mice had average tumor weight of about 0.55 g; PX-866 treated mice had an average tumor weight of about 0.4 g; PLX-4720 treated mice had an average tumor weight of about 0.3 g; and the combination treated mice had an average tumor weight of about 0.1 g. The results were statistically significant (p <0.005, comparing combo with vehicle and combo with the two single treatments).

Phase 1/2 Study of PX-866 and Vemurafenib

[088] Enhanced PI-3K pathway signaling via IGF-1R or PDGFR activation appears to be one of the by-pass pathways activated in BRAF inhibitor resistant melanoma. The loss of PTEN in melanoma derived tumor cell lines confers resistance to BRAF inhibitor mediated apoptosis.

Accordingly, Applicants tested a combination of PI-3K inhibition with vemurafenib to test whether the combination provided improved outcomes in patients with melanoma harboring PTEN deletions or other changes that increase PI-3K pathway activation.

[089] Tumor profiling of baseline patient biopsies together with molecular analysis of pre- treatment and on-treatment biopsies will allow expansion of our knowledge concerning patterns of resistance to selective BRAF inhibitors generally as well as facilitating interpretation of the clinical effects of adding a pan-PI-3K inhibitor to BRAF inhibitor therapy.

Treatment Cycles

[090] Cycle 1

[091] Cycle 1 Day 1 Predose

[092] Pre-dose assessments should be performed pre-dose on Cycle 1 Day 1 unless a patient was screened within 96 hours of scheduled dosing. In this case, these evaluations do not need to be repeated.

• Physical examination

Vital signs

ECG

• ECOG performance status

• Blood samples for hematology, clinical chemistry, and LFT

• Coagulation panel

Urinalysis

• Collection of concomitant medications

• Collection of AEs

[093] Cycle 1 Day 1

• For Phase 1 patients, begin PX-866 given orally once per day on Days 1 to 28 followed by Day 1 PK sampling per PK Blood Sample Schedule

• For Phase 2 patients, begin PX-866 given orally once per day on Days 1 to 28 • For Phase 2 patients, begin vemurafenib given orally twice per day on Days 1 to 28 Vital signs

• Collection of concomitant medications

• Collection of AEs

[094] Cycle 1 Day 8 (-24 hours; Phase 1 only)

Vital signs

• Blood samples for hematology, clinical chemistry, and LFT

• Collection of concomitant medications

• Collection of AEs

Required tumor biopsy (4 to 8 hours after the dose of PX-866; biopsy may be done on any day from Day 4 to Day 8)

[095] Cycle 1 Day 9

• Begin vemurafenib given orally twice per day on Days 9 to 28 (Phase 1 only)

[096] Cycle 1 Day 15

ECG

Vital signs

• Blood samples for hematology, clinical chemistry, and LFT

• Collection of concomitant medications

• Collection of AEs

Tumor biopsy (Phase 2 only), when possible (+ 2 weeks) (4 to 8 hours after the dose of PX-866)

[097] Cycle 2

[098] Cycle 2 Day 1 (± 72 hours)

Tumor biopsy, when possible (Phase 1 only) (-1 week/+ 2 weeks) (4 to 8 hours after the dose of PX-866)

• PX-866 given orally once per day on Days 1 to 28

• Vemurafenib given orally twice per day on Days 1 to 28

• Physical examination to include skin and anus

• In females, examination of external genitalia, if clinically indicated

Vital signs

ECG

• ECOG performance status

• Blood samples for hematology, clinical chemistry, and LFT

• Coagulation panel Urinalysis

• Collection of concomitant medications

• Collection of AEs

[099] Cycle 2 Day 15 (± 72 hours)

For Phase 1 patients, pre-dose PK sampling per PK Blood Sample Schedule

• For Phase 1 patients, PX-866 and vemurafenib given orally followed by PK sampl per PK Blood Sample Schedule

Vital signs

• Blood samples for hematology, clinical chemistry, and LFT

• Collection of concomitant medications

• Collection of AEs

[0100] Cycle2 Day 28 (even numbered cycles only: ± 5 days)

[0101] CT/MRI scan and assessment of tumor burden based on RECIST 1.1

[0102] Cycle 3

[0103] Cycle 3 Day 1 (± 72 hours)

• PX-866 given orally once per day on Days 1 to 28

• Vemurafenib given orally twice per day on Days 1 to 28

• Physical examination to include skin and anus

• In females, examination of external genitalia, if clinically indicated

Vital signs

ECG

• ECOG performance status

• Blood samples for hematology, clinical chemistry, and LFT

• Coagulation panel

• Urinalysis

• Collection of concomitant medications

• Collection of AEs

[0104] Cycle 3 Day 15 (± 72 hours)

Vital signs

• Blood samples for hematology, clinical chemistry, and LFT

• Collection of concomitant medications

• Collection of AEs

[0105] Cycle 4

[0106] Cycle 4 Day 1 (± 72 hours) • PX-866 given orally once per day on Days 1 to 28

• Vemurafenib given orally twice per day on Days 1 to 28

• Physical examination to include skin and anus

• In females, examination of external genitalia, if clinically indicated

Vital signs

ECG (may be done every three cycles after Cycle 3 [i.e. Cycles 6, 9, 12, etc.] or more frequently, if clinically appropriate)

• ECOG performance status

• Blood samples for hematology, clinical chemistry, and LFT

• Coagulation panel

• Urinalysis

• Collection of concomitant medications

• Collection of AEs

[0107] Cycle 4 Day 15 (± 72 hours)

Vital signs

• Blood samples for hematology, clinical chemistry, and LFT

• Collection of concomitant medications

• Collection of AEs

[0108] Cycle 4 Day 28 (even numbered cycles only: ± 5 days)

• CT/MRI scan and assessment of tumor burden based on RECIST 1.1

[0109] Cycles 5+

[0110] Cycle 5+ Day 1 (± 72 hours)

• PX-866 given orally once per day on Days 1 to 28

• Vemurafenib given orally twice per day on Days 1 to 28

• Physical examination to include skin and anus

• In females, examination of external genitalia, if clinically indicated

Vital signs

ECG (may be done every three cycles after Cycle 3 [i.e. Cycles 6, 9, 12, etc.] or more frequently, if clinically appropriate)

• ECOG performance status

• Blood samples for hematology, clinical chemistry, and LFT

• Coagulation panel

Urinalysis

• Collection of concomitant medications • Collection of AEs

[0111] Cycle 6+

[0112] Cycle 6+ Day 28 (even numbered cycles only: ± 5 days)

• CT/MRI scan and assessment of tumor burden based on RECIST 1.1 (may be done every 3 cycles after Cycle 6 [i.e. Cycles 9, 12, 15 etc.])

[0113] Tumor Biopsy at Time of Progression

When possible, a tumor biopsy should be collected as close as possible to the time that progressive disease is identified

[0114] End of Treatment (30 + 7days after last dose of PX-866 or vemurafenib, whichever is later)

• Physical examination to include skin, anus

• Pelvic exam of external genitalia, women only, as clinically appropriate

Vital signs

• ECOG performance status

• Blood samples for hematology, clinical chemistry, and LFT

• Coagulation panel

Urinalysis

• Collection of concomitant medications

• Collection of AEs

CT/MRI scan and assessment of tumor burden based on RECIST 1.1 as needed (discuss with medical monitor)

[0115] Efficacy Measures

[0116] Following initiation of study treatment, CT/MRI scans will be obtained at the end of every two treatment cycles for the initial six cycles and then every three treatment cycles thereafter until PD, initiation of a new therapy, or withdrawal of consent.

[0117] Primary Efficacy Measures

[0118] The primary efficacy measure will be progression-free survival (PFS), measured from the date of randomization to the date of progression (radiologically or symptomatically), or until death from any cause. For each patient that is not known to have progressed or died, PFS will be censored at the date that the patients was last known to be alive and progression free (typically the date of their last radiographic assessment).

[0119] Secondary Efficacy Measures

[0120] Secondary efficacy measures will include objective response rate (ORR) defined as a best response of complete remission (CR) or partial remission (PR). For the analysis of ORR, all patients who do not meet the criteria for a CR or PR as specified by RECIST 1.1, will be analyzed as not having a response. Secondary efficacy measures will also include disease control rate (DCR), defined as the proportion of patients with CR, PR, and SD.

[0121] Duration of response will be measured in patients with complete or partial response from the date that the patient first meets the criteria of CR or PR to the date that the patient progresses (radiologically or symptomatically), or until death from any cause. For each patient that is not known to have progressed or died, duration of response will be censored at the date that the patient was last known to be alive and progression-free.

[0122] Pharmacokinetic Measures

[0123] The effect of combination treatment on the PK of both PX-866 and vemurafenib will be assessed in Phase 1. Plasma samples will be collected in all patients participating in Phase 1 to measure levels of PX-866 and related metabolites as well as levels of vemurafenib. PX-866 PK will be assessed as a single agent at the start of treatment (Cycle 1 Day 1) and in combination with vemurafenib after vemurafenib levels have reached steady state (Cycle 2 Day 15) to evaluate whether PX-866 and related metabolite levels may be decreased following prolonged exposure to vemurafenib. Vemurafenib PK will be assessed in combination with PX-866 on Cycle 2 Day 15 and Cycle 2 Day 16. Vemurafenib PK will be compared to published data.

[0124] Biomarker Assessments

[0125] Biomarker assessments will be performed to identify molecular signatures that may be associated with response or resistance to treatment. Initial assessments will be done using archived tumor specimens and/or pretreatment biopsies. Tissue sections may be used to assess genetic mutations in tumor DNA that may confer resistance or sensitivity to PX-866 therapy, including but not limited to expression of PTEN, CRAF, MAP3K8/COT, platelet-derived growth factor β (PDGFRP), insulin- like growth factor 1 receptor (IGF-1R), as well as assessments of genetic alteration of PTEN, NRAS, PI-3K, CDK4, CDK2NA, Cyclin D, AKT, MEK and BRAF.

Additional studies may include full genome copy number profiling from paraffin embedded blocks. Fresh frozen tissue obtained pre -treatment and at the time of progression may also be used for whole exome or genome sequencing, reverse phase protein arrays (RPPA), or mRNA/miRNA expression profiling to evaluate potential mechanisms of resistance to treatment. Fresh tumor biopsy tissue may also be used to establish melanoma cell lines, enabling preclinical studies which would be directly correlative to patient response.

[0126] Pharmacodynamic Assessments

[0127] Pharmacodynamic assessments of PX-866 and/or vemurafenib activity will be made using fresh tumor biopsies obtained pretreatment (Phase 1 and Phase 2), following treatment with PX- 866 alone (Phase 1 only), after at least two weeks of combination therapy (Phase 1 and Phase 2 when feasible), and at the time of progression (Phase 1 and Phase 2 when feasible). Formalin fixed tissue sections may be stained to assess the presence of drug target activity in the PI-3K and MAPK signaling pathways, including downstream targets. Fresh tissue used for RPPA may also be used for pharmacodynamic assessment.

[0128] Safety Evaluations

[0129] The investigator is responsible for the appropriate medical care and the safety of patients who have entered this study. The investigator must document any AE experienced by patients who have entered this study and report all SAEs

[0130] Identification of Mechanisms of and Predictive Biomarkers for Acquired Resistance to BRAF and PI-3K Inhibitor therapy

[0131] Tissue from consenting patients should be obtained and processed according to the tissue collection tumor processing manual. When possible, fresh tissue will be collected in culture medium to establish melanoma cell lines that will be used for pre-clinical studies.

[0132] Rationale: The identification of molecular and protein expression changes in tumors prior to treatment and at the time of progression after BRAF and PI-3K inhibitor therapy may help to identify mechanisms of resistance, which may ultimately be used to predict response and optimize combination or sequential therapy approaches.

[0133] Hypothesis: Genes or pathways may be up-regulated through mutations or through amplification leading to disease progression or primary treatment resistance.

[0134] Hierarchical analyses:

1. DNA genetic/genomic analysis, in paired samples from both pre-treatment and time of progression

The availability of tissues will determine the prioritization of correlative studies. All samples will undergo somatic genetic profiling using a customized melanoma iPlex

(Sequenom). As possible, we will do either whole exome or genome sequencing, or targeted re-sequencing (including genes such as BRAF, NRAS, MEK1/2, PTEN, CDKN2A, PI-3K) in samples. PTEN mutations and/or deletions status will be determined for all samples. Based on DNA amount, analyses may include copy number profiling of specific genes (e.g. CDKN2A, CDK4) or the full genome on DNA extracted from paraffin embedded or fresh frozen tissues to identify other genomic changes associated with response.

2. Protein based analyses in samples from pre-treatment, on treatment and time of progression may include immunohistochemistry studies to evaluate pathway activation, focusing on the MAPK and PI-3K pathways or a more unbiased approach such as reverse phase protein array (RPPA). The specific analyses proposed will depend on tissue availability.

3. Expression profiling - RNA based

mPvNA or miRNA expression profiling will be done as fresh frozen tissues are available. The results will be combined with those from the DNA and protein based studies to put together a fulsome picture of the tumor and determinants of response and progression.

Phase 1/2 Study of PX-866 and PLX-4720

[0135] The above Phase 1/2 study of PX-866 and vemurafenib is adapted for the study of PX-866 and PLX-4720. The study parameters are the same, with the only difference that vemurafenib is substituted with PLX-4720.

Synthesis of Wortmannin Analogs

[0136] Wortmannin analogs described herein may be synthesized using standard synthetic techniques known to those of skill in the art or using methods known in the art in combination with methods described herein. In additions, solvents, temperatures and other reaction conditions presented herein may vary according to the practice and knowledge of those of skill in the art.

[0137] The starting material used for the synthesis of wortmannin analogs described herein can be obtained from commercial sources, such as Aldrich Chemical Co. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.), or the starting materials can be synthesized. The wortmannin analogs described herein, and other related compounds having different substituents can be synthesized using techniques and materials known to those of skill in the art, such as described, for example, in March, ADVANCED ORGANIC CHEMISTRY 4th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4th Ed., Vols. A and B (Plenum 2000, 2001), and Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 3rd Ed., (Wiley 1999) (all of which are incorporated by reference in their entirety). General methods for the preparation of wortmannin analogs as disclosed herein may be derived from known reactions in the field, and the reactions may be modified by the use of appropriate reagents and conditions, as would be recognized by the skilled person, for the introduction of the various moieties found in the formulae as provided herein.

[0138] Additional synthesis methods and schemes for the wortmannin analogs described herein can be found in, for example, U.S. Patent No. 5,480,906, U.S. Patent No. 7,335,679, and U.S. Patent Appl. Pub. No. 2007/0191466, each of which is incorporated herein by reference for synthesis of wortmannin analogs. Pharmaceutical Compositions of PI3 kinase Inhibitors

[0139] Pharmaceutical compositions containing PI3 kinase inhibitors (e.g., wortmannin analogs, e.g., PX-866) can be administered in therapeutically effective amounts as pharmaceutical compositions by any conventional form and route known in the art including, but not limited to: oral and intravenous administration. In addition, the pharmaceutical composition containing PI3 kinase inhibitors may be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation.

[0140] For oral administration, PI3 kinase inhibitors can be formulated by combining the active compounds with pharmaceutically acceptable carriers or excipients well known in the art. Such carriers enable the compounds described herein to be formulated as tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a patient to be treated. For intravenous administration, PI3 kinase inhibitors are formulated in suitable pharmacologically acceptable vehicles. The PI3 kinase inhibitor (e.g., PX-866) is present as a crystalline form or as an amorphous form in the composition.

[0141] A summary of pharmaceutical compositions is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton,

Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999), herein incorporated by reference in their entirety. Certain PI3 kinase inhibitor compositions (e.g., PX-866 compositions) are described in WO 2011/153488, WO 2011/153495, and WO 2012/092288 and are incorporated herein by reference.

Wortmannin Analogs Dosages

[0142] Dosages of wortmannin analogs described herein (e.g., compounds of Formula IA, IB, IIA or IIB or any other PI-3 kinase inhibitor and/or wortmannin analog described herein) can be determined by any suitable method. Maximum tolerated doses (MTD) and maximum response doses (MRD) can be determined via established animal and human experimental protocols as well as in the examples described herein. For example, toxicity and therapeutic efficacy of wortmannin analogs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, for determining the LD₅o (the dose lethal to 50% of the population) and the ED₅o (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD₅o and ED₅o. Wortmannin analogs exhibiting high therapeutic indices are of interest. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅o with minimal toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Additional relative dosages, represented as a percent of maximal response or of maximum tolerated dose, are readily obtained via the protocols.

[0143] In some embodiments, the amount of a given wortmannin analog that corresponds to such an amount varies depending upon factors such as the particular compound, disease condition and its severity, the identity (e.g., weight, sex) of the subject or host in need of treatment, but can nevertheless be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0144] In other embodiments, however, doses employed for adult human treatment are typically in the range of about 0.0 lmg to about 5000 mg per day, or about lmg to about 1500 mg per day. In one embodiment, the desired dose is conveniently presented in a single dose or in divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[0145] In some embodiments, wortmannin analogs are provided in a dose per day from about 0.01 mg to 1000 mg, from about 0.1 mg to about 100 mg, from about 1 to about 20, from about 2 mg to about 12 mg. In certain embodiments, wortmannin analogs are provided in a daily dose of about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.4 mg, about 0.6 mg, about 0.8 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 500, mg, about 750 mg, about 1000 mg, or more, or any range derivable therein. In certain instances, wortmannin analogs are provided in a dose per day of about 1 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 2 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 3 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 4 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 5 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 6 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 7 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 8 mg. In certain instances,

wortmannin analogs are provided in a dose per day of about 9 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 10 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 11 mg. In certain instances, wortmannin analogs are provided in a dose per day of about 12 mg. The dose per day described herein can be given once per day or multiple times per day in the form of sub-doses given b.i.d., t.i.d., q.i.d., or the like where the number of sub-doses equal the dose per day.

[0146] In further embodiments, the daily dosages appropriate for the compound of Formula IA, IB, IIA or IIB or any other PI-3 kinase inhibitor and/or wortmannin analog described herein are from about 0.001 to about 100 mg/kg per body weight. In one embodiment, the daily dosages appropriate for the compound of Formula IA, IB, IIA or IIB or any other PI-3 kinase inhibitor and/or wortmannin analog described herein are from about 0.01 to about 10 mg/kg per body weight. In some embodiments, an indicated daily dosage in a large mammal, including, but not limited to, humans, is in the range from about 0.02 mg to about 1000 mg, conveniently administered in divided doses, including, but not limited to, up to four times a day. In one embodiment, the daily dosage is administered in extended release form. In certain embodiments, suitable unit dosage forms for oral administration comprise from about 1 to 500 mg active ingredient. In other embodiments, the daily dosage or the amount of active in the dosage form are lower or higher than the ranges indicated herein, based on a number of variables in regard to an individual treatment regime. In various embodiments, the daily and unit dosages are altered depending on a number of variables including, but not limited to, the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0147] In other embodiments wortmannin analogs are provided at the maximum tolerated dose (MTD). In other embodiments, the amount of wortmannin analogs administered is from about 10% to about 90% of the maximum tolerated dose (MTD), from about 25% to about 75% of the MTD, or about 50% of the MTD. In particular embodiments, the amount of wortmannin analogs administered is from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher, or any range derivable therein, of the MTD.

EXAMPLES

Example 1

[0148] Phase 1/2 study of PI-3K inhibition with PX-866 combined with vemurafenib (BRAF inhibitor) in patients with BRAF mutant cancer including advanced melanoma

[0149] Study Objectives [0150] Phase 1

[0151] Primary:

[0152] To evaluate the safety and tolerability of PX-866 in combination with twice daily oral administration of vemurafenib in patients with any advanced BRAF-mutant cancer

[0153] Secondary:

[0154] To evaluate the pharmacokinetics (PK) of vemurafenib when given in combination with PX-866

[0155] To evaluate the effect of vemurafenib on the PK of PX-866

[0156] Exploratory:

[0157] To describe any antitumor activity of PX-866 administered in combination with

vemurafenib

[0158] Phase 2

[0159] Primary:

[0160] To compare the progression-free survival (PFS) of PX-866 administered in combination with vemurafenib vs. vemurafenib alone in patients with advanced BRAF-mutant melanoma

[0161] Secondary:

[0162] To compare the objective response rate (ORR) of PX-866 administered in combination with vemurafenib vs. vemurafenib alone in patients with advanced BRAF-mutant melanoma

[0163] To compare the disease control rate (DCR) of PX-866 administered in combination with vemurafenib vs. vemurafenib alone in patients with advanced BRAF-mutant melanoma

[0164] To evaluate the safety and tolerability of PX-866 in combination with vemurafenib in patients with advanced BRAF-mutant melanoma

[0165] Exploratory

[0166] To evaluate, using a cross-over design, the antitumor effects of PX-866 when given in combination with vemurafenib at the time of progression on treatment with vemurafenib alone

[0167] Phase 1 and 2

[0168] Exploratory:

[0169] To evaluate potential pharmacodynamic markers of PX-866 and vemurafenib activity in serial tumor biopsies

[0170] To evaluate potential predictive biomarkers of response and resistance to PX-866 and vemurafenib

[0171] Study Population

[0172] Phase 1 : Patients with any advanced BRAF-mutant cancers who meet the study eligibility criteria [0173] Phase 2: Patients with advanced (defined as unresectable Stage IIIC or IV) BRAF-mutant melanomas who meet the study eligibility criteria

[0174] Inclusion Criteria

1. > 18 years at time of consent.

2. If a sexually active male or a sexually active female of child-bearing potential, agrees to use a medically accepted form of contraception from the time of consent to completion of all follow-up study visits

3. If female of child-bearing potential, negative pregnancy test (not required for

post-menopausal females)

4. Has signed an informed consent document that has been approved by an institutional review board or independent ethics committee (IRB/IEC)

5. For Phase 1: must have histologically or cyto logically-confirmed advanced cancer, including melanoma, that is BRAF mutation-positive (V600E or V600K) for which there is no remaining standard therapy with curative potential. Patients must have disease sites amenable to biopsy

For Phase 2: must have histologically or cyto logically-confirmed BRAF mutation-positive (V600E or V600K) advanced (defined as unresectable Stage IIIC or IV) melanoma that has not been treated with a selective BRAF inhibitor

6. For Phase 1 : must have measurable or non-measurable disease per Response Evaluation Criteria In Solid Tumors (RECIST 1.1)

For Phase 2: must have measurable disease per RECIST 1.1, defined as at least one lesion that can be accurately measured in at least one dimension (longest diameter to be recorded for non-nodal lesions and short axis for nodal lesions) as >20 mm using conventional techniques or as >10 mm using spiral computed tomography (CT) scans

7. For Phase 1 : no restriction on number of prior therapy regimens. Patients who were previously treated with a selective BRAF inhibitor may initiate treatment on this protocol 14 days after the selective BRAF inhibitor has been stopped. For Phase 2: the following restrictions on prior therapy apply: 1) must not have been treated with a selective BRAF inhibitor and must not have had more than 2 prior treatment regimens; 2) must have completed prior cytotoxic chemotherapy a minimum of 4 weeks prior to starting PX-866 and/or vemurafenib (except for BCNU and/or mitomycin C, which must have been completed a minimum of 6 weeks prior to starting therapy). Prior biologic therapy and localized radiation therapy must have been completed a minimum of 2 weeks prior to starting therapy . All toxicities related to prior cancer therapies other than alopecia must have resolved to Grade 1 or less

. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1

0. In the opinion of the clinical investigator, life expectancy > 3 months

1. Adequate hematologic function as defined by:

a. Hemoglobin > 9 g/dL (patients may be transfused to achieve level)

b. Absolute neutrophil count (ANC) > 1,000 cells/μΕ

c. Platelets > 90,000 cells^L

2. Adequate hepatic function as defined by the following:

a. Total bilirubin < 1.5 mg/dL (unless known Gilbert's disease)

b. Aspartate aminotransaminase (AST/SGOT) and alanine aminotransferase (ALT/SGPT) < 1.5 x upper limit of normal (ULN)

3. S erum creatinine < 2.0 mg/ dL

4. Corrected (Fridericia's) QTcF must be < 480 milliseconds ] Exclusion Criteria

. May not be receiving any other investigational agents

. Active central nervous system (CNS) metastases are excluded. Patients with a history of CNS metastasis, who have been treated prior to enrollment, must be stable for eight weeks after completion of treatment. These patients must have undergone appropriate imaging studies and currently be on a stable, lowest possible dose of steroids

. History of allergic reactions attributed to compounds of similar chemical or biologic composition to PX-866 or vemurafenib

. Uncontrolled intercurrent illness including, but not limited to: ongoing or active

infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements

. Uncorrectable electrolyte abnormalities, long QT syndrome, or taking medicinal products known to prolong the QT interval

. Poorly controlled diabetes mellitus

. Pregnant, breastfeeding, or planning to become pregnant

. Known to be human immunodeficiency virus (HlV)-positive

. Inability to swallow pills

0. Previous treatment with a phosphatidylinositol-3 -kinase (PI-3K) inhibitor 11. Any other significant medical or psychiatric condition that in the opinion of the investigator renders the patient inadequate for participation

[0176] Test Product, Dose, and Mode of Administration

[0177] All treatments will be administered on a 28-day cycle.

[0178] Phase 1 : For Cycle 1 only, PX-866 will be administered orally at doses of either 6 or 8 mg or other dose to be determined (TBD) once per day on Days 1-28; vemurafenib will be administered orally twice per day at doses of either 720 mg or 960 mg on Days 9-28. For all subsequent cycles, both PX-866 and vemurafenib will be administered on Days 1-28.

[0179] Phase 2: Treatment for all patients will begin on Cycle 1 Day 1. For patients randomized to treatment with vemurafenib alone, vemurafenib will be administered orally twice per day at the approved dose of 960 mg. For patients randomized to treatment with vemurafenib and PX-866, vemurafenib will be administered twice per day at the dose recommended from Phase 1 , and PX- 866 will be administered once per day at the dose recommended from Phase 1.

[0180] Number of Planned Patients

[0181] Planned enrollment for this study is up to 146 patients.

[0182] Phase 1 : Up to 30 DLT evaluable patients may be enrolled in up to 5 dose escalation cohorts, and up to 6 additional patients may be enrolled in an optional MTD expansion cohort.

[0183] Phase 2: Up to 110 patients may be enrolled and randomized 2: 1 to receive both PX-866 and vemurafenib versus vemurafenib alone.

Duration of Treatment

[0184] Patients will be evaluated for progression approximately every 8 weeks for the initial 24 weeks and every 12 weeks thereafter. All patients with stable disease (SD) or better, as per investigator assessment, will receive repeat cycles of treatment on a 28 -day schedule until disease progression (PD), unacceptable toxicity, or withdrawal of consent. In Phase 2, patients randomized to the vemurafenib alone arm may be allowed to receive PX-866 in combination with vemurafenib at the time of disease progression.

Study Design

[0185] This is a Phase 1/2 open-label study of PX-866 given in combination with vemurafenib to patients with advanced BRAF -mutant cancers (Phase 1) or patients with advanced (defined as unresectable Stage IIIC or IV) V600E or V600K BRAF-mutant melanoma (Phase 2).

[0186] Phase 1 will use a 3+3 dose escalation design to evaluate up to three dose levels of PX-866 in combination with up to two dose levels of vemurafenib in order to identify the maximal tolerated dose/recommended dose (MTD/RD) of both PX-866 and vemurafenib to be used in Phase 2. A minimum of 6 evaluable patients will be treated at the MTD/RD prior to advancing to Phase 2. To evaluate the effects of vemurafenib on PX-866 PK, and the effects of combination treatment on vemurafenib PK, PK assessments will be performed in all Phase 1 patients. An additional 6 patients may be treated in an optional MTD/RD expansion cohort (for a total of 12 patients total treated at the MTD/RD) as recommended by the study safety monitoring committee to further evaluate the PK and pharmacodynamic profile of combination treatment with PX-866 and vemurafenib.

[0187] Phase 2 will evaluate the antitumor activity and safety of PX-866 given in combination with vemurafenib at the doses recommended from Phase 1 compared with vemurafenib alone administered at the approved dose of 960 mg orally BID.

[0188] Efficacy Assessments

[0189] For patients with measurable disease, efficacy will be evaluated as per RECIST 1.1.

[0190] Safety Assessments

[0191] Safety assessments will include surveillance and documentation of adverse events, including adverse events of special interest, laboratory assessments and physical exam findings. Pharmacokinetic Assessments

[0192] Pharmacokinetic assessments will include measurement of plasma levels of PX-866 and/or metabolites as well as plasma levels of vemurafenib.

Pharmacodynamic Assessments

[0193] Pharmacodynamic assessments of PX-866 and/or vemurafenib activity will aso be made using fresh tumor biopsies obtained pretreatment (Phase 1 and Phase 2), following treatment with PX-866 alone (Phase 1 only), after at least two weeks of combination therapy (Phase 1 and Phase 2 when feasible), and at the time of progression (Phase 1 and Phase 2 when feasible). The initial pretreatment biopsy is mandatory for Phase l patients and strongly encouraged for Phase 2 patients, and should be obtained prior to the start of study drug treatment and after the patient has signed informed consent and has been found to meet study eligibility requirements. In Phase 1, patients will also undergo a second mandatory tumor biopsy in Cycle 1. Patients in Phase 1 are also strongly encouraged to provide two additional biopsies (one on combination therapy and one at time of progression). In Phase 2, patients will be strongly encouraged to undergo a second tumor biopsy (Cycle 1 Day 15), as well as a third biopsy at the time of progression.

[0194] Pharmacodynamic assessments of PX-866 and vemurafenib activity in tumor biopsy samples may include, but not be limited to, the evaluation of phosphorylated extracellular signal- regulated kinase (ERK), cyclin Dl, Ki-67, p-mammalian target of rapamycin (mTOR), pAKT, and pS6 in tumor biopsy samples.

Biomarker Assessments

[0195] All patients will be asked to provide archived tumor biopsy specimens for assessment of potential biomarkers of PX-866 and vemurafenib response and resistance. Assessments may include but not be limited to evaluation of expression of PTEN, CRAF, MAP3K8/COT, platelet- derived growth factor β (PDGFRP), insulin- like growth factor 1 receptor (IGF-1R), as well as assessments of genetic alteration of PTEN, NRAS, PI-3K, CDK4, CDK2NA, Cyclin D, AKT, MEK and BRAF. Tumor biopsies obtained prior to the start of treatment for pharmacodynamic assessments may also be used for biomarker assessments.

[0196] Endpoints

[0197] Phase 1

[0198] Primary Endpoint

[0199] Incidence and severity of adverse events (AEs)

[0200] Secondary Endpoints

[0201] Plasma concentrations of PX-866 and metabolites

[0202] Plasma concentrations of vemurafenib [0203] Exploratory Endpoint

[0204] Objective response rate (ORR)

[0205] Phase 2

[0206] Primary Endpoint

[0207] Progression-free survival (PFS)

[0208] Secondary Endpoints

[0209] ORRs for each treatment arm

[0210] Disease control rate (DCR) (the proportion of patients with complete response [CR], partial response [PR], or stable disease [SD])

[0211] Duration of response

[0212] Incidence and severity of AEs

[0213] Incidence and severity of AEs of special interest (e.g., squamous cell carcinomas [SCCs] of the skin and kerato acanthomas)

[0214] Exploratory Endpoints

[0215] ORR following addition of PX-866 at time of progression in patients initially treated on the vemurafenib alone control arm

[0216] Phase 1 and Phase 2

[0217] Secondary Endpoints

[0218] Frequency of dose reductions in vemurafenib

[0219] Frequency of dose reductions in PX-866

[0220] Exploratory Endpoints

[0221] Pharmacodynamic changes of expression by immunohistochemistry (IHC) in downstream signaling proteins of PX-866 and vemurafenib inhibition, possibly including, but not limited to phosphorylated extracellular signal -regulated kinase (pER ), pMEK, cyclin Dl, Ki-67, PRAS40, 4EBP1, TSC2, GSK3, p-mammalian target of rapamycin (mTOR), pAKT, and pS6 in serial tumor biopsies

[0222] Genetic and genomic profiles in serial tumor biopsies, possibly including but not limited to somatic mutations and gene alterations in the MAP kinase pathway (PTEN, NRAS, PI-3K, CDK4, CDK2NA, CyclinD, AKT, MEKl/2 and BRAF), and potentially including whole genome or exome analyses and copy number analyses (either full genome or more limited to include genes such as RAF, PTEN, CDKN2A, CDK4, and MITF)

[0223] Amplification, over-expression, and /or activation of receptor tyrosine kinases (RTKs) possibly including PDGFR, IGF-1R, as well as PI-3K pathway signaling proteins possibly including but not limited to AKT, mTOR, and S6, by IHC and/or array based protein profiling (using techniques such as Reverse Phase Protein Array [RPPA]), as well as PTEN expression by IHC

[0224] Statistical Methods

[0225] Phase 1

[0226] Phase 1 will enroll up to 30 DLT evaluable patients in up to 5 different cohorts using a standard 3+3 dose escalation design to determine the MTD/RD of PX-866 and vemurafenib. Up to

6 additional patients may also be enrolled in an optional MTD/RD expansion cohort to further evaluate the PK and pharmacodynamic effects of combination treatment with PX-866 and vemurafenib.

[0227] Phase 2

[0228] The primary efficacy endpoint, PFS, will be compared between PX-886 + vemurafenib and vemurafenib alone by a logrank test. Assuming a vemurafenib alone (control arm) median PFS of

7 months, a 1 -sided 0.20 false positive error rate, a 1 year enrollment period with an additional 1 year of follow-up before analysis, and a control over experimental hazard ratio of 1.5, as well as a 2: 1 randomization ratio, a total of 100 evaluable patients are required for the logrank test to detect with 0.80 power a statistically significant PX-886 + vemurafenib PFS benefit over vemurafenib alone. Assuming a drop-out rate of 10%, a total of 110 patients will be enrolled.

[0229] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for treating cancer in an individual in need thereof comprising administration of a B-raf inhibitor and a PI3 kinase inhibitor to the individual.

2. The method of claim 1, wherein the PI3 kinase inhibitor is a wortmannin analog.

3. The method of claim 1, wherein the PI3 kinase inhibitor is a compound selected from

Formula IIA Formula IIB

wherein Y is a heteroatom selected from nitrogen and sulfur and R 1 and R 2 are

independently selected from an unsaturated alkyl, cyclic alkyl, or R 1 and R 2 together with Y form a heterocycle.

4. The method of claim 1, wherein the PI3 kinase inhibitor is an irreversible PI3 kinase inhibitor.

5. The method of claim 1 wherein the PI3 kinase inhibitor is

6. The method of claim 1 , wherein the B-raf inhibitor is selected from

(sorafenib), (PLX4032),

7. The method of claim 1 , wherein the B-raf inhibitor is vemurafenib.

8. The method of claim 1 , wherein the B-raf inhibitor is PLX 4720.

9. The method of claim 1, wherein the cancer is a BRAF mutant cancer.

10. The method of claim 1, wherein the cancer is selected from the group consisting of head and neck cancer, lung cancer, ovarian cancer, colon cancer, breast cancer, pancreatic cancer, cervical cancer, prostate cancer, and melanoma.

11. The method of claim 1 , wherein the cancer is melanoma.

12. The method of claim 1, wherein the cancer is advanced-BRAF mutant melanoma.

13. The method of any of claims 1-12, wherein the individual has not been treated with a selective BRAF inhibitor.

14. The method of any of claims 1-12, wherein the individual does not have any active central nervous system metastases.

15. The method of any of claims 1-12, wherein the individual does not have poorly controlled diabetes.

16. The method of any of claims 1-12, wherein the individual is not HIV-positive.

17. The method of any of claims 1-12, wherein PX-866 is administered orally at doses of 6 mg or 8 mg once per day on days 1-28 of the first 28-day cycle of therapy.

18. The method of any of claims 1-12, wherein vemurafenib is administered orally at doses of 720 mg or 960 mg twice per day on days 9-28 of the first 28-day cycle of therapy.

19. The method of claims 17 and 18, wherein both PX-866 and vemurafenib are administered on days 1-28 of subsequent cycles of therapy.

20. The method of claim 19, wherein PX-866 is administered orally at doses of 6 mg or 8 mg once per day and vemurafenib is administered orally at doses of 720 mg or 960 mg twice per day.

21. The method of claims 17-20, wherein 1-6 cycles of therapy are administered to the individual.

22. The method of claims 17-20, wherein more than 6 cycles of therapy are

administered to the individual.

23. A method for treating a BRAF mutant cancer in an individual in need thereof comprising administration of PX-866 and vemurafenib to the individual in need thereof.

24. A method for treating a BRAF mutant cancer in an individual in need thereof comprising administration of PX-866 and PLX 4720 to the individual in need thereof.