HK40001148A - Combination therapy for the treatment of pancreatic cancer - Google Patents

Description

Combination therapy for treating pancreatic cancer

Technical Field

The subject matter described herein relates to the treatment of pancreatic cancer with a combination of a MEK inhibitor and a multi-target agent that targets the RTK, S6 and JAK/STAT.

Referencing sequence lists submitted as text files through EFS-WEB

The official copy of the sequence listing was submitted electronically as an ASCII formatted sequence listing through EFS-Web with a file name of 501159 sequalt. txt, created on 8 months and 10 days 2017, 471 bytes in size, and submitted simultaneously with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is incorporated by reference herein in its entirety.

Background

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most malignant cancers, and many therapeutic approaches have been explored based on drug response findings of pancreatic cancer cells tested in culture models. Most PDAC tumors are driven by activation of MAPK signaling driven by KRAS mutations (Journal S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenerdt P, Core signaling pathway in human systemic cancers associated by globalgenic analytes, Science, 2008; 321 (5897): 1801-6, epipub 2008/09/06; Biankin AV, Waddell N, Kassahn KS, Gingras MC, Muthousmy LB, Johns, Panstanding tumors associated in axon guiding genes, Nature, 2012; 491 7424) -405, epipub 2012/10/30, Cid-Areange A, Jukura area in molecular tumors, Peruch molecular tumors 2015 important genes, MEK 52, MEK mutation of Japanese scientific patients, MEK 52, and MEK mutation of Japanese scientific patients, MEK 31, Japan scientific tumors, Japanese scientific tumors, and MEK mutation 97, thus being important candidates for the treatment of PDAC (MEK) patients, MEK-related to be referred to as a clinical diagnosis of Japanese scientific research. However, preclinical and clinical studies have revealed to a large extent that inhibition of the MAPK pathway alone lacks efficacy, probably due to rapid development of resistance to MAPK inhibitors through various compensatory mechanisms, thereby limiting the efficacy of the inhibitor and leading to the emergence of drug-resistant tumors. (Baines AT, Xu D, Der CJ, Inhibition of Ras for registration: the search constraints, Future medical Chemistry, 2011; 3 (14): 1787. sub.808, epub.2011/10/19; McCubree JA, Steelman LS, Chappell WH, Abrams SL, Franklin RA, Montalto G, Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cassette inhibitors: homutation can rest in thermal resistance and stress resistance, Oncotarget 2012, 3 (10): 1068. sub.111, Epub 2012/10/23; Juntilia MR, Deviatili V, Molecular resistance, and mutation of reaction, MEK 7, model AC 36, model of culture J14; sample AC 36, model of culture of Ma).

Enhancing the understanding of the underlying mechanisms of sensitivity and resistance to MEK inhibitors would identify inhibitors that are effective in targeting pancreatic cancer cells for use in combination with MEK inhibitors.

Disclosure of Invention

In one aspect, the subject matter described herein relates to a method of treating pancreatic cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a combination of active agents, wherein the combination comprises a MEK inhibitor and a multikinase that targets PDGFR α, S6, and STAT 3.

In another aspect, the subject matter described herein relates to a combination of a) a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and b) a multi-kinase inhibitor targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, for use in the treatment of pancreatic cancer.

In one aspect, the subject matter described herein relates to a pharmaceutical composition comprising an effective amount of a MEK inhibitor, or a pharmaceutically acceptable salt thereof, in combination with a multikinase agent targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the composition is for use in the treatment of pancreatic cancer.

In another aspect, the subject matter described herein relates to a kit for treating pancreatic cancer comprising a) a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multi-kinase inhibitor targeting PDGFR α, S6, and STAT3, or a pharmaceutically acceptable salt thereof, and b) a package insert or label indicating treatment for pancreatic cancer.

These and other aspects are described more fully below.

Drawings

Figure 1 depicts the differential response of KP4 PDAC cells to MEK inhibition in 2D monolayer and 3D sphere cultures: small molecule inhibitor screening of KP4 cancer cells in 2D and 3D cultures.

Figure 2 depicts the differential response of KP4 PDAC cells to MEK inhibition in 2D monolayer and 3D sphere cultures: phosphokinase array layout, the position of differentially activated proteins in panel C darkens.

Figure 3 depicts the differential response of KP4 PDAC cells to MEK inhibition in 2D monolayer and 3D sphere cultures: phosphorylation kinase arrays of 2D and 3D KP4 cells.

Figure 4 depicts the differential response of KP4 PDAC cells to MEK inhibition in 2D monolayer and 3D sphere cultures: protein kinase arrays determined by RT-PCR of KP4 cells cultured in 2D or 3D and treated with cobicistinib (cobimetinib) for 24 hours. Samples were normalized to the DMSO-treated KP42D control.

Figure 5 depicts the differential response of KP4 PDAC cells to MEK inhibition in 2D monolayer and 3D sphere cultures: luciferase reporter assay in KP4 cells cultured in 2D or 3D and treated with cobicistinib for 24 hours. Transcription factors with significant differences in fold-change in luciferase activity are shown in red.

Figures 6A-B depict MEK inhibition inducing activation of PDGFR α, S6 and STAT3 (figure 6A) immunoblotting confirming PDGFR α, S6 and STAT 3activation after cobicistinib treatment in KP4 cells (figure 6B) confirming by western blotting an increase in p-STAT3 after cobicistinib treatment in PDAC cell lines and KPP GEMM derived cell lines.

Figure 7 depicts MEK inhibition induced activation of PDGFR α, S6 and STAT3 by measuring IL-6 secretion levels in 2D culture supernatants of PDAC cell lines treated with cobitinib for 24 hours.

Fig. 8 depicts MEK inhibition induced PDGFR α, S6, and STAT 3activation CTG assay measures the effect of Dox-induced STAT3 knockdown in combination with cobinib on KP4 and MIA-PACA2 cell viability.

Figure 9 depicts MEK inhibition induced activation of PDGFR α, S6 and STAT 3. western blot confirms effective knock-down of STAT3 after 72 hours of treatment with Dox (1 μ g/ml).

Figure 10 depicts MEK inhibition induces PDGFR α, S6 and STAT 3activation quantifying the invasive potential of pancreatic cancer cells cultured in 2D and 3D in response to 10% FBS in Matrigel invasion assay.

Figure 11 depicts that inhibition of S6 and STAT3in combination with cobicistinib increased pancreatic cancer cell death: the effect of the combination of the multiple RTK inhibitors ponatinib (ponatinib) and cobitinib on the activation of signaling receptors and downstream targets was confirmed by western blotting.

Figure 12 depicts that inhibition of S6 and STAT3in combination with cobicistinib increased pancreatic cancer cell death: the effect of cobitinib +/-ponatinib or JAK inhibitors on STAT 3activation and induction of cleaved PARP was confirmed by western blotting.

Figure 13 depicts that inhibition of S6 and STAT3in combination with cobitinib increased pancreatic cancer cell death: PathScan multiplex Western blot analysis of KP4 cells pretreated with Coptitinib +/-ponatinib in 2D and 3D cultures.

FIG. 14 depicts the effect of Coptinib +/-Ponatinib/S6/PDGFR α inhibitors on STAT 3activation and induced cleaved PARP as confirmed by Western blotting that inhibition of S6 and STAT3in combination with Coptinib increased pancreatic cancer cell death.

Figure 15 depicts that inhibition of S6 and STAT3in combination with cobitinib increased pancreatic cancer cell death: the effect of the PDGFR inhibitor creolanib (crenolanib) in combination with cobitinib and ruxolitinib (ruxolitinib) on the viability of KP4 cells was measured using the CTG assay.

Figures 16 and 17 depict that inhibition of S6 and STAT3in combination with cobicistinib increased pancreatic cancer cell death: synergy between cobicistinib and ponatinib was analyzed in pancreatic cancer cell lines using the Bliss assay.

Figure 18 depicts that inhibition of S6 and STAT3in combination with cobicistinib increased pancreatic cancer cell death: bliss analysis of the synergistic effect of cobicistinib and the S6 inhibitor rapamycin (rapamycin) in KP4 cells.

Figure 19 depicts that inhibition of S6 and STAT3in combination with cobicistinib increased pancreatic cancer cell death: bliss analysis of the synergistic effect of cobicisinib and GDC-0980 in KP4 cells.

Figures 20A-B depict inhibition of PDGFR α/S6/STAT3 and MEK impairs tumor growth and reduces serum PDGF α (figure 20A) tumor growth curves (mean ± s.e.m) for KP4 xenograft models (n ═ 10 per cohort) treated every 24 hours with cobicistinib (5mg/kg, oral, once daily) and/or ponatinib (30mg/kg, oral, once daily) (figure 20B) tumor growth curves (mean ± SEM) for KPP xenograft models treated as in (figure 20A).

FIG. 21 depicts inhibition of PDGFR α/S6/STAT3 and MEK impairs tumor growth and reduces serum PDGF α representative tumor sections from KP4 xenografts stained by IHC to detect p-STAT3, p-Erk and cleaved caspase 3, or co-stained by IF with p-STAT3 and F4/80 or p-PDGFR α antibodies.

Figure 22 depicts inhibition of PDGFR α/S6/STAT3 and MEK impairs tumor growth and reduces serum PDGF α immunoblotting confirming PDGFR α, S6 and STAT 3activation after treatment of KP4 xenograft tumors with cobicistinib/ponatinib.

Figure 23 depicts the Luminex assay of inhibition of PDGFR α/S6/STAT3 and MEK impaired tumor growth and reduced growth factors and cytokines in plasma samples from serum PDGF α: KP4 xenograft mice.

FIG. 24 depicts an analysis of p-Erk and p-STAT 3in pancreatic tumors and metastases from PDAC patient samples: distribution of p-Erk-and p-STAT 3-positive samples in different stages of primary and metastatic malignancy from 82 PDAC patients.

FIG. 25 depicts an analysis of p-Erk and p-STAT 3in pancreatic tumors and metastases from PDAC patient samples: representative primary tumors and liver sections stained for p-Erk and p-STAT 3in PDAC patient samples. Scale bar: 20 μm.

FIG. 26 depicts an analysis of p-Erk and p-STAT 3in pancreatic tumors and metastases from PDAC patient samples: microarray analysis of PDGFR and IL-6R expression in tumor samples from PDAC patients.

Fig. 27A-B depict KP 4-derived 3D spheroids with histopathological features of micrometastases showing differential sensitivity to small molecule inhibitors: KP4 cells were cultured for 3 days in RPMI medium containing 10% normal FBS (for 2D culture) or 10% precooked FBS (for 3D culture) and IHC was performed on 2D cell pellets or 3D spheroids for Ki67, p-STAT3 and cleaved caspase-3.

Figure 28 depicts KP 4-derived 3D spheroids with histopathological features of micrometastases showing differential sensitivity to small molecule inhibitors: spheroids formed using 10% precooked FBS showed similar drug sensitivity characteristics as seen with other conventional 3D culture methods.

Figure 29 depicts KP 4-derived 3D spheroids with histopathological features of micrometastases showing differential sensitivity to small molecule inhibitors: cell viability assay and colony count assay of spheroids formed by different 3D culture methods.

Figure 30 depicts cobicistinib/ponatinib co-treatment impaired tumor growth and reduced tumor-associated macrophage infiltration: representative tumor sections from KPP xenografts were examined by IHC with p-Erk and p-STAT3 antibodies or co-stained by immunofluorescence with anti-p-STAT 3 and anti-F4/80. Scale bar: IHC slides 20 μm, IF slides 50 μm.

Fig. 31A-B depict cobicistinib/ponatinib co-treatment impaired tumor growth and reduced tumor-associated macrophage infiltration: body weight changes in mice treated with cobicistinib +/-ponatinib in KP4 and KPP xenograft models.

Figure 32 depicts cobicistinib/ponatinib co-treatment impaired tumor growth and reduced tumor-associated macrophage infiltration: luminex assay of growth factors for plasma samples of KP4 xenografted mice.

Figure 33 depicts cobicistinib/ponatinib co-treatment impaired tumor growth and reduced tumor-associated macrophage infiltration immunoblotting confirmed PDGFR α, S6, and STAT 3activation following cobicistinib/ponatinib treatment of KPP xenograft tumors.

Data in fig. 6-23 are expressed as mean ± s.e.m. P < 0.05, student t-test.

Detailed Description

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most fatal human diseases, and most remain refractory to available drug therapies. The KRAS-driven MEK pathway is activated mutally in most pancreatic cancers and is an important target for therapeutic agents. However, under-targeting and compensatory feedback loop activation of oncogenic drivers and failure to prevent metastatic spread are known to lead to poor prognosis of the disease.

Activation of transmembrane Receptor Tyrosine Kinases (RTKs) leads to a number of biochemical cascades that ultimately lead to the regulation of cell fate. This activation is regulated by molecules in the extracellular environment, mainly growth factors, extracellular matrix (ECM) proteins and adhesion molecules present on the surface of adjacent cells. Cellular responses to these signals occur when the molecule binds to a specific receptor present on the cell surface. Many Growth Factors (GF) bind to and activate transmembrane glycoproteins of the Receptor Tyrosine Kinase (RTK) family.

RTKs contain an extracellular ligand binding domain, a single transmembrane domain, and an intracellular component containing a tyrosine kinase domain and several regulatory tyrosines. RTKs can phosphorylate and activate cytoplasmic STAT family transcription factors. These activated STATs are translocated to the nucleus. Once inside the nucleus, STATs promote transcription of genes involved in cell proliferation.

Platelet-derived growth factor receptors (PDGFRs) are cell surface tyrosine kinase receptors for members of the platelet-derived growth factor (PDGF) family PDGFR α and PDGFR β isoforms are factors that regulate cell proliferation, cell differentiation, cell growth and development after binding, the two isoforms dimerize to activate kinase activity and PDGFR phosphorylation.

As described herein, using a 2-dimensional monolayer culture system and a 3-dimensional sphere culture system, it has been found that combination therapy with a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multi-target agent, or a pharmaceutically acceptable salt thereof, that targets PDGFR α, S6, and STAT3, effectively targets pancreatic cancer cells in both the monolayer and the spheroid, without being bound by theory, this combination can effectively prevent signaling via PDGFR α and MEK kinase, while also preventing activation of compensatory feedback loops mediated by STAT 38stat 32 and S6 in cancer cells.

The survival pathways identified as PDGFR β, MEK, S6 and STAT3 involve tumorigenesis and metastasis in various Cancer models (tissue M, Kurkowski MU, Ludes K, Rose-John S, Treiber M, Kloppel G, State 3/Socs3 activity by IL-6 metastasis promoter and maintenance of pancreas tumor, Cancer Cell, 2011; 19(4) 456-69, Epcan 2011/04/13; Yen TW, Aardal NP, Bronner MP, Thorning DR, Savard CE, Lee SP, Myofibre stresses for pancreas Cancer or PDG 11, percutaneous lung Cancer cells, PDG, VEGF-103, percutaneous lung tissue, PDG, VEGF-11, prostate tissue, VEGF-11, PDG 2, VEGF-11, VEGF-2, VEGF-9, VEGF-2, Cell, VEGF-6, Cell.

The pancreatic duct Cancer (PDAC) Is associated with obvious involvement of various stromal elements, such as tumor growth, metastasis, immunosuppression and resistance of tumor cells (Cid-areguigu A et al, (2015), Hidalgo M. pancreaticoccus, The New engand tumor of Medicine 2010, 362, 1605-17, epip 8, Korc M. pancreaticoccus Cancer-2015, tissue expression, protein expression and tumor metastasis, such as tumor growth.

The presently disclosed subject matter is described more fully below. However, many modifications and other embodiments of the presently disclosed subject matter described herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein encompasses all alternatives, modifications, and equivalents. If one or more of the incorporated documents, patents, and similar materials differ or contradict the present application, including but not limited to defined terms, term usage, described techniques, and the like, the present application controls. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

I. Definition of

The term "pancreatic cancer" refers to a neoplasm that originates in the pancreas. Pancreatic cancer includes exocrine and endocrine cancers. Most pancreatic cancers are exocrine tumors. Pancreatic endocrine tumors are also known as islet cell tumors. Unfortunately, the prognosis for pancreatic cancer is currently poor, even if detected early. Pancreatic cancers that can be treated with the methods described herein include, but are not limited to, exocrine pancreatic cancers and endocrine pancreatic cancers. Exocrine pancreatic cancers include, but are not limited to, adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, colloid-like carcinoma, undifferentiated carcinoma with osteoclastoid giant cells, hepatoid carcinoma, intraductal papillary mucinous tumors, mucinous cystic tumors, pancreatoblastoma, serous cystadenoma, signet ring cell carcinoma, solid and pseudopapillary tumors, pancreatic ductal carcinoma, and undifferentiated carcinoma. In some embodiments, the exocrine pancreatic cancer is pancreatic ductal carcinoma. Endocrine pancreatic cancers include, but are not limited to, insulinomas and glucagonoma.

In some embodiments, the pancreatic cancer is any one of early stage pancreatic cancer, non-metastatic pancreatic cancer, primary pancreatic cancer, resectable pancreatic cancer, advanced pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer, unresectable pancreatic cancer, remission pancreatic cancer, recurrent pancreatic cancer, pancreatic cancer under adjuvant therapy, or pancreatic cancer under neoadjuvant therapy. In some embodiments, the pancreatic cancer is locally advanced pancreatic cancer, unresectable pancreatic cancer, or metastatic ductal pancreatic cancer. In some embodiments, the pancreatic cancer is resistant to gemcitabine-based therapy. In some embodiments, the pancreatic cancer is refractory to gemcitabine-based therapy.

The methods described herein may be used for any one or more of the following purposes: alleviating one or more symptoms of pancreatic cancer, delaying progression of pancreatic cancer, reducing pancreatic cancer tumor size, disrupting (e.g., destroying) pancreatic cancer stroma, inhibiting pancreatic cancer tumor growth, extending overall survival, extending disease-free survival, extending the time to pancreatic cancer disease progression, preventing or delaying pancreatic cancer tumor metastasis, reducing (e.g., eradicating) pre-existing pancreatic cancer tumor metastasis, reducing the occurrence or burden of pre-existing pancreatic cancer tumor metastasis, preventing pancreatic cancer recurrence, and/or improving the clinical benefit of a pancreatic cancer patient.

The pancreatic cancer treatment methods described herein comprise administering to a subject (human or animal) a therapeutically effective amount of a combination of a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multi-kinase inhibitor, or a pharmaceutically acceptable salt thereof, that targets PDGFR α, S6, and STAT3 to inhibit, slow, or reverse the growth, development, or spread of pancreatic cancer, including primary and metastatic tumors.

The term "subject" refers to an animal, such as a mammal, including, but not limited to, a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, and the like. In certain embodiments, the subject is a human.

The term "prevention" or "prophylactic" refers to the continued absence of symptoms of a disease or disorder that is expected in the absence of administration of the combination.

The term "synergistic" as used herein refers to a synergistic combination that is more effective than the additive effect of two or more single agents, as measured by the synergistic interaction between a MEK inhibitor and a multikinase inhibitor targeting PDGFR α, S6 and STAT3 can be based on the results obtained from the assays described herein, e.g., the in vivo or in vitro methods disclosed herein, the results of these assays can be analyzed using Chou and Talalay combination methods and dose effect analysis with CalcuSyn software to obtain a combination index (Chou and Talalay, 1984, adv. enzyme Regul.22: 27-55), the combinations provided can be evaluated in one or more assay Systems, and data can be analyzed using standard procedures to quantify the synergistic, additive, and antagonistic effects between anti-cancer agents, an exemplary procedure is that Chou and Talalay are formulated in New Avenus in development therapy when the synergistic, additive, or antagonistic, as a single agent, or synergistic effect when the combination is administered in a single dose of a single agent, the combination index (when the combination index is greater than 0, the synergistic effect is greater than 0, the sum of the synergistic effect when the combination index is greater than the sum of the single agent alone, the sum of the synergistic effect when the synergistic effect is greater than the sum of the single agent, the synergistic effect is greater than the sum of the synergistic effect of the single agent when the sum of the single agent, the sum of the single agent, the sum of.

"spheroid" refers to a cell culture, as known in the art as a three-dimensional (3D) cell culture in an artificial environment, where biological cells grow in all three dimensions or interact with their surroundings. Unlike a 2D environment, such as a petri dish, 3D cell culture allows cells to grow in vitro in all directions, similar to the way they grow in vivo. The artificial environment comprises a medium that provides certain nutrients and other factors that allow the cells to grow. The media described herein provide an environment for rapid cell culture. The term "rapid" refers to a time period less than that required for the same cells in a control medium to expand.

The term "contacting" refers to an enzyme target or survival pathway that allows the active agent to come into sufficiently close proximity such that the active agent is able to bind and inhibit, reduce, or reduce the activity of the target.

As used herein, the term "mammal" refers to humans as well as all other mammals. As used herein, the term "mammal" includes "subject" or "patient" and refers to a warm-blooded animal. It is understood that guinea pigs, dogs, cats, rats, mice, horses, goats, cattle, sheep, zoo animals, livestock, primates, and humans are all examples of animals within the meaning of this term.

As used herein, a "mammal in need thereof" is a subject that has been diagnosed as having a particular condition intended to be treated, e.g., pancreatic cancer or a particular type of pancreatic cancer.

The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an adverse physiological change or disorder, such as the development or spread of cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, palliation (whether partial or total), whether detectable or undetectable. "treatment" also means an increase in survival compared to the expected survival when not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those predisposed to the condition or disorder or those for which the condition or disorder is to be prevented.

The use of the term "inhibitor" herein means a molecule that inhibits the activity of a target enzyme. By "inhibit" is meant herein a decrease in the activity of the target enzyme compared to the activity of the enzyme in the absence of the inhibitor. In some embodiments, the term "inhibit" means a reduction in MEK activity or a reduction in a particular survival pathway of a cancer cell by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In other embodiments, inhibition means a decrease in activity of about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to 100%. In some embodiments, inhibition means a decrease in activity of about 95% to 100%, e.g., a decrease in activity of 95%, 96%, 97%, 98%, 99%, or 100%. This decrease can be measured using a variety of techniques recognizable to those skilled in the art, including in vitro kinase assays.

As used herein, a "MEK inhibitor" is a molecule that reduces, inhibits, or otherwise impairs one or more biological activities of MEK (MEK1 and/or MEK 2). Activity can be reduced by a statistically significant amount, including, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 63%, 70%, 75%, 80%, 85%, 95%, or 100% reduction in MEK activity as compared to a suitable control.

As used herein, a "multi-kinase inhibitor" is a molecule that reduces, inhibits, or otherwise impairs the biological activity of PDGFR α, S6, and STAT 3a suitable multi-kinase inhibitor exhibits activity against each of these three targets, and may have activity against other targets.

The presently disclosed compounds may or may not beIs a specific inhibitor. By "specific inhibitor" is meant an agent that reduces, inhibits, or otherwise impairs the activity of a defined target over an unrelated target. For example, a MEK-specific antagonist reduces at least one biological activity of MEK by an amount that is statistically greater than the inhibitory effect of the antagonist on any other protein (e.g., other serine/threonine kinases). In some embodiments, IC of the target antagonist₅₀Is a non-target antagonist IC₅₀About 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or less of. Specific MEK inhibitors reduce the biological activity of one or more family members of MEK in amounts statistically greater than the inhibitory effect of the antagonist on any other protein (e.g., other serine/threonine kinases). In some of these embodiments, the IC of the MEK inhibitor of RAF₅₀IC of MEK inhibitor being another serine/threonine kinase, other MEK family member or other type of kinase (e.g., tyrosine kinase)₅₀About 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 0.1%, 0.01%, 0.001% or less.

The term "targeting" refers to the ability of an active agent to specifically act on a specific MEK enzyme or a specific enzyme associated with a survival pathway. The ability of an active agent to target an enzyme can be determined by routine experimentation as described herein or using other known methods.

As used herein, the "survival pathway" refers to the adaptive mechanism of a cancer cell or the ability to promote its survival and proliferation and to be resistant to an active agent as used herein, the survival pathway is associated with PDGFR α, S6, and STAT 3.

The phrase "therapeutically effective amount" means an amount of an active agent that (i) treats or prevents pancreatic cancer, (ii) alleviates, ameliorates, or eliminates one or more symptoms of pancreatic cancer, or (iii) prevents or delays the onset of one or more symptoms of pancreatic cancer. The therapeutically effective amount of the drug may reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or relieve to some extent one or more symptoms associated with cancer. To the extent that the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and/or determining Response Rate (RR).

The term "pharmaceutically acceptable salt" refers to salts that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include acid addition salts and base addition salts. The phrase "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. The phrase "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable organic or inorganic salts of the molecule. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1' -methylenebis- (2-hydroxy-3-naphthoate)). Pharmaceutically acceptable salts can include salts comprising another molecule, such as an acetate, succinate, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Examples where the plurality of charged atoms are part of a pharmaceutically acceptable salt can have a plurality of counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions. Examples include, but are not limited to, the fumarate or hemifumarate salts of cobicistinib and ponatinib HCl.

Other definitions are provided below as appropriate.

In vivo methods

The presently disclosed combination of MEK inhibitors and multi-kinase inhibitors targeting PDGFR α, S6 and STAT3 are useful for the treatment of pancreatic cancer.

In an embodiment disclosed herein is a method of treating pancreatic cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a combination of active agents, wherein the combination comprises a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multikinase agent, or a pharmaceutically acceptable salt thereof, that targets PDGFR α, S6, and STAT 3.

Examples of MEK inhibitors that may be used include cobicistinib, GDC-0623, trametinib (trametinib), bimetinib (binimetinib), semetinib (selumetinib), pimatinib (pimasetinib), rafatinib (refametinib), PD-0325901, and BI-847325, or a pharmaceutically acceptable salt thereof. In one embodiment, the MEK inhibitor is cobicistinib, or a pharmaceutically acceptable salt thereof. Cobitinib has the following structure:

cobitinib asSold and presented as the hemifumarate salt in commercial preparations.

A useful multikinase agent targeting PDGFR α, S6, and STAT3 is ponatinib, which has the following structure:

ponatinib toSold and presented as the hydrochloride salt in commercial preparations.

Pancreatic cancer includes endocrine and exocrine cancers. In embodiments, the pancreatic cancer is selected from the group consisting of: adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, colloid-like carcinoma, undifferentiated carcinoma with osteoclastoid giant cells, hepatoid, intraductal papillary mucinous tumors, mucinous cystic tumors, pancreatoblastoma, serous cystadenoma, signet ring cell carcinoma, solid and pseudopapillary tumors, pancreatic ductal carcinoma, and undifferentiated carcinoma. In one embodiment, the pancreatic cancer is pancreatic ductal adenocarcinoma.

In embodiments, the MEK inhibitor, or a pharmaceutically acceptable salt thereof, and the multi-kinase inhibitor, or a pharmaceutically acceptable salt thereof, targeting PDGFR α, S6, and STAT3 are synergistic.

In embodiments, the MEK inhibitor, or a pharmaceutically acceptable salt thereof, and the multi-kinase inhibitor, or a pharmaceutically acceptable salt thereof, are administered as a combined preparation. In embodiments, the combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. Co-administration includes co-administration, use of separate formulations or a single pharmaceutical formulation, and sequential administration in either order, wherein preferably there is a period of time during which both (or all) active agents exert their biological activity simultaneously.

Any method known in the art for measuring MEK activity or PDGFR α, S6, STAT3, and tumor-associated macrophage activation levels can be used to determine the activity of the combination, including in vitro kinase assays, immunoblotting with antibodies specific for phosphorylated targets, or measuring biological effects downstream of activity.

Pancreatic cancer may be early or late, or may have metastasized. The combinations described herein can be used to treat cancer at any stage, including cancer that has metastasized.

Treatment of a subject with an effective amount of a combination MEK inhibitor and a multi-kinase inhibitor targeting PDGFR α, S6, and STAT3 may comprise a monotherapy or may comprise a series of therapies.

Useful amounts of active agents are as described elsewhere herein. In particular embodiments, the MEK inhibitor is administered in an amount from about 5mg to about 100mg, or from about 45mg to about 75mg, and the multi-kinase inhibitor is administered in an amount from about 5mg to about 100mg, or from about 30mg to about 60 mg. In embodiments, the MEK inhibitor is administered in an amount of about 60mg, and the multi-kinase inhibitor is administered in an amount of about 45 mg. In embodiments, the administration of the combination therapy is once daily oral administration. In embodiments, it is preferred that the MEK inhibitor is cobicistinib, or a pharmaceutically acceptable salt thereof, and the multi-kinase inhibitor is ponatinib, or a pharmaceutically acceptable salt thereof. In some embodiments, each active agent is administered to the subject at a dose ranging from about 0.001 μ g/kg to about 1000mg/kg, including but not limited to about 0.001 μ g/kg, 0.01 μ g/kg, 0.05 μ g/kg, 0.1 μ g/kg, 0.5 μ g/kg, 1 μ g/kg, 10 μ g/kg, 25 μ g/kg, 50 μ g/kg, 100 μ g/kg, 250 μ g/kg, 500 μ g/kg, 1mg/kg, 5mg/kg, 10mg/kg, 25mg/kg, 30mg/kg, 40mg/kg, 50mg/kg, 100mg/kg, and 200 mg/kg. The amount of each active agent present in the combination composition to be administered may be from about 1mg to about 1000mg, or from about 5mg to about 100mg, or from about 10mg to about 80mg, or from about 45mg to about 75mg, or from about 30mg to about 60 mg. It will be appreciated that the appropriate dosage of the active compound will depend on a number of factors within the knowledge of the ordinarily skilled physician or veterinarian. The dosage of the active agent may vary, for example, depending on the age, body weight, general health, sex and diet of the subject, time of administration, route of administration, rate of excretion and any drug combination. It is also understood that the effective dose for treatment may be increased or decreased during a particular course of treatment. Dose variation may result and become apparent from the results of the diagnostic assay.

In some embodiments, described herein is a method of treating pancreatic cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a combination MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multikinase agent that targets PDGFR α, S6, and STAT3, or a pharmaceutically acceptable salt thereof, further comprising administering an additional therapy.

In some embodiments, the treatment elicits a sustained response in the subject after the treatment is stopped. By "sustained response" is meant a sustained effect on the reduction of tumor growth after cessation of treatment. For example, the tumor size may remain the same or smaller than the size at the beginning of the administration phase. In some embodiments, the duration of the sustained response is at least the same as the duration of treatment, at least 1.5 times, 2.0 times, 2.5 times, or 3.0 times the length of the duration of treatment.

The methods of treatment disclosed herein may result in a partial or complete response. As used herein, "complete response" or "CR" refers to the disappearance of all target lesions; "partial response" or "PR" means that the sum of the longest diameter (SLD) of the target lesion is reduced by at least 30% with reference to the baseline SLD; and "stable disease" or "SD" means neither sufficient reduction to meet PR requirements nor sufficient increase to meet PD requirements for the target lesion, with reference to the smallest SLD since the start of treatment. As used herein, "total reaction rate" (ORR) refers to the sum of the rate of Complete Reaction (CR) and the rate of Partial Reaction (PR).

The treatment methods disclosed herein can result in an increase in progression-free survival and overall survival in subjects administered the combination therapy. As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the treated disease (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient experiences a complete response or a partial response, as well as the amount of time a patient experiences stable disease.

As used herein, "overall survival" refers to the percentage of subjects in a group that are likely to survive for a particular duration of time.

In some embodiments, the subject to which the combination is administered is a mammal, such as a domestic animal (e.g., cattle, sheep, cats, dogs, and horses), a primate (e.g., human and non-human primates, such as monkeys), a rabbit, and a rodent (e.g., mouse and rat). In some embodiments, the subject treated is a human.

A subject in need of treatment for pancreatic cancer can be a human exhibiting symptoms of cancer, a human already diagnosed with cancer, a subject in remission from pancreatic cancer, or a subject at increased risk of developing cancer (e.g., genetic predisposition, certain dietary or environmental exposures).

Many of the combinations described herein will be identified as synergistic.

In vitro formulations as described hereinThe method uses a method of preparing a spherical cell culture comprising culturing cells in a medium containing serum that has been boiled prior to addition to the medium for said culturing. It has been found that the medium can be modified to rapidly culture 3-D spheroids. The modified medium is prepared by boiling Fetal Bovine Serum (FBS) before contacting it with the cells to be cultured. In embodiments, the FBS is boiled at 95-100 ℃ for 10 minutes, at which time the serum turns yellow in color. The boiled FBS was then centrifuged at 8000Xg for 20 minutes and filtered through a sterile Corning vacuum filter unit having a filter pore size of 0.22 μm. Useful media comprise about 5% to about 20% (v/v) fetal bovine serum. The medium for the spheroid culture was prepared by adding 10% (v/v) boiled FBS to 100 unit ml supplemented with 2mM L-glutamine^-1Penicillin and streptomycin in RPMI medium. The spherical medium needs to be prepared fresh before use. Boiled FBS can be stored at 4 ℃ for 2-3 months. The medium for monolayer culture was prepared by adding 10% (v/v) of normal FBS to 100 unit ml supplemented with 2mM L-glutamine^-1Penicillin and streptomycin in RPMI medium. The medium used for the migration assay was prepared by adding 20% (v/v) of normal FBS to 100 unit ml supplemented with 2mM L-glutamine^-1Penicillin and streptomycin in RPMI medium.

Combination pharmaceutical composition

In embodiments, the subject matter described herein relates to a combination of a) a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and b) a multi-kinase inhibitor targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, for use in the prophylactic or therapeutic treatment of pancreatic cancer.

In embodiments, the subject matter described herein relates to a pharmaceutical composition comprising an effective amount of a MEK inhibitor, or a pharmaceutically acceptable salt thereof, in combination with a multikinase agent targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

In embodiments, the composition comprises a MEK inhibitor selected from: cobicistinib, GDC-0623, trametinib, bimetinib, semetinib, pimatinib, rifatinib, PD-0325901, and BI-847325, or a pharmaceutically acceptable salt thereof. In one embodiment, the MEK inhibitor is cobicistinib, or a pharmaceutically acceptable salt thereof. In a particular embodiment, the MEK inhibitor is cobicistinib hemifumarate.

In embodiments, the multikinase agent is ponatinib or a pharmaceutically acceptable salt thereof. In one embodiment, the multi-kinase inhibitor is ponatinib hydrochloride.

In embodiments, the composition comprises cobicistinib or a pharmaceutically acceptable salt thereof and ponatinib or a pharmaceutically acceptable salt thereof. In embodiments, the composition comprises cobicistinib hemifumarate and ponatinib hydrochloride.

The amount of each active agent present in the combination may be from about 1mg to about 1000mg, or from about 5mg to about 100mg, or from about 10mg to about 80mg, or from about 45mg to about 75mg, or from about 30mg to about 60 mg. In embodiments, the MEK inhibitor is present in an amount from about 45mg to about 75mg, and the multi-kinase inhibitor is present in an amount from about 30mg to about 60 mg.

Combination therapy may provide "synergy" and prove "synergistic," i.e., the effect achieved when the active agents are used together is greater than the sum of the effects produced by the separate use of the active agents. When the active agent: (1) co-formulated and administered or delivered simultaneously in a combined unit dose formulation; (2) delivered as separate formulations, alternately or concurrently; or (3) a synergistic effect may be obtained by other schemes. When delivered in alternation therapy, a synergistic effect may be obtained when the active agents are administered or delivered sequentially, e.g. by different injections in separate syringes, separate pills or capsules, or separate infusions. Generally, during alternation therapy, the effective dose of each active agent is administered sequentially, i.e., continuously, while in combination therapy, the effective doses of two or more active agents are administered together.

MEK inhibitors and/or multi-kinase inhibitors, each also referred to herein as active agents, may be formulated as pharmaceutical compositions according to standard pharmaceutical practice. An exemplary multi-kinase inhibitor, ponatinib, can be used as tablets for oral administration at 15mg, 30mg, and 45mg strengths. An exemplary MEK inhibitor, cobicistinib, may be used as an orally administered tablet at a strength of 20 mg. However, according to this aspect, there is provided further pharmaceutical compositions comprising a MEK inhibitor or a multi-kinase inhibitor, or both, in combination with a pharmaceutically acceptable excipient, such as a carrier or diluent.

Typical formulations are prepared by mixing the active agent and one or more excipients. Suitable excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like. The particular excipients used will depend on the mode and purpose of administering the active agent. Solvents are generally selected based on the knowledge of those skilled in the art to administer to a mammal safe (GRAS) solvents. Generally, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG 400, PEG 300), and the like, and mixtures thereof. The formulations may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, light-protecting agents, glidants, processing aids, colorants, sweeteners, fragrances, flavoring agents and other known additives to provide a refined presentation of the active agent or aid in the manufacture of the pharmaceutical product (i.e., medicament).

The formulations may be prepared using conventional dissolution and mixing procedures. For example, bulk drug substances (i.e., active agents or active agents in a stable form (e.g., complexes with cyclodextrin derivatives or other known complexing agents) are dissolved in a suitable solvent in the presence of one or more of the above-mentioned excipients.

The pharmaceutical composition (or formulation) for administration may be packaged in a variety of ways depending on the method used to administer the drug. Typically, the article of manufacture for dispensing comprises a container in which the pharmaceutical formulation is deposited in a suitable form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), pouches, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-proof assembly to prevent inadvertent access to the package contents. In addition, the container has deposited thereon a label describing the contents of the container. The tag may also contain appropriate warnings. In one embodiment, the container is a blister pack.

Pharmaceutical formulations can be prepared for various routes and types of administration. For example, an active agent having a desired purity can optionally be mixed with pharmaceutically acceptable excipients (Remington's Pharmaceutical Sciences (1980) 16 th edition, Osol, a.ed., Mack Publishing co., Easton, PA) in the form of a lyophilized formulation, a milled powder, or an aqueous solution. Formulation may be carried out by mixing at ambient temperature at an appropriate pH and desired purity with a physiologically acceptable carrier, i.e., a carrier that is non-toxic to the recipient at the dosages and concentrations employed. The pH of the formulation depends primarily on the particular use and compound concentration, but can range from about 3 to about 8. Formulation in acetate buffer at pH 5 is a suitable embodiment.

The active agent may be sterile. In particular, formulations for in vivo administration must be sterile. Such sterilization can be readily accomplished by filtration through sterile filtration membranes.

The active agent can be stored as a solid composition, a lyophilized formulation, or an aqueous solution.

Pharmaceutical compositions comprising active agents may be formulated, administered and administered in a manner, i.e., amount, concentration, schedule, course of treatment, vehicle and route of administration, consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the individual patientClinical condition, cause of the condition, site of delivery of the agent, method of administration, administration regimen, and other factors known to the physician. A "therapeutically effective amount" of a compound to be administered will be governed by such considerations. Acceptable excipients are non-toxic to the recipient at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, e.g. methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as polysorbates (e.g., TWEEN)^TM) Poloxamers (e.g., PLURONICS)^TM) Or polyethylene glycol (PEG) the active agent may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, these techniques are disclosed in Remington's pharmaceutical α l Sciences.

Sustained release formulations of the active agent can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the active agent, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methylpropylene)Acid esters) or poly (vinyl alcohol)), polylactide (US3773919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as copolymers of lactic acid-glycolic acid and leuprolide acetate (LUPRON DEPOT)^TM) And poly-D- (-) -3-hydroxybutyric acid.

The formulations include those suitable for the routes of administration detailed herein. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are commonly found in Remington's pharmaceutical sciences. Such methods include the step of bringing into association the active agent with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of active agents suitable for oral administration may be prepared as discrete units, such as pills, capsules, cachets, or tablets, each containing a predetermined amount of the active agent.

Compressed tablets may be prepared by compressing in a suitable machine a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered active agent moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally formulated so as to provide sustained or controlled release of the active agent therefrom.

Tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules (e.g., gelatin capsules), syrups or elixirs may be prepared for oral use. Formulations of active agents intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents, including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide palatable preparations. Tablets containing the active agent in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques, including microencapsulation, to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

Aqueous suspensions of the active agents contain the agents in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, for example sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, as well as dispersing or wetting agents, for example naturally-occurring phosphatides (for example lecithin), condensation products of an alkylene oxide with fatty acids (for example polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (for example heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (for example polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives (e.g., ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweetening agents (e.g., sucrose or saccharin).

The pharmaceutical compositions of the active agents may be in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. Such suspensions may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Sterile injectable formulations may also be prepared as lyophilized powders. Acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The amount of active agent that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a timed release formulation for oral administration to humans may contain about 1 to 1000mg of the active agent, combined with an appropriate and convenient amount of carrier material, which may vary from about 5 to about 95% (weight: weight) of the total composition. The pharmaceutical compositions can be prepared to provide an easily measurable amount for administration. For example, an aqueous solution intended for intravenous infusion may contain about 3 to 500 μ g of active agent per mL of solution so that a suitable volume can be infused at a rate of about 30 mL/hour.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for intrapulmonary or intranasal administration have a particle size, for example, in the range of 0.1 to 500 microns (including particle sizes in the range of 0.1 to 500 microns, with micron increments of, for example, 0.5, 1, 30 microns, 35 microns, etc.), which are administered by rapid inhalation through the nasal cavity or by inhalation through the oral cavity in order to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active agent. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents, such as compounds heretofore used in the treatment or prevention of the conditions described below.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active agent such carriers as are known in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dose formulations are those containing a daily dose or unit daily sub-dose, or an appropriate fraction thereof, of the active agent as described above.

The subject matter also provides a veterinary composition comprising at least one active agent as defined above and a veterinary carrier. Veterinary carriers are materials that can be used to administer the compositions, and can be solid, liquid, or gaseous materials that are inert or acceptable in the veterinary art and are compatible with the active agent. These veterinary compositions may be administered parenterally, orally or by any other desired route.

IV. product

In another aspect, described herein is an article of manufacture, such as a "kit" containing materials useful for treating pancreatic cancer, the kit comprising a container comprising a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multikinase inhibitor, or a pharmaceutically acceptable salt thereof, targeting PDGFR α, S6, and STAT3, for treating pancreatic cancer, the kit may further comprise a label or package insert on or associated with the container the term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products containing information regarding indications, usage, dosage, administration, contraindications, and/or warnings for using such therapeutic products.

The kit may further include instructions for administering the MEK inhibitor, or a pharmaceutically acceptable salt thereof, and if formulated alone, a formulation of the multi-kinase inhibitor, or a pharmaceutically acceptable salt thereof. For example, if a kit comprises a first composition comprising a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a second pharmaceutical formulation comprising a multi-kinase inhibitor, or a pharmaceutically acceptable salt thereof, the kit may further comprise instructions for administering the first and second pharmaceutical compositions simultaneously, sequentially or separately to a patient in need thereof.

In another embodiment, the kit is suitable for delivering a solid oral form of a MEK inhibitor, or a pharmaceutically acceptable salt thereof, in combination with a multi-kinase inhibitor, or a pharmaceutically acceptable salt thereof. Such kits preferably comprise a plurality of unit doses. Such kits may include cards with the doses oriented in the order of their intended use. One example of such a kit is a "blister pack". Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. If desired, a memory aid can be provided, for example in the form of numbers, letters or other indicia or with a calendar insert, specifying the date in the treatment plan on which the dose can be administered.

According to one embodiment, a kit may comprise (a) a first container having a MEK inhibitor, or a pharmaceutically acceptable salt thereof; and optionally (b) a second container having incorporated therein a formulation of a multi-kinase inhibitor or a pharmaceutically acceptable salt thereof. Alternatively or additionally, the kit may further comprise a third container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may also include other materials that are commercially and user friendly, including other buffers, diluents, filters, needles and syringes.

In certain other embodiments in which the kit comprises a composition of a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multikinase inhibitor, or a pharmaceutically acceptable salt thereof, the kit may comprise a container for holding the individual compositions, such as a separate bottle or a separate foil packet, however, the individual compositions may also be held in a single, undivided container. Typically, the kit includes instructions for administering the individual components. The kit form is particularly advantageous when the individual components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when the prescribing physician desires to titrate the individual components of the combination.

In yet another embodiment, the subject matter described herein relates to the use of a medium in which the serum has been pre-boiled, i.e., boiled for a certain time before it is used in the medium. This medium provides for the rapid formation of spheroids compared to a medium using normal serum that has not been boiled. In embodiments, the medium used to culture the cells comprises about 10% boiled fetal bovine serum, wherein the serum is boiled at 95-100 ℃ for 10 minutes prior to its addition to the medium.

The subject matter described herein includes the following specific embodiments:

1. a method of treating pancreatic cancer in a subject in need thereof comprising administering to the subject an effective amount of a combination of active agents, wherein the combination comprises a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multikinase agent, or a pharmaceutically acceptable salt thereof, that targets PDGFR α, S6, and STAT 3.

2. The method of embodiment 1, wherein the MEK inhibitor is selected from the group consisting of: cobicistinib, GDC-0623, trametinib, bimetinib, semetinib, pimatinib, rifatinib, PD-0325901, and BI-847325, or a pharmaceutically acceptable salt thereof.

3. The method according to any preceding embodiment, wherein the MEK inhibitor is selected from cobicistinib and trametinib, or a pharmaceutically acceptable salt thereof.

4. The method according to any preceding embodiment, wherein the MEK inhibitor is cobicistinib, or a pharmaceutically acceptable salt thereof.

5. The method of any preceding embodiment, wherein the multikinase agent is ponatinib, or a pharmaceutically acceptable salt thereof.

6. The method of any preceding embodiment, wherein the pancreatic cancer is endocrine.

7. The method of any of the above embodiments, wherein the pancreatic cancer is exocrine.

8. The method of any of the above embodiments, wherein the pancreatic cancer is adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, colloid-like carcinoma, undifferentiated carcinoma with osteoclastoid giant cells, hepatoid carcinoma, intraductal papillary mucinous tumors, mucinous cystic tumors, pancreatoblastoma, serous cystadenoma, signet ring cell carcinoma, solid and pseudopapillary tumors, pancreatic ductal carcinoma, and undifferentiated carcinoma.

9. The method of any preceding embodiment, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

10. The method according to any of the above embodiments, wherein the combination is synergistic.

11. The method of any preceding embodiment, wherein the active agents are administered sequentially.

12. The method of any preceding embodiment, wherein the active agents are administered simultaneously.

13. The method according to any preceding embodiment, wherein the MEK inhibitor and the multi-kinase inhibitor are administered as a combined preparation.

14. The method of any of the above embodiments, wherein the MEK inhibitor is administered in an amount of about 45mg to about 75mg and the multikinase inhibitor is administered in an amount of about 30mg to about 60 mg.

15. The method of any of the above embodiments, wherein the amount is administered once daily.

16. The method according to any preceding embodiment, wherein the MEK inhibitor is cobicistinib or a pharmaceutically acceptable salt thereof, such as cobicistinib hemifumarate, and the multi-kinase inhibitor is ponatinib or a pharmaceutically acceptable salt thereof, such as ponatinib HCl.

17. A combination of a) a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and b) a multi-kinase inhibitor targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, for use in the prophylactic or therapeutic treatment of pancreatic cancer.

18. A pharmaceutical composition comprising a combination of an effective amount of a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multikinase agent targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the composition is for use in the treatment of pancreatic cancer.

19. The composition of any preceding embodiment, wherein the MEK inhibitor is selected from: cobicistinib, GDC-0623, trametinib, bimetinib, semetinib, pimatinib, rifatinib, PD-0325901, and BI-847325, or a pharmaceutically acceptable salt thereof.

20. The composition according to any of the above embodiments, wherein the MEK inhibitor is selected from cobicistinib and trametinib, or a pharmaceutically acceptable salt thereof.

21. The composition according to any preceding embodiment, wherein the MEK inhibitor is cobicistinib, or a pharmaceutically acceptable salt thereof.

22. The composition of any preceding embodiment, wherein the multikinase agent is ponatinib, or a pharmaceutically acceptable salt thereof.

23. A composition according to any of the above embodiments, comprising cobicistinib or a pharmaceutically acceptable salt thereof and ponatinib or a pharmaceutically acceptable salt thereof.

24. The composition according to any of the above embodiments, wherein the MEK inhibitor, or a pharmaceutically acceptable salt thereof, is present in an amount of from about 45mg to about 75mg and the multi-kinase inhibitor, or a pharmaceutically acceptable salt thereof, is present in an amount of from about 30mg to about 60 mg.

25. A kit comprising a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multikinase agent targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, a container and a package insert or label.

The following examples are provided for the purpose of illustration and not for the purpose of limitation.

Examples

Materials and methods

Cell lines and reagents:

all cell lines were from ATCC except PA-TU-8988T cells from DSMZ and SUIT-2 cells from JCRB. All cell lines were stored in Genentech cell line core facility, which routinely performed SNP and STR analyses to confirm cell line identity. All cell lines were routinely supplemented with 10% FBS, 2mM L-glutamine, 100 unit ml^-1Penicillin and streptomycin in RPMI medium (Gibco). Rapamycin (Rapamycin) and ponatinib are from Selleckchem, ClaraNib is from Arog Pharmaceuticals, and cobitinib, GDC-0941 and GDC-0980 are synthesized in Genentech. Human phosphokinase array kit from R&D Systems (ARY003B), Cignal-45-Pathway reporter array from Qiagen (CCA-901L), human tyrosine kinase RT²The Profiler PCR array was from Qiagen (PAHS-161Z) and the MDSC isolation kit was from Miltenyi Biotec. All assays using these kits were performed according to the manufacturer's instructions.

Western blot and cell viability assay:

cells were seeded in 10cm dishes and treated with 1 μ M small molecule inhibitor for 24 hours. cell lysates were prepared in RIPA lysis buffer (Thermo Scientific) containing protease inhibitor cocktail (Thermo Scientific), SDS-PAGE was performed and proteins were transferred to nitrocellulose membranes. immunoblots were performed using standard methods.Primary antibodies were quantitated for protein bands using ImageJ software p-Erk, p-STAT3, p-S6, Total S6, p-PDGFR α, p-PDGFR β, p-RSK3, cleaved PARP, Bcl-xL, Mcl, survivin, Rab11, p-AkhA, β -actin, GAPDH (cell signaling), Total 3, Total-PDGFR α (Santa Cruz Biotechnology), p-hA 4 (Gene Tex) and p-hA Tex 23/365 (Myou 365/My).

Cell viability assays were performed by treating cells with dose titrations (0.001 to 10 μ M) or fixed doses (1 μ M) of various pharmacological inhibitors for 72 hours, and measuring viability using CellTiter Glo (Promega). The Bliss score was calculated as described previously. (Borisy AA, Elliott PJ, Hurst NW, Lee MS, Lehar J, Price ER, Systematic discovery of multicomponent therapeutics, Proceedings of the national Academy of sciences of the United States of America, 2003; 100 (13): 7977-82, Epub 2003/06/12).

Lentivirus infection:

lentivirus particles of sh STAT3 were generated as described previously. (Moffat J, Grueneberg DA, Yang X, KimSY, Kloepfer AM, Hinkle G, A lentiviral RNAi library for human and mouse gene applied to an arrayed viral high-content screen, Cell, 2006; 124 (6): 1283-98.Epub 2006/03/28). shSTAT 3in the Dox inducible system was obtained from LakePharma. The target sequence for shSTAT3 was AATCTTAGCAGGAAGGTGCCT. KP4 and MIA-PACA2 cells were transduced with optimized titers of lentivirus and selected with 1. mu.g/ml puromycin.

Luminex assay and ELISA:

culture supernatants from cancer cells cultured for 72 hours or plasma samples collected at the end of the animal study were subjected to a Luminex assay using a multiplex kit (Bio-Rad) according to the manufacturer's instructions PDGF α ELISA was performed using a kit from R & D Systems.

Invasion assay:

cells in RPMI containing 2% FBS were seeded on inverted inserts of a BD BioCoat matrix invasion chamber (BDBiosciences). The lower chamber was filled with RPMI supplemented with 10% FBS as a chemoattractant and the number of cells entering the bottom chamber after 18 hours of incubation was measured using SpectraMax M5(Molecular Devices).

Animal studies:

all animal studies were conducted according to the national institutes of health laboratory animal care and use guidelines. All dosing regimens were well tolerated in xenografts. Two xenograft models were used: the human PDAC-derived cell line KP4 and cells derived from tumors isolated from KPP GEMM mice. Mix 5x10⁶Individual cells were implanted subcutaneously in the right flank of immunodeficient mice (charles river Laboratories) without any Matrigel. When the tumor reaches-200 mm³Mice were then randomized into groups and then treated with vehicle (MCT +25mM citrate buffer, pH 2.75), cobicistinib (5mg/kg) +/-ponatinib (30mg/kg) orally once daily. Tumor volumes were measured at the indicated intervals using an Ultra-Cal IV caliper. To properly analyze repeated measurements of tumor volume over time from the same animal, a hybrid modeling approach was used. (Josepinnheiro DB, Saikat DebRoy, Deepayan Sarkar, R Core teamPackage 'nlme' 2008). This approach addresses repeated measurements and moderate dropouts as any treatment-independent animal died before the study ended. Fitting the log of each dose level using a cubic regression spline₂Non-linear curve of the time course of the tumor volume. These non-linear curves are then correlated with the dose within the mixed model. Tumor growth inhibition as a percentage of vehicle (% TGI) was calculated as a percentage of the area under the fitted curve (AUC) of each dose group versus vehicle per day using the following formula:

％TGI＝100x(1-AUC_{dosage form}/AUC_Media)。

To determine the Uncertainty Interval (UI) of% TGI, random samples were generated as an approximation of the% TGI distribution using a fitted curve and a fitted covariance matrix. The random sample consisted of 1,000 simulated realizations of the fitted mixture model, with the% TGI recalculated for each realization. The reported UI is the value at which the 95% time recalculated% TGI value would fall within the region given the fitted model. The 2.5 and 97.5 percentiles of the simulated distribution were used as the up-down UI. Tumor growth inhibition > 60% was considered significant. (Wong H, Choo EF, Alicke B, Ding X, La H, McNamara E, anti activity of targeted and cytoxic agents in human sub environmental sources or models with a Clinical response, Clinical Cancer Research: and scientific resource of the American Association for Cancer Research, 2012; 18 (14): 3846-55.Epub 2012/06/01).

Plots were performed and generated using R version 2.8.1(R Development Core Team 2008; R Foundation for statistical Computing; Vienna, Austria) and Excel version 12.0.1(Microsoft Corporation). Data were analyzed using R version 2.8.1 and a mixed model was fitted to R using nlme package version 3.1-89 (41).

Immunohistochemistry and immunofluorescence:

for formalin-fixed paraffin-embedded cell pellets, whole tissue sectionsAnd sections 4 μm thick cut with TMA block (USBiomax, Inc) were subjected to immunohistochemistry and immunofluorescence. Slides were dewaxed and antigen exposure was performed on PT module (Thermo Scientific) with target retrieval solution (Dako). With 3% H₂O₂Primary antibodies against p-Erk and p-STAT3 (cell signaling) were used at 1 μ g/ml, p-PDGFR α (cell signaling) was 0.4 μ g/ml, F4/80(Serotec) was 10 μ g/ml, Gr-1(BD Pharmingen) was 1 μ g/ml, cleaved caspase 3 (cell signaling) was 0.6 μ g/ml and CD8 α (genetech) was 5 μ g/ml.

Histology:

images of the entire slide were obtained at 200x final magnification using a nanobromometer XR automated slide scanning platform (Hamamatsu, Hamamatsu City, shizuoka pref., Japan).

Microarray and RNA sequencing analysis:

gene expression in normal and PDAC human tissues was measured by Gene Logic using the Affymetrix HGU133P array expression probe set 203131_ at for human PDGFR α, 202273_ at for human PDGFR β, 205945_ at for human IL-6-R, and 204171_ at for PRS6KB 1.

Statistical analysis:

all data are expressed as mean ± standard error of mean (s.e.m). Student's t-test (two-tailed) was used to compare 2 groups and to calculate P-values using Prism or Excel. P < 0.05 was considered significant. Survival curves were plotted using the Kaplan-Meier method.

Example 1: sensitivity to MEK inhibition in pancreatic cancer cell spheroid cultures

To compare how pancreatic cancer cells cultured in two-dimensional (2D) and three-dimensional (3D) cultures responded to various cancer therapeutics, we treated the KRAS mutant PDAC cancer cell line KP4 with a panel of 203 small molecule inhibitors of cancer-associated targets and known chemotherapeutic agents (table 1).

TABLE 1 Small molecule inhibitors

For 3D culture, methods are described herein that facilitate relatively rapid "high throughput" analysis. It has been observed that culturing cancer cells in a medium containing 10% fetal bovine serum that has been pre-boiled at 95-100 ℃ for 10 minutes before its addition to the medium always results in the rapid formation of 3D "spheroids". Spheroids formed using this method showed all the typical features of the previously described spheroids, including low level proliferation, major localization of Ki67 staining in the peripheral layer, and necrotic/apoptotic centers with apoptosis marked by cleaved caspase-3 by 72 hours (fig. 27). Their drug sensitivity pattern was also similar to the spheroids described previously (fig. 28 and 29).

Treatment of KP4 cells with a panel of 203 compounds under 2D and 3D culture conditions showed different sensitivity to several agents (fig. 1). The 3D cultures were generally more resistant to drug treatment, and KP4 spheroids did be significantly more resistant than the 2D cultures to a subset of 18 inhibitors from this group, which mainly included chemotherapeutic agents (fig. 1). Unexpectedly, we also identified 11 inhibitors that were more effective on 3D cultures than 2D cultures (fig. 1). These 11 inhibitors are composed of a variety of molecules and reported activity to target a variety of signaling pathways. Four of these inhibitors (cobitinib, GDC-0623, AZD6244, PD901) targeted the MEK pathway, 3 targeted the PI3K pathway (GDC-0941, GDC-0980, G38390), and one targeted the MET pathway (G45203).

Example 2: differentially regulated multiple signaling pathways in 2D versus 3D cultures in pancreatic cancer cells

To identify signaling pathways that may contribute to the differential treatment sensitivity of pancreatic cancer cells observed in 2D versus 3D, we performed phosphokinase arrays, gene expression arrays, and luciferase expression reporter gene assays, and compared changes in the expression levels of signaling proteins in KP4 cells under 2D and 3D conditions phosphokinase arrays showed increased tyrosine phosphorylation of several signaling proteins in KP4 spheroids, including p38, ERK, EGFR, MSK, Akt, TOR, CREB, HSP-27, STAT2, STAT5 α, Hck, Chk-2, c-Jun, RSK 23/2/3, eNOS, p5, and PLC- γ 1, compared to KP4 cells cultured in 2D, suggesting that signaling pathways under 3D conditions are substantially "reconnected" (figures 2 and 3) in KP4 monolayer cultures, MEK inhibits ERK, RSK and PLC- γ 1, and increases phosphorylation of family members and activation proteins (Akt family activation inhibition, STAT activation inhibition of Akt family, STAT activation of protein activation is shown in contrast to the inhibition map of MEK, STAT-5, STAT activation of Akt family, STAT-t activation, STAT-387 family, and STAT activation of protein activation, STAT-11.

qRT-PCR analysis of mRNA expression corresponding to protein kinases in KP4 monolayer and globular cultures indicated increased expression of several RTKs in KP4 spheroids, notably FGFR3, EPHA1, EPHB6 and INSR (FIG. 4). MEK inhibition caused a down-regulation of most of these RTKs in KP4 monolayers and spheroids, however PDGFR α and PDGFR β expression was up-regulated after Coptitinib treatment in KP4 monolayer cultures, in contrast EPHA1 and EPHB1 were selectively increased in KP4 spheroids after MEK inhibition (FIG. 4). these findings reveal drug response characteristics specific to 3D culture conditions for PDAC-derived cells in general, which may contribute to the observed differential sensitivity to drug treatment relative to that observed in standard 2D conditions.

Further evaluation of changes in transcription factor pathway activation following cobitinib treatment using a number of luciferase reporter assays showed that KP4 monolayer cultures exhibited increased activation of STAT3, c-Myc, GRE, KLF4, ISRE and GAS transcription factors (fig. 5). In contrast, KP4 spheroids did not show similar levels of activation of these transcription factors. These results suggest that differential regulation of multiple transcription factors may contribute to the activation of alternative pro-survival signaling pathways in response to MEK pathway inhibition under both monolayer and spheroid conditions.

The phosphokinase array and gene expression array results were validated by western blotting of p-PDGFR α, p-STAT3, p-Erk, p-S6 and p-RSK after cobitinib treatment on KP4 cells and increased levels of activation of p-PDGFR α, p-STAT3 and p-S6 were observed (fig. 6A), increased p-STAT3 was also repeatedly detected after cobitinib treatment in multiple PDAC human cancer cell lines (fig. 6B), significantly, increased secretion of IL-6 ligand was also observed in multiple PDAC cell lines, IL-6 ligand could promote STAT 3activation (fig. 7) consistent with the requirement for STAT 3activation, RNAi knockdown of KP4 and in MIA-PACA2 cells induced cell death after cobitinib treatment (fig. 8 and 9), additionally, inhibition of cell invasion by multiple PDAC cell lines and gep GEMM cell lines after MEK treatment could be demonstrated to be insufficient to inhibit primary tumor cell invasion as a result of MEK 8945, inhibition of MEK cell invasion of MEK cells after MEK 9, suggesting that inhibition of MEK cell invasion by multiple PDAC cell lines and gpt-S6 could be insufficient to inhibit primary cancer cell invasion in vitro as a single inhibition of MEK cell lines.

Example 3 PDGFR α, S6 and STAT3 inhibition together with MEK inhibition effectively promoted apoptosis of PDAC cells

Previous studies have reported that MEK inhibition can lead to upregulation and activation of various RTKs and IL6/JAK/STAT pathways as "alternative" survival mechanisms (Duncan JS, Whittle MC, Nakamura K, Abell AN, Midland AA, Zawistowski JS, Dynamic reprogramming of the hormone in response to targetedMEK inhibition in tri-negative Research Cancer, Cell, 2012; 149 (2): 307-21.Epub 2012/04/17; Lee HJ, Zhuang G, Cao Y, Ducan P, Kim HJ, Settlemenman J, Drug resistance vitamin activity of State 3in oncogene-addided cells, Cancer receptor, 26-2011 kinase, and the inhibition of tumor receptor activation of protein kinase alone and/or in combination with the protein of the protein kinase 7, mRNA expression of protein of prostate-mediated receptor, VEGF-mediated receptor 3, VEGF-mediated receptor kinase, VEGF-mediated kinase, Cell activation of the protein kinase, and the receptor activation of the protein of the tumor receptor activation of the protein of the prostate receptor kinase alone (see the protein of the protein kinase alone, the protein of the tumor receptor kinase, the protein of the tumor receptor of the protein of the prostate receptor of the prostate, prostate receptor of the prostate, prostate receptor of the prostate, kidney, prostate, kidney, prostate, kidney.

Since ponatinib inhibits PDGFR α in addition to FGFR, VEGFR, ephrin receptors, and S6 and JAK2/STAT 3signaling, we further explored the selective activity of ponatinib by examining the effect of other PDGFR α, S6 and JAK2 inhibitors in combination with cobitinib unlike the EGFR inhibitor erlotinib or PDGFR inhibitor, downregulating S6 activation by combination therapy with cobitinib and S6 inhibitors, rapamycin or GDC-0980 promoted cell death in KP4 cells (fig. 14, 18 and 19). following triple combination therapy with PDGFR α inhibitors claratinib, JAK2 inhibitors ruxolitinib and cobitinib, downregulation of PDGFR α and STAT 3activation also inhibited KP4 cell growth and caused significant cell death (fig. 15). however, we observed synergistic treatment with cobitinib/ponatinib, which had the strongest synergistic effect on inhibition of cell growth in pdfr 16, PDAC 16, and potently observed as a combined therapy with JAK ac 16, or map of cell growth in a synergistic combination therapy with cobitinib/ac 16, whereas it was shown to be potent in cell growth inhibition of cells in JAK cells, whereas co-therapy with cobitinib/ac 16, and/16.

Example 4: cocobicistinib/ponatinib co-treatment induced tumor regression in mouse models

To test the effectiveness of the combitinib/ponatinib combination in vivo, we performed xenograft studies with KP4 and KPP GEMM-derived tumor cells, both combitinib and ponatinib were ineffective as single agents in KP4 xenografts, but co-treatment with combitinib and ponatinib caused significant inhibition of tumor growth and increased cell death with increased cleaved caspase 3 (fig. 20(KP4 cells), 21 and 22), similar results were obtained with KPP GEMM-derived cell line xenograft tumors, although we observed predominantly significant delay of tumor growth as opposed to tumor regression (fig. 20(KPP cells)). combitinib/ponatinib combination treatment also produced 103% TGI tumor growth inhibition in KP4 xenografts) and produced TGI% TGI in KPP 4 xenografts (table 2) in KP 636 xenograft studies, did not show significant loss of PDGF growth in mice treated with KP p 6335, but did not show a decrease in serum, PDGF cd 55, cd 55% PDGF + p + 5 + p5 + p5 + p5 + p5 + p5 + p.

Analysis of the myeloid cell compartment within PDAC tumors showed that F4/80+ tumor-associated macrophages (TAMs) were the most predominant myeloid cell population (fig. 21). cobitinib/ponatinib co-treatment significantly reduced the number of TAMs in tumors, induced serum levels of macrophage inhibitory factor GCSF, and reduced levels of macrophage activating factors GM-CSF and MCSF (fig. 22 and 23). these results indicate that cobitinib/ponatinib co-treatment effectively abolished tumor cells and myeloid cell populations by targeting the PDGFR α pathway in addition to downstream MEK, S6, and STAT3 survival pathways.

TABLE 2 tumor growth inhibition (% TGI) of KP4 and KPP xenografts

AUC ═ area under the curve

UI-uncertainty interval

Time to tumor progression (2X original volume)

Example 5: differential activation of Erk and STAT3in liver metastases in PDAC patients

To test the potential clinical relevance of these findings, we performed IHC analysis of p-Erk and p-STAT3 on 76 human PDAC tissue samples and 4 liver metastases, Erk was activated in 13% of PDAC tissue samples (10/76), primarily in stage I and II malignancy patients, STAT 3activation was observed in 26% of samples (20/76), and 6.5% of samples (5/76) exhibited activated Erk and STAT3, primarily in stage I malignancies (fig. 24 and 25). furthermore, 50% of liver metastases exhibited Erk activation (2/4), and STAT 3activation was observed in 25% of samples (1/4) (fig. 24 and 25). additionally, gene expression analysis further revealed increased levels of PDAC tumors from human patients relative to RTK fr α, PDGFR β, and IL6-R expressed in normal tissues (fig. 26, table 3).

TABLE 3 Gene expression analysis data

The data in table 3 indicate that PDAC tumors can be driven by PDGFR, IL-6 and STAT3signaling, and that these pathways can limit the response to MEK pathway inhibition. These findings also suggest that the MEK pathway may play an important role in tumor cell survival in liver metastases, which does not switch to a STAT 3-mediated survival cascade after metastasis in human pancreatic cancer patients.

All technical and scientific terms used herein have the same meaning. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for.

Throughout this specification and the claims, unless the context requires otherwise, the word "comprise" is used in a non-exclusive sense. It is to be understood that the embodiments described herein include embodiments that "consist of and/or" consist essentially of.

As used herein, the term "about" when referring to a value is intended to encompass a difference from the specified amount of ± 50% in some embodiments, 20% in some embodiments, 10% in some embodiments, 5% in some embodiments, 1% in some embodiments, 0.5% in some embodiments, and 0.1% in some embodiments, as such a difference is suitable for performing the disclosed method or using the disclosed composition.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limit in the stated range. If a stated range includes one or both of the limits, ranges excluding either or both of those limits are also included.

Many modifications and other embodiments of the subject matter set forth herein will come to mind to one skilled in the art to which this subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims (27)

1. A method of treating pancreatic cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a combination of active agents, wherein the combination comprises a MEK inhibitor and a multikinase agent that targets PDGFR α, S6, and STAT 3.

2. The method of claim 1, wherein the MEK inhibitor is selected from the group consisting of: cobicistinib, GDC-0623, trametinib, bimetinib, semetinib, pimatinib, rifatinib, PD-0325901, and BI-847325, or a pharmaceutically acceptable salt thereof.

3. The method of claim 2, wherein the MEK inhibitor is selected from cobicistinib and trametinib, or a pharmaceutically acceptable salt thereof.

4. The method of claim 3, wherein the MEK inhibitor is cobicisinib or a pharmaceutically acceptable salt thereof.

5. The method of claim 1, wherein the multikinase agent is ponatinib or a pharmaceutically acceptable salt thereof.

6. The method of claim 1, wherein the pancreatic cancer is endocrine.

7. The method of claim 1, wherein the pancreatic cancer is exocrine.

8. The method of claim 7, wherein the pancreatic cancer is adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, colloid-like carcinoma, undifferentiated carcinoma with osteoclastoid giant cells, hepatoid carcinoma, intraductal papillary mucinous tumors, mucinous cystic tumors, pancreatoblastoma, serous cystadenoma, signet ring cell carcinoma, solid and pseudopapillary tumors, pancreatic ductal carcinoma, or undifferentiated carcinoma.

9. The method of claim 8, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

10. The method of claim 1, wherein the combination is synergistic.

11. The method of claim 1, wherein the active agents are administered sequentially.

12. The method of claim 1, wherein the active agents are administered simultaneously.

13. The method of claim 1, wherein the MEK inhibitor and the multi-kinase inhibitor are administered as a combined preparation.

14. The method of claim 1, wherein the MEK inhibitor is administered in an amount from about 45mg to about 75mg and the multikinase inhibitor is administered in an amount from about 30mg to about 60 mg.

15. The method of claim 14, wherein the amount is administered once daily.

16. The method of claim 1, wherein the MEK inhibitor is cobicistinib, or a pharmaceutically acceptable salt thereof, and the multi-kinase inhibitor is ponatinib, or a pharmaceutically acceptable salt thereof.

17. A combination of a) a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and b) a multi-kinase inhibitor targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, for use in the treatment of pancreatic cancer.

18. A pharmaceutical composition comprising a combination of an effective amount of a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multikinase agent targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the composition is for use in the treatment of pancreatic cancer.

19. The composition of claim 18, wherein the MEK inhibitor is selected from: cobicistinib, GDC-0623, trametinib, bimetinib, semetinib, pimatinib, rifatinib, PD-0325901, and BI-847325, or a pharmaceutically acceptable salt thereof.

20. The composition of claim 19, wherein the MEK inhibitor is selected from cobicistinib and trametinib, or a pharmaceutically acceptable salt thereof.

21. The composition according to claim 20, wherein the MEK inhibitor is cobicistinib, or a pharmaceutically acceptable salt thereof.

22. The composition of claim 18, wherein the multikinase agent is ponatinib or a pharmaceutically acceptable salt thereof.

23. The composition of claim 18, comprising cobicistinib or a pharmaceutically acceptable salt thereof and ponatinib or a pharmaceutically acceptable salt thereof.

24. The composition of claim 18, wherein the MEK inhibitor, or a pharmaceutically acceptable salt thereof, is present in an amount of from about 45mg to about 75mg and the multi-kinase inhibitor, or a pharmaceutically acceptable salt thereof, is present in an amount of from about 30mg to about 60 mg.

25. A kit comprising a MEK inhibitor, or a pharmaceutically acceptable salt thereof, and a multikinase agent targeting PDGFR α, S6 and STAT3, or a pharmaceutically acceptable salt thereof, a container and a package insert or label.

26. The method of claim 1, wherein the MEK inhibitor is administered at a dose of about 1mg/kg to about 50mg/kg and the multi-kinase inhibitor is administered at a dose of about 1mg/kg to about 50 mg/kg.

27. The method of claim 26, wherein the MEK inhibitor is administered at a dose of about 1mg/kg to about 10mg/kg and the multi-kinase inhibitor is administered at a dose of about 10mg/kg to about 40 mg/kg.