CN117083283A

CN117083283A - Combination therapy for cancer treatment

Info

Publication number: CN117083283A
Application number: CN202180093091.6A
Authority: CN
Inventors: 莱努斯·马丁; 莱斯利·哈里斯·布雷尔; 罗伯特·菲尔德·舒梅克
Original assignee: Yirui Shikang Pharmaceutical Research And Development Co
Current assignee: Yirui Shikang Pharmaceutical Research And Development Co
Priority date: 2020-12-11
Filing date: 2021-12-10
Publication date: 2023-11-17

Abstract

The present disclosure provides methods of treating cancer with a combination therapy of an SHP2 inhibitor, such as a compound of formula I, with an FGFR inhibitor, a B-Raf inhibitor, a MEK inhibitor, or a MET inhibitor.

Description

Combination therapy for cancer treatment

Cross reference

The present application claims U.S. provisional patent application No. 63/124,663 filed on 11/12/2020; U.S. provisional patent application No. 63/124,667, filed 12/11/2020; U.S. provisional patent application No. 63/124,671, filed on 12/11/2020; and U.S. provisional patent application No. 63/124,674, filed on 12/11/2020; each of which is incorporated by reference in its entirety.

Background

Src homology-2 phosphatase (SHP 2) is a non-receptor protein phosphatase that is widely expressed in various tissues and cell types (see for review: tajan M et al, eur J Med Genet 2016 58 (10): 509-25; grossmann KS et al, advanced Cancer Res 2010 106:53-89). SHP2 consists of two Src homology 2 (N-SH 2 and C-SH 2) domains located at its NH2 terminus, a catalytic PTP (protein tyrosine phosphatase) domain, and a C-terminal tail with regulatory properties. In the ground state, intermolecular interactions between SH2 domains and PTP domains prevent the entry of the substrate into the catalytic pocket, keeping SHP2 in a closed self-inhibiting conformation. In response to the stimulus, the SHP2 activator protein with the phospho-tyrosine motif binds to the SH2 domain, resulting in active site exposure and enzymatic activation of SHP 2.

Disclosure of Invention

Embodiments disclosed herein relate generally to compositions and methods related to combination therapies (including while providing an unexpected degree of synergy) for treating cancer using SHP2 inhibitors in combination with FGFR inhibitors, B-Raf inhibitors, MEK inhibitors, or MET inhibitors.

SHP2 plays an important role in basic cellular functions including proliferation, differentiation, cell cycle maintenance and motility. SHP2 regulates a variety of intracellular signaling pathways in response to a wide range of growth factors, cytokines and hormones by dephosphorylating its associated signaling molecule. The cell signaling processes involved in SHP2 include RAS-MAPK (mitogen activated protein kinase), PI3K (phosphoinositide 3-kinase) -AKT and JAK-STAT pathways.

SHP2 also plays a signal enhancing role on this path, acting downstream of the RTK and upstream of the RAS. One common resistance mechanism involves the activation of RTKs that stimulate reactivation of MAPK signaling. RTK activation recruits SHP2 via direct binding and through an adapter protein. These interactions result in the transition of SHP2 from a closed (inactive) conformation to an open (active) conformation. SHP2 is an important contributor to RAS signaling reactivation, bypassing drug inhibition in primary and secondary resistance. Inhibition of SHP2 achieves the effect of comprehensively attenuating upstream RTK signaling, which generally drives oncogenic signaling and adaptive tumor escape (see prahalad, a. Et al Cell Reports 12,1978-1985 (2015); chen YN, nature 535,148-152 (2016)), all of the teachings of which, including but not limited to all methods, compounds, compositions, data, etc., are incorporated herein by reference in their entirety for use with any of the embodiments and disclosures herein.

Fibroblast Growth Factor Receptor (FGFR) binds to members of the fibroblast growth factor protein family and also affects the RAS-MAPK signaling pathway upstream of the RAS. The opportunity to target the signal transduction pathway from multiple angles and potentially improve the feedback loop upstream of Ras via SHP2 provides an opportunity to develop methods employing combination therapies. The present disclosure provides such methods while providing an unexpected degree of synergy.

The RAS-MAPK signal transduction pathway includes the Raf protein family. This family consists of three related kinases (A-, B-and C-Raf) that act as downstream effectors of Ras. B-Raf is in particular a serine/threonine protein kinase which activates the MAP kinase/ERK signaling pathway. It is well known that constitutively active B-Raf mutants cause cancer by excessively signalling to cells for their growth. For example, activating the B-Raf V600E kinase mutation occurs in about 7% of human malignancies and about 50-60% of melanomas.

The RAS-MAPK signaling pathway also includes MEK1 and MEK2.MEKl and MEK2 are bifunctional serine/threonine and tyrosine protein kinases, also known as MAP kinase kinases. MEK plays a key role in RAS-mediated RAF-MEK-ERK signaling pathways (pathways that transmit signals from growth factor receptors to the nucleus) to regulate, among other things, cell proliferation, differentiation, survival and invasion.

Finally, extracellular MET (or c-MET) acts as a key protein tyrosine kinase upstream of the RAS-MAPK signaling pathway. The opportunity to target the signal transduction pathway from multiple angles and potentially improve the feedback loop upstream of Ras via SHP2 provides an opportunity to develop methods employing combination therapies. Embodiments disclosed herein provide such methods while providing an unexpected degree of synergy.

In a first aspect, the present disclosure provides a method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and FGFR inhibitors. In some embodiments, FGFR in a subject is constitutively active. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the inhibitor is selected from the group consisting of erdasatinib (erdafitinib), AZD4547, ly2874455, CH5183284, NVP-BGJ398, INCB054828, luo Jiati ni (rogatainib), PRN1371, TAS-120, BLU-554, H3B-6527, and FGF401. In some embodiments, the FGFR inhibitor is erdasatinib. In some embodiments, the FGFR inhibitor is pemigatinib (pemigatinib), inflatinib (infigatinib), previtinib (dovitinib), panatinib (ponatinib), nipanib (nintenidanib), and fexotinib (fesatinib). In some embodiments, the method comprises administering a third MAPK pathway inhibitor. In some embodiments, the administration is oral. In some embodiments, the amount of the compound of formula I administered is in the range of 20mg to 400mg per day. In some embodiments, the FGFR inhibitor is administered in an amount ranging from 1mg to 500mg per day.

In a second aspect, the present disclosure provides a method of treating liver cancer in a subject, the method comprising orally co-administering to the subject a therapeutically effective amount of a compound of formula I:

and erdasatinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, erdasatinib is administered once or twice daily. In some embodiments, the subject is a human.

In a third aspect, the present disclosure provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and an FGFR inhibitor. In some embodiments, the compound of formula I and the FGFR inhibitor are in separate packages. In some embodiments, the kit further comprises instructions for administering the contents of the kit to a subject for cancer treatment. In some embodiments, the FGFR inhibitor is one or more of erdasatinib, AZD4547, ly2874455, CH5183284, NVP-BGJ398, INCB054828, luo Jiati ni, PRN1371, TAS-120, BLU-554, H3B-6527, FGF401, pemitinib, inflicterib, dolichos Wei Tini, panatinib, nilaninib, and fexotinib.

In a fourth aspect, the present disclosure provides a method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and inhibitors of the B-Raf protein with class 1 mutations. In some embodiments, the class 1 mutation is V600E. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the inhibitor is selected from Kang Naifei ni (encorafenib), vitamin Mo Feini (vemurafenib), dabrafenib (dabrafenib), sorafenib (sorafenib), and regrafenib (regorafenib). In some embodiments, the inhibitor is Kang Naifei ni. In some embodiments, the inhibitor is vitamin Mo Feini. In some embodiments, the inhibitor is dabrafenib. In some embodiments, the inhibitor is sorafenib. In some embodiments, the inhibitor is regorafenib. In some embodiments, the method comprises administering a third MAPK pathway inhibitor. In some embodiments, the administration is oral. In some embodiments, the amount of the compound of formula I administered is in the range of 20mg to 400mg per day. In some embodiments, the B-Raf inhibitor is administered in an amount ranging from 1mg to 500 mg.

In another aspect, the present disclosure provides a method of treating colorectal cancer in a subject, the method comprising orally co-administering to the subject a therapeutically effective amount of a compound of formula I:

and B-Raf inhibitor Kang Naifei. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, kang Naifei ni is administered once or twice daily. In some embodiments, the subject is a human.

In another aspect, the present disclosure provides a method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and Kang Naifei Ni.

and dimension Mo Feini.

And dabrafenib.

and sorafenib.

and regorafenib.

In various embodiments of the methods described herein, the cancer is colorectal cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the amount of B-Raf inhibitor administered is less than the amount required for monotherapy with the B-Raf inhibitor. In some embodiments, the amount of the compound of formula I administered is less than the amount required for monotherapy with the compound of formula I.

In another aspect, the present disclosure provides a method of inhibiting ERK1/2 phosphorylation in a cell population, the method comprising contacting the cell population with a compound of formula I:

And regorafenib in combination. In some embodiments, the concentration of the compound of formula I is in the range of 1nM to 500 nM. In some embodiments, the concentration of Kang Naifei ni is in the range of 10nM to 20 nM.

In another aspect, the present disclosure provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a B-Raf inhibitor. In some embodiments, the compound of formula I and the B-Raf inhibitor are in separate packages. In some embodiments, the kit further comprises instructions for administering the contents of the kit to a subject for cancer treatment. In some embodiments, the B-Raf inhibitor is one or more of Kang Naifei ni, vitamin Mo Feini, dabrafenib, sorafenib, and regorafenib.

and MEK inhibitors. In some embodiments, the MEK inhibitor selectively inhibits MEK1 or selectively inhibits MEK2 or selectively inhibits both MEK1 and MEK 2. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib (trametinib), cobimetinib (cobimetinib), bemetinib (binimetinib), PD-0325901, semetinib (selumetinib), and CI-1040. In some embodiments, the MEK inhibitor is trimetinib. In some embodiments, the MEK inhibitor is cobicitinib. In some embodiments, the MEK inhibitor is bemetinib. In some embodiments, the MEK inhibitor is PD-325901. In some embodiments, the MEK inhibitor is CI-1040. In some embodiments, the method comprises administering another MAPK pathway inhibitor. In some embodiments, the administration is oral. In some embodiments, the amount of the compound of formula I administered is in the range of 20mg to 400mg per day. In some embodiments, the amount of MEK inhibitor administered is in the range of 1mg to 500mg per day.

In another aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising orally co-administering to the subject a therapeutically effective amount of a compound of formula I:

and the MEK inhibitor bemetinib or trametinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, bemetinib or Qu Meiti ni is administered once or twice daily. In some embodiments, the subject is a human.

and bemetinib.

and trametinib.

In various embodiments of the methods described herein, the cancer is colorectal cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the amount of MEK inhibitor administered is less than the amount required for monotherapy with the MEK inhibitor. In some embodiments, the amount of the compound of formula I administered is less than the amount required for monotherapy with the compound of formula I.

In another aspect, the present disclosure provides a method of inhibiting ERK1/2 phosphorylation, the method comprising contacting a population of cells with formula I:

and bemetinib or trimetinib in combination. In some embodiments, the concentration of the compound of formula I is in the range of 1nM to 1,000 nM. In some embodiments, the concentration of the MEK inhibitor is in the range of 10nM to 500 nM.

In another aspect, the present disclosure provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a MEK inhibitor. In some embodiments, the compound of formula I and the MEK inhibitor are in separate packages. In some embodiments, the kit further comprises instructions for administering the contents of the kit to a subject for cancer treatment. In some embodiments, the MEK inhibitor is one or more of trimetinib or bemetinib.

and MET inhibitors. In some embodiments, the MET inhibitor is also an ALK inhibitor, a ROS1 inhibitor, or both. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is gastric adenocarcinoma. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the MET inhibitor is selected from crizotinib (crizotinib), tepontinib (tepontinib), cerclatinib (savoliinib), cabozantinib (cabozantinib), and tivantinib (tivantinib). In some embodiments, the MET inhibitor is crizotinib. In some embodiments, the MET inhibitor is tepontinib. In some embodiments, the inhibitor is cerutinib. In some embodiments, the inhibitor is cabozantinib. In some embodiments, the inhibitor is tivantinib. In some embodiments, the method comprises administering a third MAPK pathway inhibitor. In some embodiments, the administration is oral. In some embodiments, the amount of the compound of formula I administered is in the range of 10mg to 500mg per day. In some embodiments, the amount of inhibitor administered is in the range of 20mg to 400mg per day.

In another aspect, the present disclosure provides a method of treating gastric cancer in a subject, the method comprising orally co-administering to the subject a therapeutically effective amount of a compound of formula I:

and crizotinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, crizotinib is administered once or twice daily. In some embodiments, the subject is a human.

In a final aspect, the present disclosure provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a MET inhibitor. In some embodiments, the compound of formula I and the MET inhibitor are in separate packages. In some embodiments, the kit further comprises instructions for administering the contents of the kit to a subject for cancer treatment. In some embodiments, the MET inhibitor is one or more of crizotinib, tezotinib, sivortinib, caboztinib, and tivantinib.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is formulated as a pharmaceutical composition.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is formulated as an oral composition.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered once or twice a day.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 28 consecutive days.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in an amount of about 10mg to about 140mg once a day.

In some embodiments, the compound or pharmaceutically acceptable salt thereof is administered once a day for a period of 3 weeks, including 2 weeks of administration of the compound followed by 1 week of no administration of the compound.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered once a day for a period of 4 weeks, including 3 weeks of administration of the compound followed by 1 week of no administration of the compound.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 6 weeks.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 8 weeks.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered 3 times a week.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered on day 1, day 3, and day 5 of the week.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered 4 times a week.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered for a period of 3 weeks, including 2 weeks of administration of the compound followed by 1 week of no administration of the compound.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered for a period of 4 weeks, including 3 weeks of administration of the compound followed by 1 week of no administration of the compound.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered twice a day, two days a week.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered on day 1 and day 2 of the week.

In some embodiments, the cancer is selected from lung cancer, stomach cancer, liver cancer, colon cancer, kidney cancer, breast cancer, pancreatic Ductal Adenocarcinoma (PDAC), juvenile myelomonocytic leukemia, neuroblastoma (neuroblastoma), melanoma, and acute myelogenous leukemia.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Fig. 1A shows data indicating that the combination of the compound of formula I and the FGFR inhibitor erdasatinib exhibits in vitro synergy. This data indicates that there is a significant degree of synergy in the combination of the compound of formula I and erdasatinib.

Fig. 1B shows synergy data in Hep3B cancer cell lines using a combination of a compound of formula I and erdasatinib. This data indicates that there is a significant degree of synergy in the combination of the compound of formula I and erdasatinib.

Fig. 2 shows a graph of tumor volumes treated with a compound of formula I alone, erdasatinib alone, and a combination of a compound of formula I and erdasatinib for a period of time in a liver cancer CDX model KATO III.

Fig. 3 shows a graph of tumor volumes treated with a compound of formula I alone, erdasatinib alone, and a combination of a compound of formula I and erdasatinib for a period of time in FGFR2 amplified gastric cancer CDX SNU-16.

FIG. 4 shows a graph of tumor volumes treated with a compound of formula I alone, erdasatinib alone, and a combination of a compound of formula I and erdasatinib for a period of time in FGF19-FGFR4 dependent liver cancer CDX model Huh-7.

Fig. 5 shows data indicating that the combination of the compound of formula I and Kang Naifei ni exhibits synergy across multiple BRAF V600E mutant cells.

FIG. 6 shows the use of a combination of a compound of formula I and the BRAF inhibitor Kang Naifei Ni in RKO BRAF ^V600E Synergy data in CRC cell lines. This data indicates that there is a significant degree of synergy in the combination of the compound of formula I and Kang Naifei.

FIG. 7 shows the use of a combination of a compound of formula I and the BRAF inhibitor Kang Naifei Ni in WiDr BRAF ^V600E Synergy data in CRC cell lines. The data are shown in the formulaThere was a significant degree of synergy in the combination of compound I and Kang Naifei.

Fig. 8 shows the use of a combination of a compound of formula I and the BRAF inhibitor Kang Naifei ni in HT29 BRAF ^V600E Synergy data in CRC cell lines. This data indicates that there is a significant degree of synergy in the combination of the compound of formula I and Kang Naifei.

FIG. 9A shows a gel demonstrating synergistic inhibition of ERK1/2 phosphorylation in RKO colorectal cancer cell lines. FIG. 9A shows a robust decrease in pERK1/2 using a combination of a compound of formula I and Kang Naifei Ni.

Fig. 9B shows a gel demonstrating robust inhibition of ERK1/2 phosphorylation in the WiDr colorectal cancer cell line. FIG. 9B shows a robust decrease in pERK1/2 using a combination of a compound of formula I and Kang Naifei Ni.

Figure 9C shows a graph of the antiproliferative effect of a compound of formula I alone or in combination with Kang Naifei ni in RKO colorectal cancer cell lines. Figure 9C shows that the combination of the compound of formula I and Kang Naifei ni increases the inhibitory activity of the compound of formula I.

Fig. 9D shows a graph of the antiproliferative effect of a compound of formula I or a compound of formula I in combination with Kang Naifei ni in a WiDr colorectal cancer cell line. Figure 9D shows that the combination of the compound of formula I and Kang Naifei ni increases the inhibitory activity of the compound of formula I.

FIG. 10A shows a gel comparing synergistic inhibition of ERK1/2 phosphorylation in RKO colorectal cancer cell lines with the following combinations: compound of formula I + Kang Naifei; TNO155+ Kang Naifei; and RMC-4550+ Kang Naifei, indicating that the combination of the compound of formula I and Kang Naifei of the SHP2 inhibitor is most effective in inhibiting ERK1/2 phosphorylation.

Fig. 10B shows at 1. Control; 2. (a compound of formula I); 3. kang Naifei Ni; bar graph of pERK as a percentage of control in the case of (compound of formula I) + Kang Naifei ni, showing that the combination of the SHP2 inhibitor compound of formula I and Kang Naifei ni is most effective in inhibiting ERK1/2 phosphorylation.

Fig. 10C shows at 1. Control; tno155;3. kang Naifei Ni; bar graph of pERK as a percentage of control with tno155+ Kang Naifei, showing that the combination of SHP2 inhibitor compound of formula I and Kang Naifei is most effective in inhibiting ERK1/2 phosphorylation.

Fig. 10D shows at 1. Control; RMC-4550;3. kang Naifei Ni; bar graph of pERK as a percentage of control with rmc-4550+ Kang Naifei ni, showing that the combination of SHP2 inhibitor compound of formula I and Kang Naifei ni is most effective in inhibiting ERK1/2 phosphorylation.

Fig. 11 shows the method in BRAF ^V600E Plots of tumor volumes treated with the compound of formula I alone, kang Naifei ni alone, and a combination of the compound of formula I and Kang Naifei ni for a period of time in mutant CRC PDX model CR 0029.

Fig. 12 shows the method in BRAF ^V600E Plots of tumor volumes treated with the compound of formula I alone, kang Naifei ni alone, and a combination of the compound of formula I and Kang Naifei ni for a period of time in mutant CRC PDX model CR 004.

Fig. 13 shows the method in BRAF ^V600E Plots of tumor volumes in mutant CRC CDX model WiDr treated with compound of formula I alone, kang Naifei ni alone, and a combination of compound of formula I and Kang Naifei ni for a period of time.

Fig. 14 shows the method in BRAF ^V600E Plots of tumor volumes in mutant CRC CDX model HT-29 treated with compound of formula I alone, kang Naifei Ni alone, and a combination of compound of formula I and Kang Naifei Ni for a period of time.

Fig. 15 shows the method in BRAF ^V600E Graphs of tumor volumes in mutant thyroid cancer CDX model BHT-101 treated with compound of formula I alone, kang Naifei ni alone, and a combination of compound of formula I and Kang Naifei ni for a period of time.

Fig. 16 shows the method in BRAF ^V600E Graphs of tumor volumes in mutant CRC CDX model RKO treated with compound of formula I alone, kang Naifei Ni alone, and a combination of compound of formula I and Kang Naifei Ni for a period of time.

Figure 17A shows synergy data in NCI-H508 cancer cell lines using a combination of a compound of formula I and trimetinib.

Fig. 17B shows synergy data in NCI-H508 cancer cell lines using a combination of a compound of formula I and bemetinib.

Figure 17C is graphical synergy data in NCI-H1666 cancer cell lines using a combination of a compound of formula I and trimetinib.

Fig. 17D shows synergy data in NCI-H1666 cancer cell lines using a combination of a compound of formula I and bemetinib.

Fig. 18A shows synergy data in MeWo cancer cell lines using a combination of a compound of formula I and trimetinib.

Fig. 18B shows synergy data in MeWo cancer cell lines using a combination of a compound of formula I and bemetinib.

Fig. 18C shows synergy data in NCI-H1838 cancer cell lines using a combination of a compound of formula I and trimetinib.

Fig. 18D shows synergy data in NCI-H1838 cancer cell lines using a combination of a compound of formula I and bemetinib.

Figure 19A shows a graph of percent activity versus inhibitor concentration (log M) in NCI-H508 cells treated with compounds of formula I alone and in combination with bemetinib. List of NCI-H508 cells treated with compounds of formula I alone and in combination with bemetinib ₅₀ Data.

Figure 19B shows a graph of percent activity versus inhibitor concentration (log M) in MeWo cells treated with compounds of formula I alone and in combination with bemetinib. Table IC50 data for MeWo cells treated with compounds of formula I alone and in combination with bemetinib.

FIG. 20A shows Western blot gel demonstrating synergistic inhibition of ERK1/2 phosphorylation in NCI-H508 cancer cell lines.

Fig. 20B shows bar graph quantification of western blot of fig. 20A.

FIG. 20C shows Western blot gels demonstrating synergistic inhibition of ERK1/2 phosphorylation in MeWo (NF 1 LoF) cancer cell lines.

Fig. 20D shows bar graph quantification of western blot of fig. 20C.

Fig. 21A shows synergy data in NCI-H2009 (KRAS G12A) cancer cell lines using a combination of a compound of formula I and trimetinib.

Fig. 21B shows synergy data in LS513 (KRAS G12D) cancer cell line using a combination of a compound of formula I and trimetinib.

Fig. 21C shows synergy data in an a549 (KRAS G12S) cancer cell line using a combination of a compound of formula I and trimetinib.

Fig. 21D shows synergy data in NCI-H727 (KRAS G12V) cancer cell lines using a combination of a compound of formula I and trimetinib.

Fig. 22A shows synergy data in NCI-H2009 (KRAS G12A) cancer cell lines using a combination of a compound of formula I and bemetinib.

Fig. 22B shows synergy data in LS513 (KRAS G12D) cancer cell lines using a combination of a compound of formula I and bemetinib.

Fig. 22C shows synergy data in an a549 (KRAS G12S) cancer cell line using a combination of a compound of formula I and bemetinib.

Fig. 22D shows synergy data in NCI-H727 (KRAS G12V) cancer cell lines using a combination of a compound of formula I and bemetinib.

Fig. 23A shows a graph of percent activity versus inhibitor concentration (log M) in LS513 (KRAS G12D) cells treated with a compound of formula I alone and in combination with trimetinib.

Fig. 23B shows a graph of percent activity versus inhibitor concentration (log M) in NCI-H2009 (KRAS G12D) cells treated with a compound of formula I alone and in combination with trimetinib. List data for NCI-H508 cells treated with compounds of formula I alone and in combination with trimetinib.

Fig. 23C shows a bar graph of CTG activity percentage indicating that either formula I or trimetinib alone had minimal effect on cell viability. Taken together, the data demonstrate that the combination of the compound of formula I and the MEK inhibitor provides synergistic inhibition of cancer cell viability in BRAF class III, NF1 LoF and KRAS G12X mutant cancers.

Fig. 24 shows a graph of tumor volumes treated with a compound of formula I alone, trimetinib alone, and a combination of a compound of formula I and trimetinib for a period of time in NF1 LoF mutant melanoma CDX model MeWo.

Fig. 25 shows a graph of tumor volumes treated with a compound of formula I alone, bemetinib alone, and a combination of a compound of formula I and bemetinib for a period of time in NF1 LoF mutant melanoma CDX model MeWo.

Fig. 26 shows a graph of tumor volumes treated with a compound of formula I alone, trimetinib alone, and a combination of a compound of formula I and trimetinib for a period of time in the BRAF class III mutant CRC CDX model NCI-H508.

Figure 27 shows a graph of tumor volumes treated with a compound of formula I alone, trimetinib alone, and a combination of a compound of formula I and trimetinib for a period of time in NF1 LoF mutant NSCLC CDX model NCI-H1838.

Fig. 28A shows synergy data in Hs746T cancer cell lines using a combination of a compound of formula I and crizotinib.

Fig. 28B shows synergy data in MKN-45 cancer cell lines using a combination of a compound of formula I and crizotinib.

Fig. 28C shows synergy data in EBC-1 cancer cell lines using a combination of a compound of formula I and crizotinib.

FIG. 29 shows a graph of tumor volumes treated with a compound of formula I alone, crizotinib alone, and a combination of a compound of formula I and crizotinib for a period of time in a c-MET amplified gastric cancer CDX model SNU-5.

FIG. 30 shows a graph of tumor volumes treated with a compound of formula I alone, crizotinib alone, and a combination of a compound of formula I and crizotinib for a period of time in the c-MET amplified NSCLC CDX model NCI-H1993.

Detailed Description

I. Overview of the invention

The present disclosure provides a method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and FGFR inhibitors. The following examples demonstrate the unexpected synergy of such combinations. Combination therapies using compounds of formula I or pharmaceutically acceptable salts thereof as disclosed herein may exhibit better results than alternative SHP2 inhibitors used in combination with FGFR inhibitors. Furthermore, the combination of SHP2 inhibitor and FGFR inhibitor of formula I provides a lower dose method that allows the use of either agent used alone in monotherapy, which may help reduce potential side effects. In particular, combination therapies may be effective in cancer cells that express mutations, including but not limited to FGFR4 mutations and amplified expression of FGFR.

and FGFR inhibitors. In some embodiments, FGFR in a subject is constitutively active. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the inhibitor is selected from the group consisting of erdastinib, AZD4547, ly2874455, CH5183284, NVP-BGJ398, INCB054828, luo Jiati, PRN1371, TAS-120, BLU-554, H3B-6527, and FGF401. In some embodiments, the FGFR inhibitor is erdasatinib. In some embodiments, the FGFR inhibitor is pemitinib, infliximab, duo Wei Tini, panatinib, niladinib, and fexotinib. In some embodiments, the method comprises administering a third MAPK pathway inhibitor. In some embodiments, the administration is oral. In some embodiments, the amount of the compound of formula I administered is in the range of 20mg to 400mg per day. In some embodiments, the FGFR inhibitor is administered in an amount ranging from 1mg to 500mg per day.

and inhibitors of the B-Raf protein with class 1 mutations. In some embodiments, the class 1 mutation is V600E. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the inhibitor is selected from Kang Naifei, vitamin Mo Feini, dabrafenib, sorafenib, and regorafenib. In some embodiments, the inhibitor is Kang Naifei ni. In some embodiments, the inhibitor is vitamin Mo Feini. In some embodiments, the inhibitor is dabrafenib. In some embodiments, the inhibitor is sorafenib. In some embodiments, the inhibitor is regorafenib. In some embodiments, the method comprises administering a third MAPK pathway inhibitor. In some embodiments, the administration is oral. In some embodiments, the amount of the compound of formula I administered is in the range of 20mg to 400mg per day. In some embodiments, the B-Raf inhibitor is administered in an amount ranging from 1mg to 500 mg.

and inhibitors of class 1 mutant B-Raf. The following examples demonstrate the unexpected significant synergy of such combinations. Combination therapies disclosed herein employing a compound of formula I, or a pharmaceutically acceptable salt thereof, may exhibit better results than the combination of alternative SHP2 inhibitors used in combination with inhibitors of class 1 mutant B-Raf. Furthermore, the combination of the SHP2 inhibitor of formula I and the class 1 mutated B-Raf inhibitor provides a lower dose method that allows for the use of either agent used alone in monotherapy, which may help reduce potential side effects. In particular, combination therapies may be effective in cancer cells expressing BRAF V600E mutations.

and Kang Naifei Ni.

and dimension Mo Feini.

And dabrafenib.

and sorafenib.

and regorafenib.

Embodiments of the present invention provide methods of treating a subject having cancer comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

/>

and MEK inhibitors. The following examples demonstrate the unexpected synergy of such combinations. Combination therapies using compounds of formula I or pharmaceutically acceptable salts thereof as disclosed herein may exhibit better results than alternative SHP2 inhibitors used in combination with MEK inhibitors. Furthermore, the combination of SHP2 inhibitor and MEK inhibitor of formula I provides a lower dose method that allows for the use of either agent used alone in monotherapy, which may help reduce potential side effects. In particular, combination therapies may be effective in cancer cells expressing mutations including, but not limited to, group III B-raf mutations and KRAS G12X mutations.

and MEK inhibitors. In some embodiments, the MEK inhibitor selectively inhibits MEK1 or selectively inhibits MEK2 or selectively inhibits both MEK1 and MEK 2. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobicitinib, bemetinib, PD-0325901, semetinib, and CI-1040. In some embodiments, the MEK inhibitor is trimetinib. In some embodiments, the MEK inhibitor is cobicitinib. In some embodiments, the MEK inhibitor is bemetinib. In some embodiments, the MEK inhibitor is PD-325901. In some embodiments, the MEK inhibitor is CI-1040. In some embodiments, the method comprises administering another MAPK pathway inhibitor. In some embodiments, the administration is oral. In some embodiments, the amount of the compound of formula I administered is in the range of 20mg to 400mg per day. In some embodiments, the amount of MEK inhibitor administered is in the range of 1mg to 500mg per day.

and bemetinib.

and trametinib.

In various embodiments of the methods described herein, the cancer is colorectal cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the amount of MEK inhibitor administered is less than the amount required for monotherapy with the MEK inhibitor. In some embodiments, the amount of the compound of formula I administered is less than the amount required for monotherapy with the compound of formula I.

and MET inhibitors. The following examples demonstrate the unexpected synergy of such combinations. Combination therapies using compounds of formula I or pharmaceutically acceptable salts thereof as disclosed herein may exhibit better results than alternative SHP2 inhibitors used in combination with MET inhibitors. Furthermore, the combination of SHP2 inhibitor and MET inhibitor of formula I provides a lower dose method that allows for the use of either agent used alone in monotherapy, which may help reduce potential side effects. In particular, combination therapies may be effective in cancer cells that express MET aberrant mutations.

and MET inhibitors. In some embodiments, the MET inhibitor is also an ALK inhibitor, a ROS1 inhibitor, or both. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is gastric adenocarcinoma. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the MET inhibitor is selected from the group consisting of crizotinib, tezotinib, sivortinib, caboztinib, and tivantinib. In some embodiments, the MET inhibitor is crizotinib. In some embodiments, the MET inhibitor is tepontinib. In some embodiments, the inhibitor is cerutinib. In some embodiments, the inhibitor is cabozantinib. In some embodiments, the inhibitor is tivantinib. In some embodiments, the method comprises administering a third MAPK pathway inhibitor. In some embodiments, the administration is oral. In some embodiments, the amount of the compound of formula I administered is in the range of 10mg to 500mg per day. In some embodiments, the amount of inhibitor administered is in the range of 20mg to 400mg per day.

Thus, such treatments are suitable for aiding in the proper patient population selection using concomitant diagnostics. These and other advantages will be recognized by those skilled in the art.

II. Definition of

Unless specifically indicated 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 these embodiments are directed. In addition, any method or material similar or equivalent to those described herein can be used in the practice of the embodiments herein. For purposes of the embodiments disclosed herein, the following terms are defined.

As used herein, "a," "an," or "the" includes aspects having not only one member, but also more than one member. For example, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the agent" includes reference to one or more agents known to those skilled in the art, and so forth.

By "pharmaceutically acceptable excipient" is meant a substance that aids in the administration of an active agent to a subject and absorption by the subject. Pharmaceutical excipients that may be used in embodiments of the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, surfactants, coatings, sweeteners, flavoring agents, and coloring agents. Those skilled in the art will recognize that other pharmaceutical excipients are also useful in embodiments of the present invention.

"treating" refers to successfully treating or ameliorating any sign of injury, pathology or condition, including any objective or subjective parameter, such as alleviation; relief; alleviation of symptoms or making injury, pathology or condition more tolerable to the patient; the rate of degradation or decay is slowed; less debilitating the end point of degradation; improving physical or mental health of the patient. Treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of physical examination, neuropsychiatric examination, and/or mental assessment.

"administration" refers to oral administration, administration as a suppository, topical contact, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal administration or subcutaneous administration, intrathecal administration, or implantation of a sustained release device, such as a micro-osmotic pump, into a subject. In the context of the combination therapies disclosed herein, administration may be at different times or simultaneously or substantially simultaneously.

By "therapeutically effective amount" is meant the amount that produces a therapeutic effect upon administration. The exact dosage will depend on The purpose of The treatment and will be determined by one skilled in The Art using known techniques (see, e.g., lieberman, pharmaceutical Dosage Forms (volumes 1-3, 1992); lloyd, the Art, science and Technology of Pharmaceutical Compounding (1999); pickar, dosage Calculations (1999); and Remington: the Science and Practice of Pharmacy, 20 th edition, 2003, gennaro editions, lippincott, williams & Wilkins). In sensitized cells, the therapeutically effective dose may generally be lower than conventional therapeutically effective doses for non-sensitized cells.

"inhibit" and "inhibitor" refer to a compound or method of partially or completely blocking or inhibiting a particular action or function.

"cancer" refers to all types of cancers, neoplasms, or malignant tumors found in mammals (e.g., humans), including, but not limited to, leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that can be treated with the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, hodgkin's disease, and non-hodgkin's lymphoma. Exemplary cancers that may be treated with the compounds or methods provided herein include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, stomach cancer, and uterine cancer. Further examples include thyroid cancer, cholangiocarcinoma, pancreatic adenocarcinoma, skin melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, pre-cancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine or exocrine pancreatic tumor, medullary thyroid cancer, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, ductal adenocarcinoma of the Pancreas (PDAC), or prostate cancer.

By "FGFR inhibitor" is meant an inhibitor of any wild-type FGFR or FGFR mutant. FGFR mutations include, but are not limited to, single nucleotide polymorphisms, exon insertions and deletions, multimers, and the like. Specific examples of mutations and inhibitors include, but are not limited to, FGFR1 gene copy gain, FGFR1 gene amplification, FGFR2 gene copy gain, FGFR2 gene amplification, FGFR3 gene copy gain, FGFR3 gene amplification, FGFR4 gene copy gain, FGFR4 gene amplification, FGFR 1T 141R 445 1N 546 1K 656G 818 2S 252 2P 253 a315 2D 336 2Y 375C 382 2V 395 2D 471I 547 2N 549 2K 659S 131 3R 248 3S 249 3G 370 3S371 3Y 373 3G 380 3G 3K 627 3K 650 3V 677D 785R 183 4R 394D 4V 510D 4V 610H and FGFR fusion (e.g., FGFR3-TACC3, FGFR2-NPM1, FGFR2-TACC2, FGFR2-BICC1, FGFR2-C10orf68, FGFR 3-kmip 1, FGFR 2-pdc 2-1598, FGFR 2-pdc 2, and ppa3-pdc 3, and FGFR 3-tumor 3, and FGFR 3-containing 3. In some embodiments, one or more of the mutant forms listed above may be specifically excluded from the embodiments set forth herein, including, but not limited to, any method, kit, composition of matter, and the like. Inhibitors include, but are not limited to, erdasatinib, pemitinib, infliximab, duo Wei Tini, panatinib, nilanib, and fexotinib. In some embodiments, one or more of the mutant forms listed above may be specifically excluded from the embodiments set forth herein, including, but not limited to, any method, kit, composition of matter, and the like.

"class 1 mutant B-Raf" or "B-Raf protein having class 1 mutation" generally refers to any mutation that deviates from the wild-type B-Raf protein at V600 (valine 600). In particular, such mutant B-Raf proteins include mutations including V600E mutations. Other class 1 BRAF mutations include, but are not limited to, V600K, V600D, V600L, V M and V600R. In some embodiments, one or more of the mutations listed above may be specifically excluded from the embodiments set forth herein, including, but not limited to, any method, kit, and composition of matter.

"MEK inhibitor" generally refers to any inhibitor that selectively inhibits MEK1 or MEK2 or inhibits both MEK1 and MEK 2. Examples of inhibitors include, but are not limited to, trametinib, cobicitinib, bemetinib, PD-0325901, semetinib, and CI-1040.

"MET inhibitor" refers to an inhibitor of any wild-type MET or MET mutant. MET mutations include, but are not limited to, single nucleotide polymorphisms, exon insertions and deletions, multimers, and the like. Specific examples of mutations and inhibitors include, but are not limited to, MET gene copy gain, MET gene amplification, MET E34K, MET H150Y, MET E168D, MET L269V, MET L299F, MET S323G, MET M362T, MET N375S, MET C385Y, MET R970C, MET R988C, MET P1009S, MET T1010I, MET S1058P, MET exon 14 skip mutation, MET exon 14 splice variant, MET a1108S, MET V1110I, MET H1112R, MET H1112L, MET H1112I, MET HJ1124D, MET G1137V, MET M1149T, MET T1I, MET V1206L, MET L1213V, MET D1228V, MET Y1230C, MET Y1230H, MET Y1230D, MET Y1235D, MET V1238D, MET V1248D, MET Y1248D, MET M1268M, MET M1248. In some embodiments, one or more of the mutant forms listed in this paragraph and elsewhere herein may be specifically excluded from the embodiments set forth herein, including, but not limited to, any methods, kits, and compositions of matter, and the like. Examples of inhibitors include, but are not limited to, crizotinib, carbamatinib (capmatinib), terpontinib, sivantinib, tivantinib, cabatinib, foretinib (foretinib), el Mo Tuo mab (amivantmamab), onatuzumab (onartuzumab), ematuzumab (emibetuzumab), and non-lattuzumab (ficlatuzumab). In some embodiments, one or more of the inhibitors listed in this paragraph and elsewhere herein may be specifically excluded from the embodiments set forth herein, including, but not limited to, any methods, kits, and compositions of matter, and the like.

A "subject" refers to a living organism that has or is susceptible to a disease or condition that can be treated by administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cows, deer, horses, and other non-mammals. In some embodiments, the patient is a human.

III methods of administration

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is formulated as a pharmaceutical composition. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is formulated as an oral composition.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered once or twice a day. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered once a day. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered twice a day. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 28 consecutive days.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered once a day for a period of 3 weeks, including 2 weeks of administration of the compound followed by 1 week of no administration of the compound.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 6 weeks. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 8 weeks.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered 3 times a week. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered on day 1, day 3, and day 5 of the week.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered twice a day, two days a week. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 8 weeks. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered on day 1 and day 2 of the week.

In some embodiments, the cancer is selected from lung cancer, stomach cancer, liver cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, and acute myelogenous leukemia. In some embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

Combination method

In some embodiments, the method comprises administering a third MAPK pathway inhibitor. Without being bound by theory, inhibition of MAPK signaling in cancer cells may lead to down-regulation of PD-L1 expression and increase the likelihood that cancer cells will be detected by the immune system. Such third MAPK pathway inhibitors may be based on other mutations of the protein in the MAPK pathway. In some embodiments, any MAPK pathway inhibitor may be employed, including those that target K-Ras, N-Ras, H-Ras, PDGFRA, PDGFRB, MET, FGFR, ALK, ROS1, TRKA, TRKB, TRKC, EGFR, IGF1R, GRB2, SOS, ARAF, BRAF, RAF1, MEK2, c-Myc, CDK4, CDK6, CDK2, ERK1, and ERK 2. Non-limiting examples of MEK inhibitors include trametinib, cobicitinib, bemetinib, PD-0325901, semetinib, and CI-1040. Exemplary MAPK pathway inhibitors include, but are not limited to, afatinib (afatinib), octreotide (osiertinib), erlotinib (erlotinib), gefitinib (gefitinib), lapatinib (lapatinib), lenatinib (neratinib), dacatinib (dacominib), vandetanib (vanretanib), cetuximab (cetuximab), panitumumab (panitumumab), nimotuzumab (nimotuzumab), cetuximab (necitumumab), trimetinib, bemetinib, cobratinib, sematinib, ulixenib (ulixotinib), LTT462, and LY3214996. In some embodiments, one or more of the inhibitors listed in this paragraph and elsewhere herein may be specifically excluded from one or more of the embodiments set forth herein, including, but not limited to, any method, kit, composition of matter, and the like.

The methods disclosed herein may be combined with other chemotherapeutic agents. Examples of such agents can be found in v.t. devita and s.hellman (editions) Cancer Principles and Practice of Oncology, 6 th edition (15/2/2001), lippincott Williams & Wilkins Publishers; all teachings of the documents, including but not limited to all methods, compounds, compositions, data, and the like, are incorporated herein by reference in their entirety for use with any of the embodiments and disclosures herein. One of ordinary skill in the art will be able to discern which combinations of agents will be useful based on the drug and the specific characteristics of the disease involved.

In some embodiments, the method may comprise co-administering at least one cytotoxic agent. As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., radioisotopes of At211, I131, I125, Y90, re186, re188, sm153, bi212, P32, pb212, and Lu); a chemotherapeutic agent; a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.

Examples of cytotoxic agents may be selected from the group consisting of anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogues, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, LDH-a inhibitors; inhibitors of fatty acid biosynthesis; inhibitors of cell cycle signaling; HDAC inhibitors, proteasome inhibitors; and inhibitors of cancer metabolism.

Chemotherapeutic agents include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib @Genentech/osipanm.), bortezomib (++>Millennium pharm), disulfiram, epigallocatechin gallate, salinomycin A (salinosporamide A), carfilzomib (carfilzomib), 17-AAG (geldanamycin), radicicol (radicicol), lactate dehydrogenase a (LDH-a), fulvestrant (pex)>Astrazeneca), sunitinib (sunitinib)>Pfizer/Sugen), letrozole (+.>Novartis), imatinib mesylate (je)>Novartis), phenazona (finasiate) (-j->Novartis), oxaliplatin (++>Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus,) >Wyeth), lapatinib (+.>GSK572016, glaxo Smith Kline), ronafani (SCH 66336), sorafenib (+.>Bayer Labs), gefitinib (>AstraZeneca), AG1478, alkylating agents such as thiotepa and +.>Cyclophosphamide; alkyl sulfonates such as busulfan, ciprofloxacin, and piposulfan; aziridines such as benzotepa, carbaquinone, mitotepa and uratepa; ethyleneimine and methyl melamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylol melamine; acetogenin (especially bullatacin and bullatacin ketone); camptothecins (including topotecan and irinotecan); bryostatin; calistatin; CC 1065 (including adorinol, calzerinol and bizerinol synthetic analogues thereof); nostoc (in particular, nostoc 1 and nostoc 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5-alpha-reductase including finasteride and dutasteride); vorinostat, romidepdipine, panobinostat, valproic acid, mo Xisi he, dolastatin; aldesleukin, talc, sesquialter (including synthetic analogs, KW-2189 and CB1-TM 1); acanthopanaxgenin; a podophylline; stoloniferol; spongosine; nitrogen mustards such as chlorambucil, napthalene mustards, cyclophosphamide, estramustine, ifosfamide, dichloromethyldiethylamine, nitrogen mustards oxide hydrochloride, melphalan, new enbicine, chlorambucil cholesterol, prednisolone mustards, triamcinolone, uramustine; nitrosoureas such as carmustine, chlorourea, fotemustine, lomustine, nimustine and ramustine; antibiotics, e.g. enediyne antibiotics The antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega 1I (Angew chem. Intl. Ed. Engl.1994 33: 183-186), daptomycin, including daptomycin A, bisphosphonates, such as chlorophosphonate, epothilone, and neocarcinomycin chromophores and related chromene diyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, diazoserine, bleomycin, actinomycin C, karastill, carminomycin, acidophilic, chromomycin, actinomycin D, daunomycin, ditetracycline, 6-diazon-5-oxo-L-norleucine,>(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, eldroubicin, idarubicin, doxycycline, mitomycin such as mitomycin C, mycophenolic acid, norgamycin, olivomycin, perlomycin, methylmitomycin, puromycin, trifoliacin, rodubicin, streptozocin, streptozotocin, tubercidin, ubenimex, cilostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as, for example, dimethyl folic acid, methotrexate, pterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioadenine, thioguanine; pyrimidine analogs such as ambcitabine, azacytidine, 6-azauridine, carmofur, arabinosporine, dideoxyuridine, doxifluridine, enocitabine, fluorouridine; androgens such as carbosterone, drotasone propionate, cyclothioandrostanol, emaandran, testosterone; anti-epinephrine such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as leucovorin; acetoglucurolactone; aldehyde phosphoramide glycosides; aminoketoglutaric acid; enuracil; amsacrine; amoustine; a specific group; eda traxas; a phosphoramide; dimecoxin; deaquinone; enonisole; ammonium elegance; epothilones; an ethyleneoxy pyridine; gallium nitrate; hydroxyurea; lentinan; luo Nida Ning; maytansinoids such as maytansine and ansamitocins; mitoguandine Hydrazone; mitoxantrone; mo Pai dar alcohol; diamine nitroacridine; prastatin; egg ammonia nitrogen mustard; pirarubicin; losoxantrone; podophylloic acid; 2-ethyl hydrazide; methyl benzyl hydrazine; />Polysaccharide complex (JHS Natural Products, eugene, oreg.); carrying out a process of preparing the raw materials; rhizobia element; cilzofuran; germanium spiroamine; alternaria tenuissima acid; triiminoquinone; 2,2',2 "-trichlorotriethylamine; trichothecene toxins (especially T-2 toxin, wart a, cyclosporin a, and serpentine; a urethane; vindesine; dacarbazine; mannitol nitrogen mustard; dibromomannitol; dibromodulcitol; pipobromine; ganciclovir; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. TAXOL (paclitaxel; bristol-Myers Squibb Oncology, princeton, n.j.),>albumin engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, schaumberg, ill.) without cremophor>(docetaxel, docetaxel; sanofi-Aventis); chlorambucil; />(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; (vinorelbine); norxiaoling (novantrone); teniposide; eda traxas; daunomycin; aminopterin; capecitabine->Ibandronate; CPT-11; topoisomeraseEnzyme inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Chemotherapeutic agents also include (i) anti-hormonal agents such as antiestrogens and Selective Estrogen Receptor Modulators (SERMs) for modulating or inhibiting hormonal effects on tumors, including, for example, tamoxifen (includingTamoxifen citrate), raloxifene, droloxifene, idoxifene, 4-hydroxy tamoxifen, trazoxifene, keoxifene (keoxifene), LY117018, onapristone, and->(tomiphene citrate); (ii) Aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland, e.g. 4 (5) -imidazole, aminoglutethimide,/->(megestrol acetate),>(exemestane; pfizer), formestane, fadrozole,(Fucloxazole),>(letrozole; novartis) and +.>(anastrozole; astraZeneca); (iii) Antiandrogens such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin; buserelin, triptorelin (tripterelin), medroxyprogesterone acetate, diethylstilbestrol, pra Lei Malin, fluoxymesterone, all-trans retinoic acid, fenretinide and troxacitabine (1, 3-dioxolane nucleoside cells) Pyrimidine analogs); (iv) a protein kinase inhibitor; (v) a lipid kinase inhibitor; (vi) Antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways associated with abnormal cell proliferation, such as, for example, PKC- α, ralf, and H-Ras; (vii) Ribozymes, such as inhibitors of VEGF expression (e.g., +.>) And an inhibitor of HER2 expression; (viii) Vaccines, such as gene therapy vaccines, e.g. +.>And->rIL-2; topoisomerase 1 inhibitors such as +.>rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

The chemotherapeutic agent also includes antibodies such as alemtuzumab (Campath), bevacizumab @, andgenentech); cetuximab (+)>Imclone); panitumumab (+)>Amgen), rituximab (+.>Genentech/Biogen Idec), pertuzumab (/ i)>2C4, genentech), trastuzumab (++>Genentech), tositumomab (Bexxar, corixia) and the antibody drug conjugate gemtuzumab ozagrel (>Wyeth). Other humanized monoclonal antibodies having therapeutic potential as agents in combination with the compounds of the invention include: abellizumab (apolizumab), alemtuzumab (aselizumab), alemtuzumab (atlizumab), bapirizumab (bapineuzumab), bivalizumab me Sha Danjian (bivatuzumab mertansine), katuzumab me Sha Danjian (cantuzumab mertansine), cetuzumab (cedelizumab), cetuzumab (certolizumab pegol), cetuximab (cidfuzumab), cetuximab (cidtuzumab), dactyluzumab (daclizumab), eculizumab (eclipzumab), efalizumab (efalizumab), epratuzumab (epratuzumab), panuzumab (felvizumab), aryluzumab (fontuzumab), valuzumab (getuzumab) and oxuzumab (gemtuzumab ozogamicin), oxuzumab (inotuzumab ozogamicin) ipilimumab (ipilimumab), la Bei Zhushan (labtuzumab), rituximab (lintuzumab), matuzumab (matuzumab), meperizumab (mepolizumab), mevaluzumab (movuzumab), mo Tuozhu mab (motovizumab), natalizumab (natalizumab), nituzumab (nitaizumab), nimuzumab (nimotuzumab), nolovizumab (nolovizumab), nu Ma Weizhu mab (numuzumab), oreuzumab (ocrelizumab), omalizumab (omalizumab), palivizumab (palivizumab), pacuzumab (paskolizumab), pezizumab (pecfuzumab), tuzumab (petuzumab), petuzumab (petuzumab), kelizumab (pexolizumab), and (lizumab) other than lizumab (lizumab), ranibizumab (ranibizumab), retimizumab (reliuzumab), rayleigh zumab (relizumab), resyvinzumab, luo Weizhu mab (rovelizumab), lu Lizhu mab (ruplizumab), sibrotuzumab (sibrotuzumab), cetiriuzumab (siplizumab), solituzumab (solituzumab), tettantuzumab (tacatuzumab tetraxetan), tazizumab (taduzumab), taluzumab (talizumab), tefeizumab (tefibazumab), tolizumab (tocilizumab), tolizumab (toralizumab), cetuximab (tucotuzumab celmoleukin), toxiuzumab (tucusituzumab), wu Mawei-zuizumab (umavizumab), wu Zhushan-antibody (urtoxazumab), ulizumab (ustekinumab), visuzumab (vislizumab) and anti-interleukin-12 (ABT-874/J695 Wyeth Research and Abbott Laboratories) as recombinant pure human sequence full length IgG1 lambda antibody genetically modified to recognize interleukin-12 p40 protein.

Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise interact directly with EGFR or mutant forms thereof and prevent or reduce its signaling activity, and are alternatively referred to as "EGFR antagonists. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. patent No. 4,943,533, mendelsohn et al) and variants thereof, such as chimeric 225 (C225 or cetuximab;) And remodelling human 225 (H225) (see WO 96/40210,Imclone Systems Inc); IMC-11F8, fully human EGFR targeting antibody (Imclone); antibodies that bind type II mutant EGFR (U.S. patent No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in U.S. patent No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF or panitumumab (see WO98/50433, abgenix/Amgen); EMD 55900 (Straglitoto et al Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a humanized EGFR antibody against EGFR that competes with EGF and TGF-alpha for EGFR binding (EMD/Merck); human EGFR antibodies, huMax-EGFR (GenMab); fully human antibodies, termed E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and are described in US 6,235,883; MDX-447 (Medarex Inc.); and mAb 806 or humanized mAb 806 (Johns et al, J. Biol. Chem.279 (29): 30375-30384 (2004)). The anti-EGFR antibody can be conjugated with a cytotoxic agent to produce an immunoconjugate (see example Such as EP659,439A2, merck Patent GmbH). EGFR antagonists include small molecules such as the compounds described in U.S. patent nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008 and 5,747,498, and PCT publications below: WO98/14451, WO98/50038, WO99/09016 and WO99/24037. Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib,Genentech/OSIPharmaceuticals); PD 183805 (CI 1033,2-acrylamide, N- [4- [ (3-chloro-4-fluorophenyl) amino group]-7- [3- (4-morpholinyl) propoxy]-6-quinazolinyl]-dihydrochloride, pfizer inc.); ZD1839, gefitinib +.>4- (3 '-chloro-4' -fluoroanilino) -7-methoxy-6- (3-morpholinopropoxy) quinazoline, astraZeneca; ZM 105180 ((6-amino-4- (3-methylphenylamino) -quinazoline, zeneca); BIBX-1382 (N8- (3-chloro-4-fluoro-phenyl) -N2- (1-methyl-piperidin-4-yl) -pyrimido [5, 4-d) ]Pyrimidine-2, 8-diamine, boehringer Ingelheim); PKI-166 ((R) -4- [4- [ (1-phenylethyl) amino group]-1H-pyrrolo [2,3-d]Pyrimidin-6-yl]-phenol); (R) -6- (4-hydroxyphenyl) -4- [ (1-phenethyl) amino]-7H-pyrrolo [2,3-d]Pyrimidine); CL-387785 (N- [4- [ (3-bromophenyl) amino)]-6-quinazolinyl]-2-butynamide); EKB-569 (N- [4- [ (3-chloro-4-fluorophenyl) amino group]-3-cyano-7-ethoxy-6-quinolinyl]-4- (dimethylamino) -2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as lapatinib (/ -)>GSK572016 or N- [ 3-chloro-4- [ (3-fluorophenyl) methoxy group]Phenyl group]6[5[ [2 ] AAlkylsulfonyl radical]Ethyl group]Amino group]Methyl group]-2-furyl group]-4-quinazolinamine). All teachings (including, but not limited to, all methods, compounds, compositions, data, etc.) of each of the above references are incorporated herein by reference in their entirety for use with any embodiment and disclosure herein.

Chemotherapeutic agents also include "tyrosine kinase inhibitors," including EGFR-targeting drugs mentioned in the preceding paragraphs; small molecule HER2 tyrosine kinase inhibitors such as TAK165 available from Takeda; CP-724,714, oral selective inhibitors of ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors, such as EKB-569 (purchased from Wyeth), which preferentially bind EGFR but inhibit both HER2 and EGFR over-expressed cells; lapatinib (GSK 572016; available from Glaxo-SmithKline), HER2 and EGFR tyrosine kinase inhibitors orally; PKI-166 (available from Novartis); pan HER inhibitors such as Kanettinib (CI-1033; pharmacia); raf-1 inhibitors, such as antisense agent ISIS-5132 from ISIS Pharmaceuticals that inhibits Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate Purchased from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib @, for examplePurchased from Pfizer); VEGF receptor tyrosine kinase inhibitors such as, for example, betaranin (PTK 787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (from Pharmacia); quinazolines, such as PD 153035,4- (3-chloroanilino) quinazoline; pyridopyrimidine; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261, and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2,3-d]Pyrimidine; curcumin (diferuloylmethane, 4, 5-bis (4-fluoroanilino) phthalimide); tyrosine containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. patent No. 5,804,396); trypsin (U.S. Pat. No. 5,804)Number 396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan HER inhibitors such as CI-1033 (Pfizer); affinitac (ISIS 3521; ISIS/Lilly); imatinib mesylate->PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); sesaminib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone), rapamycin (sirolimus,) >) The method comprises the steps of carrying out a first treatment on the surface of the Or as described in any of the following patent publications: U.S. patent No. 5,804,396; WO 1999/09016 (American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983 (Warner Lambert); WO 1999/06678 (Warner Lambert); WO 1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, inc.); WO 1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca). All teachings (including, but not limited to, all methods, compounds, compositions, data, etc.) of each of the above references are incorporated herein by reference in their entirety for use with any embodiment and disclosure herein.

Chemotherapeutic agents also include dexamethasone, interferon, colchicine, chlorpheniramine, cyclosporin, amphotericin, metronidazole, alemtuzumab, aliskiren, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene, cladribine, clofarabine, dabigatran alpha, diniinterleukin, dexrazoxane, epoetin alpha, erlotinib, filigirtine, histrelin acetate, temozolomab, interferon alpha-2 a, interferon alpha-2 b, lenalidomide, levamisole, mesna, methoxalin, nandrolone, nipagin, nafamolmustine, opril, palivesmide, disodium aminodiphosphate, gargasan, gabexastim, fluperzine, quinine, flupirtine, prazophos, 6, and ATRADISARG, R-26, and ATRADISAL, and ATRAMITIR-PIX.

Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tiecoat pivalate, triamcinolone, mometasone, ambroxide, budesonide, fluocinolone acetonide, betamethasone sodium phosphate, dexamethasone sodium phosphate, fludrolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclava Luo Misong dipropionate, betamethasone valerate, betamethasone dipropionate, prednisolide, clobetasone-17-butyrate, clobetasol-17-propionate, flucoumarone hexanoate, flucoulone pivalate, and fluprednisodine acetate; immunoselective anti-inflammatory peptides (ImSAID) such as phenylalanine-glutamine-glycine (FEG) and D-isomer forms (feG) thereof (IMULAN BioTherapeutics, LLC); antirheumatic drugs such as imidazothioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide Mi Temi, sulfasalazine, tumor necrosis factor alpha (TNF alpha) blockers such as etanercept (Enbrel), infliximab (Remicode), adalimumab (Humira), cetuzumab (Cimzia), golimumab (Simpli), interleukin 1 (IL-1) blockers such as anakinra (Kineret), T cell co-stimulatory blockers such as Abelip (Orencia), interleukin 6 (IL-6) blockers such as tolizumab Interleukin 13 (IL-13) blockers such as Leimumab; interferon alpha (IFN) blocking agents such as Long Li group mab; beta 7 integrin blockers such as rhuMAb beta 7; igE pathway blockers such as anti-M1; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1/β2 blockers, such as anti-lymphotoxin α (LTa); radioisotopes (e.g., at ²¹¹ 、I ¹³¹ 、I ¹²⁵ 、Y ⁹⁰ 、Re ¹⁸⁶ 、Re ¹⁸⁸ 、Sm ¹⁵³ 、Bi ²¹² 、P ³² 、Pb ²¹² And a radioisotope of Lu); other research agents, such asSuch as thioplatin, PS-341, phenylbutyrate, ET-18-OCH ₃ Or a farnesyl transferase inhibitor (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanol, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol,)>) The method comprises the steps of carrying out a first treatment on the surface of the Beta-lapachone; lapaol; colchicine; betulinic acid; acetylcamptothecin, scopolamine, and 9-aminocamptothecin); podophyllotoxin; tegafur->BexaroteneBisphosphonates, such as chlorophosphonate (e.g.,/->Or->) Etidronate->NE-58095, zoledronic acid/zoledronate->Alendronate->Pamidronate- >Tiludronate saltOr risedronate->And epidermal growth factor receptor (EGF-R); vaccines, such as->A vaccine; pirifaxin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS 341); CCI-779; tipifanib (R11577); orafenib, ABT510; bcl-2 inhibitors such as Olimsenna +.>Peking raw agar; farnesyl transferase inhibitors, such as lenafani (SCH 6636, SARASAR) ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the And pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the foregoing, such as CHOP, cyclophosphamide, doxorubicin, vincristine, and abbreviations for prednisolone combination therapies; FOLFOX, oxaliplatin (ELOXATIN) ^TM ) Abbreviations for treatment regimen combined with 5-FU and leucovorin.

Chemotherapeutic agents also include non-steroidal anti-inflammatory drugs with analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxic, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid and COX-2 inhibitors such as celecoxib, etoricoxib, lomecoxib, parecoxib and valdecoxib. NSAIDs are useful in symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory joint diseases, ankylosing spondylitis, psoriatic arthritis, rette's syndrome, acute gout, dysmenorrhea, bone transfer pain, headache and migraine, postoperative pain, mild to moderate pain caused by inflammation and tissue damage, fever, ileus and renal colic.

In certain embodiments, chemotherapeutic agents include, but are not limited to, doxorubicin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, interferons, platinum derivatives, taxanes (e.g., paclitaxel, docetaxel), vinca alkaloids (e.g., vinblastine), anthracyclines (e.g., doxorubicin), epipodophyllotoxins (e.g., etoposide), cisplatin, mTOR inhibitors (e.g., rapamycin), methotrexate, actinomycin D, cervacporin 10, colchicine, trimesate, chloramphenicol, cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents (e.g., chlorambucil), 5-fluorouracil, camptothecine, cisplatin, metronidazole, imatinib mesylate, and the like. In other embodiments, the compounds disclosed herein are administered in combination with a biologic agent such as bevacizumab or panitumumab.

In certain embodiments, a compound disclosed herein, or a pharmaceutically acceptable composition thereof, is administered in combination with an antiproliferative agent or chemotherapeutic agent selected from any one or more of the following: abarybacil, aldesleukin, alemtuzumab, alisavic acid, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacytidine, live BCG, bevacizumab, fluorouracil, betasaltine, bleomycin, bortezomib, busulfan, carbosterone, capecitabine, camptothecine, carboplatin, carmustine, cetuximab, chlorambucil, cladribine, clofarabine, cyclophosphamide, cytarabine, actinomycin D, dapoxetine alpha, daunorubicin, dimesleukin, dextroamide, docetaxel, doxorubicin (neutral), doxorubicin hydrochloride, droxithrone propionate, epirubicin, epoetin alpha, erlotinib, estramustine, etoposide, exemestane, febuxine, fludarabide, fludarabine fulvestrant, gefitinib, gemcitabine, gemtuzumab, goserelin acetate, histrelin acetate, hydroxyurea, temozolomide, idarubicin, ifosfamide, imatinib mesylate, interferon alpha-2 a, interferon alpha-2 b, irinotecan, lenalidomide, letrozole, leucovorin, leuprorelin acetate, levamisole, lomustine, megestrol acetate, melphalan, mercaptopurine, 6-MP, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, nol, nilamide, norfinasteride, olpride, oxaliplatin, paclitaxel, paliferamine, pamidronate, pernase, colupitaconate, peginkgetin, meltrazocine, pravastatin, poise, pramipexole, promethazine, phenomer sodium, methyl benzyl hydrazine, quinicline, labyrine, rituximab, saxitin, sorafenib, streptozotocin, sunitinib malate, talc, tamoxifen, temozolomide, teniposide, VM-26, testosterone, thioguanine, 6-TG, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, retinoic acid, ATRA, uramestin, pentarubicin, vinblastine, vincristine, vinorelbine, zoledronate, or zoledronic acid.

In some embodiments, the amount of the compound of formula I administered may be any suitable amount for treating cancer. For example, the amount administered may be a daily dose of between 1mg body weight and 500 mg. As another example, the daily dose may be in the range of about 20mg to 400mg (or any subrange or sub-value therebetween, inclusive). In some embodiments, the amount of the compound of formula I administered may range from 10mg to 300mg. In some embodiments, the amount of the compound of formula I administered may range from 10mg to 100mg. In some embodiments, the amount of the compound of formula I administered may range from 5mg to 50mg. Daily dosages may be achieved by administering a single administered dose (e.g., QD) over a day or via multiple administrations (e.g., BID, TID, QID, etc.) to provide a total daily dose. In some embodiments, the amount of MEK inhibitor administered is any suitable amount. For example, it may be an amount in the range of 1mg to 500mg per day (or any subrange or sub-value therebetween, inclusive). The amount of MEK inhibitor administered may be the same or less than the approved amount of any given MEK inhibitor and may depend on the given indication. In some embodiments, trametinib may be administered at a dose in the range of about 1mg to about 10mg once daily. For example, trametinib is approved as 2mg once daily. It is also approved to reduce doses such as 1.5mg QD and 1mg QD. In some embodiments, bemetinib may be administered at a dose ranging from about 30mg to about 100mg. For example, bemetinib is approved as a 45mg dose twice daily. Bemetinib is also approved for reduced dose, such as about 30mg BID. It should be understood that each of the ranges recited above may include any subrange or sub-point therein, including endpoints. It should be understood that each of the ranges recited above may include any subrange or sub-point therein, including endpoints. The usual dosage range for adults is usually 5mg to 2 g/day. Tablets or other dosage forms provided in discrete units may conveniently contain an amount of one or more compounds which are effective at that dose or multiples thereof, for example, containing units of from 5mg to 500mg, typically from about 10mg to 200 mg. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. In some embodiments, the administration is oral.

In some embodiments, there is provided a method of treating colorectal cancer and NSCLC cancer in a subject, the method comprising orally administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with trimetinib or bemetinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, the trimetinib or bemetinib may be administered once or twice daily. For example, the drugs may be co-administered as described herein.

In some embodiments, the subject is a human. In some embodiments, the subject is a mammal other than a human, such as a primate, rodent, dog, cat, or other small animal.

In some embodiments, a method of inhibiting ERK1/2 phosphorylation is provided, the method comprising contacting a population of cells with a combination of formula I or a pharmaceutically acceptable salt thereof and trimetinib or bemetinib. In some embodiments, the concentration of the compound of formula I is in the range of 1nM to 1 micromolar or 1nM to 500nM or 1nM to 20 nM. In some embodiments, the concentration of trimetinib or bemetinib is in the range of 10nM to 1 micromolar, or 10nM to 500 nM.

Composition and method for producing the same

The compounds of formula I disclosed herein may exist as salts. Embodiments of the present invention include such salts, which may be pharmaceutically acceptable salts. Examples of suitable salt forms include hydrochloride, hydrobromide, sulfate, mesylate, nitrate, maleate, acetate, citrate, fumarate, tartrate (e.g., (+) -tartrate, (-) -tartrate or mixtures thereof, including racemic mixtures), succinate, benzoate and salts with amino acids such as glutamate. These salts can be prepared by methods known to those skilled in the art. Also included are base addition salts such as sodium, potassium, calcium, ammonium, organic amino or magnesium salts or the like. When the compounds of embodiments of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid (neat or in a suitable inert solvent). Examples of acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrocarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and the like, as well as salts derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Salts of amino acids such as arginine and the like, and salts of organic acids such as glucuronic acid or galacturonic acid and the like are also included. Certain specific compounds of embodiments of the present invention contain both basic and acidic functionalities, which enable the compounds to be converted into base or acid addition salts.

Other salts include acid or base salts of the compounds used in the methods of embodiments of the present invention. Illustrative examples of pharmaceutically acceptable salts are inorganic acid (hydrochloric acid, hydrobromic acid, phosphoric acid, etc.) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid, etc.) salts, and quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts. It should be understood that pharmaceutically acceptable salts are non-toxic. Additional information regarding suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17 th edition, mack Publishing Company, easton, pa.,1985, all of the teachings of which (including but not limited to all methods, compounds, compositions, data, etc.) are incorporated herein by reference in their entirety for use with any of the embodiments and disclosures herein.

Pharmaceutically acceptable salts include salts of the active compounds which are prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the compounds described herein. When the compounds of embodiments of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base (sodium carbonate or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compounds of embodiments of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid (neat or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrocarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydroiodic or phosphorous acids and the like, as well as salts derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic and the like. Also included are salts of amino acids such as arginine and the like, as well as salts of organic acids such as glucuronic acid or galacturonic acid and the like (see, e.g., berge et al, "Pharmaceutical Salts", journal of Pharmaceutical Science,1977,66,1-19), all teachings of which (including but not limited to all methods, compounds, compositions, data, and the like) are incorporated herein by reference in their entirety for use with any of the embodiments and disclosures herein. Certain specific compounds of embodiments of the present invention contain both basic and acidic functionalities, which enable the compounds to be converted into base or acid addition salts.

The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

Certain compounds of embodiments of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of embodiments of the present invention. Certain compounds of embodiments of the present invention may exist in a variety of crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated for embodiments of the invention and are intended to fall within the scope of embodiments of the invention.

Certain compounds of embodiments of the invention have asymmetric carbon atoms (optical centers) or double bonds; in absolute stereochemistry, enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms of the amino acids may be defined as (R) -or (S) -or as (D) -or (L) -, and individual isomers are all encompassed within the scope of embodiments of the present invention. The compounds of embodiments of the present invention do not include those known in the art that are too unstable to synthesize and/or isolate. Embodiments of the present invention are meant to include both racemic and optically pure forms of the compounds. Optically active (R) -and (S) -or (D) -and (L) -isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

Unless otherwise indicated, compounds of embodiments of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms making up such compounds. For example, compounds of embodiments of the invention may be labeled with radioactive or stable isotopes, such as, for example, deuterium @, for example ² H) The tritium is ³ H) Iodine-125% ¹²⁵ I) Fluorine-18% ¹⁸ F) Nitrogen-15% ¹⁵ N) and oxygen-17% ¹⁷ O) and oxygen-18% ¹⁸ O), C-13% ¹³ C) Or C-14% ¹⁴ C) A. The invention relates to a method for producing a fibre-reinforced plastic composite All isotopic variations of the compounds of the embodiments of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the embodiments of the present invention.

In addition to salt forms, embodiments of the invention also provide compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide compounds of embodiments of the present invention. Furthermore, prodrugs can be converted to the compounds of embodiments of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of embodiments of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical agent.

In some embodiments, pharmaceutical compositions comprising a compound of formula I and a pharmaceutically acceptable excipient are provided. In some embodiments, the pharmaceutical composition is configured as an oral tablet formulation.

The compounds of embodiments of the present invention may be prepared and administered in a variety of oral, parenteral and topical dosage forms. Oral formulations include tablets, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions and the like, suitable for ingestion by a patient. The compounds of embodiments of the present invention may also be administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection. Furthermore, the compounds described herein may be administered by inhalation, e.g., intranasal inhalation. Furthermore, the compounds of embodiments of the present invention may be administered transdermally. The compounds of formula I disclosed herein may also be administered by intraocular, intravaginal, and intrarectal routes, including suppositories, insufflation, powder, and aerosol formulations (see Rohatag, J.Clin. Pharmacol.35:1187-1193,1995;Tjwa,Ann.Allergy Asthma Immunol.75:107-111,1995 for examples of steroid inhalants), all teachings of which, including but not limited to all methods, compounds, compositions, data, and the like, are incorporated herein by reference in their entirety for use with any of the embodiments and disclosures herein. Thus, embodiments of the present invention also provide pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and/or excipients and a compound of formula I or a pharmaceutically acceptable salt of a compound of formula I.

For preparing pharmaceutical compositions from the compounds of embodiments of the present invention, the pharmaceutically acceptable carrier may be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier may be one or more substances which may also be used as diluents, flavouring agents, surfactants, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details concerning formulation and administration techniques are well described in the scientific and patent literature, see, for example, remington's Pharmaceutical Sciences, maack Publishing Co, the latest versions of Easton PA ("Remington's"), all teachings of which, including but not limited to, all methods, compounds, compositions, data, etc., are incorporated herein by reference in their entirety for use with any of the embodiments and disclosures herein.

In powders, the carrier is a finely divided solid which is admixed with the finely divided active component. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties and the required additional excipients in suitable proportions and compacted in the shape and size desired.

Powders, capsules and tablets preferably contain 5% or 10% to 70% of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "formulation" is intended to include the formulation of the active compound with an encapsulating material as a carrier, thereby providing a capsule in which the active ingredient, with or without other excipients, is surrounded by the carrier, thereby associating therewith. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets and lozenges can be used as solid dosage forms suitable for oral administration.

Suitable solid excipients are carbohydrate or protein fillers including, but not limited to, sugars including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato or other plants; cellulose, such as methyl cellulose, hydroxypropyl methyl cellulose, or sodium carboxymethyl cellulose; and gums including gum arabic and gum tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for product identification or to characterize the amount (i.e., dosage) of active compound. The pharmaceutical formulations disclosed herein may also be used orally, for example using push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. The push-fit capsules may contain a compound of formula I in admixture with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the compounds of formula I may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin or liquid polyethylene glycol, with or without a stabilizer.

Formulations in liquid form include solutions, suspensions and emulsions, for example water or water/propylene glycol solutions. For parenteral injection, liquid formulations may be formulated as solutions in aqueous polyethylene glycol solutions.

Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers, and thickeners as desired. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active component in water with: viscous materials such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and gum acacia, and dispersing or wetting agents such as natural phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain fatty alcohols (e.g., heptadecaethyleneoxycetyl alcohol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate) or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose, aspartame or saccharin. The formulation may be adjusted for osmotic pressure.

Also included are solid form preparations which are intended to be converted shortly before use into liquid form preparations for oral administration. Such liquid forms include solutions, suspensions and emulsions. In addition to the active ingredient, these formulations may contain coloring agents, flavoring agents, stabilizing agents, buffering agents, artificial and natural sweeteners, dispersants, thickening agents, solubilizing agents, and the like.

The oil suspension may be prepared by suspending a compound of formula I in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. The oil suspension may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweeteners may be added to provide a palatable oral preparation, such as glycerin, sorbitol or sucrose. These formulations may be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil carrier, see Minto, j.pharmacol.exp.ther.281:93-102,1997, all teachings of which, including but not limited to all methods, compounds, compositions, data, etc., are incorporated herein by reference in their entirety for use with any of the embodiments and disclosures herein. The pharmaceutical formulations disclosed herein may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil or mineral oil as described above, or a mixture thereof. Suitable emulsifying agents include natural gums, such as gum acacia and gum tragacanth, natural phospholipids, such as soy bean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweetening and flavoring agents, such as in the preparation of syrups and elixirs. Such formulations may also contain a demulcent, a preservative or a colorant.

Pharmaceutical formulations of the compounds of formula I disclosed herein may be provided in the form of salts and may be formed with bases, i.e. cationic salts, such as alkali metal and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, and ammonium salts such as ammonium, trimethyl-ammonium, diethylamine and tris- (hydroxymethyl) -methyl-ammonium salts.

The pharmaceutical formulation is preferably in unit dosage form. In such forms, the formulation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form may be a packaged formulation containing discrete amounts of the formulation, such as packaged tablets, capsules and powders in vials or ampoules. Furthermore, the unit dosage form may be a capsule, tablet, cachet, or lozenge itself, or it may be any of these in a suitable number of packaged forms.

The amount of active ingredient in a unit dosage formulation may vary or be adjusted from 0.1mg to 10000mg, more typically from 1.0mg to 1000mg, and most typically from 10mg to 500mg, depending on the particular application and potency of the active ingredient. The composition may also contain other compatible therapeutic agents, if desired.

The dosage regimen also takes into account pharmacokinetic parameters well known in the art, namely absorption rate, bioavailability, metabolism, clearance, etc. (see, e.g., hidalgo-Argones (1996) J. Steroid biochem. Mol. Biol.58:611-617; groning (1996) Pharmazie51:337-341; fotherby (1996) Contraceon 54:59-69; johnson (1995) J. Pharm. Sci.84:1144-1146; rohattagi (1995) Pharmazie 50:610-613; brophy (1983) Eur. J. Pharmin. 24:103-108; most recent Remington's; see above), all teachings of each of the above references, including but not limited to all methods, compounds, compositions, data, etc., are incorporated herein by reference in their entirety for use in any of the embodiments and disclosures herein). The prior art allows clinicians to determine dosage regimens for each individual patient, GR and/or MR modulator, and the disease or condition being treated.

Single or multiple administrations of the compound formulation of formula I may be administered in accordance with the dosage and frequency desired and tolerated by the patient. The formulation should provide a sufficient amount of active agent to be effective in treating the disease state. Thus, in one embodiment, the daily dosage of the pharmaceutical formulation for oral administration of a compound of formula I is from about 0.5 to about 30 mg/kg body weight/day, including all subranges and sub-values therein, inclusive. In alternative embodiments, a dosage of about 1mg to about 20mg per kg of patient body weight per day is used. Lower doses may be used compared to oral administration, particularly when the drug is administered to anatomically concealed sites, such as the cerebrospinal fluid (CSF) space, into the blood stream, body cavity or lumen of an organ. Topical application may use significantly higher doses. Practical methods for preparing formulations comprising compounds of formula I for parenteral administration are known or obvious to those skilled in the art and are described in more detail in publications such as Remington's, supra. See also Nieman, in "Receptor Mediated Antisteroid Action," Agarwal et al, edit, de Gruyter, new York (1987), all teachings of which references, including but not limited to, all methods, compounds, compositions, data, etc., are incorporated herein by reference In their entirety for use with any of the embodiments and disclosures herein.

In some embodiments, co-administration includes administration of one active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours (or any sub-time range or sub-time value within a 24 hour period) of the second active agent. Co-administration includes administration of the two active agents simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes (or any sub-time range or sub-time value, e.g., 0-30 minutes) of each other), or sequentially in any order. In some embodiments, co-administration may be achieved by co-formulation, i.e., preparing a single pharmaceutical composition comprising both active agents. In some embodiments, the active agents may be formulated separately. In some embodiments, the active agents and/or adjuvants may be linked or conjugated to each other. For example, at least one administered dose of the drug may be administered simultaneously. For example, at least one administered dose of the drug may be administered within a few minutes or less than one hour of each other. For example, at least one administered dose of the drug may be administered at a different time, but on the same day or on different days.

After a pharmaceutical composition comprising a compound of formula I disclosed herein is formulated in one or more acceptable carriers, it may be placed in a suitable container and labeled for treatment of the indicated condition. For administration of a compound of formula I, such a label will include instructions for, for example, the amount administered, the frequency of administration, and the method of administration.

Drug dosage

Of course, the dosage regimen of the compounds herein will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health condition, medical condition and weight of the recipient; the nature and extent of the symptoms; the type of contemporaneous treatment; the frequency of treatment; the route of administration, the renal function and hepatic function of the patient, and the desired effect. The clinician may determine and prescribe an effective amount of the drug required to prevent, counter or arrest the progress of the disease or condition.

As a general guidance, when used to indicate an effect, the daily oral dosage of each active ingredient will be between about 0.001 to about 1000mg/kg body weight, preferably between about 0.01 to about 100mg/kg body weight/day, and most preferably between about 0.1 to about 20 mg/kg/day. In some embodiments, the compound of formula (I) may be administered at a dose of about 10 mg/day to about 200 mg/day. In some embodiments, the compound of formula (I) may be administered at a dose of about 10 mg/day, 20 mg/day, 30 mg/day, 40 mg/day, 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/day, 100 mg/day, 110 mg/day, 120 mg/day, 130 mg/day, 140 mg/day, 150 mg/day, 160 mg/day, 170 mg/day, 180 mg/day, 190 mg/day, or 200 mg/day. The dose may be any value or subrange within the stated range.

The frequency of administration of the therapeutic agent may vary, for example, from once per day to six times per day, depending on the condition of the patient and the desired therapeutic effect. That is, the dosing frequency may be QD, i.e., once per day, BID, i.e., twice per day; TID, three times per day; QID, four times per day; five times per day, or six times per day. In another embodiment, the dosing frequency may be BIW, i.e., twice a week, TIW, i.e., three times a week, or QIW, i.e., four times a week.

Depending on the condition of the patient and the desired therapeutic effect, the treatment cycle may have a period of time without the administration of the therapeutic agent. As used herein, "intermittent administration" refers to administration of a therapeutic agent immediately followed by a blank day or week. For example, the treatment cycle may be 3 weeks long, including 2 weeks of administration of the therapeutic agent, followed by 1 week of no therapeutic agent administration. In some embodiments, the treatment period is 4 weeks long, including 3 weeks of administration followed by 1 week of no therapeutic agent.

As used herein, the term "treatment cycle" refers to a predetermined period of time for which a therapeutic agent is administered. Typically, the patient is examined at the end of each treatment cycle to assess the effect of the therapy.

In one embodiment, each of the treatment cycles has about 3 or more days. In another embodiment, each of the treatment cycles has from about 3 days to about 60 days. In another embodiment, each of the treatment cycles has from about 5 days to about 50 days. In another embodiment, each of the treatment cycles has from about 7 days to about 28 days. In another embodiment, each of the treatment cycles has 28 days. In one embodiment, the treatment cycle has about 29 days. In another embodiment, the treatment cycle has about 30 days. In another embodiment, the treatment cycle has about 31 days. In another embodiment, the treatment cycle has a treatment cycle of about one month. In another embodiment, the treatment period is any length of time from 3 weeks to 8 weeks. In another embodiment, the treatment period is any length of time from 3 weeks to 6 weeks. In yet another embodiment, the treatment period is 3 weeks. In another embodiment, the treatment period is one month. In another embodiment, the treatment period is 4 weeks. In another embodiment, the treatment period is 5 weeks. In another embodiment, the treatment period is 6 weeks. In another embodiment, the treatment period is 7 weeks. In another embodiment, the treatment period is 8 weeks. The duration of the treatment cycle may include any value or subrange within the range, including the endpoints.

As used herein, the term "co-administration" or "co-administration" refers to the administration of (a) an additional therapeutic agent and (b) a compound of formula (I) or a salt, solvate, ester and/or prodrug thereof together in a synergistic manner. For example, co-administration may be simultaneous administration, sequential administration, overlapping administration, intermittent administration, continuous administration, or a combination thereof.

In some embodiments, the dosing regimen of the compound of formula (I) is once daily over a period of 28 consecutive days. In some embodiments, a once-a-day dosing regimen of a compound of formula (I) may be, but is not limited to, 20 mg/day, 30 mg/day, 40 mg/day, 50 mg/day, 60 mg/day. The compound of formula (I) may be administered in any dose from 20mg to 60mg once a day. The dose may be any value or subrange within the stated range.

In some embodiments, the dosing regimen of the compound of formula (I) is twice daily over a period of 28 consecutive days. In some embodiments, the twice-daily dosing regimen of a compound of formula (I) may be, but is not limited to, 10 mg/day, 20 mg/day, 30 mg/day, 40 mg/day, 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/day, 100 mg/day. The compound of formula (I) may be administered in any dose of 20mg to 80mg twice a day. In some embodiments, the compound of formula (I) may be administered at any dose from 10 mg/day to 100 mg/day. The dose may be any value or subrange within the stated range.

In some embodiments, the dosing regimen of the compound of formula (I) may be any dose of 20mg to 60mg once a day for two weeks over a period of 6 weeks, followed by a discontinuation of the administration for one week (e.g., 2 weeks of administration, 1 week of cessation). In some embodiments, the dosing regimen of the compound of formula (I) may be any dose of 10mg to 100mg twice daily for two weeks and then discontinued for one week (e.g., 2 weeks dosing, 1 week discontinuation) over a period of 6 weeks.

In some embodiments, the dosing regimen of the compound of formula (I) may be any dose of 20mg to 60mg once a day for three weeks over a period of 8 weeks, followed by a discontinuation of the administration for one week (e.g., 3 weeks of administration, 1 week of cessation). In some embodiments, the dosing regimen of the compound of formula (I) may be any dose of 10mg to 100mg twice daily for three weeks twice a day over a period of 8 weeks, followed by a discontinuation of one week (e.g., 8 week dosing, 1 week discontinuation).

In some embodiments, the dosing regimen of the compound of formula (I) may be twice daily on days 1 and 2 of the week for 8 weeks. In some embodiments, the amount of the compound of formula (I) administered may be, but is not limited to, 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg.

When the compound of formula I is administered multiple times a week, the dose may be administered on any one or a combination of days during the week. For example, three times per week may include administration at the following times: day 1, day 3 and day 5; day 1, day 2, and day 3; day 1, day 3 and day 5; etc. Two days of weekly administration may include administration at the following times: day 1 and day 2; day 1 and day 3; day 1 and day 4; day 1 and day 5; day 1 and day 6; day 1 and day 7; etc.

Kit and product

Some embodiments of the present disclosure relate to kits and products comprising a compound of formula I and/or at least one FGFR inhibitor. For example, the kit or product may comprise a package or container containing the compound of formula I. Such kits and products may further include a product insert or label with approved drug administration and indication information, including how to use the compounds of formula I in combination with the FGFR inhibitor provided alone. The kit can be used in the methods of treating cancer described herein.

In some aspects, the kit or product may comprise a compound of formula I and at least one FGFR inhibitor. In some embodiments, the FGFR inhibitor is, for example, erdasatinib. Such kits may comprise one or more containers or packages containing one or both of the combined medicaments together in a single container and/or package, or in separate packages/containers. In some cases, the two medicaments are separately packaged, but contained in a single package, container or box. Such kits and products may further include a product insert or label with approved drug administration and indication information, including how to use the compounds of formula I in combination with FGFR inhibitors. The kit can be used in the methods of treating cancer described herein.

Some embodiments of the present disclosure relate to kits and products comprising a compound of formula 1 and/or at least one B-Raf inhibitor. For example, the kit or product may comprise a package or container containing the compound of formula I. Such kits and products may further include a product insert or label with approved drug administration and indication information including how to use the compound of formula 1 in combination with a B-Raf inhibitor provided separately. The kit can be used in the methods of treating cancer described herein.

In some aspects, the kit or product may comprise a compound of formula 1 and at least one B-Raf inhibitor. In some embodiments, the B-Raf inhibitor is, for example, kang Naifei ni. Such kits may comprise one or more containers or packages containing one or both of the combined medicaments together in a single container and/or package, or in separate packages/containers. In some cases, the two medicaments are separately packaged, but contained in a single package, container or box. Such kits and products may further include a product insert or label with approved drug administration and indication information, including how to use the compound of formula 1 in combination with a B-Raf inhibitor. The kit can be used in the methods of treating cancer described herein.

Some embodiments of the present disclosure relate to kits and products comprising a compound of formula I and/or at least one MEK inhibitor. For example, the kit or product may comprise a package or container containing the compound of formula I. Such kits and products may further include a product insert or label with approved drug administration and indication information including how to use the compound of formula 1 in combination with a MEK inhibitor provided alone. The kit can be used in the methods of treating cancer described herein.

In some aspects, a kit or product may comprise a compound of formula 1 and at least one MEK inhibitor. In some embodiments, the MEK inhibitor is, for example, trimetinib or bemetinib. Such kits may comprise one or more containers or packages containing one or both of the combined medicaments together in a single container and/or package, or in separate packages/containers. In some cases, the two medicaments are separately packaged, but contained in a single package, container or box. Such kits and products may further include a product insert or label with approved drug administration and indication information, including how to use the compound of formula 1 in combination with a MEK inhibitor. The kit can be used in the methods of treating cancer described herein.

Some embodiments of the present disclosure relate to kits and products comprising a compound of formula I and/or at least one MET inhibitor. For example, the kit or product may comprise a package or container containing the compound of formula I. Such kits and products may further include a product insert or label with approved drug administration and indication information, including how to use the compounds of formula I in combination with a MET inhibitor provided separately. The kit can be used in the methods of treating cancer described herein.

In some aspects, the kit or product may comprise a compound of formula I and at least one MET inhibitor. In some embodiments, the MET inhibitor is, for example, crizotinib, terbutatinib, cerwatinib, caboztinib, or tivantinib. Such kits may comprise one or more containers or packages containing one or both of the combined medicaments together in a single container and/or package, or in separate packages/containers. In some cases, the two medicaments are separately packaged, but contained in a single package, container or box. Such kits and products may further include a product insert or label with approved drug administration and indication information, including how to use the compound of formula I in combination with a MET inhibitor. The kit can be used in the methods of treating cancer described herein.

Examples

Example 1 synergistic combinations of Compounds of formula I and FGFR inhibitors

This example demonstrates the synergistic combination of a compound of formula I with an FGFR inhibitor.

Combined cell proliferation assay

Cells (2000 cells per well) were plated on 96-well plates in 100 μl of cell culture medium. Cells were treated with compounds of formula I and erdasatinib at concentrations ranging from 0 to 10 μm by using a Tecan D300e digital dispenser combinatorial matrix protocol. On day 5, 50 μl CellTiter-Glo (CTG) reagent (Promega) was added and the plates incubated under gentle shaking for 10 minutes. After incubation for 10 minutes, the luminescence signal was determined according to the manufacturer's instructions (Promega) and combined data was generated by standard HSA model using combeneflit software. The combined synergy is represented by a positive number in the results table. Negative numbers represent antagonism of the combination.

Results

Figures 1A-1B show data indicating that the combination of the compound of formula I and the FGFR inhibitor erdasatinib exhibits in vitro synergy. Fig. 1A shows 3D graphical synergy data in Hep3B cancer cell lines using a combination of a compound of formula I and erdasatinib. Fig. 1B shows 3D graphical synergy data in JHH-7 cancer cell lines using a combination of a compound of formula I and erdasatinib.

Fig. 1A and 1B show data indicating that the combination of the compound of formula I and the FGFR inhibitor erdasatinib exhibits in vitro synergy. Fig. 1A shows synergy data in Hep3B cancer cell lines using a combination of a compound of formula I and erdasatinib. Fig. 1B shows synergy data in JHH-7 cancer cell lines using a combination of a compound of formula I and erdasatinib.

Example 2-combination therapy of Compounds of formula I and erdasatinib in FGFR2 amplified liver cancer CDX model KATO III

Material

Throughout the 28 day administration in mice, a vehicle/control preparation, 100mM acetic acid in deionized water, was prepared, pH adjusted to 4.8-5.0, and stored under ambient conditions.

Test preparations of the compounds of formula I were freshly prepared weekly in 100mM acetate buffer vehicle and stored under ambient conditions. The combination agent erdasatinib was prepared in a carrier of 20% hp-beta-CD and stored at 2-8 ℃.

Female Balb/c nude mice were purchased from beijing velarihua laboratory animal technologies limited (Beijing Vital River Laboratory Animal Technology co., ltd.). Mice were 6-8 weeks old at the time of implantation. According to the IACUC protocol, mice are placed in an pathogen free (SPF) environment of an animal feeding facility and allowed to adapt to the new environment for at least 3 days prior to starting any experiments.

All procedures related to animal handling, care and treatment in this study were performed according to guidelines approved by the committee for animal care and use (IACUC) of the tin-free AppTec institution. During the course of the study, care and use of animals was performed as prescribed by the laboratory animal care assessment and certification association (AAALAC). Furthermore, all parts of this study conducted in tin-free AppTec followed the study protocols approved by the study taker and the applicable Standard Operating Procedure (SOP).

Preparation of xenograft models

The KATO-III cell line is a human hepatoma cell with FGFR amplification. KATO-III cell line was purchased from ATCC [ (]HTB-103 ^TM ). Will contain 5 x 10 using a syringe ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. When the tumor volume reaches 220mm after subcutaneous implantation ³ Tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started the following day after the randomized group. The treatment start date is indicated as treatment day 1. Mice were dosed by oral administration of a vehicle control solution, 10mg/kg BID of the compound of formula I alone, and 10mg/kg QD of erdasatinib alone. Another group received a combination treatment of a compound of formula I and erdasatinib, wherein the compound of formula I was administered at 10mg/kg BID and the erdasatinib was administered at 10mg/kg QD. The dosing volume was 5mL/kg and the BID protocol was 8 hours apart. In the combination group, erdasatinib was administered one hour after the first BID dose of the compound of formula I. The study was terminated on day 28 of treatment as defined in the study protocol.

Results

Fig. 2 shows a graph of tumor volumes treated with a compound of formula I alone, erdasatinib alone, and a combination of a compound of formula I and erdasatinib for a period of time in a liver cancer CDX model KATO III. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 2, the combination of the compound of formula I and erdasatinib exhibited superior tumor growth inhibition in the FGFR2 amplified liver cancer CDX model KATO III relative to treatment with the compound of formula I alone or with erdasatinib alone.

Example 3-combination therapy of Compounds of formula I and erdasatinib in FGFR 2-amplified gastric cancer CDX SNU-16

Material

Test preparations of the compounds of formula I were freshly prepared weekly in 100mM acetate buffer vehicle and stored under ambient conditions. The combination agent erdasatinib was freshly prepared weekly in 20% HP-beta-CD carrier and stored at 2-8deg.C.

Female Balb/c nude mice were purchased from Beijing Vietnam Lihua laboratory animal technologies Co. Mice were 6-8 weeks old at the time of implantation. According to the IACUC protocol, mice are placed in an pathogen free (SPF) environment of an animal feeding facility and allowed to adapt to the new environment for at least 3 days prior to starting any experiments.

All procedures related to animal handling, care and treatment in this study were performed according to guidelines approved by the committee for animal care and use (IACUC) of the tin-free AppTec institution. During the course of the study, care and use of animals was performed as prescribed by the laboratory animal care assessment and certification association (AAALAC). Furthermore, all parts of this study were conducted in tin-free AppTec and adhered to the study protocols and applicable Standard Operating Procedures (SOP) approved by the study taker.

Preparation of xenograft models

The SNU16 cell line is a human gastric cancer cell with FGFR amplification. SNU16 cell line was purchased from ATCC [ ]CRL-1420 ^TM ). Will contain 5 x 10 using a syringe ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. When the tumor volume reaches 180mm after subcutaneous implantation ³ Tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started on the day of randomized block. The treatment start date is indicated as day 0 of treatment. Mice were dosed by oral administration of a vehicle control solution, 10mg/kg BID of the compound of formula I alone, 30mg/kg QD of the compound of formula I alone, and 10mg/kg QD of erdasatinib alone. Two other groups received a combination treatment of the compound of formula I and erdasatinib, one group received 10mg/kg BID of the compound of formula I and 10mg/kg QD of erdasatinib, and the other group received 30mg/kg QD of the compound of formula I and 10mg/kg QD of erdasatinib. The dosing volume of each compound was 5mL/kg and the BID regimen was 8 hours apart. In the combination group, erdasatinib is administered once after QD administration of the compound of formula I or after the first BID dose of the compound of formula I. The study was terminated on day 28 of treatment as defined in the study protocol.

Results

Fig. 3 shows a graph of tumor volumes treated with a compound of formula I alone, erdasatinib alone, and a combination of a compound of formula I and erdasatinib for a period of time in FGFR2 amplified gastric cancer CDX model SNU-16. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 3, the combination of the compound of formula I and erdasatinib exhibited superior tumor growth inhibition in FGFR2 amplified gastric cancer CDX model SNU-16 relative to treatment with the compound of formula I alone or with erdasatinib alone.

Example 4-combination therapy of Compounds of formula I and erdasatinib in FGF19-FDFR4 dependent liver cancer CDX model Huh-7

Material

Preparation of xenograft models

The Huh-7 cell line is a human hepatoma cell with FGFR overexpression. Huh-7 cell line was purchased from Japanese research bioresources cell bank (Japanese Collection of Research Bioresources Cell Bank) (JCRB cell bank, JCRB 0403). Huh-7 cells were cultured in a medium containing Dulbecco's Modified Eagle Medium, DMEM plus 10% Fetal Bovine Serum (FBS) and 1% antibiotic-Antifungal Agent (AA) at 37℃in air with 5% CO ₂ Is cultured in an atmosphere of (2). The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation. Huh-7 tumor cells were implanted subcutaneously into mice. Will contain 5 x 10 using a syringe ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. When the tumor volume reaches about 500-1000 mm ³ When harvesting tumor fragments (15-30 mm) ³ ) The right flank of the mouse was then implanted subcutaneously with an 18g trocar. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reaches 146mm after subcutaneous implantation ³ At the mean value of (2), tumor-bearing mice were randomly divided into different groups8 mice per group. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started the following day after the randomized group. The treatment start date is indicated as treatment day 1. Mice were dosed by oral administration of a vehicle control solution, 10mg/kg BID alone, 30mg/kg QD alone, and 10mg/kg QD alone of erdasatinib to the monotherapy treatment group. Two other groups received combination treatment of the compound of formula I and erdasatinib, one group being dosed with 10mg/kg BID of the compound of formula I and 10mg/kg QD of erdasatinib, and the other group being dosed with 30mg/kg QD of the compound of formula I and 10mg/kg QD of erdasatinib. The dosing volume of each compound was 5mL/kg and the BID regimen was 8 hours apart. In the combination group, erdasatinib was administered one hour after QD administration of the compound of formula I or one hour after the first BID dose of the compound of formula I. The study was terminated on day 21 of treatment.

Results

FIG. 4 shows a graph of tumor volumes treated with a compound of formula I alone, erdasatinib alone, and a combination of a compound of formula I and erdasatinib for a period of time in FGF19-FGFR4 dependent liver cancer CDX model Huh-7. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 4, the combination of the compound of formula I and erdasatinib exhibited superior tumor growth inhibition relative to treatment with the compound of formula I alone or with erdasatinib alone in FGF19-FGFR 4-dependent liver cancer CDX model Huh-7.

Example 5-synergistic in vitro combination of Compounds of formula I and class 1 mutant B-Raf protein inhibitors

Cell proliferation assay

Cells (2000 cells per well) were plated on 96-well plates in 100 μl of cell culture medium and treated with either the compound of formula I alone or the compound of formula I and a fixed concentration of Kang Naifei ni. On day 5, 50 μl CellTiter-Glo (CTG) reagent (Promega) was added and the plates incubated under gentle shaking for 10 minutes. After incubation for 10 minutes, the luminescence signal was determined according to the manufacturer's instructions (Promega) and a chart was drawn using Prism GraphPad.

Combined cell proliferation assay

Cells (2000 cells per well) were plated on 96-well plates in 100 μl of cell culture medium. Cells were treated with compounds of formula I and Kang Naifei ni at concentrations ranging from 0 to 10 μm by using a Tecan D300e digital dispenser combinatorial matrix protocol. On day 5, 50 μl CellTiter-Glo (CTG) reagent (Promega) was added and the plates incubated under gentle shaking for 10 minutes. After incubation for 10 minutes, the luminescence signal was determined according to the manufacturer's instructions (Promega) and combined data was generated by standard HSA model using combeneflit software. The combined synergy is represented by a positive number in the results table. Negative numbers represent antagonism of the combination.

Western blot of pERK and pERK

Cells were treated with the compound for 4 hours. After treatment, cells were lysed with Thermo Fisher RIPA lysis buffer with protease and phosphatase inhibitors for 10 min on ice. Cells were centrifuged at 4℃for 10 min with a microcentrifuge. The supernatant was transferred to a pre-chilled microcentrifuge tube and the protein concentration of the lysate was measured using BCA method. Immunoblots were performed against pERK and total ERK using equal amounts of protein cell lysate supernatant.

Results

FIG. 7 shows the use of a combination of a compound of formula I and the BRAF inhibitor Kang Naifei Ni in WiDr BRAF ^V600E Synergy data in CRC cell lines. This data indicates that there is a significant degree of synergy in the combination of the compound of formula I and Kang Naifei.

FIG. 9A shows a gel demonstrating robust inhibition of ERK1/2 phosphorylation in RKO colorectal cancer cell lines. Fig. 9B shows a gel demonstrating robust inhibition of ERK1/2 phosphorylation in the WiDr colorectal cancer cell line. Figure 9C shows a graph of the antiproliferative effect of a compound of formula I alone or in combination with Kang Naifei ni in RKO colorectal cancer cell lines. Fig. 9D shows a graph of the antiproliferative effect of a compound of formula I or a compound of formula I in combination with Kang Naifei ni in a WiDr colorectal cancer cell line. FIGS. 9A-9B show robust inhibition of pERK1/2 using a combination of a compound of formula I and Kang Naifei Ni. Figures 9C-9D demonstrate that the combination of the compound of formula I and Kang Naifei ni increases the inhibitory activity of the compound of formula I.

FIGS. 10A-10D show comparative studies of the efficacy of SHP2 inhibitors in combination with Kang Naifei Ni in RKO colorectal cancer cell lines. FIG. 10A shows a gel comparing inhibition of ERK1/2 phosphorylation in RKO colorectal cancer cell lines with the following combinations: compound of formula I + Kang Naifei; TNO155+ Kang Naifei; and RMC-4550+ Kang Naifei Ni. Fig. 10B shows at 1. Control; 2. (a compound of formula I); 3. kang Naifei Ni; bar graph of pERK as a percentage of control in case of (compound of formula I) + Kang Naifei. Fig. 10C shows at 1. Control; tno155;3. kang Naifei Ni; bar graph of pERK as a percentage of control for the case of tno155+ Kang Naifei. Fig. 10D shows at 1. Control; RMC-4550;3. kang Naifei Ni; bar graph of pERK as a percentage of control for rmc-4550+ Kang Naifei. As shown in fig. 10A-10D, the combination of the SHP2 inhibitor compound of formula I and Kang Naifei ni was most effective in inhibiting ERK1/2 phosphorylation.

Example 6-in BRAF ^V600E Combination therapy of a compound of formula I and Kang Naifei Ni in mutant CRC PDX model CR0029

Material

Test preparations of the compounds of formula I were freshly prepared weekly in 100mM acetate buffer vehicle and stored under ambient conditions. The combination agent Kang Naifei was freshly prepared weekly in 0.5% cmc and 0.5% tween 80 in vehicle and stored at 2-8 ℃.

Female Balb/c nude mice were purchased from si Bei Fu (Beijing) laboratory animal technologies limited (SPF (beijin) Laboratory Animal Technology Co, ltd.). Mice were 7-9 weeks old at the time of implantation. According to the IACUC protocol, mice are placed in an pathogen free (SPF) environment of an animal feeding facility and allowed to adapt to the new environment for at least 3 days prior to starting any experiments.

All procedures related to animal handling, care and treatment in this study were performed according to guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Crown Bioscience (tai ku, china). During the course of the study, care and use of animals was performed as prescribed by the laboratory animal care assessment and certification association (AAALAC). In addition, all parts of this study conducted in Crown Bioscience (tai ku, china) followed the study protocols approved by the study taker and the applicable Standard Operating Program (SOP)

Preparation of xenograft models

The CR0029 PDX model was established at CrownBio for preclinical efficacy studies. The PDX model was derived from chinese female CRC patients. BRAF in PDX model CR0029 was confirmed by both RNA sequencing and exome sequencing ^V600E Mutation. The skin of the mice was cleaned on the right flank with an appropriate surgical scrub and iodophor. Tumor fragments (2-3 mm diameter) harvested from the PDX model were subcutaneously implanted into the right flank of female Balb/c nude mice using an 18g trocar.

Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reaches approximately 141mm ³ Average value (in the range of 110-176 mm) ³ ) At this time, tumor-bearing mice were randomly divided into 7 different groups of 8 mice each. Random grouping date representationDay 0 for treatment.

Treatment of

Treatment was started on the day of randomized block. The treatment start date is indicated as day 0 of treatment. Mice were dosed by oral administration of a vehicle control solution, 10mg/kg BID alone of the compound of formula I, and 90mg/kg QD alone of Kang Naifei ni. The other group received combination therapy in which the compound of formula I was administered at 10mg/kg BID and Kang Naifei Ni was administered at 90mg/kg QD. The dosing volume of each compound was 5mL/kg and the BID regimen was 8 hours apart. In the combination group, kang Naifei Ni was administered one hour after the administration of the compound of formula I. The study was terminated on day 28 of treatment as defined in the study protocol.

Results

Fig. 11 shows the method in BRAF ^V600E Plots of tumor volumes treated with the compound of formula I alone, kang Naifei ni alone, and a combination of the compound of formula I and Kang Naifei ni for a period of time in mutant CRC PDX model CR 0029. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 11, in BRAF ^V600E The combination of the compound of formula I and Kang Naifei ni showed superior tumor growth inhibition relative to treatment with the compound of formula I alone or Kang Naifei ni alone in mutant CRC PDX model CR 0029.

Example 7-in BRAF ^V600E Combination therapy of a compound of formula I and Kang Naifei Ni in mutant CRC PDX model CR004

Material

Female Balb/c nude mice were purchased from St Bei Fu (Beijing) laboratory animal technologies Co., ltd. Mice were 9-11 weeks of age at the time of implantation. According to the IACUC protocol, mice are placed in an pathogen free (SPF) environment of an animal feeding facility and allowed to adapt to the new environment for at least 3 days prior to starting any experiments.

All procedures related to animal handling, care and treatment in this study were performed according to guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Crown Bioscience (beijing, china). During the course of the study, care and use of animals was performed as prescribed by the laboratory animal care assessment and certification association (AAALAC). In addition, all parts of this study conducted in Crown Bioscience (beijing, china) followed the study protocols approved by the study taker and the applicable Standard Operating Program (SOP).

Preparation of PDX

The CR0004 PDX model was established at CrownBio for preclinical efficacy studies. The PDX model was derived from a 73 year old chinese male CRC patient. The BRAFV600E mutation in PDX model CR0004 was confirmed by both RNA sequencing and exome sequencing. The skin of the mice was cleaned on the right flank with an appropriate surgical scrub and iodophor. Tumor fragments (2-3 mm diameter) harvested from the PDX model were subcutaneously implanted into the right flank of female Balb/c nude mice using an 18g trocar. When the average tumor size reached 141mm3 (range 121-180mm 3), tumor-bearing mice were randomly divided into 6 study groups of 8 mice each.

Treatment of

Results

Fig. 12 shows the method in BRAF ^V600E Plots of tumor volumes treated with the compound of formula I alone, kang Naifei ni alone, and a combination of the compound of formula I and Kang Naifei ni for a period of time in mutant CRC PDX model CR 004. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 12, in BRAF ^V600E The combination of the compound of formula I and Kang Naifei ni showed superior tumor growth inhibition relative to treatment with the compound of formula I alone or Kang Naifei ni alone in mutant CRC PDX model CR 004.

Example 8-in BRAF ^V600E Combination therapy of a compound of formula I and Kang Naifei Ni in a mutant CRC CDX model WiDr

Material

Preparation of xenograft models

WiDrIs a human CRC tumor cell line with BRAVV 600E mutation. The WiDr cell line was purchased from european collection of certified cell cultures (European Collection of Authenticated Cell Cultures) (ECACC, 85111501). WiDr cells were cultured in medium containing EMEM (EBSS) plus 10% Fetal Bovine Serum (FBS), 2mM glutamine and supplemented with 1% non-essential amino acids (NEAA) at 37deg.C in air with 5% CO ₂ Is cultured in an atmosphere of (2). The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation.

The WiDr tumor cells were implanted subcutaneously into mice. Will contain 5 x 10 using a syringe ⁶ 200 μl of cell suspension of individual tumor cells was subcutaneously implanted into the right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reached an average value of 189mm3 (ranging from 139 to 240mm 3), tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started on the day of randomized block. The treatment start date is indicated as day 0 of treatment. Mice were dosed by oral administration of a vehicle control solution, 10mg/kg BID of the compound of formula I alone, 30mg/kg QD of the compound of formula I alone, and 90mg/kg QD of Kang Naifei ni alone. The other two groups received combination therapy of the compound of formula I and Kang Naifei Ni, with the first group being administered with 10mg/kg BID of the compound of formula I and 90mg/kg QD of Kang Naifei Ni and the second group being administered with 30mg/kg QD of the compound of formula I and 90mg/kg QD of Kang Naifei Ni. The compound of formula I and Kang Naifei Ni were administered in a volume of 5mL/kg and the BID protocol was 8 hours apart. In the combination group, kang Naifei Ni was administered one hour after QD administration of the compound of formula 1. The study was terminated on day 28 of treatment as defined in the study protocol.

Results

Fig. 13 shows the method in BRAF ^V600E In mutant CRC CDX model WiDr, the individual compounds of formula I are usedGraph of tumor volume for a period of time for the combination treatment of compound, kang Naifei ni alone, compound of formula I and Kang Naifei ni. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 13, in BRAF ^V600E The combination of the compound of formula I and Kang Naifei ni demonstrated superior tumor growth inhibition in mutant CRC CDX model WiDr relative to treatment with the compound of formula I alone or Kang Naifei ni alone.

Example 9-in BRAF ^V600E Combination therapy of a compound of formula I and Kang Naifei Ni in mutant CRC CDX model HT-29

Material

The test preparation of formula 1 was freshly prepared weekly in 100mM acetate buffer carrier and stored under ambient conditions. The combination agent Kang Naifei was freshly prepared weekly in 0.5% cmc and 0.5% tween 80 in vehicle and stored at 2-8 ℃.

Female Balb/c nude mice were purchased from Beijing Vietnam Lihua laboratory animal technologies Co. Prior to starting any experiments, mice were placed in an pathogen free (SPF) environment of an animal feeding facility and allowed to adapt to the new environment for at least 3 days. Mice were 6-8 weeks old at the time of implantation.

All procedures related to animal handling, care and treatment in this study were performed according to protocols and guidelines approved by the committee for animal care and use (IACUC) of the genedesign institution. Animal facilities and procedures are operated according to the standard of care and use guidelines for laboratory animals (NRC, 2011) and are approved by the laboratory animal care assessment and certification association (AAALAC). In particular, all parts of this study conducted at genedesign are in compliance with IACUC review and approval protocols and applicable Standard Operating Procedures (SOP).

Preparation of xenograft models

HT-29 is a human possessing BRAIV 600E mutationCRC-like tumor cell lines. HT-29 cell line was purchased from American type culture Collection (American Type Culture Collection).)CRL-2577 ^TM ). HT-29 cells were cultured in McCoy's 5a medium plus 10% Fetal Bovine Serum (FBS) at 37℃in an atmosphere of 5% CO2 in air. The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation.

HT-29 tumor cells were implanted subcutaneously into mice. Will contain 2 x 10 using a syringe ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reached an average value approaching 200mm3 (ranging from 146 to 259mm 3), tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started on the day of randomized block. The treatment start date is indicated as day 0 of treatment. Mice were dosed by oral administration of a vehicle control solution, 10mg/kg BID of the compound of formula I alone, 30mg/kg QD of the compound of formula I alone, and 90mg/kg QD of Kang Naifei ni alone. The other two groups received combination therapy of the compound of formula I and Kang Naifei Ni, with the first group being administered with 10mg/kg BID of the compound of formula I and 90mg/kg QD of Kang Naifei Ni and the second group being administered with 30mg/kg QD of the compound of formula I and 90mg/kg QD of Kang Naifei Ni. The dosing volume of each compound was 5mL/kg and the BID regimen was 8 hours apart. In the combination group, kang Naifei Ni was administered one hour after the administration of the compound of formula I. The study was terminated on day 28 of treatment as defined in the study protocol.

Results

Fig. 14 shows the method in BRAF ^V600E In mutant CRC CDX model HT-29, a single nucleotide sequence was usedA plot of tumor volumes for compounds of formula I alone, kang Naifei ni alone, and a combination of compounds of formula I and Kang Naifei ni for a period of time. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 14, in BRAF ^V600E The combination of the compound of formula I and Kang Naifei ni demonstrated superior tumor growth inhibition in mutant CRC CDX model HT-29 relative to treatment with the compound of formula I alone or Kang Naifei ni alone.

Example 10-in BRAF ^V600E Combination therapy of a compound of formula I and Kang Naifei Ni in mutant thyroid cancer CDX model BHT-101

Material

Throughout the 20 day administration in mice, a vehicle/control preparation, 100mM acetic acid in deionized water, was prepared, pH adjusted to 4.8-5.0, and stored under ambient conditions.

Preparation of xenograft models

BHT-101 is a human thyroid cancer cell line harboring a BRAFV600E mutation. The BHT-101 cell line was purchased from China academy of sciences cell Bank (Cell Bank of Chinese Academy of Sciences) (initially from DSMZ-German microorganism and cell culture Collection Co., ltd. (DSMZ-German Collection of Microorganisms and Cell Cultures GmbH)). BHT-101 cells were cultured in DMEM medium containing 20% Fetal Bovine Serum (FBS) and supplemented with 1 XGlutamax solution and 1mM sodium pyruvate, at 37℃in air with 5% CO ₂ Is cultured in an atmosphere of (2). The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation.

BHT-101 tumor cells were subcutaneously implanted in mice. Will contain 2 x 10 using a syringe ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reached an average value of 190mm3 (ranging from 146 to 258mm 3), tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started on the day of randomized block. The treatment start date is indicated as day 0 of treatment. Mice were dosed by oral administration of a vehicle control solution, 10mg/kg BID of the compound of formula I alone, 30mg/kg QD of the compound of formula I alone, and 90mg/kg QD of Kang Naifei ni alone. The other two groups received combination therapy of the compound of formula I and Kang Naifei Ni, with the first group being administered with 10mg/kg BID of the compound of formula I and 90mg/kg QD of Kang Naifei Ni and the second group being administered with 30mg/kg QD of the compound of formula I and 90mg/kg QD of Kang Naifei Ni. The dosing volume of each compound was 5mL/kg and the BID regimen was 8 hours apart. In the combination group, kang Naifei Ni was administered one hour after the administration of the compound of formula I. Because of the rapid growth of the tumor, the study terminated on day 20 of treatment, which is earlier than the initial termination day defined in the study protocol. On day 20 of treatment, half of the tumors in the vehicle control group exceeded the tumor volume threshold (2,000 mm 3) obtained according to the IACUC protocol.

Results

Fig. 15 shows the method in BRAF ^V600E Graphs of tumor volumes in mutant thyroid cancer CDX model BHT-101 treated with compound of formula I alone, kang Naifei ni alone, and a combination of compound of formula I and Kang Naifei ni for a period of time. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 15, in BRAF ^V600E The combination of the compound of formula I and Kang Naifei ni demonstrated superior tumor growth inhibition in mutant thyroid cancer CDX model BHT-101 relative to treatment with the compound of formula I alone or Kang Naifei ni alone.

Example 11-in BRAF ^V600E Combination therapy of a compound of formula I and Kang Naifei Ni in mutant CRC CDX model RKO

Material

Throughout the 16 day administration in mice, a vehicle/control preparation, 100mM acetic acid in deionized water, was prepared, pH adjusted to 4.8-5.0, and stored under ambient conditions.

Preparation of xenograft models

RKO is a human CRC tumor cell line harboring BRAVV 600E mutations. RKO cell lines were purchased from the American type culture Collection @CRL-2577 ^TM ). RKO cells were cultured in medium containing MEM plus 10% Fetal Bovine Serum (FBS) supplemented with nonessential amino acids at 37℃in an atmosphere of 5% CO2 in air. The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation.

RKO tumor cells were subcutaneously implanted into mice. Will contain 2 x 10 using a syringe ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reached an average value of 217mm3 (ranging from 163-262mm 3), tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started on the day of randomized block. The treatment start date is indicated as day 0 of treatment. Mice were dosed by oral administration of a vehicle control solution, 10mg/kg BID of the compound of formula I alone, 30mg/kg QD of the compound of formula I alone, and 90mg/kg QD of Kang Naifei ni alone. The other two groups received combination treatment with the compound of formula I, wherein the first group was dosed with 10mg/kg BID of the compound of formula I and 90mg/kg QD of Kang Naifei Ni, and the second group was dosed with 30mg/kg QD of the compound of formula I and 90mg/kg QD of Kang Naifei Ni. The dosing volume of each compound was 5mL/kg and the BID regimen was 8 hours apart. In the combination group, kang Naifei Ni was administered one hour after the QD dose of the compound of formula I. Because of the rapid growth of the tumor, the study terminated on day 16 of treatment, which is earlier than the initial termination day defined in the study protocol. On day 16 of treatment, the majority of tumors in the vehicle control group exceeded the tumor volume threshold (2,000 mm 3) obtained according to the IACUC protocol.

Results

Fig. 16 shows the method in BRAF ^V600E Graphs of tumor volumes in mutant CRC CDX model RKO treated with compound of formula I alone, kang Naifei Ni alone, and a combination of compound of formula I and Kang Naifei Ni for a period of time. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 16, in BRAF ^V600E In mutant CRC CDX model RKO, the combination of the compound of formula I and Kang Naifei Ni demonstrated superior tumor growth inhibition relative to treatment with the compound of formula I alone or Kang Naifei Ni alone.

Example 12 synergistic combinations of Compounds of formula I and MEK inhibitors

This example demonstrates the synergistic combination of a compound of formula I with a MEK inhibitor.

Cell proliferation assay

Cells (2000 cells per well) were plated on 96-well plates in 100 μl of cell culture medium and treated with either compound of formula I alone or compound of formula I and a fixed concentration of trimetinib or bemetinib. On day 5, 50 μl CellTiter-Glo (CTG) reagent (Promega) was added and the plates incubated under gentle shaking for 10 minutes. After incubation for 10 minutes, the luminescence signal was determined according to the manufacturer's instructions (Promega) and a chart was drawn using Prism GraphPad.

Combined cell proliferation assay

Cells (2000 cells per well) were plated on 96-well plates in 100 μl of cell culture medium. Cells were treated with compounds of formula I and trimetinib or bemetinib at concentrations varying from 0 to 10 μm by using a Tecan D300e digital dispenser combination matrix protocol. On day 5, 50 μl CellTiter-Glo (CTG) reagent (Promega) was added and the plates incubated under gentle shaking for 10 minutes. After incubation for 10 minutes, the luminescence signal was determined according to the manufacturer's instructions (Promega) and combined data was generated by standard HSA model using combeneflit software. The combined synergy is represented by a positive number in the results table. Negative numbers represent antagonism of the combination.

Western blot of pERK and pERK

NCI-H508 cells were treated with the compounds for 4 hours. After treatment, cells were lysed with Thermo Fisher RIPA lysis buffer with protease and phosphatase inhibitors for 10 min on ice. Cells were centrifuged at 4℃for 10 min with a microcentrifuge. The supernatant was transferred to a pre-chilled microcentrifuge tube and the protein concentration of the lysate was measured using BCA method. Immunoblots were performed against pERK and total ERK using equal amounts of protein cell lysate supernatant.

MeWo cells were treated with the compound for 4 hours. After treatment, cells were lysed with Thermo Fisher RIPA lysis buffer with protease and phosphatase inhibitors for 10 min on ice. Cells were centrifuged at 4℃for 10 min with a microcentrifuge. The supernatant was transferred to a pre-chilled microcentrifuge tube and the protein concentration of the lysate was measured using BCA method. Immunoblots were performed against pERK and total ERK using equal amounts of protein cell lysate supernatant.

Results

Figure 17A shows synergy data in NCI-H508 cancer cell lines using a combination of a compound of formula I and trimetinib. Fig. 17B shows synergy data in NCI-H508 cancer cell lines using a combination of a compound of formula I and bemetinib. Figure 17C is graphical synergy data in NCI-H1666 cancer cell lines using a combination of a compound of formula I and trimetinib. Fig. 17D shows synergy data in NCI-H1666 cancer cell lines using a combination of a compound of formula I and bemetinib.

Fig. 18A shows synergy data in MeWo cancer cell lines using a combination of a compound of formula I and trimetinib. Fig. 18B shows synergy data in MeWo cancer cell lines using a combination of a compound of formula I and bemetinib. Fig. 18C shows synergy data in NCI-H1838 cancer cell lines using a combination of a compound of formula I and trimetinib. Fig. 18D shows synergy data in NCI-H1838 cancer cell lines using a combination of a compound of formula I and bemetinib.

Figure 19A shows a graph of percent activity versus inhibitor concentration (log M) in NCI-H508 cells treated with compounds of formula I alone and in combination with bemetinib. List of NCI-H508 cells treated with compounds of formula I alone and in combination with bemetinib ₅₀ Data. Fig. 19B shows a graph of percent activity versus inhibitor concentration (log M) in MeWo cells treated with compounds of formula I alone and in combination with bemetinib. Table IC50 data for MeWo cells treated with compounds of formula I alone and in combination with bemetinib.

FIG. 20A shows Western blot gel demonstrating synergistic inhibition of ERK1/2 phosphorylation in NCI-H508 cancer cell lines. Fig. 20B shows bar graph quantification of western blot of fig. 20A. FIG. 20C shows Western blot gels demonstrating synergistic inhibition of ERK1/2 phosphorylation in MeWo (NF 1 LoF) cancer cell lines. Fig. 20D shows bar graph quantification of western blot of fig. 20C.

Fig. 21A shows synergy data in NCI-H2009 (KRAS G12A) cancer cell lines using a combination of a compound of formula I and trimetinib. Fig. 21B shows synergy data in LS513 (KRAS G12D) cancer cell line using a combination of a compound of formula I and trimetinib. Fig. 21C shows synergy data in an a549 (KRAS G12S) cancer cell line using a combination of a compound of formula I and trimetinib. Fig. 21D shows synergy data in NCI-H727 (KRAS G12V) cancer cell lines using a combination of a compound of formula I and trimetinib.

Fig. 22A shows synergy data in NCI-H2009 (KRAS G12A) cancer cell lines using a combination of a compound of formula I and bemetinib. Fig. 22B shows synergy data in LS513 (KRAS G12D) cancer cell lines using a combination of a compound of formula I and bemetinib. Fig. 22C shows synergy data in an a549 (KRAS G12S) cancer cell line using a combination of a compound of formula I and bemetinib. Fig. 22D shows synergy data in NCI-H727 (KRAS G12V) cancer cell lines using a combination of a compound of formula I and bemetinib.

Fig. 23A shows a graph of percent activity versus inhibitor concentration (log M) in LS513 (KRAS G12D) cells treated with a compound of formula I alone and in combination with trimetinib. Fig. 23B shows a graph of percent activity versus inhibitor concentration (log M) in NCI-H2009 (KRAS G12D) cells treated with a compound of formula I alone and in combination with trimetinib. List data for NCI-H508 cells treated with compounds of formula I alone and in combination with trimetinib. Fig. 23C shows a bar graph of CTG activity percentage indicating that either formula I or trimetinib alone had minimal effect on cell viability. Taken together, the data demonstrate that the combination of the compound of formula I and the MEK inhibitor provides synergistic inhibition of cancer cell viability in BRAF class III, NF1 LoF and KRAS G12X mutant cancers.

Example 13-combination therapy of Compounds of formula I and trametinib in the NF1 LoF mutant melanoma CDX model MeWo

Material

Test preparations of the compounds of formula I were freshly prepared weekly in 100mM acetate buffer vehicle and stored under ambient conditions. The combination agent trametinib was freshly prepared weekly in 0.5% hpmc and 0.2% tween 80 in a vehicle and stored under ambient conditions.

Preparation of xenograft models

MeWo is a human melanoma cell line possessing NF 1Q 1336 mutations. MeWo cell line was purchased from american type culture collection @HTB-65 ^TM ). MeWo cells were cultured in medium containing Minimal Essential Medium (MEM) plus 10% Fetal Bovine Serum (FBS), 1% nonessential amino acids (NEAA) and 1% antibiotic-Antifungal Agent (AA) at 37 ℃ in air at 5% co ₂ Is cultured in an atmosphere of (2). The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation.

MeWo tumor cells were implanted subcutaneously into mice. Will contain 5 x 10 using a syringe ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reached an average value of 191mm3 (ranging from 150 to 242mm 3), tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started the following day after the randomized group. The treatment start date is indicated as treatment day 1. Mice were dosed by oral administration of vehicle control, 10 mg/kg/dose BID of the compound of formula I alone, 30mg/kg QD of the compound of formula I alone, and 0.4mg/kg QD of trimetinib alone. Two groups received combination therapy of the compound of formula I and trimetinib, one group being administered with 10 mg/kg/dose of the compound of formula I of BID and the other group being administered with 30mg/kg of the compound of formula I of QD. Both combination groups were dosed with 0.4mg/kg QD of trimetinib. The dosing volume was 5mL/kg and the BID protocol was 8 hours apart. In the combination group, trimetinib is administered one hour after the first dose of the compound BID or QD regimen of formula I.

Results

Fig. 24 shows a graph of tumor volumes treated with a compound of formula I alone, trimetinib alone, and a combination of a compound of formula I and trimetinib for a period of time in NF1 LoF mutant melanoma CDX model MeWo. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 24, the combination of the compound of formula I and trimetinib exhibited superior tumor growth inhibition in NF1 LoF mutant melanoma CDX model MeWo relative to treatment with the compound of formula I alone or treatment with trimetinib alone.

Example 14-combination therapy of a Compound of formula I and Bemetinib in the NF1 LoF mutant melanoma CDX model MeWo

Material

Test preparations of the compounds of formula I were freshly prepared weekly in 100mM acetate buffer vehicle and stored under ambient conditions. The combination drug bemetinib was freshly prepared weekly in 1.0% mc and 0.5% tween 80 in a vehicle and stored at 2-8 ℃.

All procedures related to animal handling, care and treatment in this study were performed according to guidelines approved by the committee for animal care and use (IACUC) of the genedesign institution. During the course of the study, care and use of animals was performed as prescribed by the laboratory animal care assessment and certification association (AAALAC). Furthermore, all parts of this study conducted at genedesign followed the study protocols approved by the study taker and the applicable Standard Operating Procedure (SOP).

Preparation of xenograft models

MeWo is a human melanoma cell line possessing NF 1Q 1336 mutations. MeWo cell line was purchased from american type culture collection @HTB-65 ^TM ). MeWo cells were cultured in medium containing Minimal Essential Medium (MEM) plus 10% Fetal Bovine Serum (FBS), 1% nonessential amino acids (NEAA) and 1% antibiotic-Antifungal Agent (AA) at 37 ℃ in an atmosphere of 5% co2 in air. The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation.

MeWo tumor cells were implanted subcutaneously into mice. In brief, using a syringe will contain 5X 10 ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reached an average value of 195mm3 (range 141-267mm 3), tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started the following day after the randomized group. The treatment start date is indicated as treatment day 1. Mice were dosed with vehicle control solution, 15mg/kg QD of the compound of formula I alone, 30mg/kg QD of the compound of formula I alone, 6mg/kg BID of bemetinib alone, and 9 mg/kg/dose BID of bemetinib alone by oral administration. Two other groups received combination treatment of the compound of formula I and bemetinib, one group being dosed with 15mg/kg of the compound of formula I and 6mg/kg of BID of bemetinib, and the other group being dosed with 15mg/kg of the compound of formula I and 9 mg/kg/dose of BID of bemetinib. The dosing volume was 5mL/kg and the BID protocol was 8 hours apart. In the combination group, bemetinib was administered one hour after QD dosing of the compound of formula I. The study was terminated on day 28 of treatment as defined in the study protocol.

Results

Fig. 25 shows a graph of tumor volumes treated with a compound of formula I alone, bemetinib alone, and a combination of a compound of formula I and bemetinib for a period of time in NF1 LoF mutant melanoma CDX model MeWo. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 25, the combination of the compound of formula I and bemetinib exhibited superior tumor growth inhibition in NF1 LoF mutant melanoma CDX model MeWo relative to treatment with the compound of formula I alone or bemetinib alone.

Example 15-combination therapy of Compounds of formula I and Bemetinib in BRAF class III mutant CRC CDX model NCI-H508

Material

Preparation of xenograft models

NCI-H508 is a human CRC cell line with a BRAF class III mutation (BRAF G596R). NCI-H508 cell line was purchased from the American type culture Collection @CCL-253 ^TM ). NCI-H508 cells were cultured in medium containing RPMI-1640 plus 10% Fetal Bovine Serum (FBS) and 1% antibiotic-antifungal (AA) at 37 ℃ in an atmosphere of 5% co2 in air. The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation.

NCI-H508 tumor cells were subcutaneously implanted in mice. In brief, using a syringe will contain 10X 10 ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reached an average value of 182mm3 (range 108-287mm 3), tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started the following day after the randomized group. The treatment start date is indicated as treatment day 1. Mice were dosed by oral administration of vehicle control, 10mg/kg BID of the compound of formula I alone, 30mg/kg QD of the compound of formula I alone, and 0.4mg/kg QD of trimetinib alone. Two groups received combination therapy of a compound of formula I and trimetinib, one group being administered with 10mg/kg BID of a compound of formula I and 0.4mg/kg QD of trimetinib, and the other group being administered with 30mg/kg QD and 0.4mg/kg QD of trimetinib. The dosing volume was 5mL/kg and the BID protocol was 8 hours apart. In the combination group, trimetinib is administered one hour after the first dose of the compound BID or QD administration of formula (la).

Results

Fig. 26 shows a graph of tumor volumes treated with a compound of formula I alone, trimetinib alone, and a combination of a compound of formula I and trimetinib for a period of time in the BRAF class III mutant CRC CDX model NCI-H508. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 26, the combination of the compound of formula I and trimetinib exhibited superior tumor growth inhibition in the BRAF class III mutant CRC CDX model NCI-H508 relative to treatment with the compound of formula I alone or treatment with trimetinib alone.

Example 16-combination therapy of Compounds of formula I and trametinib in the NF1 LoF mutant NSCLC CDX model NCI-H1838

Material

Female SCID Beige mice (Cat # 405) were purchased from beijing villous laboratory animal technologies limited. Mice were 6-8 weeks old at the time of implantation. According to the IACUC protocol, mice are placed in an pathogen free (SPF) environment of an animal feeding facility and allowed to adapt to the new environment for at least 3 days prior to starting any experiments.

Preparation of xenograft models

NCI-H1838 is a human lung adenocarcinoma cell line harboring the NF1LOF mutation NF 1N 184 fs. NCI-H1838 cell line was purchased from the American type culture Collection @CRL-5899 ^TM ). NCI-H1838 cells were cultured in a medium containing RPMI-1640 plus 10% Fetal Bovine Serum (FBS) and 1% antibiotic-antifungal (AA) at 37 ℃ in an atmosphere of 5% co2 in air. The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation.

NCI-H1838 tumor cells were subcutaneously implanted into mice. In brief, using a syringe will contain 10X 10 ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When the tumor volume reached an average value of 254mm3 (range 149-503mm 3), tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started the following day after the randomized group. The treatment start date is indicated as treatment day 1. Mice were dosed by oral administration of vehicle control, 10mg/kg BID of the compound of formula I alone, 30mg/kg QD of the compound of formula I alone, and 0.4mg/kg QD of trimetinib alone. Two groups received combination treatment of the compound of formula I and trimetinib, one group was dosed with 10mg/kg BID of the compound of formula I and 0.4mg/kg QD of trimetinib, and the other group was dosed with 30mg/kg QD of the compound of formula I and 0.4mg/kg QD of trimetinib. The dosing volume was 5mL/kg and the BID protocol was 8 hours apart. In the combination group, trametinib is administered one hour after the first dose of the compound of formula I or QD administration.

Results

Figure 27 shows a graph of tumor volumes treated with a compound of formula I alone, trimetinib alone, and a combination of a compound of formula I and trimetinib for a period of time in NF1 LoF mutant NSCLC CDX model NCI-H1838. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 27, the combination of the compound of formula I and trimetinib exhibited superior tumor growth inhibition in NF1 LoF mutant NSCLC CDX model NCI-H1838 relative to treatment with the compound of formula I alone or treatment with trimetinib alone.

Example 17 synergistic combination of a Compound of formula I and a MET inhibitor

Combined cell proliferation assay

Cells (2000 cells per well) were plated on 96-well plates in 100 μl of cell culture medium. Cells were treated with compounds of formula I and crizotinib at concentrations varying from 0 to 10 μm by using a Tecan D300e digital dispenser combinatorial matrix protocol. On day 5, 50 μl CellTiter-Glo (CTG) reagent (Promega) was added and the plates incubated under gentle shaking for 10 minutes. After incubation for 10 minutes, the luminescence signal was determined according to the manufacturer's instructions (Promega) and combined data was generated by standard HSA model using combeneflit software. The combined synergy is represented by a positive number in the results table. Negative numbers represent antagonism of the combination.

Results

Fig. 28A shows synergy data in Hs746T cancer cell lines using a combination of a compound of formula I and crizotinib. Fig. 28B shows synergy data in MKN-45 cancer cell lines using a combination of a compound of formula I and crizotinib. Fig. 28C shows synergy data in EBC-1 cancer cell lines using a combination of a compound of formula I and crizotinib.

Example 18-combination therapy of a Compound of formula I and crizotinib in a c-MET amplified gastric cancer CDX model SNU-5

Material

Test preparations of the compounds of formula I were freshly prepared weekly in 100mM acetate buffer vehicle and stored under ambient conditions. The combination agent crizotinib is prepared in a carrier of 0.5% methylcellulose and stored at 2-8 ℃.

Female Balb/c nude mice were purchased from Beijing Vietnam Lihua laboratory animal technologies Co. Mice were 6-8 weeks old at the time of implantation. According to the IACUC protocol, mice are placed in an pathogen free (SPF) environment of an animal feeding facility and allowed to adapt to the new environment for at least 3 days prior to starting any experiments. All procedures related to animal handling, care and treatment in this study were performed according to guidelines approved by the committee for animal care and use (IACUC) of the tin-free AppTec institution. During the course of the study, care and use of animals was performed as prescribed by the laboratory animal care assessment and certification association (AAALAC). Furthermore, all parts of this study conducted in tin-free AppTec followed the study protocols approved by the study taker and the applicable Standard Operating Procedure (SOP).

Preparation of xenograft models

SNU-5 is a c-MET amplified gastric cancer cell line. SNU-5 cell line was purchased from the American type culture Collection @CRL-5973 ^TM ). SNU-5 cells were cultured in Medium containing IMDM (Iscove's Modified Dulbecco's Medium) plus 20% Fetal Bovine Serum (FBS) and 1% antibiotic-Antifungal Agent (AA) at 37℃in an atmosphere of 5% CO2 in air. The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation. SNU-5 tumor cells (generation 13) were subcutaneously implanted in mice. Will contain 10 x 10 using a syringe ⁶ Subcutaneous implantation of 200 μl of cell suspension of individual tumor cells into right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is palpable and measurable, tumor volume is measured by calipers twice a week. When tumor volumes reached an average of 227mm3 on day 34 after subcutaneous implantation, tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started the following day after the randomized group. The treatment start date is indicated as treatment day 1. Mice were dosed with vehicle control solution, 10mg/kg BID alone, 30mg/kg QD alone, formula I alone, and 50mg/kg BID alone. The other two groups received a combination treatment of the compound of formula I and crizotinib, one group being administered with a combination of 5mg/kg BID of the compound of formula I and 50mg/kg BID of crizotinib, and the other group being administered with a combination of 15mg/kg QD of the compound of formula I and 50mg/kg BID of crizotinib. The dosing volume was 5mL/kg and the BID protocol was 8 hours apart. In the combination group, crizotinib is administered one hour after QD administration of the compound of formula I or after the first dose of the BID dose regimen. The study was terminated on day 28 of treatment as defined in the study protocol.

Results

FIG. 29 shows a graph of tumor volumes treated with a compound of formula I alone, crizotinib alone, and a combination of a compound of formula I and crizotinib for a period of time in a c-MET amplified gastric cancer CDX model SNU-5. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 29, the combination of the compound of formula I and crizotinib exhibited superior tumor growth inhibition in the c-MET amplified gastric cancer CDX model SNU-5 relative to treatment with the compound of formula I alone or with crizotinib alone.

Example 19-combination therapy of Compounds of formula I and crizotinib in the c-MET amplified NSCLC CDX model NCI-H1993

Material

Preparation of xenograft models

NCI-H1993 is a c-MET expanded NSCLC cell line. NCI-H1993 cell line was purchased from the American type culture Collection @CRL-5909 ^TM ). NCI-H1993 cells were cultured in a medium containing RPMI-1640 plus 10% Fetal Bovine Serum (FBS) and 1% antibiotic-Antifungal Agent (AA) at 37℃in an atmosphere of 5% CO2 in air. The medium was changed every 2 to 3 days and tumor cells were routinely subcultured with trypsin-EDTA at 80-90% confluence. Cells in exponential growth phase were harvested and counted for inoculation. NCI-H1993 tumor cells (passage 13) were subcutaneously implanted into mice. Will contain 5 x 10 using a syringe ⁶ 200 μl of cell suspension of individual tumor cells mixed with 50% matrigel was subcutaneously implanted into the right flank of mice. Animals were monitored daily for health and tumor growth. When the tumor is accessible and measurable,tumor volumes were measured by calipers twice a week. When tumor volume reached an average of 201mm3 on day 10 post-subcutaneous implantation, tumor-bearing mice were randomly divided into different groups of 8 mice each. The randomized block date is expressed as day 0 of treatment.

Treatment of

Treatment was started the following day after the randomized group. The treatment start date is indicated as treatment day 1. Mice were dosed with vehicle control solution, 10mg/kg BID alone, 30mg/kg QD alone, formula I alone, and 50mg/kg BID alone. The other group received a combination therapy of 5mg/kg BID of a compound of formula I and 50mg/kg BID of crizotinib. The dosing volume was 5mL/kg and the BID protocol was 8 hours apart. In the combination group, crizotinib is administered one hour after the first dose of BID administration of the compound of formula I. The study was terminated on day 28 of treatment as defined in the study protocol.

Results

FIG. 30 shows a graph of tumor volumes treated with a compound of formula I alone, crizotinib alone, and a combination of a compound of formula I and crizotinib for a period of time in the c-MET amplified NSCLC CDX model NCI-H1993. No significant body weight change was observed in the control and treatment groups.

Conclusion(s)

As shown in fig. 30, the combination of the compound of formula I and crizotinib demonstrated superior tumor growth inhibition in the c-MET amplified NSCLC CDX model NCI-H1993 relative to treatment with the compound of formula I alone or crizotinib alone.

Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety as if each reference were individually incorporated by reference. In the event of conflict between the present disclosure and the references provided herein, the present disclosure will control.

Claims

1. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

And FGFR inhibitors.

2. The method of claim 1, wherein FGFR in the subject is constitutively active.

3. The method of claim 1 or 2, wherein the cancer is lung cancer.

4. The method of claim 1 or 2, wherein the cancer is hepatocellular carcinoma.

5. The method of claim 1 or 2, wherein the cancer is cholangiocarcinoma.

6. The method of claim 1 or 2, wherein the cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

7. The method of any one of claims 1 to 5, wherein the inhibitor is selected from the group consisting of erdastinib, AZD4547, ly2874455, CH5183284, NVP-BGJ398, INCB054828, luo Jiati ni, PRN1371, TAS-120, BLU-554, H3B-6527, and FGF401.

8. The method of any one of claims 1-5, wherein the FGFR inhibitor is erdasatinib.

9. The method of any one of claims 1-5, wherein the FGFR inhibitor is pemitinib, infliximab, duo Wei Tini, panatinib, niladinib, and fexotinib.

10. The method of any one of claims 1 to 9, wherein the method comprises administering a third MAPK pathway inhibitor.

11. The method of any one of claims 1 to 10, wherein the administration is oral.

12. The method according to any one of claims 1 to 11, wherein the amount of the compound of formula I administered is in the range of 20mg to 400mg per day.

13. The method of any one of claims 1-12, wherein the FGFR inhibitor is administered in an amount ranging from 1mg to 500mg per day.

14. A method of treating liver cancer in a subject, the method comprising orally co-administering to the subject a therapeutically effective amount of a compound of formula I:

and erdasatinib.

15. The method of claim 14, wherein the compound of formula I is administered once or twice daily.

16. The method of claim 14 or 15, wherein erdasatinib is administered once or twice daily.

17. The method of claim 14, wherein the subject is a human.

18. A kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and an FGFR inhibitor.

19. The kit of claim 18, wherein the compound of formula I and the FGFR inhibitor are in separate packages.

20. The kit of claim 18 or 19, wherein the kit further comprises instructions for administering the contents of the kit to a subject for cancer treatment.

21. The kit of any one of claims 18-20, wherein the FGFR inhibitor is one or more of erdasatinib, AZD4547, ly2874455, CH5183284, NVP-BGJ398, INCB054828, luo Jiati ni, PRN1371, TAS-120, BLU-554, H3B-6527, FGF401, petomitinib, inflictinib, doxatinib Wei Tini, panatinib, nilaninib, and fexotinib.

22. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and inhibitors of the B-Raf protein with class 1 mutations.

23. The method of claim 22, wherein the class 1 mutation is V600E.

24. The method of claim 22 or 23, wherein the cancer is colorectal cancer.

25. The method of claim 22 or 23, wherein the cancer is melanoma.

26. The method of claim 22 or 23, wherein the cancer is thyroid cancer.

27. The method of claim 22 or 23, wherein the cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

28. The method of any one of claims 22 to 27, wherein the inhibitor is selected from Kang Naifei ni, vitamin Mo Feini, dabrafenib, sorafenib and regorafenib.

29. The method of any one of claims 22 to 27, wherein the inhibitor is Kang Naifei ni.

30. The method of any one of claims 22 to 27, wherein the inhibitor is vitamin Mo Feini.

31. The method of any one of claims 22 to 27, wherein the inhibitor is dabrafenib.

32. The method of any one of claims 22 to 27, wherein the inhibitor is sorafenib.

33. The method of any one of claims 22 to 27, wherein the inhibitor is regorafenib.

34. The method of any one of claims 22-33, wherein the method comprises administering a third MAPK pathway inhibitor.

35. The method of any one of claims 22-34, wherein the administration is oral.

36. The method of any one of claims 22 to 35, wherein the amount of the compound of formula I administered is in the range of 20mg to 400mg per day.

37. The method of any one of claims 22 to 36, wherein the B-Raf inhibitor is administered in an amount ranging from 1mg to 500 mg.

38. A method of treating colorectal cancer in a subject, the method comprising orally co-administering to the subject a therapeutically effective amount of a compound of formula I:

And B-Raf inhibitor Kang Naifei.

39. The method of claim 38, wherein the compound of formula I is administered once or twice daily.

40. The method of claim 38 or 39, wherein Kang Naifei ni is administered once or twice daily.

41. The method of any one of claims 38 to 40, wherein the subject is a human.

42. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and Kang Naifei Ni.

43. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and dimension Mo Feini.

44. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and dabrafenib.

45. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and sorafenib.

46. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and regorafenib.

47. The method of any one of claims 42 to 46, wherein the cancer is colorectal cancer.

48. The method of any one of claims 42 to 46, wherein the cancer is thyroid cancer.

49. The method of any one of claims 42 to 46, wherein the cancer is melanoma.

50. The method of any one of claims 42 to 46, wherein the cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

51. The method of any one of claims 22 to 50, wherein the amount of B-Raf inhibitor administered is less than the amount of B-Raf inhibitor administered required for monotherapy with the B-Raf inhibitor.

52. The method of any one of claims 22 to 51, wherein the amount of the compound of formula I administered is less than the amount of the compound of formula I administered required for monotherapy with the compound of formula I.

53. A method of inhibiting ERK1/2 phosphorylation in a cell population, the method comprising contacting the cell population with a compound of formula I:

And regorafenib in combination.

54. The method of claim 53, wherein the concentration of the compound of formula I is in the range of 1nM to 500 nM.

55. The method of claim 53 or 54, wherein the concentration of Kang Naifei n is in the range of 10nM to 20 nM.

56. A kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a B-Raf inhibitor.

57. The kit of claim 56, wherein the compound of formula I and the B-Raf inhibitor are in separate packages.

58. The kit of claim 56 or 57, wherein the kit further comprises instructions for administering the contents of the kit to a subject for cancer treatment.

59. The kit of any one of claims 56-58, wherein the B-Raf inhibitor is one or more of Kang Naifei ni, vitamin Mo Feini, dabrafenib, sorafenib and regorafenib.

60. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and MEK inhibitors.

61. The method of claim 60, wherein the MEK inhibitor selectively inhibits MEK1 or selectively inhibits MEK2 or selectively inhibits both MEK1 and MEK 2.

62. The method of claim 60, wherein the cancer is metastatic.

63. The method of any one of claims 60-62, wherein the cancer is colorectal cancer.

64. The method of any one of claims 60-62, wherein the cancer is melanoma.

65. The method of any one of claims 60-62, wherein the cancer is lung cancer.

66. The method of any one of claims 60-62, wherein the cancer is pancreatic cancer.

67. The method of any one of claims 60-62, wherein the cancer is breast cancer.

68. The method of any one of claims 60-62, wherein the cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

69. The method of any one of claims 60 to 68, wherein the MEK inhibitor is selected from the group consisting of trametinib, cobicitinib, bemetinib, PD-0325901, semetinib, and CI-1040.

70. The method of any one of claims 60-68, wherein the MEK inhibitor is trametinib.

71. The method of any one of claims 60-68, wherein the MEK inhibitor is cobicitinib.

72. The method of any one of claims 60-68, wherein the MEK inhibitor is bemetinib.

73. The method of any one of claims 60-68, wherein the MEK inhibitor is PD-325901.

74. The method of any one of claims 60-68, wherein the MEK inhibitor is CI-1040.

75. The method according to any one of claims 60 to 74, wherein the method comprises administering another MAPK pathway inhibitor.

76. The method of any one of claims 60-75, wherein the administration is oral.

77. The method of any one of claims 60 to 76, wherein the amount of the compound of formula I administered is in the range of 20mg to 400mg per day.

78. The method of any one of claims 60-77, wherein the amount of MEK inhibitor administered is in the range of 1mg to 500mg per day.

79. A method of treating cancer in a subject, the method comprising orally co-administering to the subject a therapeutically effective amount of a compound of formula I:

and the MEK inhibitor bemetinib or trametinib.

80. The method of claim 79, wherein the compound of formula I is administered once or twice daily.

81. The method of claim 79 or 80, wherein bemetinib or Qu Meiti ni is administered once or twice daily.

82. The method of any one of claims 79 to 81, wherein the subject is a human.

83. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and bemetinib.

84. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

and trametinib.

85. The method of claim 83 or 84, wherein the cancer is colorectal cancer.

86. The method of claim 83 or 84, wherein the cancer is lung cancer.

87. The method of claim 83 or 84, wherein the cancer is melanoma.

88. The method of claim 83 or 84, wherein the cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

89. The method of any one of claims 60-88, wherein the amount of MEK inhibitor administered is less than the amount of MEK inhibitor administered required for monotherapy with the MEK inhibitor.

90. The method of any one of claims 60-89, wherein the amount of the compound of formula I administered is less than the amount of the compound of formula I administered required for monotherapy with the compound of formula I.

91. A method of inhibiting ERK1/2 phosphorylation, the method comprising contacting a population of cells with formula I:

and bemetinib or trimetinib in combination.

92. The method of claim 91, wherein the concentration of the compound of formula I is in the range of 1nM to 1,000 nM.

93. The method of claim 91 or 92, wherein the concentration of the MEK inhibitor is in the range of 10nM to 500 nM.

94. A kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a MEK inhibitor.

95. The kit of claim 94, wherein the compound of formula I and the MEK inhibitor are in separate packages.

96. The kit of claim 94 or 95, wherein the kit further comprises instructions for administering the contents of the kit to a subject for cancer treatment.

97. The kit of any one of claims 94-96, wherein the MEK inhibitor is one or more of trametinib or bemetinib.

98. A method of treating a subject having cancer, the method comprising co-administering to the subject a therapeutically effective amount of a compound of formula I:

And MET inhibitors.

99. The method of claim 98, wherein the MET inhibitor is also an ALK inhibitor, a ROS1 inhibitor, or both.

100. The method of claim 98 or 99, wherein the cancer is non-small cell lung cancer.

101. The method of claim 98 or 99, wherein the cancer is gastric cancer.

102. The method of claim 98 or 99, wherein the cancer is gastric adenocarcinoma.

103. The method of claim 98 or 99, wherein the cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

104. The method of any one of claims 98-103, wherein the MET inhibitor is selected from the group consisting of crizotinib, teposinib, sivoratinib, cabozatinib, and tivanitinib.

105. The method of any one of claims 98-103, wherein the MET inhibitor is crizotinib.

106. The method of any one of claims 98-103, wherein the MET inhibitor is teposinib.

107. The method of any one of claims 98-103, wherein the MET inhibitor is sivoratinib.

108. The method of any one of claims 98-103, wherein the MET inhibitor is cabozantinib.

109. The method of any one of claims 98-103, wherein the MET inhibitor is tivantinib.

110. The method of any one of claims 98-109, wherein the method comprises administering a third MAPK pathway inhibitor.

111. The method of any one of claims 98-110, wherein the administration is oral.

112. The method of any one of claims 98-111, wherein the amount of the compound of formula I administered is in the range of 10mg to 500mg per day.

113. The method of any one of claims 98 to 112, wherein the inhibitor is administered in an amount ranging from 20mg to 400mg per day.

114. A method of treating gastric cancer in a subject, the method comprising orally co-administering to the subject a therapeutically effective amount of a compound of formula I:

and crizotinib.

115. The method of claim 114, wherein the compound of formula I is administered once or twice daily.

116. The method of claim 114 or 115, wherein crizotinib is administered once or twice daily.

117. The method of any one of claims 114-116, wherein the subject is a human.

118. A kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a MET inhibitor.

119. The kit of claim 118, wherein the compound of formula I and the MET inhibitor are in separate packages.

120. The kit of claim 118 or 119, wherein the kit further comprises instructions for administering the contents of the kit to a subject for cancer treatment.

121. The kit of any one of claims 118-120, wherein the MET inhibitor is one or more of crizotinib, teposinib, sivoratinib, cabozatinib, and tivanitinib.

122. The method of any one of claims 1-121, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is formulated into a pharmaceutical composition.

123. The method of any one of claims 1-122, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is formulated as an oral composition.

124. The method of any one of claims 1-123, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered once or twice a day.

125. The method of any one of claims 1 to 124, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 28 consecutive days.

126. The method of any one of claims 1-125, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in an amount of about 10mg to about 140mg once a day.

127. The method of any one of claims 1-126, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered once a day for a period of 3 weeks, comprising administering the compound for 2 weeks, followed by not administering the compound for 1 week.

128. The method of any one of claims 1-126, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered once a day for a period of 4 weeks, comprising administration of the compound for 3 weeks followed by no administration of the compound for 1 week.

129. The method of any one of claims 1 to 128, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 6 weeks.

130. The method of any one of claims 1 to 128, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 8 weeks.

131. The method of any one of claims 1-130, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered 3 times a week.

132. The method of claim 131, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered on days 1, 3, and 5 of the week.

133. The method of any one of claims 1-132, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered 4 times a week.

134. The method of claim 133, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered for a period of 3 weeks comprising administering the compound for 2 weeks followed by not administering the compound for 1 week.

135. The method of claim 133, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered for a period of 4 weeks, comprising administering the compound for 3 weeks, followed by not administering the compound for 1 week.

136. The method of any one of claims 1-125, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered twice a day, two days a week.

137. The method of any one of claims 1-126, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered over a period of 8 weeks.

138. The method of claim 136 or 137, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered on days 1 and 2 of the week.

139. The method of any one of claims 1-138, wherein the cancer is selected from lung cancer, stomach cancer, liver cancer, colon cancer, kidney cancer, breast cancer, pancreatic Ductal Adenocarcinoma (PDAC), juvenile myelomonocytic leukemia, neuroblastoma, melanoma, and acute myelogenous leukemia.