阅读说明：本技术 具有braf突变的癌症的组合疗法 (Combination therapy for cancers with BRAF mutations ) 是由 陈一友 C·蒋 于 2019-04-08 设计创作，主要内容包括：本公开文本提供了用于治疗具有BRAF突变的癌症的组合疗法,所述组合疗法包括向受试者给予有效量的(a)表皮生长因子受体(EGFR)抑制剂；(b)丝裂原激活蛋白激酶(MEK)1/2抑制剂；(c)细胞周期蛋白依赖性激酶(CDK)4/6抑制剂。还提供了与所述组合疗法有关的组合物和试剂盒。(The present disclosure provides a combination therapy for treating a cancer with a BRAF mutation, comprising administering to a subject an effective amount of (a) an Epidermal Growth Factor Receptor (EGFR) inhibitor; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; (c) cyclin Dependent Kinase (CDK)4/6 inhibitors. Compositions and kits related to the combination therapy are also provided.)

1. A composition, comprising:

(a) epidermal Growth Factor Receptor (EGFR) inhibitors;

(b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and

(c) inhibitors of Cyclin Dependent Kinase (CDK) 4/6;

wherein the composition does not comprise a BRAF inhibitor.

2. The composition of claim 1, wherein the EGFR inhibitor is oxitinib, or a salt thereof, wherein the MEK1/2 inhibitor is cobicistinib, trametinib, TAK-733, or a salt of any of the foregoing, and wherein the CDK4/6 inhibitor is palbociclib, or a salt thereof.

3. A composition comprising (a) oxitinib, or a salt thereof, (b) cobinib, trametinib, TAK-733, or a salt of the foregoing, and (c) palbociclib, or a salt thereof.

4. The composition of any one of claims 1-3, further comprising a pharmaceutically acceptable carrier, excipient, binder, or diluent.

5. The composition of any one of claims 1-4, wherein the composition is formulated for oral administration to a subject.

6. A method of treating or delaying progression of cancer in a subject, the method comprising administering to the subject an effective amount of

(a) Epidermal Growth Factor Receptor (EGFR) inhibitors;

(b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and

(c) inhibitors of Cyclin Dependent Kinase (CDK) 4/6;

wherein the subject has or is at risk of having cancer with a BRAF mutation.

7. The method of claim 6, wherein the subject is not administered a BRAF inhibitor.

8. The method of claim 6 or claim 7, wherein the EGFR inhibitor is oxitinib or a salt thereof or cetuximab, wherein the MEK1/2 inhibitor is cobicistinib, trametinib, TAK-733, or a salt of the foregoing, and wherein the CDK4/6 inhibitor is palbociclib or a salt thereof.

9. The method of any one of claims 6-8, wherein the method comprises administering an effective amount of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof.

10. The method of any one of claims 6-8, wherein the method comprises administering to the subject an effective amount of oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof.

11. The method of any one of claims 6-8, wherein the method comprises administering to the subject an effective amount of oxitinib or a salt thereof, trametinib or a salt thereof, and palbociclib or a salt thereof.

12. The method of any one of claims 6-8, wherein the method comprises administering to the subject an effective amount of cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof.

13. The method of any one of claims 6-8, wherein the method comprises administering to the subject an effective amount of cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof.

14. The method of any one of claims 6-8, wherein the method comprises administering to the subject an effective amount of cetuximab, tremetinib, or a salt thereof, and palbociclib or a salt thereof.

15. The method according to claim 9, wherein oxitinib or a salt thereof, cobicistinib or a salt thereof and palbociclib or a salt thereof are administered in one composition.

16. The method of claim 9, wherein the subject is administered otitinib or a salt thereof, cobicistinib or a salt thereof, and palbociclib or a salt thereof simultaneously.

17. The method of claim 9, wherein the subject is administered oxitinib or a salt thereof, cobicistinib or a salt thereof, and palbociclib or a salt thereof intermittently.

18. The method of claim 12, wherein cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are administered in two or more compositions.

19. The method of claim 12, wherein cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are administered intermittently to the subject.

20. The method of claim 12, wherein cobicistinib or a salt thereof and palbociclib or a salt thereof are administered in one composition.

21. The method of claim 12, wherein cobicistinib or a salt thereof and palbociclib or a salt thereof are formulated for oral administration.

22. The method of claim 12, wherein cobicisinib or a salt thereof and palbociclib or a salt thereof are administered to the subject simultaneously.

23. The method of any one of claims 6-22, wherein the cancer has a BRAF V600 mutation or a BRAF D581V mutation.

24. The method of claim 23, wherein the BRAF V600 mutation is V600E, V600D, or V600K.

25. The method of any one of claims 6-24, wherein the cancer is a malignant epithelial tumor or carcinoma.

26. The method of claim 25, wherein the cancer is selected from one or more of: adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma.

27. The method of claim 25 or claim 26, wherein the cancer is selected from one or more of: epithelial tumors, squamous cell tumors, basal cell tumors, transitional cell carcinomas, adenocarcinomas, adnexal or cutaneous adnexal tumors, mucoepidermoid tumors, cystic, mucinous or serous tumors, ductal, lobular or medullary tumors, acinar cell tumors, and complex epithelial tumors.

28. The method of any one of claims 6-27, wherein the cancer is a cancer selected from one or more of: colon cancer, gastric cancer, lung cancer, breast cancer, pancreatic cancer, oral cancer, prostate cancer, germline cancer, rectal cancer, liver cancer, kidney cancer, papillary thyroid cancer, and ovarian cancer.

29. The method of any one of claims 6-28, wherein the cancer is colorectal cancer.

30. The method of claim 29, wherein the cancer is stage IV colorectal cancer.

31. The method of claim 30, wherein the colorectal cancer has a BRAF V600E or D581V mutation.

32. The method of any one of claims 7-31, wherein the method has about 20-99% more reduction in cancer cell growth and/or increase in cancer cell killing than administration of (a) the EGFR inhibitor, (b) the MEK1/2 inhibitor, or (c) the CDK4/6 inhibitor alone.

33. The method of any one of claims 6-32, wherein the method reduces the average tumor volume by about 20-95%.

34. The method of claim 9, wherein the oxcininib or salt thereof is administered to the subject in a daily dose of about 40-80 mg.

35. The method of claim 9, wherein cobicistinib, or a salt thereof, is administered to the subject at a daily dose of about 20-60 mg.

36. The method of claim 9, wherein palbociclib, or a salt thereof, is administered to the subject at a daily dose of about 75-125 mg.

37. The method of claim 12, wherein cetuximab is administered to the subject in the following mannerThe following steps: about 400mg/m infused over 120 minutes at a maximum infusion rate of 10mg/min²Followed by 250mg/m over 60 minutes at a maximum infusion rate of 10mg/min²The weekly dose of (c).

38. The method of any one of claims 6-37, wherein the subject is a human.

39. The method of any one of claims 6-38, wherein the method reduces tumor volume by at least about 85%.

40. A kit, comprising:

(a) epidermal Growth Factor Receptor (EGFR) inhibitors;

(b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and

(c) inhibitors of Cyclin Dependent Kinase (CDK) 4/6;

wherein the kit does not comprise a BRAF inhibitor.

41. The kit of claim 40, wherein the EGFR inhibitor is oxitinib, or a salt thereof, or cetuximab, wherein the MEK1/2 inhibitor is cobicistinib, trametinib, TAK-733, or a salt of the foregoing, and wherein the CDK4/6 inhibitor is palbociclib, or a salt thereof.

42. A kit comprising (a) oxitinib or a salt thereof or cetuximab, (2) cobicistinib, trametinib, TAK-733, or a salt of the foregoing, and (3) palbociclib or a salt thereof.

43. The kit according to claim 41 or claim 42, wherein the kit comprises a pharmaceutical composition comprising oxitinib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

44. The kit of claim 41 or claim 42, wherein the kit comprises a pharmaceutical composition comprising cobicistinib or a salt thereof and a pharmaceutically acceptable carrier, excipient, binder or diluent.

45. The kit of claim 41 or claim 42, wherein the kit comprises a pharmaceutical composition comprising palbociclib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

46. The kit of claim 41 or claim 42, wherein the kit comprises a pharmaceutical composition comprising trametinib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

47. The kit of claim 41 or claim 42, wherein the kit comprises a pharmaceutical composition comprising TAK-733 or a salt thereof and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

48. The kit of claim 41 or claim 42, wherein the kit comprises a pharmaceutical composition comprising cetuximab and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

49. The kit of claim 41 or claim 42, wherein the ocitinib or salt thereof is formulated for oral administration to a subject.

50. The kit of claim 41 or claim 42, wherein cobicistinib, or a salt thereof, is formulated for oral administration to a subject.

51. The kit of claim 41 or claim 42, wherein the palbociclib, or salt thereof, is formulated for oral administration to a subject.

52. The kit of claim 41 or claim 42, wherein trametinib, or a salt thereof, is formulated for oral administration to a subject.

53. The kit of claim 41 or claim 42, wherein TAK-733, or a salt thereof, is formulated for oral administration to a subject.

54. The kit of claim 41 or claim 42, wherein the kit comprises oxitinib or a salt thereof, cobicistinib or a salt thereof, and palbociclib or a salt thereof.

55. The kit according to claim 54, wherein oxitinib or a salt thereof, cobitinib or a salt thereof and palbociclib or a salt thereof are formulated in one composition.

56. The kit of claim 54, wherein oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated for simultaneous administration to a subject.

57. The kit of claim 54, wherein oxitinib or a salt thereof, cobicistinib or a salt thereof, and palbociclib or a salt thereof are formulated for intermittent administration to a subject.

58. The kit of claim 41 or claim 42, wherein kit comprises cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof.

59. The kit of claim 58, wherein cetuximab is formulated for intravenous administration to a subject.

60. The kit of claim 58, wherein cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated in two or more compositions.

61. The kit of claim 58, wherein cobicistinib or a salt thereof and palbociclib or a salt thereof are formulated for oral administration.

62. The kit of claim 58, wherein cobicistinib or a salt thereof and palbociclib or a salt thereof are administered in one composition.

63. The kit of claim 58, wherein cobicistinib or a salt thereof and palbociclib or a salt thereof are formulated for oral administration.

64. The kit of claim 58, wherein cobicisinib or a salt thereof and palbociclib or a salt thereof are administered to the subject simultaneously.

Technical Field

The present disclosure relates generally to combination therapies and related compositions and kits for treating or delaying progression of cancers with BRAF mutations.

Background

The v-raf murine sarcoma virus oncogene homolog B1(BRAF) is a potent activator of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway, which is involved in regulating cell proliferation, differentiation, and survival in response to extracellular signaling. Cancer ther.10,385, to Cantwell-Dorris et al (2011) mol. This pathway can be activated when extracellular ligands bind to Epidermal Growth Factor Receptor (EGFR), initiating a cascade of ERK signaling by RAS gtpase. Once RAS participates, it recruits and activates RAFs (such as ARAF, BRAF, and CRAF) which phosphorylate MAPK/ERK kinases (MEK), triggering downstream ERK pathways. Oikonomouu et al (2014) Oncotarget 5,11752.

BRAF mutations have been detected in a variety of cancers, including about 59% malignant melanoma, about 45% papillary thyroid carcinoma, about 18% colorectal cancer (CRC), about 4% ovarian cancer, about 2% breast cancer, and about 3% lung cancer, making them potential therapeutic targets. Davies et al (2002) Nature 417,949; xing et al (2005) endothelial. relat. cancer 12,245. Mutations in BRAF occur most often at nucleotide 1976, resulting in a change at the valine residue (V600). Davies et al (2002) Nature 417,949. Mutant BRAF may cause constitutive activation of MAPK/ERK kinase (MEK), which in turn phosphorylates ERK and leads to transcription of target genes, thereby promoting tumor cell growth and survival in the absence of any extracellular stimulus. Hall et al (2014) Cancer Control 21,221; cancer ther.10,385, to Cantwell-Dorris et al (2011) mol.

Chemotherapy, even a highly aggressive regimen, does not respond well in treating cancers with BRAF mutations. For example, in clinical trials, patients with metastatic CRC received FOLFOXIRI plus bevacizumab (a highly aggressive regimen), but patients with BRAF mutant CRC had less than half the survival (overall survival median, 19.0 months versus 41.7 months) of patients with wild-type tumors. Loupakis F et al (2014) N Engl J Med.371, 1609.

The united states Food and Drug Administration (FDA) approves BRAF inhibitors alone or in combination with MEK inhibitors for the treatment of metastatic melanoma cancer with BRAF mutations. Larkin et al (2014) n.engl.j.med.371, 1867; robert et al (2015) n.engl.j.med.372, 30. To date, melanoma cancers with BRAF mutations V600K or V600E are the only cancer types with BRAF mutations with approved targeted therapies. However, the duration of the reaction is limited due to the development of acquired and adaptive resistance. Manzano et al (2016) ann.trans.med.4, 237; barras (2015) Biomarkers in Cancer 7, 9. Studies have shown that ERK can be reactivated through EGFR-mediated RAS and CRAF activation, contributing to resistance of cancer cells to BRAF inhibitors. Barras (2015) Biomarkers in Cancer 7, 9. Furthermore, it has been reported that the use of BRAF inhibitors may lead to the development of secondary skin tumors due to abnormal activation of the MAPK pathway in cells without BRAF mutations, but that combining BRAF inhibitors with MEK inhibitors may reduce the incidence of BRAF inhibitor-induced skin tumors. Chapman et al (2011) n.engl.j.med.364, 2507; robert et al (2011) curr. opin. oncol.23, 177; larkin et al (2014) n.engl.j.med.371, 1867. Recent and current clinical trials for cancers with BRAF mutations typically involve the combination of BRAF inhibitors with other therapeutic or candidate drugs targeting different pathways (such as EGFR, MEK, PI3K) and cytotoxic chemotherapy. Morris et al (2013) F1000Prime Rep.5, 11; hyman DM et al (2015) N.Engl.J.Med.373, 726.

Thus, there remains a need for robust therapies for treating cancers with BRAF mutations.

The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety.

Disclosure of Invention

Provided herein are compositions comprising: (a) epidermal Growth Factor Receptor (EGFR) inhibitors; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor; wherein the composition does not comprise a BRAF inhibitor. In some embodiments, the composition consists of: (a) epidermal Growth Factor Receptor (EGFR) inhibitors; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor.

Also provided herein are methods for treating or delaying progression of cancer in a subject comprising administering to the subject an effective amount of (a) an Epidermal Growth Factor Receptor (EGFR) inhibitor; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor, wherein the subject has or is at risk of having a cancer with a BRAF mutation. In some embodiments, the methods do not include administering (a) an Epidermal Growth Factor Receptor (EGFR) inhibitor; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) administering a BRAF inhibitor to the subject during the period of administering the inhibitor of Cyclin Dependent Kinase (CDK) 4/6. In some embodiments, the methods do not include administering (a) an Epidermal Growth Factor Receptor (EGFR) inhibitor; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) administering an additional therapeutic agent to the subject during the period of the Cyclin Dependent Kinase (CDK)4/6 inhibitor.

Also provided herein are methods for treating or delaying the progression of cancer in a subject comprising administering to the subject an effective amount of oxitinib or a salt thereof, cobicistinib or a salt thereof, and palbociclib or a salt thereof, wherein the subject has or is at risk of having a cancer with a BRAF mutation. In some embodiments, the method does not comprise administering a BRAF inhibitor to the subject during the administration of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the method does not comprise administering to the subject an additional therapeutic agent during the administration of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, ocitinib or a salt thereof, cobicistinib or a salt thereof, and palbociclib or a salt thereof are administered in one composition. In some embodiments, the oxcininib or a salt thereof, cobinib or a salt thereof, and palbociclib or a salt thereof are administered to the subject continuously. In some embodiments, the oxcininib or a salt thereof, cobinib or a salt thereof, and palbociclib or a salt thereof is administered intermittently to the subject.

In some embodiments, the oxertib or salt thereof is administered to the subject at a daily dose of about 20-160mg of oxertib. In some embodiments, cobicistinib, or a salt thereof, is administered to the subject at a daily dose of about 20-60mg cobicistinib. In some embodiments, the palbociclib, or a salt thereof, is administered to the subject at a daily dose of about 75-125mg palbociclib. In some embodiments, the subject is a human. In some embodiments, the oxertib or salt thereof is administered to the subject at a daily dose of about 0.5-3mg/kg of oxertib. In some embodiments, cobicistinib, or a salt thereof, is administered to the subject at a daily dose of about 0.25-1mg/kg cobicistinib. In some embodiments, the palbociclib, or a salt thereof, is administered to the subject at a daily dose of about 1-2.5mg/kg palbociclib.

Also provided herein are methods for treating or delaying the progression of cancer in a subject comprising administering to the subject an effective amount of cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, wherein the subject has cancer or is at risk of having cancer with a BRAF mutation. In some embodiments, the method does not comprise administering a BRAF inhibitor to the subject during the administration of cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, the method does not comprise administering to the subject an additional therapeutic agent during the administration of cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are administered in one composition. In some embodiments, the subject is administered cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, sequentially. In some embodiments, the subject is administered cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, intermittently.

In some embodiments, 400mg/m is infused over 120 minutes²Then infused 250mg/m within 60 minutes per week²Is oriented in the manner ofSubjects were given cetuximab. In some embodiments, the maximum infusion rate is about 10 mL/min. In some embodiments, cobicistinib, or a salt thereof, is administered to the subject at a daily dose of about 20-60mg cobicistinib. In some embodiments, the palbociclib, or a salt thereof, is administered to the subject at a daily dose of about 75-125mg palbociclib. In some embodiments, at about 150mg/m per subject²To the subject at a weekly dose of cetuximab. In some embodiments, the subject is a human. In some embodiments, cobicistinib, or a salt thereof, is administered to the subject at a daily dose of about 0.25-10mg/kg cobicistinib. In some embodiments, the palbociclib, or a salt thereof, is administered to the subject at a daily dose of about 5-30mg/kg palbociclib.

In some embodiments, the cancer has a BRAF V600 mutation or a BRAF D581D mutation. In some embodiments, the BRAF V600 mutation is a BRAF V600E, V600D, or V600K mutation. In some embodiments, the cancer is a malignant epithelial tumor or carcinoma. In some embodiments, the cancer is a cancer selected from one or more of: colon cancer, gastric cancer, lung cancer, breast cancer, pancreatic cancer, oral cancer, prostate cancer, germline cancer, rectal cancer, liver cancer, kidney cancer, papillary thyroid cancer, and ovarian cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is stage IV colorectal cancer. In some embodiments, the colorectal cancer has a BRAF V600E or D581V mutation.

In some embodiments, the subject has received a BRAF inhibitor in a previous treatment cycle. In some embodiments, the subject has not received a BRAF inhibitor in a previous treatment cycle.

In some embodiments, the methods provided herein reduce the mean tumor volume. In some embodiments, the methods provided herein have reduced cancer cell growth and/or increased cancer cell killing as compared to administration of (a) an Epidermal Growth Factor Receptor (EGFR) inhibitor alone; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) about 20-99% more Cyclin Dependent Kinase (CDK)4/6 inhibitor. In some embodiments, the methods provided herein have about 20-99% more reduction in cancer cell growth and/or increase in cancer cell killing than oxentinib, or a salt thereof, cobicisib, or a salt thereof, or palbociclib, or a salt thereof, administered alone. In some embodiments, the methods provided herein have about 20-99% more reduction in cancer cell growth and/or increase in cancer cell killing than cetuximab, cobicistinib, or a salt thereof, or palbociclib or a salt thereof, administered alone. In some embodiments, the method reduces tumor volume by about 20-95%. The method may comprise administering any of the compositions or kits described herein.

In another aspect, provided herein is a composition comprising: (a) epidermal Growth Factor Receptor (EGFR) inhibitors; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor. In some embodiments, the composition comprises oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the composition does not comprise a BRAF inhibitor. In some embodiments, the composition consists of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the composition is formulated for oral administration to a subject. In some embodiments, the subject has or is at risk of having cancer with a BRAF mutation.

In another aspect, provided herein is a kit comprising: (a) epidermal Growth Factor Receptor (EGFR) inhibitors; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor; wherein the kit does not comprise a BRAF inhibitor. In some embodiments, the kit consists of: (a) epidermal Growth Factor Receptor (EGFR) inhibitors; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor.

In some embodiments, the kit comprises oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the kit does not comprise a BRAF inhibitor. In some embodiments, the kit consists of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the kit comprises a pharmaceutical composition comprising oxitinib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising cobicisinib or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising palbociclib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the ocitinib, or salt thereof, is formulated for oral administration to the subject. In some embodiments, cobicistinib, or a salt thereof, is formulated for oral administration to a subject. In some embodiments, the palbociclib, or a salt thereof, is formulated for oral administration to a subject. In some embodiments, the subject has or is at risk of having cancer with a BRAF mutation. In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated in one composition. In some embodiments, the oxcininib or a salt thereof, cobinib or a salt thereof, and palbociclib or a salt thereof are administered to the subject continuously. In some embodiments, the oxcininib or a salt thereof, cobinib or a salt thereof, and palbociclib or a salt thereof is administered intermittently to the subject.

In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated into a composition. In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated as separate compositions. In some embodiments, the otitinib or salt thereof and the cobicistinib or salt thereof are formulated into one composition. In some embodiments, the ocitinib or salt thereof and the palbociclib or salt thereof are formulated into a composition. In some embodiments, cobicisinib or a salt thereof and palbociclib or a salt thereof are formulated into a composition. In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated in a liquid form. In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated in a solid form.

In some embodiments, the ocitinib or salt thereof, cobitinib or salt thereof, and palbociclib or salt thereof are administered as one composition. In some embodiments, the ocitinib or salt thereof, cobitinib or salt thereof, and palbociclib or salt thereof are administered separately. In some embodiments, the ocitinib or salt thereof, cobitinib or salt thereof, and palbociclib or salt thereof are administered simultaneously. In some embodiments, the ocitinib or salt thereof, cobicistinib or salt thereof, and palbociclib or salt thereof are administered sequentially. In some embodiments, the ocitinib or salt thereof, cobicistinib or salt thereof, and palbociclib or salt thereof are administered intermittently.

In some embodiments, the kit comprises cetuximab, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the kit does not comprise a BRAF inhibitor. In some embodiments, the kit consists of cetuximab, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the kit comprises a pharmaceutical composition comprising cetuximab and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising cobicisinib or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising palbociclib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the cetuximab is formulated for administration to the subject via intravenous infusion. In some embodiments, cobicistinib, or a salt thereof, is formulated for oral administration to a subject. In some embodiments, the palbociclib, or a salt thereof, is formulated for oral administration to a subject. In some embodiments, the subject has or is at risk of having cancer with a BRAF mutation. In some embodiments, cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated in different compositions. In some embodiments, cobicisinib or a salt thereof and palbociclib or a salt thereof are formulated in one composition. In some embodiments, the subject is administered cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, sequentially. In some embodiments, the subject is administered cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, intermittently. In some embodiments, cetuximab, cobicisinib or a salt thereof, and palbociclib or a salt thereof are administered to the subject at different dosing frequencies.

In some embodiments, cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated into a composition. In some embodiments, cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated as separate compositions. In some embodiments, cetuximab and cobicisinib, or a salt thereof, are formulated into a composition. In some embodiments, cetuximab and palbociclib, or a salt thereof, are formulated into a composition. In some embodiments, cobicisinib or a salt thereof and palbociclib or a salt thereof are formulated into a composition. In some embodiments, cetuximab, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated in a liquid form. In some embodiments, cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated in a solid form.

In some embodiments, cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are administered as one composition. In some embodiments, cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are administered separately. In some embodiments, cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, are administered simultaneously. In some embodiments, the cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, are administered sequentially. In some embodiments, the cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, are administered intermittently. In some embodiments, the cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, are administered at different dosing frequencies.

In some embodiments, the kit comprises a package insert containing instructions for: indications, usage, dosage, administration, contraindications, other drugs in combination with the packaged product, and/or warnings regarding the use of such drugs.

Drawings

Figure 1 shows representative images of stained tumor cells obtained by the High Content Screening (HCS) platform.

Figure 2 depicts the reduction in CRC (NYL132) volume following treatment with oxitinib, cobitinib, and palbociclib. The graph shows the change in mean tumor volume over time during the combination treatment: (filled circles) -untreated control; (filled triangles) -oxaliplatin 10mg/kg and capecitabine 200mg/kg daily (p.o.) for 3 weeks; (solid diamonds) -oxitinib 5mg/kg, cobitinib 2.5mg/kg and palbociclib 10mg/kg per day (p.o.) for 3 weeks; (open squares) -oxitinib 7.5mg/kg, cobitinib 3.75mg/kg and palbociclib 15mg/kg daily (p.o.) for 3 weeks; and (open circles) -oxitinib 10mg/kg, cobitinib 5mg/kg and palbociclib 20mg/kg daily (p.o.) for 3 weeks. Dosage unit mg/kg refers to the dose of the compound per kg body weight of the mouse.

Figure 3 depicts the reduction in CRC (NYL132) volume following treatment with oxitinib, cobitinib, and palbociclib. The figure shows the change in Tumor Growth Inhibition (TGI) over time during combination therapy: (filled triangles) -oxaliplatin 10mg/kg and capecitabine 200mg/kg daily (p.o.) for 3 weeks; (solid diamonds) -oxitinib 5mg/kg, cobitinib 2.5mg/kg and palbociclib 10mg/kg per day (p.o.) for 3 weeks; (open squares) -oxitinib 7.5mg/kg, cobitinib 3.75mg/kg and palbociclib 15mg/kg daily (p.o.) for 3 weeks; and (open circles) -oxitinib 10mg/kg, cobitinib 5mg/kg and palbociclib 20mg/kg daily (p.o.) for 3 weeks. Dosage unit mg/kg refers to the dose of the compound per kg body weight of the mouse. TGI ═ 100% (1- (Ti-T0)/(Vi-V0)).

Figure 4 depicts body weight of mice (NYL132) during combination therapy: (filled circles) -untreated control; (filled triangles) -oxaliplatin 10mg/kg and capecitabine 200mg/kg daily (p.o.) for 3 weeks; (solid diamonds) -oxitinib 5mg/kg, cobitinib 2.5mg/kg and palbociclib 10mg/kg per day (p.o.) for 3 weeks; (open squares) -oxitinib 7.5mg/kg, cobitinib 3.75mg/kg and palbociclib 15mg/kg daily (p.o.) for 3 weeks; and (open circles) -oxitinib 10mg/kg, cobitinib 5mg/kg and palbociclib 20mg/kg daily (p.o.) for 3 weeks.

FIG. 5 depicts the reduction in CRC (NYL-GZ-082) volume following treatment with combination therapy. The graph shows the change in mean tumor volume over time during the combination treatment: (filled triangles) -control, where no treatment lasted 14 days, cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg, and palbociclib 20mg/kg daily (p.o.) for 21 days, followed by no treatment for 1 week; the second cycle (same treatment as the first cycle (first 4 weeks)) starting from day 43; (solid squares) -oxitinib 10mg/kg, cobitinib 5mg/kg and palbociclib 20mg/kg per day (p.o.) for 3 weeks, followed by no treatment for 1 week; day 28-56 is the second cycle, and the same treatment as the first cycle (first 4 weeks) is performed; (open circles) -cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg and palbociclib 20mg/kg daily (p.o.) for 21 days, followed by no treatment for 1 week; the second cycle was started from day 28-56 and the same treatment as the first cycle (first 4 weeks) was performed. Dosage unit mg/kg refers to the dose of the compound per kg body weight of the mouse.

Figure 6 depicts the body weight change (%) of mice (NYL-GZ-082) during combination therapy: (filled triangles) -control, where no treatment lasted 14 days, cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg, and palbociclib 20mg/kg daily (p.o.) for 21 days, followed by no treatment for 1 week; the second cycle (same treatment as the first cycle (first 4 weeks)) starting from day 43; (solid squares) -oxitinib 10mg/kg, cobitinib 5mg/kg and palbociclib 20mg/kg per day (p.o.) for 3 weeks, followed by no treatment for 1 week; day 28-56 is the second cycle, and the same treatment as the first cycle (first 4 weeks) is performed; (open circles) -cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg and palbociclib 20mg/kg daily (p.o.) for 21 days, followed by no treatment for 1 week; the second cycle, day 28-56, was treated the same as the first cycle (first 4 weeks).

Figure 7 depicts the reduction in CRC (NYL132) volume following treatment with combination therapy. The graph shows the change in mean tumor volume over time during the combination treatment: (filled triangles) -untreated control; (filled squares) -cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg and palbociclib 20mg/kg daily (p.o.) for 21 days, no treatment for 7 days, followed by a second cycle, with the same treatment as the first cycle (first 4 weeks); (filled circle) -oxitinib 10mg/kg, TAK-7333 mg/kg and palbociclib 20mg/kg per day (p.o.) for 21 days without treatment for 7 days, followed by a second cycle with the same treatment as the first cycle; (filled diamonds) -cetuximab 1mg weekly (i.p.), TAK-7333 mg/kg and palbociclib 20mg/kg daily (p.o.) for 21 days, no treatment for 7 days, followed by a second cycle, with the same treatment as the first cycle; (open triangle) -cetuximab 1mg weekly (i.p.) for 21 days without treatment for 7 days, cetuximab 1mg weekly (i.p.) for 28-49 days without treatment for 7 days, followed by cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg and palbociclib 20mg/kg daily (p.o.) for 21 days; (open circle) -cetuximab 1mg weekly (i.p.), TAK-73310 mg/kg and palbociclib 20mg/kg daily (p.o.) for 21 days, no treatment for 7 days, followed by a second cycle, with the same treatment as the first cycle; (hollow diamond) -oxitinib 10mg/kg, TAK-73310 mg/kg and palbociclib 20mg/kg per day (p.o.) for 21 days, no treatment for 7 days, followed by a second cycle, with the same treatment as the first cycle.

Figure 8 depicts the reduction in CRC (HT-29) volume following treatment with combination therapy. The graph shows the change in mean tumor volume over time during the combination treatment: (filled triangles) -no treatment for 28 days control; (filled squares) -capecitabine 200mg/kg per day (p.o.) for 21 days; (filled circles) -cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg, and palbociclib 20mg/kg daily (p.o.) for 21 days; (solid diamonds) -cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg and palbociclib 13.3mg/kg daily (p.o.) for 21 days; (open triangle) -cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg and palbociclib 6.6mg/kg daily (p.o.) for 21 days; (open squares) -oxitinib 10mg/kg, cobitinib 5mg/kg and palbociclib 20mg/kg per day (p.o.) for 21 days; (open circles) -oxitinib 10mg/kg and cobitinib 5mg/kg per day (p.o.) for 21 days; (open diamonds) -cobitinib 5mg/kg per day (p.o.) for 21 days; (dotted solid triangles) -cobitinib 5mg/kg and palbociclib 20mg/kg per day (p.o.) for 21 days; (dotted filled circles) -5-FU 40mg/kg weekly for 3 weeks. On day 21, all treatments were stopped and followed by 1 week of observation.

Figure 9 depicts the reduction in CRC (HT-29) volume following treatment with combination therapy. The graph shows the change in mean tumor volume over time during the combination treatment: (filled triangles) -no treatment for 21 days control; (filled squares) -TAK-73310 mg/kg per day (p.o.) for 21 days; (filled circles) -day 1 TAK-73330 mg/kg, then 3mg/kg per day (p.o.) for 20 days; (solid diamonds) -palbociclib 20mg/kg per day (p.o.) for 21 days; (open triangles) -cetuximab 1mg weekly (i.p.) for 21 days, then 7 days to stop treatment, followed by 1mg weekly (i.p.) for 14 more days; (open squares) -capecitabine 200mg/kg daily (p.o.) and oxaliplatin 10mg/kg weekly (i.p.) for 21 days, then 7 days to stop treatment; after day 28, cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg and palbociclib 20mg/kg daily (p.o.) for 14 days; (open circles) -oxitinib 10mg/kg, TAK-73310 mg/kg and palbociclib 20mg/kg per day (p.o.) for 21 days, then treatment was discontinued for 7 days followed by 14 days at the same dose; (open diamond) -cetuximab 1mg weekly (i.p.), TAK-73310 mg/kg and palbociclib 20mg/kg daily (p.o.) for 21 days, then treatment was discontinued for 7 days, followed by 14 days at the same dose; (dotted solid triangles) -TAK-73310 mg/kg and palbociclib 20mg/kg per day (p.o.) for 21 days.

Detailed Description

The present specification is based on the inventors' data showing that the combination of an epidermal growth factor receptor inhibitor (such as oxitinib or cetuximab), a mitogen-activated protein kinase 1/2 inhibitor (such as cobitinib, trametinib or TAK-733) and a cyclin-dependent kinase 4/6 inhibitor (such as palbociclib) provides a robust therapy for methods of treating or slowing the progression of cancers with BRAF mutations. In particular, BRAF inhibitors are not required for such combination therapy. Despite the fact that none of the compounds in the combination is an inhibitor of mutant BRAF, the combination therapies described herein have surprisingly demonstrated a synergistic effect on cancers with BRAF mutations, as well as a strong efficacy in reducing tumor volume by up to about 95% in established animal models. The instructions also provide compositions and kits that can be used to perform such combination therapies.

I. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications mentioned herein are incorporated by reference in their entirety. If a definition set forth in this section conflicts with or is otherwise inconsistent with a definition set forth in a patent, application, or other publication incorporated by reference, the definition set forth in this section overrides the definition incorporated by reference.

It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments that relate to particular method steps, reagents or conditions are expressly included in the disclosure and disclosed herein as if each and every combination were individually and explicitly disclosed.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative type" limitation.

As used herein, the terms "comprising," "including," and "containing" are used in their open, non-limiting sense. It is also understood that aspects and embodiments of the invention described herein may comprise "consist of" and/or "consist essentially of" aspects and embodiments.

It is understood that each quantity given herein is meant to refer to the actual given value, and also to the approximation of that given value that would reasonably be inferred based on the ordinary skill in the art, whether or not the term "about" is explicitly used, including equivalents and approximations due to the experimental and/or measurement conditions for that given value.

As used herein, a subject "at risk" for developing a disease may or may not have a detectable disease or disease symptom, and may or may not have exhibited a detectable disease or disease symptom prior to the treatment methods described herein. A subject "at risk" has one or more risk factors that are measurable parameters associated with the development of a disease (such as cancer), as described herein and known in the art. A subject "at risk" may have one or more risk factors. A subject with one or more risk factors has a higher probability of developing a disease than a subject without one or more risk factors.

As used herein, "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include, but are not limited to, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung), peritoneal cancer, hepatocellular cancer, gastric (gastic) or stomach (stomach) cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney (kidney) or renal (renal) cancer, liver cancer (liver cancer), prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatic carcinoma) and various types of head and neck cancer, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocyte (SL) NHL, medium-grade/follicular NHL, medium-grade NHL, high-immunoblastic NHL, high-grade small-cell NHL, large-cell lymphoma, mantle NHL, and large-cell disease (ii) a AIDS-related lymphomas; and waldenstrom's macroglobulinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphocytic Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and Lymphoproliferative Disorder (PTLD) after transplantation, as well as abnormal vascular proliferation associated with nevus destructor, edema (such as that associated with brain tumors), and Meigs syndrome. Examples of cancer may include a primary tumor of any of the above types of cancer or a metastatic tumor at a second site derived from any of the above types of cancer. This definition includes benign and malignant cancers as well as dormant tumors or micrometastases.

The terms "neoplastic cell," "tumor cell," or "cancer cell," used in the singular or plural, refer to a cell that has undergone malignant transformation to render it diseased to a host organism. Primary cancer cells (i.e., cells obtained from the vicinity of the site of malignant transformation) can be readily distinguished from non-cancer-cells by established techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells, but also any cells derived from cancer progenitor cells. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that usually manifests as a solid tumor, a "clinically detectable" tumor is a tumor that is detectable based on tumor mass; for example, it can be detected by procedures such as CAT scanning, Magnetic Resonance Imaging (MRI), X-ray, ultrasound, or palpation. Biochemical or immunological findings alone may not be sufficient to satisfy this definition.

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers which are non-toxic to the cells or mammal to which they are exposed at the dosages and concentrations employed. Typically, the physiologically acceptable carrier is an aqueous pH buffered solution. Non-limiting examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; 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, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN^TMPolyethylene glycol (PEG) and PLURONICS^TM。

As used herein, "delaying the progression of a disease" means delaying, impeding, slowing, delaying, stabilizing and/or delaying the progression of a disease (such as cancer). This delay may be of varying lengths of time depending on the medical history and/or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually encompass prevention, as the individual will not suffer from the disease. For example, the development of advanced cancers, such as metastases, may be delayed. A method of "delaying" the progression of cancer is a method of reducing the probability of disease progression in a given time frame and/or reducing the extent of disease in a given time frame when compared to not using the method. Such comparisons are typically based on clinical studies using statistically significant numbers of subjects. Cancer progression may be detectable using standard methods including, but not limited to, computerized axial tomography (CAT scan), Magnetic Resonance Imaging (MRI), ultrasound, coagulation tests, arteriography, biopsy, urine cytology, and cystoscopy. Progression may also refer to cancer progression, which may initially be undetectable and includes occurrence, recurrence and onset.

As used herein, the term "effective amount" or "therapeutically effective amount" of a substance is at least the minimum concentration required to achieve measurable improvement or prevention of a particular disorder. The effective amount herein may vary depending on a variety of factors such as the disease state, age, sex, and weight of the patient, and the ability of the substance to elicit a desired response in the individual. An effective amount is also the following amount: wherein the therapeutically beneficial effect outweighs any toxic or detrimental effects of the treatment. With respect to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and/or to reduce the rate of tumor growth (such as to inhibit tumor growth) or to prevent or delay other unwanted cell proliferation in the cancer. In some embodiments, an effective amount is an amount sufficient to delay the development of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay relapse. In some embodiments, an effective amount is an amount sufficient to reduce the recurrence rate of the individual. An effective amount may be administered in one or more administrations. The effective amount of the drug or composition may be: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) to some extent inhibit, delay, slow and preferably prevent cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably prevent) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the occurrence and/or recurrence of a tumor; (vii) (viii) reducing the rate of recurrence of the tumor, and/or (viii) alleviating to some extent one or more symptoms associated with the cancer. An effective amount may be administered in one or more administrations. For the purposes of this disclosure, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment, either directly or indirectly. As understood in the clinical setting, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and administration of a single agent in an effective amount may be considered if the desired result can be achieved either in combination with one or more other agents.

As used herein, the term "inhibitor" or "antagonist" refers to a biological or chemical substance that interferes with or otherwise reduces the physiological and/or biochemical effect of another biological or chemical molecule. In some embodiments, the inhibitor or antagonist specifically binds to another molecule.

"package insert" refers to instructions typically contained in commercial packaging for pharmaceuticals that contain information about: indications, usage, dosage, administration, contraindications, other drugs to be combined with the packaged product and/or warnings regarding the use of such drugs, etc.

A "pharmaceutically acceptable salt" is a salt form that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject. See generally Berge et al (1977) j.pharm.sci.66, 1. Particular pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without undue toxicity, irritation, or allergic response. Pharmaceutically acceptable salts include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid, and the like. These salts may be derived from inorganic or organic acids. Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, benzenesulfonate, dihydrogensulfonate, dihydrogensulfate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, or octanoate, Xylene sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate and mandelate. In some embodiments, when an acidic proton present in the parent compound is replaced with a metal ion (e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion); or when coordinated to an organic base, form a pharmaceutically acceptable salt. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, the following: primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-ethylglucamine, N-methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins), amino acids (such as lysine, arginine, histidine), and the like. Examples of pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. In some embodiments, the organic non-toxic base is an L-amino acid, such as L-lysine and L-arginine, tromethamine, N-ethylglucamine, and N-methylglucamine. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. A list of other suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17 th edition, Mack Publishing Company, Iston, Pa., 1985.

A "solvate" is formed by the interaction of a solvent with a compound. Suitable solvents include, for example, water and alcohols (e.g., ethanol). Solvates include hydrates having any ratio of compound to water, such as the monohydrate, dihydrate, and hemihydrate.

A "subject," "patient," or "individual" includes a mammal, such as a human or other animal, and typically a human. In some embodiments, the subject (e.g., patient) to which the therapeutic agents and compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and may be at any suitable age, including infant, juvenile, adolescent, adult and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent, dog, cat, farm animal (such as a cow or horse), and the like.

As used herein, "tissue sample" or "cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue or cell sample may be solid tissue from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any time of pregnancy or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ (such as cancer or tumor tissue). Tissue samples may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

As used herein, the term "treatment" refers to a clinical intervention designed to have a beneficial and desired effect on the natural processes of the individual or cell being treated during clinical pathology. For the purposes of this disclosure, desirable effects of treatment include, but are not limited to, reducing the rate of disease progression, ameliorating or alleviating the disease state, and palliating or improving the prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including but not limited to, reducing the proliferation of cancer cells (or destroying cancer cells), increasing cancer cell killing, reducing symptoms caused by the disease, preventing the spread of the disease, preventing the recurrence of the disease, increasing the quality of life of those patients with the disease, reducing the dose of other drugs required to treat the disease, delaying the progression of the disease, and/or prolonging the survival of the individual.

Methods of treating and delaying progression of cancers with BRAF mutations

Provided herein are methods for treating or delaying progression of cancer in a subject, comprising administering to the subject an effective amount of (a) an Epidermal Growth Factor Receptor (EGFR) inhibitor; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor, wherein the subject has or is at risk of having a cancer with a BRAF mutation.

In some embodiments, (a) is a small molecule or antibody (or antigen-binding fragment thereof) that specifically binds to EGFR or a ligand thereof, and optionally is selected from one or more of cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, erlotinib, gefitinib, afatinib, lapatinib, oxitinib, bocatinib, and erlotinib. In some embodiments, (b) is a small molecule or antibody (or antigen-binding fragment thereof) that specifically binds to MEK1/2 or a ligand thereof, and optionally is selected from one or more of trametinib, semetinib, TAK-733, CI-1040, PD0325901, MEK162, AZD8330, GDC-0623, remetinib, pimavasertib, RO4987655, RO5126766, WX-554, HL-085, and cobitinib. See Chen et al (2017) Molecules 22,1551. In some embodiments, (c) is a small molecule or antibody (or antigen-binding fragment thereof) that specifically binds CDK4/6 or a ligand thereof, and optionally is selected from one or more of palbociclib, ribbociclib, and abbe. In some embodiments, the methods do not include administering (a) an Epidermal Growth Factor Receptor (EGFR) inhibitor; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) administering a BRAF inhibitor to the subject during the period of administering the inhibitor of Cyclin Dependent Kinase (CDK) 4/6. In some embodiments, the methods do not include administering (a) an Epidermal Growth Factor Receptor (EGFR) inhibitor; (b) inhibitors of mitogen-activated protein kinase (MEK) 1/2; and (c) administering an additional therapeutic agent during the administration of the Cyclin Dependent Kinase (CDK)4/6 inhibitor. In some embodiments, the subject has received a BRAF inhibitor in a previous treatment cycle. In some embodiments, the subject has not received a BRAF inhibitor in a previous treatment cycle. In some embodiments, the subject is a human.

In one aspect, the methods disclosed herein can be used to treat or delay progression of a cancer having a BRAF mutation. BRAF is a serine-threonine kinase, and BRAF mutations have been found in 28 primary cancers, including 6 primary melanomas, 12 short-term cultures of melanomas, colorectal cancer, ovarian cancer, glioma, lung cancer, breast cancer, sarcoma, and the like. Without wishing to be bound by theory, it is proposed that BRAF mutations lead to constitutive BRAF kinase activity, phosphorylation of MEK and ERK kinases, and sustained MAPK pathway signaling, leading to tumor cell proliferation and survival. BRAF mutations have been reported at several sites, but the vast majority of mutant BRAFs are V600E, corresponding to a T to a transversion mutation at nucleotide 1796. BRAF mutations V600K, V600D, and D581V have also been observed. Detection of these mutations can be performed using conventional methods, such as the non-limiting examples reported in Lasota et al (2015) am.J.Surg.Pathol.38, 1235. In some embodiments, the BRAF mutation is detected in a tissue or cell sample containing cancer cells from the subject. In some embodiments, the BRAF mutation is a somatic mutation. In some embodiments, the methods are used to treat or delay progression of a cancer having the BRAF V600E mutation. In some embodiments, the methods are used to treat or delay progression of cancer with BRAF V600D. In some embodiments, the methods are used to treat or delay progression of cancer with BRAF V600K. In some embodiments, the methods are used to treat or delay progression of cancer with BRAF D581V. In some embodiments, the method does not comprise administering a BRAF inhibitor to the subject during the administration of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the method does not comprise administering the additional therapeutic agent during the administration of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the subject has received a BRAF inhibitor in a previous treatment cycle. In some embodiments, the subject has not received a BRAF inhibitor in a previous treatment cycle.

In some embodiments, the cancer is adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma. In some embodiments, the cancer is melanoma and carcinoma, such as epithelial tumors, squamous cell tumors, basal cell tumors, transitional cell cancers, adenocarcinoma, adnexal or cutaneous adnexal tumors, mucoepidermoid tumors, cystic, mucinous or serous tumors, ductal, lobular or medullary tumors, acinar cell tumors, and complex epithelial tumors. In some embodiments, the cancer is colon cancer, stomach cancer, lung cancer, breast cancer, pancreatic cancer, oral cancer, prostate cancer, germline cancer, rectal cancer, liver cancer, kidney cancer, and ovarian cancer. In some embodiments, the cancer is an advanced cancer, such as stage IV colorectal cancer. In some embodiments, the cancer is advanced colorectal cancer.

Also provided herein are methods for treating or delaying the progression of cancer in a subject comprising administering to the subject an effective amount of oxitinib or a salt thereof, cobicistinib or a salt thereof, and palbociclib or a salt thereof, wherein the subject has or is at risk of having a cancer with a BRAF mutation. In another aspect, provided herein is a method for treating or delaying the progression of cancer in a subject, comprising administering to the subject an effective amount of cetuximab, cobicistinib or a salt thereof, and palbociclib or a salt thereof, wherein the subject has or is at risk of having a cancer with a BRAF mutation. In another aspect, provided herein is a method for treating or delaying the progression of cancer in a subject, comprising administering to the subject an effective amount of cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof, wherein the subject has, or is at risk of having, a cancer with a BRAF mutation. In another aspect, also provided herein is a method for treating or delaying progression of cancer in a subject, comprising administering to the subject an effective amount of oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof, wherein the subject has or is at risk of having a cancer with a BRAF mutation. In another aspect, provided herein is a method for treating or delaying the progression of cancer in a subject, comprising administering to the subject an effective amount of cetuximab, tremetinib, or a salt thereof, and palbociclib, or a salt thereof, wherein the subject has or is at risk of having a cancer with a BRAF mutation. In another aspect, also provided herein is a method for treating or delaying the progression of cancer in a subject, comprising administering to the subject an effective amount of oxitinib or a salt thereof, trametinib or a salt thereof, and palbociclib or a salt thereof, wherein the subject has or is at risk of having a cancer with a BRAF mutation.

Axitinib is an oral third-generation EGFR inhibitor that has been approved by the us FDA and European Commission (EC) for the treatment of non-small cell lung cancer with EGFR mutations. Oxitinib targets EGFR Tyrosine Kinase Inhibitor (TKI) sensitizing mutations, and in particular T790M, which generally contribute to acquiring resistance to EGFR TKI therapy. Cobinib is a MEK inhibitor approved by the FDA in the united states for use in combination with the BRAF inhibitor verafenib in the treatment of metastatic melanoma with the BRAF V600E or V600K mutation. Cobitinib and verafenib target different components of the MAPK/ERK pathway, respectively: MEK, and BRAF. Palbociclib, a CDK4/6 inhibitor, is approved by the U.S. FDA for use in the treatment of advanced or metastatic breast cancer that is Hormone Receptor (HR) positive, human epidermal growth factor receptor 2(HER2) negative, as an endocrine-based basal therapy in postmenopausal women, in combination with an aromatase inhibitor. Cetuximab is a chimeric monoclonal antibody administered by intravenous infusion and is an EGFR inhibitor that has been approved by the us FDA for the treatment of colon cancer with wild-type KRAS in 2009. TAK-733, an orally bioavailable, non-ATP-competitive small molecule inhibitor of MEK1/2, completed phase I clinical studies. Trametinib is a MEK inhibitor approved by the FDA in the united states for use in the treatment of melanoma with BRAF V600E or V600K mutations, either as a single agent or in combination with the BRAF inhibitor dabrafenib. The structures of oxitinib, TAK-733, cobitinib, trametinib and palbociclib are shown below.

In some embodiments, the method comprises administering a salt of oxitinib, cobitinib, and palbociclib. In some embodiments, the salt is a pharmaceutically acceptable salt. Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, methanesulfonate, propylsulfonate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, butanoate, 4-dioate, hexanoate, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, benzene sulfonate, xylene sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate and mandelate. In some embodiments, the pharmaceutically acceptable salt is a fumarate salt. In some embodiments, the pharmaceutically acceptable salt is a mesylate salt. In some embodiments, the method comprises administering fumarate salts of oxitinib, cobitinib, and palbociclib. In some embodiments, the method comprises administering oxcininib, a fumarate salt of cobicisib, and palbociclib. In some embodiments, the method comprises administering oxentinib and a fumarate salt of cobicisib and palbociclib. In some embodiments, the method comprises administering the mesylate salt of oxitinib and the fumarate salt of cobicistinib and palbociclib. In some embodiments, the method comprises administering oxitinib, cobitinib, and palbociclib, or a solvate or salt of any of the foregoing.

The method may comprise administering any of the compositions or kits described herein.

In another aspect, the method comprises administering an effective amount of oxitinib or a salt thereof, cobinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about 0.25-0.5mg/kg, 0.5-1mg/kg, about 1-1.5mg/kg, about 1.5-2mg/kg, about 2-2.5mg/kg, about 2.5-3mg/kg, about 3-3.5mg/kg, about 3.5-4mg/kg, or about 0.5-3mg/kg oxitinib. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about 20mg, about 40mg, about 80mg, or about 160mg of oxitinib. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of less than about 20mg, about 40mg, about 80mg, or about 160mg of oxitinib. In some embodiments, the dose of oxitinib, or a salt thereof, is an amount of about 20-240mg, about 20-40mg, about 40-80mg, about 80-160mg, about 160-240mg, or about 40-160mg of oxitinib. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about 0.6-2.7mg/kg of oxitinib. In some embodiments, oxitinib is in the form of the mesylate salt. The amount of inhibitor described herein and throughout the specification refers to the amount of inhibitor without regard to the weight of the counter ion if the inhibitor is present in salt form. For example, the term "80 mg of ocitinib" may include, but is not limited to, 80mg of ocitinib in salt-free form or 95.4mg of ocitinib mesylate.

In some embodiments, the daily dose of cobicistinib or a salt thereof is an amount of about 0.1-0.25mg/kg, about 0.25-0.5mg/kg, about 0.5-0.75mg/kg, about 0.75-1mg/kg, about 1-1.25mg/kg, about 1.25-1.5mg/kg, about 1.5-1.75mg/kg, about 1.75-2mg/kg, or about 0.25-1mg/kg cobicistinib. In some embodiments, the daily dose of cobicistinib, or a salt thereof, is an amount of about 10mg, about 20mg, about 40mg, or about 60mg cobicistinib. In some embodiments, the daily dose of cobicistinib, or a salt thereof, is an amount of less than about 10mg, about 20mg, about 40mg, or about 60mg cobicistinib. In some embodiments, the daily dose of cobicistinib or a salt thereof is an amount of about 3.5-100mg, about 3.5-10mg, about 10-20mg, about 20-40mg, about 40-60mg, about 20-60mg, or about 60-100mg cobicistinib. In some embodiments, the daily dose of cobicistinib or a salt thereof is an amount of about 0.3-1mg/kg cobicistinib. In some embodiments, cobicisinib is in its hemi-fumarate salt form.

In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 0.5-1mg/kg, about 1.5-2mg/kg, about 2-2.5mg/kg, about 2.5-3mg/kg, about 3-3.5mg/kg, about 1-2.5mg/kg, about 1-3mg/kg, or about 3-5mg/kg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount less than about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 25-225mg, about 25-50mg, about 50-75mg, about 75-125mg, about 125-150mg, about 150-200mg, or about 200-225mg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 1.25-2.1mg/kg palbociclib. In some embodiments, the method comprises administering oxitinib, or a solvate or salt thereof, cobicistinib, or a solvate or salt thereof, and palbociclib, or a solvate or salt thereof.

In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, or greater than about 65%, or greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85%, or greater than about 90% or greater than 95% by weight of the combined daily dose of oxitinib, cobitinib, and palbociclib, or the aforementioned salts. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about less than 20%, or less than about 25%, or less than about 30%, or less than about 35% or less than about 40%, or less than about 45%, or less than about 50%, or less than about 55%, or less than about 60%, or less than about 65%, or less than about 70%, or less than about 75%, or less than about 80%, or less than about 85%, or less than about 90% or less than 95% by weight of the combined daily dose of oxitinib, cobitinib, and palbociclib, or the aforementioned salts.

In some embodiments, the daily dose of cobicistinib, or a salt thereof, is an amount of greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35% or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, or greater than about 65%, or greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85%, or greater than about 90% or greater than 95% by weight of the combined daily dose of oxitinib, cobicisib, and palbociclib, or the aforementioned salts. In some embodiments, the daily dose of cobicisinib, or a salt thereof, is an amount of less than about 20%, or less than about 25%, or less than about 30%, or less than about 35% or less than about 40%, or less than about 45%, or less than about 50%, or less than about 55%, or less than about 60%, or less than about 65%, or less than about 70%, or less than about 75%, or less than about 80%, or less than about 85%, or less than about 90% or less than 95% by weight of the combined daily dose of oxitinib, cobicisib, and palbociclib, or the aforementioned salts.

In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, or greater than about 65%, or greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85%, or greater than about 90%, or greater than 95% by weight of the combined daily dose of oxitinib, cobinib, and palbociclib, or the foregoing salts. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of less than about 20%, or less than about 25%, or less than about 30%, or less than about 35% or less than about 40%, or less than about 45%, or less than about 50%, or less than about 55%, or less than about 60%, or less than about 65%, or less than about 70%, or less than about 75%, or less than about 80%, or less than about 85%, or less than about 90% or less than 95% by weight of the combined daily dose of oxitinib, cobinib, and palbociclib, or the foregoing salts.

In another aspect, the method comprises administering an effective amount of western medicineTubociclib, cobicistinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the dose of cetuximab is an amount of about 0.1-20mg/kg, about 0.1-0.5mg/kg, about 0.5-1mg/kg, about 1-2mg/kg, about 2-3mg/kg, about 3-5mg/kg, about 5-7.5mg/kg, about 7.5-10mg/kg, about 10-15mg/kg, about 15-20mg/kg, or about 0.1-10 mg/kg. In some embodiments, the dose of cetuximab is about 150-200mg/m²About 200-250mg/m²About 250-300mg/m²About 300-400mg/m²About 400mg/m²About 500-750mg/m²About 150-250mg/m²About 250-400mg/m²Or about 400-750mg/m². In some embodiments, the cetuximab is infused over 30-180 minutes, about 30-60 minutes, about 60-120 minutes, or about 120-180 minutes. In some embodiments, the maximum infusion rate is about 5mL/min or about 10 mL/min. In some embodiments, cetuximab is administered about daily, about weekly, about every two weeks, about every three weeks, or about every four weeks. In some embodiments, the dose of cetuximab is about 500mg/m infused over 60-120 minutes every two weeks². In some embodiments, the cetuximab is administered according to a schedule that includes an initial dose followed by several subsequent doses. In some embodiments, the initial dose is about 250-500mg/m²About 250-300mg/m²About 300-400mg/m or about 400-500mg/m². In some embodiments, the initial dose is about 250mg/m²About 400mg/m²Or about 500mg/m². In some embodiments, the subsequent dose is about 50-300mg/m²About 50-150mg/m²About 150-200mg/m²Or about 200-300mg/m². In some embodiments, the subsequent dose is about 50mg/m²About 150mg/m²Or about 250mg/m². In some embodiments, 400mg/m is infused over 120 minutes²Then infused 250mg/m within 60 minutes per week²The method of (a) administering cetuximab. In some embodiments, 400mg/m is infused over 120 minutes²Followed by a weekly infusion of 150mg/m over 60 minutes²The method of (a) administering cetuximab.

In some embodiments, the daily dose of cobicistinib or a salt thereof is an amount of about 0.1-0.25mg/kg, about 0.25-0.5mg/kg, about 0.5-0.75mg/kg, about 0.75-1mg/kg, about 1-1.25mg/kg, about 1.25-1.5mg/kg, about 1.5-1.75mg/kg, about 1.75-2mg/kg, or about 0.25-1mg/kg cobicistinib. In some embodiments, the daily dose of cobicistinib, or a salt thereof, is an amount of about 10mg, about 20mg, about 40mg, or about 60mg cobicistinib. In some embodiments, the daily dose of cobicistinib, or a salt thereof, is an amount of less than about 10mg, about 20mg, about 40mg, or about 60mg cobicistinib. In some embodiments, the daily dose of cobicistinib or a salt thereof is an amount of about 3.5-100mg, about 3.5-10mg, about 10-20mg, about 20-40mg, about 40-60mg, about 20-60mg, or about 60-100mg cobicistinib. In some embodiments, the daily dose of cobicistinib or a salt thereof is an amount of about 0.3-1mg/kg cobicistinib. In some embodiments, cobicisinib is in its hemi-fumarate salt form.

In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 0.5-1mg/kg, about 1.5-2mg/kg, about 2-2.5mg/kg, about 2.5-3mg/kg, about 3-3.5mg/kg, about 1-2.5mg/kg, about 1-3mg/kg, or about 3-5mg/kg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount less than about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 25-225mg, about 25-50mg, about 50-75mg, about 75-125mg, about 125-150mg, about 150-200mg, or about 200-225mg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 1.25-2.1mg/kg palbociclib. In some embodiments, the method comprises administering cetuximab, cobicistinib, or a solvate or salt thereof, and palbociclib, or a solvate or salt thereof.

In another aspect, the method comprises administering an effective amount of cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, the daily dose of TAK-733, or a salt thereof, is an amount of TAK-733, or a salt thereof, in an amount of about 0.001-1mg/kg, about 0.001-0.002mg/kg, about 0.002-0.005mg/kg, about 0.005-0.01mg/kg, about 0.01-0.05mg/kg, about 0.05-0.1mg/kg, about 0.1-0.2mg/kg, about 0.2-0.3mg/kg, about 0.3-0.4mg/kg, about 0.4-0.5mg/kg, about 0.5-0.6mg/kg, about 0.6-0.7mg/kg, about 0.7-0.8mg/kg, about 0.8-0.9mg/kg, or about 0.9-1 mg/kg. In some embodiments, the daily dose of TAK-733, or a salt thereof, is an amount of about 10mg, about 15mg, about 20mg, or about 25mg of TAK-733. In some embodiments, the daily dose of TAK-733, or a salt thereof, is an amount of less than about 10mg, about 15mg, about 20mg, or about 25mg of TAK-733. In some embodiments, the daily dose of TAK-733, or a salt thereof, is an amount of about 0.1-25mg, about 0.1-1mg, about 1-5mg, about 5-10mg, about 8-16mg, about 10-15mg, about 15-20mg, or about 20-25mg TAK-733.

In some embodiments, the dose of cetuximab is an amount of about 0.1-20mg/kg, about 0.1-0.5mg/kg, about 0.5-1mg/kg, about 1-2mg/kg, about 2-3mg/kg, about 3-5mg/kg, about 5-7.5mg/kg, about 7.5-10mg/kg, about 10-15mg/kg, about 15-20mg/kg, or about 0.1-10 mg/kg. In some embodiments, the dose of cetuximab is about 150-200mg/m²About 200-250mg/m²About 250-300mg/m²About 300-400mg/m²About 400mg/m²About 500-750mg/m²About 150-250mg/m²About 250-400mg/m²Or about 400-750mg/m². In some embodiments, the cetuximab is infused over 30-180 minutes, about 30-60 minutes, about 60-120 minutes, or about 120-180 minutes. In some embodiments, the maximum infusion rate is about 5mL/min or about 10 mL/min. In some embodiments, cetuximab is administered about daily, about weekly, about every two weeks, about every three weeks, or about every four weeks. In some embodiments, the dose of cetuximab is about 500mg/m infused over 60-120 minutes every two weeks². In some embodiments, the cetuximab is administered according to a schedule that includes an initial dose followed by several subsequent doses. In some embodiments, the initial dose is about 250-500mg/m²About 250-300mg/m²About 300-400mg/m or about 400-500mg/m². In some embodiments, the initial dose is about 250mg/m²About 400mg/m²Or about 500mg/m². In some embodiments, the subsequent dose is about 50-300mg/m²About 50-150mg/m²About 150-200mg/m²Or about 200-300mg/m². In some embodiments, the subsequent dose is about 50mg/m²About 150mg/m²Or about 250mg/m². In some embodiments, 400mg/m is infused over 120 minutes²Then infused 250mg/m within 60 minutes per week²The method of (a) administering cetuximab. In some embodiments, 400mg/m is infused over 120 minutes²Followed by a weekly infusion of 150mg/m over 60 minutes²The method of (a) administering cetuximab.

In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 0.5-1mg/kg, about 1.5-2mg/kg, about 2-2.5mg/kg, about 2.5-3mg/kg, about 3-3.5mg/kg, about 1-2.5mg/kg, about 1-3mg/kg, or about 3-5mg/kg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount less than about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 25-225mg, about 25-50mg, about 50-75mg, about 75-125mg, about 125-150mg, about 150-200mg, or about 200-225mg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 1.25-2.1mg/kg palbociclib. In some embodiments, the method comprises administering cetuximab, TAK-733, or a solvate or salt thereof, and palbociclib, or a solvate or salt thereof.

In another aspect, the method comprises administering an effective amount of oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about 0.25-0.5mg/kg, 0.5-1mg/kg, about 1-1.5mg/kg, about 1.5-2mg/kg, about 2-2.5mg/kg, about 2.5-3mg/kg, about 3-3.5mg/kg, about 3.5-4mg/kg, or about 0.5-3mg/kg oxitinib. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about 20mg, about 40mg, about 80mg, or about 160mg of oxitinib. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of less than about 20mg, about 40mg, about 80mg, or about 160mg of oxitinib. In some embodiments, the dose of oxitinib, or a salt thereof, is an amount of about 20-240mg, about 20-40mg, about 40-80mg, about 80-160mg, about 160-240mg, or about 40-160mg of oxitinib. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about 0.6-2.7mg/kg of oxitinib. In some embodiments, oxitinib is in the form of the mesylate salt.

In some embodiments, the daily dose of TAK-733, or a salt thereof, is an amount of TAK-733, or a salt thereof, in an amount of about 0.001-1mg/kg, about 0.001-0.002mg/kg, about 0.002-0.005mg/kg, about 0.005-0.01mg/kg, about 0.01-0.05mg/kg, about 0.05-0.1mg/kg, about 0.1-0.2mg/kg, about 0.2-0.3mg/kg, about 0.3-0.4mg/kg, about 0.4-0.5mg/kg, about 0.5-0.6mg/kg, about 0.6-0.7mg/kg, about 0.7-0.8mg/kg, about 0.8-0.9mg/kg, or about 0.9-1 mg/kg. In some embodiments, the daily dose of TAK-733, or a salt thereof, is an amount of about 10mg, about 15mg, about 20mg, or about 25mg of TAK-733. In some embodiments, the daily dose of TAK-733, or a salt thereof, is an amount of less than about 10mg, about 15mg, about 20mg, or about 25mg of TAK-733. In some embodiments, the daily dose of TAK-733, or a salt thereof, is an amount of about 0.1-25mg, about 0.1-1mg, about 1-5mg, about 5-10mg, about 8-16mg, about 10-15mg, about 15-20mg, or about 20-25mg TAK-733.

In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 0.5-1mg/kg, about 1.5-2mg/kg, about 2-2.5mg/kg, about 2.5-3mg/kg, about 3-3.5mg/kg, about 1-2.5mg/kg, about 1-3mg/kg, or about 3-5mg/kg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount less than about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 25-225mg, about 25-50mg, about 50-75mg, about 75-125mg, about 125-150mg, about 150-200mg, or about 200-225mg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 1.25-2.1mg/kg palbociclib. In some embodiments, the method comprises administering oxitinib, or a solvate or salt thereof, TAK-733, or a solvate or salt thereof, and palbociclib, or a solvate or salt thereof.

In another aspect, the method comprises administering an effective amount of cetuximab, tremetinib, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, the daily dose of trametinib, or a salt thereof, is an amount of about 0.01-1mg/kg, about 0.01-0.02mg/kg, about 0.02-0.03mg/kg, about 0.03-0.05mg/kg, about 0.05-0.08mg/kg, about 0.08-0.1mg/kg, about 0.1-0.2mg/kg, about 0.2-0.3mg/kg, about 0.3-0.4mg/kg, about 0.4-0.5mg/kg, about 0.5-0.6mg/kg, about 0.6-0.7mg/kg, about 0.7-0.8mg/kg, about 0.8-0.9mg/kg, or about 0.9-1mg/kg trametinib. In some embodiments, the daily dose of trametinib, or a salt thereof, is an amount of about 0.5mg, about 1mg, about 2mg, or about 4mg of trametinib. In some embodiments, the daily dose of trametinib, or a salt thereof, is an amount less than about 0.5mg, about 1mg, about 2mg, or about 4mg of trametinib. In some embodiments, the daily dose of trametinib, or a salt thereof, is an amount of about 0.1-25mg, about 0.1-0.5mg, about 0.5-1mg, about 1-2mg, about 2-4mg, about 4-10mg, or about 0.5-2mg of trametinib.

In some embodiments, the dose of cetuximab is an amount of about 0.1-20mg/kg, about 0.1-0.5mg/kg, about 0.5-1mg/kg, about 1-2mg/kg, about 2-3mg/kg, about 3-5mg/kg, about 5-7.5mg/kg, about 7.5-10mg/kg, about 10-15mg/kg, about 15-20mg/kg, or about 0.1-10 mg/kg. In some embodiments, the dose of cetuximab is about 150-200mg/m²About 200-250mg/m²About 250-300mg/m²About 300-400mg/m²About 400mg/m²About 500-750mg/m²About 150-250mg/m²About 250-400mg/m²Or about 400-750mg/m². In some embodiments, the cetuximab is infused over 30-180 minutes, about 30-60 minutes, about 60-120 minutes, or about 120-180 minutes. In some embodiments, the maximum infusion rate is about 5mL/min or about 10 mL/min. In some embodiments, cetuximab is administered about daily, about weekly, about every two weeks, about every three weeks, or about every four weeks. In some embodiments, the dose of cetuximab is about 500mg/m infused over 60-120 minutes every two weeks². In some embodiments, the subsequent ones are based on inclusion of an initial doseSchedule of subsequent doses cetuximab was administered. In some embodiments, the initial dose is about 250-500mg/m²About 250-300mg/m²About 300-400mg/m or about 400-500mg/m². In some embodiments, the initial dose is about 250mg/m²About 400mg/m²Or about 500mg/m². In some embodiments, the subsequent dose is about 50-300mg/m²About 50-150mg/m²About 150-200mg/m²Or about 200-300mg/m². In some embodiments, the subsequent dose is about 50mg/m²About 150mg/m²Or about 250mg/m². In some embodiments, 400mg/m is infused over 120 minutes²Then infused 250mg/m within 60 minutes per week²The method of (a) administering cetuximab. In some embodiments, 400mg/m is infused over 120 minutes²Followed by a weekly infusion of 150mg/m over 60 minutes²The method of (a) administering cetuximab.

In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 0.5-1mg/kg, about 1.5-2mg/kg, about 2-2.5mg/kg, about 2.5-3mg/kg, about 3-3.5mg/kg, about 1-2.5mg/kg, about 1-3mg/kg, or about 3-5mg/kg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount less than about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 25-225mg, about 25-50mg, about 50-75mg, about 75-125mg, about 125-150mg, about 150-200mg, or about 200-225mg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 1.25-2.1mg/kg palbociclib. In some embodiments, the method comprises administering cetuximab, TAK-733, or a solvate or salt thereof, and palbociclib, or a solvate or salt thereof.

In another aspect, the method comprises administering an effective amount of oxitinib or a salt thereof, trametinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about 0.25-0.5mg/kg, 0.5-1mg/kg, about 1-1.5mg/kg, about 1.5-2mg/kg, about 2-2.5mg/kg, about 2.5-3mg/kg, about 3-3.5mg/kg, about 3.5-4mg/kg, or about 0.5-3mg/kg oxitinib. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about 20mg, about 40mg, about 80mg, or about 160mg of oxitinib. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of less than about 20mg, about 40mg, about 80mg, or about 160mg of oxitinib. In some embodiments, the dose of oxitinib, or a salt thereof, is an amount of about 20-240mg, about 20-40mg, about 40-80mg, about 80-160mg, about 160-240mg, or about 40-160mg of oxitinib. In some embodiments, the daily dose of oxitinib, or a salt thereof, is an amount of about 0.6-2.7mg/kg of oxitinib. In some embodiments, oxitinib is in the form of the mesylate salt.

In some embodiments, the daily dose of trametinib, or a salt thereof, is an amount of about 0.01-1mg/kg, about 0.01-0.02mg/kg, about 0.02-0.03mg/kg, about 0.03-0.05mg/kg, about 0.05-0.08mg/kg, about 0.08-0.1mg/kg, about 0.1-0.2mg/kg, about 0.2-0.3mg/kg, about 0.3-0.4mg/kg, about 0.4-0.5mg/kg, about 0.5-0.6mg/kg, about 0.6-0.7mg/kg, about 0.7-0.8mg/kg, about 0.8-0.9mg/kg, or about 0.9-1mg/kg trametinib. In some embodiments, the daily dose of trametinib, or a salt thereof, is an amount of about 0.5mg, about 1mg, about 2mg, or about 4mg of trametinib. In some embodiments, the daily dose of trametinib, or a salt thereof, is an amount less than about 0.5mg, about 1mg, about 2mg, or about 4mg of trametinib. In some embodiments, the daily dose of trametinib, or a salt thereof, is an amount of about 0.1-25mg, about 0.1-0.5mg, about 0.5-1mg, about 1-2mg, about 2-4mg, about 4-10mg, or about 0.5-2mg of trametinib.

In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 0.5-1mg/kg, about 1.5-2mg/kg, about 2-2.5mg/kg, about 2.5-3mg/kg, about 3-3.5mg/kg, about 1-2.5mg/kg, about 1-3mg/kg, or about 3-5mg/kg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount less than about 50mg, about 75mg, about 100mg, or about 125mg of palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 25-225mg, about 25-50mg, about 50-75mg, about 75-125mg, about 125-150mg, about 150-200mg, or about 200-225mg palbociclib. In some embodiments, the daily dose of palbociclib, or a salt thereof, is an amount of about 1.25-2.1mg/kg palbociclib. In some embodiments, the method comprises administering oxitinib, or a solvate or salt thereof, trametinib, or a solvate or salt thereof, and palbociclib, or a solvate or salt thereof.

In another aspect, the methods provide for administering effective amounts of an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) until disease progression or unacceptable toxicity occurs. In some embodiments, the methods provide for administering an effective amount of an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobitinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) for at least about 1-2 weeks, about 2-3 weeks, about 3-4 weeks, about 4-5 weeks, about 5-6 weeks, about 6-7 weeks, about 7-8 weeks, about 8-9 weeks, about 9-10 weeks, about 2-3 months, about 3-4 months, about 4-5 months, about 5-6 months, about 6-12 months, or about 12-24 months. In some embodiments, an effective amount of an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) are administered for 21 consecutive days, followed by 7 days of cessation to constitute a 28 day full cycle. In some embodiments, the subject is administered an effective amount of an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) for a one, two, three, four, five, six, seven, eight, nine, ten, or more 28-day cycle. In some embodiments, an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) are administered for different durations.

In another aspect, an effective amount of an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) are formulated into a composition. In another aspect, effective amounts of an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) are formulated separately. In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated as one composition or separate compositions for oral administration. In some embodiments, oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof are formulated as one composition or separate compositions for oral administration. In some embodiments, the ocitinib or salt thereof, trametinib or salt thereof, and palbociclib or salt thereof are formulated as one composition or separate compositions for oral administration. In some embodiments, cobicisinib or a salt thereof and palbociclib or a salt thereof are formulated as one composition or separate compositions for oral administration. In some embodiments, TAK-733 or a salt thereof and palbociclib or a salt thereof are formulated into one composition or separate compositions for oral administration. In some embodiments, trametinib or a salt thereof and palbociclib or a salt thereof are formulated as one composition or separate compositions for oral administration. For oral administration, the method may comprise formulating the compound into a solid form, such as a tablet or capsule, or into a solution, emulsion or suspension. To prepare an oral composition, the compounds can be formulated to give the daily dosages described herein. In some embodiments, the cetuximab is formulated for intravenous infusion.

In some embodiments, an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) are formulated in the same form. In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof and/or palbociclib or a salt thereof is formulated into a solid form, such as a tablet or capsule. In some embodiments, oxitinib or a salt thereof, TAK-733 or a salt thereof, and/or palbociclib or a salt thereof is formulated into a solid form, such as a tablet or capsule. In some embodiments, the oxitinib or salt thereof, trametinib or salt thereof, and/or palbociclib or salt thereof is formulated into a solid form, such as a tablet or capsule. In some embodiments, cetuximab, cobicistinib or a salt thereof, and/or palbociclib or a salt thereof is formulated in a liquid form, such as a suspension, solution, emulsion, or syrup, or can be lyophilized. In some embodiments, cetuximab, TAK-733, or a salt thereof, and/or palbociclib, or a salt thereof, are formulated in a liquid form, such as a suspension, solution, emulsion, or syrup, or can be lyophilized. In some embodiments, cetuximab, tremetinib or a salt thereof, and/or palbociclib or a salt thereof is formulated in a liquid form, such as a suspension, solution, emulsion, or syrup, or may be lyophilized.

In another aspect, an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) are administered simultaneously or intermittently. In some embodiments, an EGFR inhibitor (e.g., oxitinib or cetuximab), a MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and a CDK4/6 inhibitor (e.g., palbociclib) are formulated and administered as a composition. In some embodiments, the EGFR inhibitor (e.g., oxitinib or cetuximab), the MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and the CDK4/6 inhibitor (e.g., palbociclib) are formulated separately and administered simultaneously. In some embodiments, the EGFR inhibitor (e.g., oxitinib or cetuximab), the MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and the CDK4/6 inhibitor (e.g., palbociclib) are formulated separately and administered intermittently. In some embodiments, the EGFR inhibitor (e.g., oxitinib or cetuximab), the MEK1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733), and the CDK4/6 inhibitor (e.g., palbociclib) are formulated separately and administered with different dosing frequencies.

In another aspect, the method comprises administering an effective amount of oxitinib or a salt thereof, cobinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the obitinib or salt thereof is administered prior to the cobicistinib or salt thereof and the palbociclib or salt thereof. In some embodiments, cobicistinib or a salt thereof is administered before oxitinib or a salt thereof and palbociclib or a salt thereof. In some embodiments, the palbociclib, or salt thereof, is administered before the oxitinib, or salt thereof, and the cobicistinib, or salt thereof. In some embodiments, the otitinib or salt thereof and the cobicistinib or salt thereof are administered together in the same or separate compositions. In some embodiments, the ocitinib or salt thereof and the palbociclib or salt thereof are administered together in the same or separate compositions. In some embodiments, cobicisinib or a salt thereof and palbociclib or a salt thereof are administered together in the same or separate compositions.

In another aspect, the method comprises administering an effective amount of cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, the cetuximab is administered prior to cobitinib or a salt thereof and palbociclib or a salt thereof. In some embodiments, cobicistinib or a salt thereof is administered prior to cetuximab and palbociclib or a salt thereof. In some embodiments, the palbociclib, or a salt thereof, is administered before the cetuximab and the cobicistinib, or a salt thereof. In some embodiments, cetuximab and cobinib, or a salt thereof, are administered together in the same or separate compositions. In some embodiments, the cetuximab and palbociclib, or a salt thereof, are administered together in the same or separate compositions. In some embodiments, cobicisinib or a salt thereof and palbociclib or a salt thereof are administered together in the same or separate compositions. In some embodiments, cetuximab is administered in a separate composition from cobicistinib or a salt thereof and palbociclib or a salt thereof. In some embodiments, the cetuximab is administered weekly or biweekly, and the cobicisinib or a salt thereof and palbociclib or a salt thereof are administered daily.

In another aspect, the method comprises administering an effective amount of cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, cetuximab is administered prior to TAK-733 or a salt thereof and palbociclib or a salt thereof. In some embodiments, TAK-733, or a salt thereof, is administered prior to cetuximab and palbociclib, or a salt thereof. In some embodiments, the palbociclib, or a salt thereof, is administered before the cetuximab and TAK-733, or a salt thereof. In some embodiments, cetuximab and TAK-733, or a salt thereof, are administered together in the same or separate compositions. In some embodiments, the cetuximab and palbociclib, or a salt thereof, are administered together in the same or separate compositions. In some embodiments, TAK-733, or a salt thereof, and palbociclib, or a salt thereof, are administered together in the same or separate compositions. In some embodiments, cetuximab is administered in a separate composition from TAK-733 or a salt thereof and palbociclib or a salt thereof. In some embodiments, cetuximab is administered weekly or biweekly, while TAK-733 or a salt thereof and palbociclib or a salt thereof are administered daily.

In another aspect, the method comprises administering an effective amount of oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the ositinib, or salt thereof, is administered before TAK-733, or salt thereof, and the palbociclib, or salt thereof. In some embodiments, TAK-733 or a salt thereof is administered prior to the oxitinib or a salt thereof and the palbociclib or a salt thereof. In some embodiments, the palbociclib, or salt thereof, is administered prior to the oxitinib, or salt thereof, and the TAK-733, or salt thereof. In some embodiments, the ocitinib, or salt thereof, and TAK-733, or salt thereof, are administered together in the same or separate compositions. In some embodiments, the ocitinib or salt thereof and the palbociclib or salt thereof are administered together in the same or separate compositions. In some embodiments, TAK-733, or a salt thereof, and palbociclib, or a salt thereof, are administered together in the same or separate compositions.

In another aspect, the method comprises administering an effective amount of cetuximab, tremetinib, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, the cetuximab is administered prior to the trametinib, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, the trametinib, or salt thereof, is administered prior to cetuximab and palbociclib, or salt thereof. In some embodiments, the palbociclib, or a salt thereof, is administered before the cetuximab and the trametinib, or a salt thereof. In some embodiments, cetuximab and tremetinib, or a salt thereof, are administered together in the same or separate compositions. In some embodiments, the cetuximab and palbociclib, or a salt thereof, are administered together in the same or separate compositions. In some embodiments, trametinib, or a salt thereof, and palbociclib, or a salt thereof, are administered together in the same or separate compositions. In some embodiments, the cetuximab is administered in a separate composition from trametinib, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, the cetuximab is administered weekly or biweekly, and the trametinib or salt thereof and palbociclib or salt thereof are administered daily.

In another aspect, the method comprises administering an effective amount of oxitinib or a salt thereof, trametinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the ositinib or salt thereof is administered prior to the trametinib or salt thereof and the palbociclib or salt thereof. In some embodiments, the trametinib or salt thereof is administered prior to the oxicetinib or salt thereof and the palbociclib or salt thereof. In some embodiments, the palbociclib, or salt thereof, is administered before the oxitinib, or salt thereof, and the trametinib, or salt thereof. In some embodiments, the ositinib, or salt thereof, and the trametinib, or salt thereof, are administered together in the same or separate compositions. In some embodiments, the ocitinib or salt thereof and the palbociclib or salt thereof are administered together in the same or separate compositions. In some embodiments, trametinib, or a salt thereof, and palbociclib, or a salt thereof, are administered together in the same or separate compositions.

In some embodiments, intermittent administration is at intervals of about 1-30 minutes, at intervals of about 30-60 minutes, at intervals of about 60-120 minutes, at intervals of about 120-240 minutes, at intervals of about 240-480 minutes, at intervals of about 480-720 minutes, at intervals of about 720-960 minutes, or at intervals of about 960-1440 minutes. In some embodiments, intermittent administration is at intervals of about 1-2 days, at intervals of 2-3 days, at intervals of 3-4 days, at intervals of 4-5 days, at intervals of 5-6 days, or at intervals of 6-7 days.

In another aspect, the subject has been previously treated with a BRAF inhibitor. In another aspect, the subject has not been previously treated with a BRAF inhibitor. In another aspect, the subject has been previously treated with a combination of a BRAF inhibitor and a MEK inhibitor. In another aspect, the subject has not previously been treated with a combination of a BRAF inhibitor and a MEK inhibitor. In some embodiments, the subject has developed acquired or adaptive resistance to a BRAF inhibitor or MEK inhibitor. In some embodiments, the subject has not developed acquired or adaptive resistance to a BRAF inhibitor or MEK inhibitor. MEK inhibitors include, but are not limited to, small molecules or antibodies that specifically bind to MEK1/2 or a ligand thereof, such as trametinib, semetinib, TAK-733, CI-1040, PD0325901, MEK162, AZD8330, GDC-0623, remetinib, pimavasertib, RO4987655, RO5126766, WX-554, HL-085, and cobitinib. BRAF inhibitors include, but are not limited to, small molecules or antibodies that specifically bind to wild or mutant BRAF or its ligands, such as verafenib and dabrafenib.

In another aspect, the methods described herein result in reduced cancer cell growth and/or increased cancer cell killing by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to administration of only one or both of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In another aspect, the methods described herein result in a reduction in cancer cell growth and/or an increase in cancer cell killing of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to administration of only one or both of cetuximab, cobinib, or a salt thereof, and palbociclib or a salt thereof. In another aspect, the methods described herein result in a reduction in cancer cell growth and/or an increase in cancer cell killing by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to administration of only one or two of cetuximab, TAK-733, and palbociclib, or a salt thereof. In another aspect, the methods described herein result in reduced cancer cell growth and/or increased cancer cell killing by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to administration of only one or both of oxitinib or a salt thereof, TAK-733, and palbociclib or a salt thereof. In another aspect, the methods described herein result in reduced cancer cell growth and/or increased cancer cell killing by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to administration of only one or two of cetuximab, tremetinib, and palbociclib, or a salt thereof. In another aspect, the methods described herein result in a reduction in cancer cell growth and/or an increase in cancer cell killing of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to administration of only one or two of oxitinib or a salt thereof, trametinib, and palbociclib or a salt thereof.

And the methods described herein have shown synergistic effects on the treatment of cancers with BRAF mutations. In some embodiments, the efficacy of the methods described herein is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to the cumulative efficacy of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof, administered alone. In some embodiments, the efficacy of the methods described herein is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to the cumulative efficacy of cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, administered alone. In some embodiments, the efficacy of the methods described herein is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to the cumulative efficacy of cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof, administered alone. In some embodiments, the efficacy of the methods described herein is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to the cumulative efficacy of oxitinib or a salt thereof, TAK-733, or a salt thereof, and palbociclib or a salt thereof, administered alone. In some embodiments, the efficacy of the methods described herein is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to the cumulative efficacy of cetuximab, trametinib, or a salt thereof, and palbociclib, or a salt thereof, administered alone. In some embodiments, the efficacy of the methods described herein is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more as compared to the cumulative efficacy of oxitinib or a salt thereof, trametinib or a salt thereof, and palbociclib or a salt thereof, administered alone.

In another aspect, the methods described herein reduce the average tumor volume. In some embodiments, the methods described herein reduce the average tumor volume by about 20-95%. In some embodiments, the average tumor volume is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

In another aspect, the methods described herein result in less than about 25%, less than about 20%, less than about 15%, or less than about 5% weight change in the subject. In some embodiments, the method does not cause weight change.

In yet another aspect, the methods described herein comprise administering a loading dose of a combination of: (a) epidermal Growth Factor Receptor (EGFR) inhibitors (e.g., oxitinib or cetuximab); (b) a mitogen-activated protein kinase (MEK)1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733); and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor (e.g., palbociclib), followed by multiple separate administrations of a maintenance dose of the combination. In some embodiments, each loading dose of the three inhibitors is higher than each maintenance dose. In some embodiments, the methods described herein provide for lower dosages, safety, and/or tolerability for long term administration and/or treatment.

Composition III

Also provided herein are compositions comprising: (a) epidermal Growth Factor Receptor (EGFR) inhibitors (e.g., oxitinib or cetuximab); (b) a mitogen-activated protein kinase (MEK)1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733); and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor (e.g., palbociclib); wherein the composition does not comprise a BRAF inhibitor. In some embodiments, the composition consists of: (a) epidermal Growth Factor Receptor (EGFR) inhibitors (e.g., oxitinib or cetuximab); (b) a mitogen-activated protein kinase (MEK)1/2 inhibitor (e.g., cobicistinib, trametinib, or TAK-733); and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor (e.g., palbociclib).

Also provided herein are compositions comprising oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. Also provided herein are compositions comprising oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof. Also provided herein are compositions comprising oxitinib or a salt thereof, trametinib or a salt thereof, and palbociclib or a salt thereof. These compositions may be used in the methods described herein to treat and delay the progression of cancers having BRAF mutations. In some embodiments, the composition does not comprise a BRAF inhibitor.

In one aspect, the composition may further comprise a pharmaceutically acceptable carrier, excipient, binder or diluent. Pharmaceutically acceptable excipients are substances that are non-toxic and otherwise biologically suitable for administration to a subject. Such excipients facilitate administration of the compounds described herein and are compatible with the active ingredient. Examples of pharmaceutically acceptable excipients include stabilizers, lubricants, surfactants, diluents, antioxidants, binders, colorants, fillers, emulsifiers, or taste modifiers. In some embodiments, the pharmaceutical composition according to the embodiments is a sterile composition. Pharmaceutical compositions may be prepared using dispensing techniques that are known or that become available to those skilled in the art. Embodiments also contemplate sterile compositions, including compositions that comply with national and local regulations governing such compositions.

The pharmaceutical compositions and compounds described herein can be formulated as solutions, emulsions, suspensions, dispersions, or inclusions (such as cyclodextrins) in suitable pharmaceutical solvents or carriers, or as pills, tablets, lozenges, suppositories, sachets, dragees, granules, powders for reconstitution, or capsules with solid carriers according to conventional methods for preparing various dosage forms known in the art. The pharmaceutical compositions provided herein can be administered by a suitable delivery route, such as oral, parenteral, rectal, nasal or topical route, or by inhalation. In some embodiments, the composition is formulated for intravenous or oral administration.

In another aspect, the composition is formulated for oral administration. For oral administration, the compositions may be formulated in solid form, such as tablets or capsules, or as solutions, emulsions or suspensions. Oral tablets may contain one or more active ingredients in admixture with compatible pharmaceutically acceptable excipients such as diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents and preservatives. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvinylpyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid are exemplary disintegrants. The binder may include starch and gelatin. The lubricant, if present, may be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or may be coated with an enteric coating.

Capsules for oral administration include hard and soft gelatin capsules. To prepare hard gelatin capsules, one or more active ingredients may be mixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsules may be prepared by mixing the active ingredient with: water, oil (such as peanut oil or olive oil), liquid paraffin, a mixture of mono-and diglycerides of short chain fatty acids, polyethylene glycol 400 or propylene glycol.

Liquids for oral administration may be in the form of suspensions, solutions, emulsions or syrups, or may be lyophilized or presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically acceptable excipients such as suspending agents (e.g., sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, etc.); non-aqueous vehicles such as oils (e.g., almond oil or fractionated coconut oil), propylene glycol, ethanol, or water; preservatives (e.g., methyl or propyl paraben or sorbic acid); wetting agents, such as lecithin; and, if desired, a flavoring or coloring agent.

In another aspect, the composition comprises oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the composition comprises greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, or greater than about 65%, or greater than about 70% by weight of oxitinib, or a salt thereof.

In some embodiments, the composition comprises greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50% by weight cobicistinib, or a salt thereof.

In some embodiments, the composition comprises greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, or greater than about 65%, or greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85%, or greater than about 90% by weight of palbociclib, or a salt thereof.

In some embodiments, the ratio by weight of oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof in the composition is about 1:1:1, 2:1:1, 3:1:1, 4:1:1, 1:2:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 3:1: 1:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1: 9: 1:10, 3:1: 1:1, or 10. In some embodiments, the ratio by weight of oxitinib or salt thereof, cobitinib or salt thereof, and palbociclib or salt thereof in the composition is about 2:1: 4. In some embodiments, the ratio of oxitinib, or a salt thereof, and cobicistinib, or a salt thereof, by weight, is in the range of about 2:3 to 4: 1. In some embodiments, the ratio of ocitinib or salt thereof and palbociclib or salt thereof by weight is in the range of about 40:125 to 80: 75. In some embodiments, the ratio of cobicisinib or a salt thereof and palbociclib or a salt thereof by weight is in a range of about 20:125 to 60: 75.

In another aspect, the composition comprises a salt of oxitinib, cobitinib, TAK-733, trametinib, or palbociclib. In some embodiments, the salt is a pharmaceutically acceptable salt. Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, benzenesulfonate, dihydrogensulfonate, dihydrogensulfate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, or octanoate, Xylene sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate and mandelate. In some embodiments, the composition comprises mesylate salts of oxitinib, cobitinib, and palbociclib. In some embodiments, the composition comprises the fumarate salt of oxitinib, cobitinib, and palbociclib. In another aspect, the composition comprises a solvate of oxitinib, cobitinib, TAK-722, or palbociclib.

In another aspect, the composition comprises oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the composition comprises greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, or greater than about 65%, or greater than about 70% by weight of oxitinib, or a salt thereof.

In some embodiments, the composition comprises greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, by weight, of TAK-733 or a salt thereof.

In some embodiments, the composition comprises greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, or greater than about 65%, or greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85%, or greater than about 90% by weight of palbociclib, or a salt thereof.

In some embodiments, the ratio by weight of oxitinib or salt thereof, TAK-733 or salt thereof, and palbociclib or salt thereof in the composition is about 1:1:1, 2:1:1, 3:1:1, 4:1:1, 1:2:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 3:1: 1:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1: 1:9, 3:1:10, or 3:1: 3. In some embodiments, the ratio by weight of oxitinib, or a salt thereof, and TAK-733, or a salt thereof, in the composition is in a range of about 5:2 to 10: 1. In some embodiments, the ratio by weight of oxitinib or salt thereof and palbociclib or salt thereof in the composition is in the range of about 40:125 to 80: 75. In some embodiments, the ratio of TAK-733, or salt thereof, and palbociclib, or salt thereof, by weight in the composition is in a range of about 8:125 to 16: 75.

In another aspect, the composition comprises oxitinib or a salt thereof, trametinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the composition comprises greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, or greater than about 65%, or greater than about 70% by weight of oxitinib, or a salt thereof.

In some embodiments, the composition comprises greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50% trametinib, or a salt thereof, by weight.

In some embodiments, the composition comprises greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, or greater than about 65%, or greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85%, or greater than about 90% by weight of palbociclib, or a salt thereof.

In some embodiments, the ratio by weight of oxitinib or salt thereof, trametinib or salt thereof, and palbociclib or salt thereof in the composition is about 15:1:10, 20:1:10, 25:1:10, 30:1:10, 35:1:10, 40:1:10, 50:1:10, 15:1:20, 20:1:20, 25:1:20, 30:1:20, 35:1:20, 40:1:20, 50:1:20, 15:1:30, 20:1:30, 25:1:30, 30:1:30, 35:1:30, 40:1:30, 50:1:30, or 40:1: 50. In some embodiments the ratio by weight of oxitinib or salt thereof, trametinib or salt thereof, and palbociclib or salt thereof in the composition is about 15:1: 12. In some embodiments, the ratio by weight of oxitinib or salt thereof and trametinib or salt thereof in the composition is in the range of about 20:1 to 160: 1. In some embodiments, the ratio by weight of oxitinib or salt thereof and palbociclib or salt thereof in the composition is in the range of about 40:125 to 80: 75. In some embodiments, the ratio of trametinib or a salt thereof and palbociclib or a salt thereof by weight in the composition is in a range of about 1:250 to 2: 75.

IV. reagent kit

Also provided herein are kits comprising: (a) epidermal Growth Factor Receptor (EGFR) inhibitors (e.g., oxitinib or cetuximab); (b) a mitogen-activated protein kinase (MEK)1/2 inhibitor (e.g., TAK-733, trametinib, or cobitinib); and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor (e.g., palbociclib); wherein the kit does not comprise a BRAF inhibitor. In some embodiments, the kit consists of: (a) epidermal Growth Factor Receptor (EGFR) inhibitors (e.g., oxitinib or cetuximab); (b) a mitogen-activated protein kinase (MEK)1/2 inhibitor (e.g., TAK-733, trametinib, or cobitinib); and (c) a Cyclin Dependent Kinase (CDK)4/6 inhibitor (e.g., palbociclib). The kits may be used in the methods described herein to treat and delay progression of cancers having BRAF mutations. In some embodiments, the kit does not comprise a BRAF inhibitor. The kit may comprise any of the compositions described herein.

Also provided herein are kits comprising oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the kit comprises a pharmaceutical composition comprising oxitinib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising cobicisinib or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising palbociclib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated into one composition in a kit. In some embodiments, the ocitinib or salt thereof, cobitinib or salt thereof, and palbociclib or salt thereof are formulated separately. In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof, and/or palbociclib or a salt thereof is formulated for oral administration. In some embodiments, oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof are formulated into the same form, such as a solid or liquid form. In some embodiments, oxitinib or a salt thereof, cobinib or a salt thereof, and/or palbociclib or a salt thereof is formulated into a solution, emulsion, suspension, dispersion or inclusion compound (such as cyclodextrin) in a suitable pharmaceutical solvent or carrier, or is formulated into a pill, tablet, lozenge, suppository, sachet, dragee, granule, powder for reconstitution, or capsule with a solid carrier according to conventional methods for preparing various dosage forms known in the art.

In some embodiments, the kit provides oxitinib or a salt thereof, cobitinib or a salt thereof, and palbociclib or a salt thereof in a ratio of about 1:1:1, 2:1:1, 3:1:1, 4:1:1, 1:2:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 3:1: 1:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1: 1:9, 3:1:10, or 3:1: 3:1: 3. In some embodiments, the ratio of ocitinib or salt thereof, cobicistinib or salt thereof, and palbociclib or salt thereof in the kit is about 2:1:4 by weight. In some embodiments, the ratio of ocitinib or salt thereof and cobicistinib or salt thereof in the kit is in the range of about 2:3 to 4:1 by weight. In some embodiments, the ratio of ocitinib or salt thereof and palbociclib or salt thereof in the kit is in the range of about 40:125 to 80:75 by weight. In some embodiments, the ratio of cobicisinib or a salt thereof and palbociclib or a salt thereof in the kit is in the range of about 20:125 to 60:75 by weight.

Also provided herein are kits comprising cetuximab, cobicistinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the kit comprises a pharmaceutical composition comprising cetuximab and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising cobicisinib or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising palbociclib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

In some embodiments, cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated into two or more compositions in a kit. In some embodiments, the cetuximab, cobitinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated separately. In some embodiments, cobicisinib or a salt thereof and/or palbociclib or a salt thereof is formulated for oral administration. In some embodiments, cobicistinib or a salt thereof and palbociclib or a salt thereof are formulated in the same form, such as a solid or liquid form. In some embodiments, cetuximab, cobicistinib or a salt thereof, and/or palbociclib or a salt thereof, are formulated into a solution, emulsion, suspension, dispersion, or inclusion compound (such as a cyclodextrin) in a suitable pharmaceutical solvent or carrier, or a pill, tablet, lozenge, suppository, sachet, dragee, granule, powder for reconstitution, or capsule with a solid carrier, according to conventional methods known in the art for preparing various dosage forms. In some embodiments, the cetuximab is formulated for administration via intravenous infusion. In some embodiments, cobicisinib or a salt thereof and palbociclib or a salt thereof are formulated into a composition for oral administration.

In some embodiments, the kit provides cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, in a ratio of about 1:1:1:, 2:1:1, 3:1:1, 4:1:1:, 1:2:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1:9, 3:1:10, 4:1:2, 4:1:3, 4:1:4, 4:1:5, 4:1:6, 4:1:7, 4:1:8, 4:1:9, 4:1:10, 5:1: 1:1, 5:1:2, 5:1:3, 5:1:4, 5:1:5, 5:1:6, 5:1:7, 5:1:8, 5:1: 1:10, 5:1: 1:1, 5:2, 5: 3, 5:1:5: 6, 5:1:7, 5:1: 1:8, 5:1: 16: 1:3, 6:1:4, 6:1:5, 6:1:6, 6:1:7, 6:1:8, 6:1:9, or 6:1: 10. In some embodiments, the ratio of cetuximab, cobicistinib, or a salt thereof, and palbociclib, or a salt thereof, in the kit is about 20:3:6 by weight. In some embodiments, the ratio of cetuximab and cobicisinib, or a salt thereof, in the kit is in the range of about 85:60 to 85:2 by weight. In some embodiments, the ratio of cetuximab and palbociclib, or a salt thereof, in the kit is in the range of about 17:25 to 34:3 by weight. In some embodiments, the ratio of cobicisinib or a salt thereof and palbociclib or a salt thereof in the kit ranges from about 20:125 to 60:75 by weight.

Also provided herein are kits comprising cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, the kit comprises a pharmaceutical composition comprising cetuximab and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising TAK-733 or a salt thereof and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising palbociclib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

In some embodiments, cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof, are formulated into one composition in a kit. In some embodiments, cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof, are formulated into two or more compositions in a kit. In some embodiments, cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof, are formulated separately. In some embodiments, TAK-733 or a salt thereof and/or palbociclib or a salt thereof is formulated for oral administration. In some embodiments, TAK-733 or a salt thereof and palbociclib or a salt thereof are formulated into the same form, such as a solid or liquid form. In some embodiments, cetuximab, TAK-733, or a salt thereof, and/or palbociclib, or a salt thereof, are formulated in a suitable pharmaceutical solvent or carrier as a solution, emulsion, suspension, dispersion, or inclusion compound (such as a cyclodextrin) or with a solid carrier as a pill, tablet, lozenge, suppository, sachet, dragee, granule, powder for reconstitution, or capsule, according to conventional methods for preparing various dosage forms known in the art. In some embodiments, the cetuximab is formulated for administration via intravenous infusion. In some embodiments, TAK-733 or a salt thereof and palbociclib or a salt thereof are formulated into a composition for oral administration.

In some embodiments, the kit provides cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof, in a ratio of about 5:1:5, 10:1:5, 15:1:5, 20:1:5, 10:2:1, 10:1:2, 10:1:3, 10:1:4, 10:1:5, 10:1:6, 10:1:7, 10:1:8, 5:1:9, 10:1:10, 15:1:2, 15:1:3, 15:1:4, 15:1:5, 15:1:6, 15:1:7, 15:1:8, 15:1:9, 15:1:10, 20:1:1, 20:1:2, 20:1:3, 20:1:4, 20:1:5, 20:1:6, 20:1:7, 20:1:8, 20:1:9, 20:1:10, or 20:1:3, by weight. In some embodiments, the ratio of cetuximab, TAK-733, or a salt thereof, and palbociclib, or a salt thereof, in the kit is about 20:1:6 by weight. In some embodiments, the ratio of cetuximab and TAK-733, or a salt thereof, in the kit is in the range of about 85:16 to 850:8 by weight. In some embodiments, the ratio of cetuximab and palbociclib, or a salt thereof, in the kit is in the range of about 17:25 to 34:3 by weight. In some embodiments, the ratio of TAK-733 or a salt thereof and palbociclib or a salt thereof in the kit is in the range of about 8:125 to 16:75 by weight.

Also provided herein are kits comprising oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the kit comprises a pharmaceutical composition comprising oxitinib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising TAK-733 or a salt thereof and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising palbociclib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

In some embodiments, oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof are formulated into one composition in a kit. In some embodiments, oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof are formulated separately. In some embodiments, oxitinib or a salt thereof, TAK-733 or a salt thereof, and/or palbociclib or a salt thereof is formulated for oral administration. In some embodiments, oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof are formulated into the same form, such as a solid or liquid form. In some embodiments, oxitinib or a salt thereof, TAK-733 or a salt thereof, and/or palbociclib or a salt thereof is formulated into a solution, emulsion, suspension, dispersion, or inclusion compound (such as cyclodextrin) in a suitable pharmaceutical solvent or carrier, or with a solid carrier, into a pill, tablet, lozenge, suppository, sachet, dragee, granule, powder for reconstitution, or capsule, according to conventional methods known in the art for preparing various dosage forms.

In some embodiments, the kit provides oxitinib or a salt thereof, TAK-733 or a salt thereof, and palbociclib or a salt thereof in a ratio of about 1:1:1, 2:1:1, 3:1:1, 4:1:1, 1:2:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 3:1: 1:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1: 1:9, 3:1:10, or 3:1: 3:1: 3. In some embodiments the ratio by weight of oxitinib or salt thereof, TAK-733 or salt thereof, and palbociclib or salt thereof in the composition is about 3:1: 6. In some embodiments, the ratio of ocitinib, or a salt thereof, and TAK-733, or a salt thereof, in the kit is in the range of about 5:2 to 10:1, by weight. In some embodiments, the ratio of ocitinib or salt thereof and palbociclib or salt thereof in the kit is in the range of about 40:125 to 80:75 by weight. In some embodiments, the ratio of TAK-733 or a salt thereof and palbociclib or a salt thereof in the kit is in the range of about 8:125 to 16:75 by weight.

Also provided herein are kits comprising cetuximab, tremetinib, or a salt thereof, and palbociclib, or a salt thereof. In some embodiments, the kit comprises a pharmaceutical composition comprising cetuximab and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising trametinib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising palbociclib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

In some embodiments, cetuximab, tremetinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated into one composition in a kit. In some embodiments, cetuximab, tremetinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated into two or more compositions in a kit. In some embodiments, the cetuximab, trametinib, or a salt thereof, and palbociclib, or a salt thereof, are formulated separately. In some embodiments, the trametinib or salt thereof and/or palbociclib or salt thereof is formulated for oral administration. In some embodiments, trametinib or a salt thereof and palbociclib or a salt thereof are formulated into the same form, such as a solid or liquid form. In some embodiments, cetuximab, tremetinib or a salt thereof, and/or palbociclib or a salt thereof, are formulated in a suitable pharmaceutical solvent or carrier as a solution, emulsion, suspension, dispersion or inclusion compound (such as cyclodextrin), or with a solid carrier as a pill, tablet, lozenge, suppository, sachet, dragee, granule, powder for reconstitution, or capsule, according to conventional methods for preparing various dosage forms known in the art. In some embodiments, the cetuximab is formulated for administration via intravenous infusion. In some embodiments, trametinib or a salt thereof and palbociclib or a salt thereof are formulated into a composition for oral administration.

Also provided herein are kits comprising oxitinib or a salt thereof, trametinib or a salt thereof, and palbociclib or a salt thereof. In some embodiments, the kit comprises a pharmaceutical composition comprising oxitinib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising trametinib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent. In some embodiments, the kit comprises a pharmaceutical composition comprising palbociclib, or a salt thereof, and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

In some embodiments, the ocitinib or salt thereof, trametinib or salt thereof, and palbociclib or salt thereof are formulated as a composition in a kit. In some embodiments, the ocitinib or salt thereof, trametinib or salt thereof, and palbociclib or salt thereof are formulated separately. In some embodiments, the ocitinib or salt thereof, trametinib or salt thereof, and/or palbociclib or salt thereof is formulated for oral administration. In some embodiments, the oxitinib or salt thereof, trametinib or salt thereof, and palbociclib or salt thereof are formulated into the same form, such as a solid or liquid form. In some embodiments, oxitinib or a salt thereof, trametinib or a salt thereof, and/or palbociclib or a salt thereof is formulated into a solution, emulsion, suspension, dispersion, or inclusion compound (such as cyclodextrin) in a suitable pharmaceutical solvent or carrier, or is formulated with a solid carrier into a pill, tablet, lozenge, suppository, sachet, dragee, granule, powder for reconstitution, or capsule, according to conventional methods known in the art for preparing various dosage forms.

In some embodiments, the kit provides the ratio by weight of oxitinib or a salt thereof, trametinib or a salt thereof, and palbociclib or a salt thereof is about 15:1:10, 20:1:10, 25:1:10, 30:1:10, 35:1:10, 40:1:10, 50:1:10, 15:1:20, 20:1:20, 25:1:20, 30:1:20, 35:1:20, 40:1:20, 50:1:20, 15:1:30, 20:1:30, 25:1:30, 30:1:30, 35:1:30, 40:1:30, 50:1:30, or 40:1: 50. In some embodiments, the ratio by weight of oxitinib or salt thereof, trametinib or salt thereof, and palbociclib or salt thereof in the composition is about 15:1: 12. In some embodiments, the ratio of ocitinib or salt thereof and trametinib or salt thereof in the kit is in the range of about 20:1 to 160:1 by weight. In some embodiments, the ratio of ocitinib or salt thereof and palbociclib or salt thereof in the kit is in the range of about 40:125 to 80:75 by weight. In some embodiments, the ratio of trametinib or a salt thereof and palbociclib or a salt thereof in the kit is in the range of about 1:250 to 2:75 by weight.

In another aspect, the kit further comprises a package insert including, but not limited to, appropriate instructions for preparing and administering the formulation, side effects of the formulation, and any other relevant information. The instructions may be in any suitable format, including but not limited to printed matter, video tape, computer readable disk, optical disk, or directions for internet-based instructions.

In another aspect, there is provided a kit for treating an individual suffering from or susceptible to a condition described herein, comprising a first container comprising a dose of a composition or formulation as disclosed herein and a package insert for use. The container may be any container known in the art and suitable for storing and delivering intravenous formulations. In certain embodiments, the kit further comprises a second container comprising a pharmaceutically acceptable carrier, diluent, adjuvant, etc., for preparing the formulation to be administered to the subject.

In another aspect, kits can also be provided that contain a sufficient dose of a composition described herein (including pharmaceutical compositions thereof) to provide effective treatment to an individual for an extended period of time, such as 1-3 days, 1-5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, or longer.

In some embodiments, the kit may also include a plurality of doses and may be packaged in quantities sufficient for storage and use in pharmacies (e.g., hospital pharmacies and pharmacy pharmacies). In certain embodiments, the kit can include a dose of at least one composition as disclosed herein.

Examples

The following examples are provided to illustrate, but not to limit, the present invention. Those skilled in the art will recognize that the following procedures may be modified using methods known to those of ordinary skill in the art.

Example 1 ex vivo drug testing using a conditionally reprogrammed cell pool

Surgical specimens from colorectal cancer patients were obtained from a hospital in Beijing after informed consent was obtained from the patients. Patient-derived xenograft tumor samples were obtained from Nod/SCID mice inoculated with patient surgical tumor samples.

For ex vivo drug sensitivity assays, colorectal tumor cells (CRC) were isolated from patient tissue samples or PDX xenograft tumor tissues. Briefly, tumor tissue is cut into small pieces less than 1mm in diameter using scissors. The tumor pieces were transferred to a sterile 100ml glass Erlenmeyer flask equipped with a magnetic stir bar. 10-15ml of digestion medium containing 0.25U/ml Liberase DH was added to the minced tumor tissue to start the enzymatic digestion. The enzyme mixture was incubated at 37 ℃ for 1-2 hours with moderate stirring. The digested tumor tissue was filtered through a 100 μm cell holder. The filtrate was filtered again through a 40 μm cell holder. The CRC pellet retained on the 40 μm cell holder was collected, washed twice with HBSS, and then resuspended in defined growth medium supplemented with several stem cell growth factors.

The CRC pellet retained on the 40- μm cell holder was collected, washed twice with HBSS, and then resuspended in defined growth medium supplemented with cell growth factors and small molecule inhibitors. The CRC pellet was recovered overnight in defined growth medium. The defined growth medium ishESC SFM (defined serum-free and trophoblast-free medium (SFM)), supplemented with: nicotinamide, Wnt3A, Noggin (a Bone Morphogenetic Protein (BMP) inhibitor), rsspondin-1 (a Wnt/β -catenin signaling agonist), and Y27632 (a Rho-associated coiled-coil containing protein kinase (ROCK-1) inhibitor).

For conditional reprogramming, recovered cancer tissue-derived spheroids (CTOS) were dissociated into single cells and seeded in a feeder cell/Rock inhibitor (Y27632) co-culture system at about 3000 cells per well in cell culture plates and grown for 3 days at 37 ℃.

For ex vivo drug testing, vaccinated CRCs were exposed to a combination of oxitinib (mesylate), cobitinib (fumarate), and palbociclib (fumarate) for 72 hours. The combination was prepared in DMSO and the final DMSO concentration in the medium was 0.1%. Typical compound concentrations for ex vivo drug testing were: 0.1-0.5 μ M of oxitinib, 0.025-0.2 μ M of cobicistinib and 0.125-1 μ M of palbociclib.

Following drug exposure, CRC was labeled with 5-ethynyl-2' -deoxyuridine (Edu) to assess tumor cell proliferation rates. In the presence of drug exposure, labeling lasted 24 hours. In the control group, epithelial tumor cells were not drug exposed with medium replacement, but were similarly labeled with Edu. Labeled CRC was fixed in blocking buffer containing 0.5% Triton X-100 and 3% BSA at 4 ℃ and stained with Hoechst 33342 overnight. Tumor cells were incubated with EpCAM antibody (1:4000) for 2 hours at room temperature and washed with PBST. Subsequently, CRC and Alexa are combined647 conjugated goat anti-mouse secondary antibody were incubated for 30 min at room temperature and washed with PBST.

Incorporated Edu was detected by a Click-iT reaction in which fixed cells were incubated in the dark with a solution containing 1 Xclick-iT Edu reaction buffer, CuSO₄Incubated with the reaction mixture of azide conjugated Alexa Fluor dye. Stained cells were washed twice with PBS before image acquisition and analysis.

For image acquisition and analysis, stained tumor cells were imaged by a High Content Screening (HCS) platform (Thermo Scientific cytologic sarrayscanxti HCS reader). Images were collected using a 10 x objective. Twenty-five fields of view were imaged per well in the analysis. From the images, three fluorescence signals were obtained from the HCS reader. Blue fluorescence signals record nuclear signals stained by Hoechst 33342, green fluorescence signals detect Edu incorporated in newly synthesized DNA, and red fluorescence signals detect EpCAM positive epithelial cell populations. A representative image is shown in fig. 1.

MI (maximum inhibition index) was calculated using EpCAM and Edu positive readings (table 1). MI ═ Edu positive cells in control/Edu positive cells in treatment. As shown in table 1, the combination therapy had the highest specificity for CRC with the BRAF V600E or D581V mutation.

TABLE 1 maximum inhibition index (MI) of CRC treated with combination therapy

CRC patient ID	KRAS mutations	BRAF mutations	NRAS mutations	MI
					CKY041	G12D			25
NYL170	G12D			39
					NYL-JN-025	G12D			106
ZKB171	G12D			908
					CKY048	G13D			3898.57
NYL170(NYP023)	G12D			39
					NYL102				309
NYL109				1085
					NYL161				175
NYL-GZ-076				73.67
					NYL-HEB-057			G12D	894.06
NYP066				20.87
					NYP069				137.86
NYL132		V600E		17882
					ZKB197		V600E		10448
ZKB040		D581V		11311
					NYL243		V600E		89

Table 2 shows the selectivity of combination therapy for CRC with or without BRAF mutation. Selectivity (%) ═ MI > -1000 CRC number/total CRC number 100%

Table 2 selectivity of combination therapy for BRAF mutations in CRC

The data presented in table 2 indicate that colorectal cancer cells with BRAF mutations are extremely sensitive to the drug combination, with 3 of 4 having a greater than 10,000-fold reduction in cell proliferation compared to no drug controls. In contrast, 2 of 11 colorectal cancer cells without BRAF mutations or KRAS/NRAS mutations had a greater than 1,000-fold reduction in cell proliferation, and none had a greater than 10,000-fold reduction.

Example 2 testing of mouse tumor volume reduction during combination therapy in PDX model using patient NYL132

The study was performed in a xenograft tumor PDX model using BRAF^V600ESurgical tumor tissue establishment of NYL132 in CRC patients. The mean tumor volumes during combination therapy are shown in table 3 and figure 2. Briefly, studies were performed using 6-8 week old female NOD/SCID mice. Tumor samples obtained from the patient were immediately transferred to tissue preservation solution and cut into small pieces. The fragment was inoculated subcutaneously on one side of the mouse to generate a xenograft called passage 1 (P1). Successive xenografts of different generations were generated using the same procedure. When the average tumor size of the mice reaches about 250-300mm³At the time, animals were randomly assigned to different groups of 5 mice each. The date of randomization was defined as study day 0. Tumor volume in mm using the following formula³Represents: v (volume) ═ a × b²) And/2, wherein a and b are the major and minor diameters of the tumor, respectively. Tumor inhibition is expressed as calculated according to the formulaTumor Growth Inhibition (TGI): TGI ═ 100% (1- (Ti-T0)/(Vi-V0)), where Ti is the mean tumor volume in the treatment groups on the day of measurement; t0 is the mean tumor volume at D0 in the treatment group; vi is the average tumor volume of the control group on the day of measurement; v0 is the tumor volume at D0 in the control group. TGI values for the combination therapy are shown in table 4 and figure 3.

Table 3 mean tumor volume during combination therapy

TABLE 4 tumor growth inhibition during combination therapy

The data presented in table 3 and table 4 show that combination therapy reduces tumor volume in a dose-dependent manner. The data demonstrate the key value of combination therapy demonstrating the ability to regress established tumors (not just to slow tumor growth rate), while standard chemotherapy regimens for colorectal cancer are completely ineffective in BRAF mutant colorectal tumor models.

Example 3 body weight of mice during combination therapy in PDX model using NYL132 patient

Using BRAF^V600ECRC patient ny 132, following the same procedure as in example 2. The average body weight of the mice is shown in table 5 and fig. 4.

TABLE 5 mouse body weight during combination therapy

The data presented in table 4 show that the combination therapy is tolerable in animals, especially at dosage levels that show significant in vivo efficacy.

Example 4 testing of tumor volume reduction and weight change in mice during combination therapy in PDX model Using patient NYL-GZ-082

Using BRAF^V600ECRC patients NYL-GZ-082, using various combinations, following the same procedure as in examples 2 and 3. Tumor volume values and body weight changes are shown in fig. 5 and 6. Two combination therapies: both oxitinib + cobinib + palbociclib and cetuximab + cobinib + palbociclib reduced tumor volume. Furthermore, in the control group (fig. 5, triangle), the tumor volume increased to 14 days after no treatment>900mm³. Beginning on day 15, combination therapy (cetuximab + cobicistinib + palbociclib) was given and tumor volume decreased rapidly, even after tumors had grown to large size. Furthermore, wet weight maintained tumor regression over 2 treatment cycles.

Example 5 BRAF treated with monotherapy and combination therapy in PDX model Using patient NYL132^V600EComparison of the maximum inhibition index (MI) of CRC and the Tumor Growth Inhibition (TGI)

Using BRAF^V600ECRC patient ny 132, following the same procedure as in example 2. MI (maximum inhibition index) was calculated using EpCAM and Edu positive readings (table 6). MI ═ Edu positive cells in control/Edu positive cells in treatment. And calculating Tumor Growth Inhibition (TGI) according to the formula: TGI ═ 100% (1- (Ti-T0)/(Vi-V0)), where Ti is the mean tumor volume in the treatment groups on the day of measurement; t0 is the mean tumor volume at D0 in the treatment group; vi is the average tumor volume of the control group on the day of measurement; v0 is the tumor volume at D0 in the control group.

TABLE 6 monotherapy and in PDX model with patient NYL132BRAF treated by combination therapy^V600EMI and TGI values of CRC

Figure 7 shows the change in tumor volume during treatment with various monotherapies and combination therapies in a PDX model using patient ny 132. All three combinations tested (1) cetuximab + cobitinib + palbociclib, (2) oxitinib + TAK-733+ palbociclib and (3) cetuximab + TAK-733+ palbociclib reduced the tumor volume by > 85% in the first treatment cycle (21 days) (see also table 7). Cetuximab treatment alone slightly inhibited tumor growth (open triangle, 2 cycles of treatment from start to day 56), and cetuximab-treated tumors were sensitive to treatment with a combination of cetuximab 1mg weekly (i.p.), cobitinib 5mg/kg, and palbociclib 20mg/kg daily (p.o.) for 21 days.

TABLE 7 from BRAF^V600ETumor growth inhibition in PDX model of NYL132 in CRC patients

Example 6 BRAF treated with monotherapy and combination therapy in a CDX model Using cell line HT-29^V600EComparison of the maximum inhibition index (MI) of CRC and the Tumor Growth Inhibition (TGI)

An in vivo efficacy study of xenografts (CDX) derived from the cell line HT-29 was performed. The experimental procedure is briefly described below. Sufficient donor cells (HT-29) were cultured and prepared for seeding. Suspending minimum 3x10 in matrigel⁶Individual cellInjected into the dorsal side of female nude mice. When the tumor volume of the mice grows to 100-300mm³In this case, mice with irregular tumor shapes and poor health were excluded according to their body weight and tumor growth, and the remaining mice were randomly divided into a plurality of groups, usually 3 to 5 mice per group. The date of randomization was defined as study day 0. Tumor volume in mm using the following formula³Represents: v (volume) ═ a × b²) And/2, wherein a and b are the major and minor diameters of the tumor, respectively. Tumor inhibition is expressed as Tumor Growth Inhibition (TGI) calculated according to the following formula: TGI ═ 100% (1- (Ti-T0)/(Vi-V0)), where Ti is the mean tumor volume in the treatment groups on the day of measurement; t0 is the mean tumor volume at D0 in the treatment group; vi is the average tumor volume of the control group on the day of measurement; v0 is the tumor volume at D0 in the control group.

MI (maximum inhibition index) was calculated using EpCAM and Edu positive readings (table 8). MI ═ Edu positive cells in control/Edu positive cells in treatment. And calculating Tumor Growth Inhibition (TGI) according to the formula: TGI ═ 100% (1- (Ti-T0)/(Vi-V0)), where Ti is the mean tumor volume in the treatment groups on the day of measurement; t0 is the mean tumor volume at D0 in the treatment group; vi is the average tumor volume of the control group on the day of measurement; v0 is the tumor volume at D0 in the control group.

TABLE 8 BRAF treated with monotherapy and combination therapy in CDX model using cell line HT-29^V600EMI and TGI values of CRC

Sample (I)	Concentration (uM)	MI	％TGI
				Oxititinib	0.5	1.20	16.9639％
Afatinib	0.2	1.24	19.2409％
				Cobicistinib	0.4	331.41	99.6983％
Trametinib	0.02	17.16	94.1714％
				Pabociclib	0.15	4.64	78.4714％
Ribocini	5	11.24	91.1053％
				Oxitinib + cobitinib + palbociclib	0.5+0.4+0.15	23332.49	99.9957％
Oxitinib + cobitinib + ribociclib	0.5+0.4+5	12790.35	99.9922％
				Oxitinib + trametinib + palbociclib	0.5+0.02+0.15	24849.10	99.9960％
Oxitinib + trametinib + ribociclib	0.5+0.02+5	24849.10	99.9960％
				Afatinib + cobitinib + palbociclib	0.2+0.4+0.15	24849.10	99.9960％
Afatinib + cobitinib + Ribociclib	0.2+0.4+5	24849.10	99.9960％
				Afatinib + trametinib + palbociclib	0.2+0.02+0.15	6598.97	99.9848％
Afatinib + trametinib + Ribociclib	0.2+0.02+5	24849.10	99.9960％

Fig. 8 and 9 show the tumor volume changes during treatment with various single agents and dual and triple combination therapies in the CDX model using cell line HT-29. All four combinations tested (1) oxitinib + cobicistinib + palbociclib, (2) cetuximab + cobicistinib + palbociclib, (3) oxitinib + TAK-733+ palbociclib and (4) cetuximab + TAK-733+ palbociclib reduced tumor volume by > 50% in the first treatment cycle (21 days). Chemotherapeutic agents of capecitabine (squares) and 5-FU (dashed circles) showed no efficacy and tumor growth trends were similar to vehicle control (triangles). Cetuximab, the triple combination of cobicistinib and palbociclib, and the triple combination of ocitinib, cobicistinib and palbociclib showed superior tumor growth inhibition over single therapy (cobicistinib) and double combinations of cobicistinib and olbcitinib and double combinations of cobicistinib and palbociclib (see also table 9).

TABLE 9 tumor growth inhibition Using CDX model of cell line HT-29

EXAMPLE 7 clinical Studies of combination therapy

In patients with any BRAF^V600EA phase 2 open label clinical study was performed in patients with mutant advanced colorectal cancer. The study was designed to evaluate the safety and efficacy of combination therapy (cetuximab + cobicistinib + palbociclib) in patients with advanced colorectal cancer. The study subject population included 18-75 year old adult males and females diagnosed as having either BRAF^V600EMutated advanced colorectal cancer and expected survival longer than 12 months.

Administering to the subject: (1) cetuximab at 400mg/m at a maximum infusion rate of 10mg/min²Infusion is carried out for 120 minutes for 1 week, followed by 250mg/m once a week²(ii) a (2) Cobicistinib, orally administered at 20-60mg per day; and (3) Pabociclib, orally administered at 75-125mg per day. Vital signs, clinical laboratory measurements, resting ECG measurements, adverse events, and severe adverse events were monitored and evaluated. Overall response rates, disease control rates, overall survival, duration of response, and quality of life were also monitored and evaluated. The study parameters are summarized in table 10.

Watch 10