Combination of CHEK1 and TOP1 inhibitors for treating colorectal cancer

Abstract

The present invention relates to a combination therapy of a CHEK1 inhibitor and a TOP1 inhibitor for use in a method of treating colorectal cancer in a patient.

Description

[Technical field]

[0001] The present invention relates to combination therapies for the treatment of cancer. [Background technology]

[0002] Despite advances in treatment, cancer continues to have a significant impact on societies, families and individuals worldwide. It is one of the leading causes of death worldwide. According to statistics provided by the National Cancer Institute, there were 18.1 million new cases and 9.5 million cancer-related deaths worldwide in 2018. By 2040, the number of new annual cancer cases is projected to rise to 29.5 million and the number of cancer-related deaths to 16.4 million. Bowel cancer is one of the most common types of cancer.

[0003] Cancer treatment has improved with the use of single-agent targeted therapy in patients with molecularly defined tumors. Nevertheless, many patients still lack effective treatments, and existing or acquired resistance limits the clinical benefit of even our most advanced medicines. Combination therapy using an increasing number of targeted anticancer drugs has the potential to overcome resistance to existing drugs, enhance response, reduce dose-limiting toxicity of single agents, and expand the scope of treatment for patients. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention is directed to the use of a therapeutic combination of active ingredients for the treatment of intestinal cancer in a patient, and in particular to the combination of a CHEK1 inhibitor with a TOP1 inhibitor, preferably camptothecin. As described herein, the inventors have observed synergistic effects for the combination of a CHEK1 inhibitor with a TOP1 inhibitor in cancer cell lines. [Means for solving the problem]

[0005] In a first aspect, the present invention can provide a combination of a CHEK1 inhibitor and a TOP1 inhibitor for use in a method of treating colon cancer in a patient. The TOP1 inhibitor irinotecan is of considerable interest for the treatment of colon cancer, but unfortunately is often associated with severe toxicity, particularly neutropenia and diarrhea. The data generated by the present inventors and described herein suggests that the combination of irinotecan with a CHEK1 inhibitor may facilitate the use of irinotecan in a dose-reduced or fractional dose approach, allowing access to irinotecan-based therapy for colon cancer patients for whom existing camptothecin-based regimens have been discontinued or are contraindicated. Thus, the present invention can provide a camptothecin derivative (e.g., irinotecan) for use in a method of treating cancer in a patient, where the camptothecin derivative is administered in a dose-reduced regimen together with the administration of a CHEK1 inhibitor. "Dose sparing" in this context is understood to refer to administration of a camptothecin derivative at a lower total dose than that used in previously approved or tested treatment regimens.

[0006] The inventors further reason that the combination may lead to better responses, particularly greater tumor regressions and / or increased survival rates at currently used clinical doses.

[0007] In some cases, the invention may provide a CHEK1 inhibitor for use in a method of treating colorectal cancer in a patient, wherein the CHEK1 inhibitor is administered to the patient in combination with a TOP1 inhibitor, and the TOP1 inhibitor is a camptothecin derivative.

[0008] In some cases, the present invention may provide a TOP1 inhibitor for use in a method of treating colorectal cancer in a patient, wherein the TOP1 inhibitor is administered to the patient in combination with a CHEK1 inhibitor, and the TOP1 inhibitor is a camptothecin derivative.

[0009] The inventors have identified the combination of a TOP1 inhibitor and a CHEK1 inhibitor as a potent combination in microsatellite stable (MSS) and / or KRAS-TP53 double mutant colon cancer cells, driving cell apoptosis and enhancing responses compared to single-agent irinotecan.

[0010] Thus, in some embodiments, the colon cancer is a KRAS-TP53 double mutant colon cancer. The cancer may be, but is not necessarily, microsatellite stable (i.e., the cancer may be microsatellite unstable).

[0011] In some embodiments, the colorectal cancer is microsatellite stable. The cancer may be, but is not necessarily, a KRAS-TP53 double mutant colorectal cancer.

[0012] In some embodiments, the colorectal cancer is KRAS-TP53 double mutant and microsatellite stable colorectal cancer.

[0013] Preferably, the TOP1 inhibitor is a camptothecin, for example selected from irinotecan, SN-38, topotecan and camptothecin. A preferred TOP1 inhibitor is irinotecan or an active metabolite thereof. SN-38 is an active metabolite of irinotecan. That is, SN-38 is a preferred CHEK1 inhibitor. Another preferred CHEK1 inhibitor is camptothecin.

[0014] In some embodiments, the CHEK1 inhibitor is selected from rabusertib, SAR-020106, AZD7762, prexasertib, MK-8776, CCT245737, CHIR-124, PF-477736, VX-803, GDC-0575, ESP-01, and BEBT-260. In some embodiments, the CHEK1 inhibitor is selected from rabusertib, SAR-020106, AZD7762, prexasertib, and MK-8776. A preferred CHEK1 inhibitor is rabusertib.

[0015] It is understood that the CHEK1 inhibitor and the TOP1 inhibitor can be administered together (concurrently) or separately, and can be administered at the same time or at different times.For example, the compounds can be administered on different days as part of a treatment cycle or treatment regimen.Preferably, but not necessarily, the CHEK1 inhibitor and the TOP1 inhibitor are formulated separately.For example, each or any compound can be formulated for oral or parenteral administration.

[0016] The claimed combination therapies can be used for both curative and symptomatic (palliative) purposes. These combination therapies may lead to better patient outcomes and / or experiences when compared to other treatment regimens and may additionally or alternatively expand the treatment options available to patients.

[0017] Suitably, the patient may be a human patient.

[0018] The present invention also relates to a method of treating colon cancer in a patient in need of such treatment, comprising administering to the patient a combination of an effective amount of a CHEK1 inhibitor and an effective amount of a TOP1 inhibitor, wherein the TOP1 inhibitor is a camptothecin derivative.

[0019] The present invention also relates to a method of treating colon cancer in a patient in need of such treatment, comprising administering to the patient an effective amount of a CHEK1 inhibitor in combination with an effective amount of a TOP1 inhibitor, wherein the TOP1 inhibitor is a camptothecin derivative.

[0020] The present invention also relates to a method of treating colon cancer in a patient in need of such treatment, comprising administering to the patient an effective amount of a TOP1 inhibitor in combination with an effective amount of a CHEK1 inhibitor, wherein the TOP1 inhibitor is a camptothecin derivative.

[0021] The present invention also relates to the use of a combination of a CHEK1 inhibitor and a TOP1 inhibitor in the manufacture of a medicament for the treatment of colon cancer in a patient, wherein the TOP1 inhibitor is a camptothecin derivative.

[0022] The present invention also relates to the use of a CHEK1 inhibitor in combination with a TOP1 inhibitor in the manufacture of a medicament for the treatment of colon cancer in a patient, wherein the TOP1 inhibitor is a camptothecin derivative.

[0023] The present invention also relates to the use of a TOP1 inhibitor in combination with a CHEK1 inhibitor in the manufacture of a medicament for the treatment of colon cancer in a patient, wherein the TOP1 inhibitor is a camptothecin derivative.

[0024] The present invention includes combinations of the described embodiments and preferred features except where such combinations are clearly unacceptable or explicitly avoided.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings. [Brief description of the drawings]

[0026] [Figure 1]Graphs showing synergy and activity of camptothecin and AZD7762 combinations in (A) colon cancer cells and (B) different colon cancer populations. See Example 1 for further details. [Diagram 2] Graphs showing synergy and activity of the reference combination AZD7762 and 5-FU in colon cancer cells. (A) compares MSI and MSS populations. (B) shows the effect of TP53 mutations in KRAS mutant populations. For further details, see Example 2. [Diagram 3] Graph showing ΔEmax and ΔIC50 values ​​of CHEK inhibitors in combination with camptothecin. See Example 3 for further details. [Figure 4-1] Figure 1 shows siRNA and Western blot results for combinations of CHEK inhibitors and camptothecin. (A) Pooled siRNA against CHEK1, CHEK2 or PLK1 (cell death positive control) + / - SN-38 (TOP1). Readout: viability (CTG), (B) Western blot of (A) confirming knockdown of CHEK1 and CHEK2, (C) Individual siRNA against CHEK1 (cell death positive control) + / - SN-38 (TOP1). Readout: viability (CTG), (D) Western blot of (C) confirming knockdown of CHEK1. See Example 3 for further details. [Figure 4-2] Figure 1 shows siRNA and Western blot results for combinations of CHEK inhibitors and camptothecin. (A) Pooled siRNA against CHEK1, CHEK2 or PLK1 (cell death positive control) + / - SN-38 (TOP1). Readout: viability (CTG), (B) Western blot of (A) confirming knockdown of CHEK1 and CHEK2, (C) Individual siRNA against CHEK1 (cell death positive control) + / - SN-38 (TOP1). Readout: viability (CTG), (D) Western blot of (C) confirming knockdown of CHEK1. See Example 3 for further details. [Diagram 5]Figure 1 shows colony formation for the claimed combinations and compounds used individually, see Example 4 for further details. [Figure 6] Figure 13 is a snapshot of cell death signals recorded at 72h for the combination of ravusertib and TOP1i. For further details see Example 5. [Figure 7] Figure 1 shows PARP cleavage and cell death for the combination of ravusertib and TOP1i in SW837 and SNU-81 cells, see Example 6 for further details. [Figure 8-1] (A) Graph showing the change in tumor volume over time compared to single agents irinotecan and ravusertib. (B) Graph showing the change in tumor volume at the endpoint. (C) Graph showing the survival rate after treatment of SNU-81-implanted mice. For further details, see Example 7. [Figure 8-2] (A) Graph showing the change in tumor volume over time compared to single agents irinotecan and ravusertib. (B) Graph showing the change in tumor volume at the endpoint. (C) Graph showing the survival rate after treatment of SNU-81-implanted mice. For further details, see Example 7. [Figure 9] Graph showing the effect of CHEK1i and TOPi on DNA double strand breaks, apoptosis and proliferation in tumors. See Example 8 for further details. [Figure 10] Graph showing the effect of siCHEK1 on the IC50 of SN-38. See Example 9 for further details. [Figure 11] 1 is a graph showing sensitivity of colon cancer cell lines to CHEK1 and topoisomerase inhibitor combinations. Colon cancer cell lines were treated with topoisomerase inhibitor SN-38 plus CHEK1 inhibitors as indicated in each dot plot. Synergy metrics, Bliss windows, and HSA windows were generated as described in Example 10. Each point is a single replicate, with 5-8 replicates per cell line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0028] CHEK1 inhibitors Checkpoint kinase 1 is commonly referred to as CHEK1 in the literature. Checkpoint kinases (CHEKs) are involved in DNA damage responses in many cancer cells, and CHEK1 inhibition is associated with slowed DNA replication, collapse of replication forks, and accumulation of DNA damage.

[0029] The term CHEK1 inhibitor (also called CHEK1i) refers to a CHEK1 inhibitor with a half-maximal inhibitory concentration (IC) of <15 nM in cell-free assays, e.g., <10 nM. 50 In some embodiments, a CHEK1 inhibitor refers to a compound that has a half maximal inhibitory concentration (IC) of <15 μM in a cell-based assay, e.g., <10 μM. 50 ) and induces cell cycle arrest, inhibits phosphorylated CHEK1 levels, or induces DNA damage in cells. For example, ravusertib has an in vitro IC of 7 nM based on an in vitro biochemical assay using recombinant CHEK1 kinase. 50 (van Ark-Otte et al., Br J Cancer, June 1998, 77(12):2171-2176).

[0030] The inventors have observed that the combination response is specific for CHEK1. The compound may also inhibit CHEK2, in which case the compound may be referred to as a CHEK1 / 2i. Preferably, the combination includes a CHEK1 specific inhibitor.

[0031] CHEK1 inhibitors include ravusertib, SAR-020106, AZD7762, prexasertib, MK-8776, CCT245737, CHIR-124, PF-477736, VX-803, GDC-0575, ESP-01, and BEBT-260. In some embodiments, the CHEK1 inhibitors include ravusertib, SAR-020106, AZD7762, prexasertib, and MK-8776.

[0032] A preferred CHEK1i is ravusertib.

[0033] Love Sertiv Rabusertib is also known as LY2603618 and IC-83. Rabusertib is a highly selective CHEK1i with IC 50 is 7 nM and has the following structure:

[0034] [ka]

[0035] In IUPAC nomenclature, ravusertib can be referred to as 1-[5-bromo-4-methyl-2-[[(2S)-morpholin-2-yl]methoxy]phenyl]-3-(5-methylpyrazin-2-yl)urea. Rabusertib is commercially available.

[0036] SAR-020106 SAR-020106 is an ATP-competitive, potent, and selective CHEK1 inhibitor with IC 50 The molecular weight of SAR-020106 is 13.3 nM and has good cellular activity. SAR-020106 has the following structure:

[0037] [ka]

[0038] In IUPAC nomenclature, SAR-020106 can be referred to as 5-(8-chloroisoquinolin-3-ylamino)-3-((R)-1-(dimethylamino)propan-2-yloxy)pyrazine-2-carbonitrile. SAR-020106 is commercially available.

[0039] AZD7762 AZD7762 is a CHEK1 / 2 inhibitor. It has an IC50 of 5 nM for CHEK1 in a cell-free assay. 50 and has the following structure:

[0040] [ka]

[0041] In IUPAC nomenclature, AZD7762 can be referred to as (S)-5-(3-fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide. AZD7762 is commercially available.

[0042] Prexasertib Prexasertib is also known as LY2606368 and ACR368. Prexasertib is a CHEK1 / 2 inhibitor with an IC of 1 nM for CHEK1 in a cell-free assay. 50 Plexasertib has the following structure:

[0043] [ka]

[0044] In IUPAC nomenclature, prexasertib can be referred to as 5-[[5-[2-(3-aminopropoxy)-6-methoxyphenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile. Prexasertib is commercially available.

[0045] MK-8776 MK-8776, also known as SCH 900776, has an IC 50 is a selective CHEK1 inhibitor at 3 nM. MK-8776 shows 500-fold selectivity over CHEK2. MK-8876 has the following structure:

[0046] [ka]

[0047] In IUPAC nomenclature, MK-8776 can be referred to as 6-bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-[(3R)-piperidin-3-yl]pyrazolo[1,5-a]pyrimidin-7-amine.MK-8776 is commercially available.

[0048] CCT245737 CCT245737, also known as SRA737 or PNT-737, has an IC 50 CCT245737 is an orally active CHEK1 inhibitor with the following structure: CCT245737 exhibits >1,000-fold selectivity over CHEK2.

[0049] [ka]

[0050] In IUPAC nomenclature, CCT245737 can be referred to as 5-methyl-N2-(5-methylpyrazin-2-yl)-N4-[[(2R)-morpholin-2-yl]methyl]pyridine-2,4-diamine. CCT245737 is commercially available.

[0051] CHIR-124 CHIR-124 is a potent CHEK1 inhibitor with IC 50 The selectivity of CHIR-124 for CHEK2 is 2,000-fold. CHIR-124 has the following structure:

[0052] [ka]

[0053] In IUPAC nomenclature, CHIR-124 can be referred to as 3-(1H-benzimidazol-2-yl)-6-chloro-4-[[(3S)-quinuclidin-3-yl]amino]-1H-quinolin-2-one. CHIR-124 is commercially available.

[0054] PF-477736 PF-477736 is also known as PF-736 or PF-00477736. PF-477736 is a selective and potent ATP-competitive CHEK1 inhibitor. PF-477736 has a K of 0.49 nM in cell-free assays. i (dissociation constant) and IC of 0.49 nM 50 PF-477736 exhibits approximately 100-fold selectivity for CHEK1 over CHEK2. PF-477736 has the following structure:

[0055] [ka]

[0056] In IUPAC nomenclature, PF-477736 is (2R)-2-amino-2-cyclohexyl-N-[2-(1-methylpyrazol-4-yl)-9-oxo-3,10,11-triazatricyclo[6.4.1.0 4,13 PF-477736 can be referred to as PF-477736, which is commercially available.

[0057] VX-803 VX-803 is also known as M4344. VX-803 is an ATP-competitive, orally active, and selective inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinases, K iVX-803 is believed to inhibit ATR-driven phosphorylation of phosphorylated CHEK1 (P-CHEK1) and has an IC 50 The dose is 8 nM. VX-803 has the following structure:

[0058] [ka]

[0059] In IUPAC nomenclature, VX-803 can be referred to as 2-amino-6-fluoro-N-[5-fluoro-4-[4-[4-(oxetan-3-yl)piperazine-1-carbonyl]-1-piperidyl]-3-pyridyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide. VX-803 is commercially available.

[0060] GDC-0575 GDC-0575 is also known as ARRY-575 or RG7741. GDC-0575 is a potent and selective CHEK1 inhibitor and is 50 is 1.2 nM. GDC-0575 has the following structure:

[0061] [ka]

[0062] In IUPAC nomenclature, GDC-0575 can be referred to as N-[4-[(3R)-3-amino-1-piperidyl]-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl]cyclopropanecarboxamide. GDC-0575 is commercially available.

[0063] ESP-01 ESP-01 (Esperas Pharma Inc) is also known as LY2880070. ESP-01 is a selective CHEK1 inhibitor being developed for the treatment of solid tumors including metastatic colorectal cancer, epithelial ovarian cancer, endometrial cancer, soft tissue sarcoma, gastrointestinal stromal tumor (GIST), pancreatic cancer and triple-negative breast cancer (TNBC). ESP-01 is administered by the oral route and is undergoing various clinical trials.

[0064] BEBT-260 BEBT-260 (Guangzhou BeBetter Medicine Technology Co Ltd) is a CHEK1 inhibitor. Related patents include EP3411036.

[0065] TOP1 inhibitors DNA topoisomerase I is commonly referred to in the literature as TOP1. DNA topoisomerase I controls and alters the topological state of DNA during transcription and is an interesting target for oncology.

[0066] TOP1 inhibitors previously used in clinical treatment include irinotecan and camptothecin. They are members of the camptothecin-based class of compounds, collectively referred to as camptothecin or camptothecin derivatives. That is, camptothecin derivatives refer to camptothecin itself or its derivatives based on the camptothecin core motif. Camptothecin derivatives include irinotecan, topotecan, belotecan, lurtotecan, exatecan, gimatecan and sinotecan. Alkylated, alkoxylated and hydroxylated derivatives have been reported in the literature. Thus, the TOP1 inhibitor can be selected from irinotecan, topotecan, belotecan, lurtotecan, exatecan, gimatecan and sinotecan, as well as their alkylated, alkoxylated and hydroxylated derivatives.

[0067] The present inventors have observed that the combination reaction may be TOP1 specific. The compound may also inhibit TOP2, in which case the compound may be called a TOP1 / 2i. Preferably, the combination includes a TOP1 specific inhibitor.

[0068] Suitably, the TOP1 inhibitor used in the method of the present invention is a camptothecin derivative, such as selected from irinotecan, topotecan and camptothecin, or their active metabolites, such as SN-38 (a metabolite of irinotecan).Preferably, the TOP1 inhibitor is camptothecin or SN-38.Even more preferably, the TOP1 inhibitor is SN-38.

[0069] Camptothecin Camptothecin is also known as NSC-100880, CPT, campatecin, and (S)-(+)-camptothecin. Camptothecin is a specific inhibitor of TOP1 and has an IC 50 The value is 0.68 μM. The structure is

[0070] [ka] It is.

[0071] In the IUPAC nomenclature, camptothecin is (19S)-19-ethyl-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 ] It can be called henicosa-1(21),2,4,6,8,10,15(20)-heptaene-14,18-dione. Camptothecin is available commercially.

[0072] Irinotecan Irinotecan is also known as CPT-11 and (+)-irinotecan. Irinotecan is a topoisomerase I inhibitor for LoVo and HT-29 cells, and is an IC 50are 15.8 μM and 5.17 μM, respectively (van Ark-Otte et al., Br J Cancer, June 1998, 77(12):2171-2176). Irinotecan is converted by carboxylesterase to SN-38, which is the active metabolite. The structure of irinotecan is:

[0073] [ka] It is.

[0074] In the IUPAC nomenclature, irinotecan is [(19S)-10,19-diethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 ]henicosa-1(21),2,4(9),5,7,10,15(20)-heptaen-7-yl]4-(1-piperidyl)piperidine-1-carboxylate. Irinotecan is commercially available, for example, as the hydrochloride salt.

[0075] Topotecan Topotecan, also known as Hycamtin and Potactasol, is an antitumor drug used to treat ovarian cancer that acts by inhibiting DNA topoisomerases, particularly DNA topoisomerase I. Its structure is:

[0076] [ka] It is.

[0077] In the IUPAC nomenclature, topotecan is (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione or (19S)-8-[(dimethylamino)methyl]-19-ethyl-7,19-dihydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2,11 .0 4,9 .0 15,20 ]Henicosa-1(21),2(11),3,5,7,9,15(20)-heptaene-14,18-dione. Topotecan is commercially available, for example, as the hydrochloride salt (which may be referred to as Topotecan HCl, Toporan, NSC609699, Nogitecan, or SKFS 104864A).

[0078] SN-38 SN-38 (also known as NK012) is also commercially available and has the structure:

[0079] [ka] It is.

[0080] SN-38 is the active metabolite of irinotecan. SN-38 inhibits DNA topoisomerase I, DNA synthesis, and frequently causes DNA single-strand breaks. SN-38 also induces autophagy. In IUPAC nomenclature, SN-38 is (19S)-10,19-diethyl-7,19-dihydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.0]. 2,11 .0 4,9 .0 15,20 ]It can be called henicosa-1(21),2,4(9),5,7,10,15(20)-heptaene-14,18-dione.

[0081] Specific CHEK1 and TOP1 inhibitor combinations In some cases, the combination of a CHEK1 inhibitor and a TOP1 inhibitor for use in the treatment methods described herein can be a combination of a specific CHEK1 inhibitor and a TOP1 inhibitor.

[0082] In some cases, the combination can be selected from AZD7762 (CHEK1 inhibitor) and camptothecin (TOP1 inhibitor), MK-8776 (CHEK1 inhibitor) and SN-38 (TOP1 inhibitor), prexasertib (CHEK1 inhibitor) and SN-38 (TOP1 inhibitor), ravusertib (CHEK1 inhibitor) and SN-38 (TOP1 inhibitor), SAR-020106 (CHEK1 inhibitor) and SN-38 (TOP1 inhibitor), and CCT245737 (CHEK1 inhibitor) and SN-38 (TOP1 inhibitor).

[0083] In some cases, the combination for use in the treatment methods described herein may be a combination of AZD7762 (CHEK1 inhibitor) and camptothecin (TOP1 inhibitor). In some cases, the combination for use in the treatment methods described herein may be a combination of MK-8776 (CHEK1 inhibitor) and SN-38 (TOP1 inhibitor). In some cases, the combination for use in the treatment methods described herein may be a combination of prexasertib (CHEK1 inhibitor) and SN-38 (TOP1 inhibitor). In some cases, the combination for use in the treatment methods described herein may be a combination of ravusertib (CHEK1 inhibitor) and SN-38 (TOP1 inhibitor). In some cases, the combination for use in the treatment methods described herein may be a combination of SAR-020106 (CHEK1 inhibitor) and SN-38 (TOP1 inhibitor). In some cases, the combination for use in the treatment methods described herein can be a combination of CCT245737 (a CHEK1 inhibitor) and SN-38 (a TOP1 inhibitor).

[0084] Pharmaceutically acceptable salts As described herein, any compound may be provided as a pharmaceutically acceptable salt, hydrate or solvate (solvate). Suitable pharmaceutically acceptable salts are known in the art and are described, for example, in Berge et al., J Pharm Sci, 1977 66(1) p. 1.

[0085] Administration of the active ingredient The compound used in the method of the present invention can be administered by any suitable route, including oral and intravenous routes.It is understood that oral administration may be preferred.The compound can be provided in a pharmaceutical composition that includes the compound and one or more pharma-ceutically acceptable excipients.The formulation for oral administration can be in the form of a tablet or a capsule that contains powder or liquid.

[0086] Administration is preferably in a "therapeutically effective amount" or "effective amount" (used interchangeably) sufficient to show benefit to an individual. The actual amount administered, as well as the rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, such as determining dosage, is within the responsibility of general practitioners and other physicians, and typically takes into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to physicians. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0087] Any art-known dosage regimen for the active ingredients described herein can be used in the present invention.

[0088] For example, ravusertib was previously administered at 150 mg / m 2 , 170 mg / m 2 or 230 mg / m 2It was administered parenterally at 100 mg / kg / day. See, for example, Weiss et al., Invest New Drugs 2013 31(1):136-44 (PMID: 22492020), ClinicalTrials.gov Identifier: NCT01341457; Scagliotti et al., Invest New Drugs 2016 Jun 27, 34(5):625-635 (PMID: 27350064).

[0089] For irinotecan as monotherapy, the recommended dose is 125 to 350 mg / m administered by intravenous infusion. 2 The dose depends on whether it is a 1-week or 2-week or 3-week regimen. When used in combination with leucovorin calcium (LV) and 5-fluorouracil (5-FU), the dose is 125-180 mg / m depending on the regimen. 2 See, e.g., https: / / www.pfizermedicalinformation.com / en-us / camptosar / dosage-admin, https: / / www.medicines.org.uk / emc / product / 481 / smpc#gref, and https: / / reference.medscape.com / drug / camptosar-irinotecan-342252.

[0090] Thus, the active ingredients described herein can be administered at a dosage of about 1 mg to about 1000 mg, for example, about 5 mg to about 500 mg, for example, about 10 mg to about 400 mg. In some embodiments, the CHEK1 inhibitor (e.g., ravusertib) can be administered at a dosage of about 1 mg to about 1000 mg, for example, about 50 mg to about 500 mg, for example, about 100 mg to about 300 mg, for example, about 170 mg to about 230 mg. In some embodiments, the TOP1 inhibitor (e.g., camptothecin, e.g., irinotecan) can be administered at a dosage of about 1 mg to about 1000 mg, for example, about 50 mg to about 500 mg, for example, about 100 mg to about 400 mg, for example, about 150 mg to about 350 mg.

[0091] In a dose reduction approach, the CHEK1 inhibitor (e.g., ravusertib) can be administered at a full dose (e.g., 230 mg) and the concentration of the TOP1 inhibitor (e.g., camptothecin, e.g., irinotecan) can be reduced to 2-10 times the full dose. For example, the full dose of irinotecan is 350 mg / m 2 A reduction in irinotecan to half the full dose is approximately 75 mg / m 2 ~about 175mg / m 2 and a reduction in irinotecan to one-tenth of the full dose is about 15 mg / m 2 ~about 35mg / m 2 The range may be:

[0092] The active ingredients described herein can be administered simultaneously or sequentially. In some embodiments of the combination therapy described herein, the CHEK1 and TOP1 inhibitors are administered sequentially. In some embodiments, the TOP1 inhibitor (e.g., camptothecin, e.g., irinotecan) is administered first, followed by the CHEK1 inhibitor (e.g., ravusertib).

[0093] Each of the CHEK1 and TOP1 inhibitors can be administered orally or parenterally independently. In some embodiments, the CHEK1 inhibitor (e.g., ravusertib) can be administered parenterally, e.g., intravenously. In some embodiments, the TOP1 inhibitor (e.g., camptothecin, e.g., irinotecan) can be administered parenterally, e.g., intravenously.

[0094] The active ingredient described herein can be administered at different times within the prescribed administration cycle.The active ingredient described herein can be administered daily, for example, once a day (QD), twice a day (BID), three times a day (TID) or four times a day (QID), or can be administered less frequently or on an intermittent schedule.

[0095] Suitably, the patient may be a human patient.

[0096] Combination with other chemotherapy agents The compound used in the method of the present invention can be administered with one or more other active agents, such as other chemotherapeutic agents, for the treatment of colon cancer.For example, the combination therapy described herein can be used in combination with other chemotherapeutic agents, such as platinum-based chemotherapy, or gemcitabine, or 5-FU, or 5-FU used in combination with leucovorin calcium.In such cases, the combination therapy (i.e., CHEK1 and TOP1 inhibitor) and additional active agent(s) can be given together (together) or separately, for example as components of the same pharmaceutical composition or formulation, or as separate formulations.

[0097] Cancer type The present invention relates to methods for the treatment of cancer in a patient, in particular the treatment of intestinal (colon) cancer. Thus, in some aspects, the present invention relates to the treatment of colon cancer in a patient.

[0098] Preferably, the colorectal cancer is microsatellite stable (MSS). That is, the tumor is classified as MSS by genomic profiling and / or immunohistochemistry. Non-MSS tumors are called MSI-rich (microsatellite instability-rich). Genomic profiling to determine MSI / MSS status is described in Kawakami et al., Curr. Treat. Options Oncol., July 2015, 16(7):30 (PMID:26031544) and test systems are commercially available, e.g., from Promega® (OncoMate™ MSI Dx Analysis System).

[0099] As can be seen from Example 1 and specifically Figure 1a, synergy and activity is evident in this patient population. That is, colon cancer patients are identified as having MSS tumor biomarkers. Estimates of the proportion of colon tumors that are MSS vary, but it is widely accepted that more than half of colon tumors are MSS.

[0100] In some embodiments, the colorectal cancer is a KRAS-TP53 double mutant colon cancer, in other words, the tumor is classified as having both a KRAS mutation and a TP53 mutation by genomic profiling.

[0101] The KRAS (Kirsten Rat Sarcoma Virus) gene is an oncogene. KRAS mutations are thought to be associated with approximately 40% of colon cancers and can be determined by tests known in the art. Many methods involve the use of PCR to amplify the appropriate region of the KRAS gene, including exons 2 and 3, and then utilize different methods to distinguish wild-type from mutant sequences at key codons, e.g., 12 and 13. Detection methods include nucleic acid sequencing, allele-specific PCR, single-strand conformation polymorphism analysis, melting curve analysis, and probe hybridization. Tests for detecting KRAS mutations, such as the Cobas® KRAS Mutation Test (Roche) and the Therascreen KRAS RGQ PCR Kit (Qiagen), are FDA approved (https: / / www.fda.gov / medical-devices / in-vitro-diagnostics / list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools).

[0102] Tumor protein p53 (TP53) is a gene that encodes a tumor suppressor protein that controls the expression of genes involved in cell cycle arrest, apoptosis, senescence, DNA repair, and changes in metabolism. TP53 mutations are associated with Li-Fraumeni syndrome and can be determined by tests known in the art. Methods for detecting and analyzing TP53 mutations include PCR assays that look for mutations in exons 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 of TP53 (https: / / www.fda.gov / medical-devices / in-vitro-diagnostics / list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools).

[0103] As shown in FIG. 1b, a significantly higher degree of potency and efficacy was observed in the KRAS-TP53 double mutant population.

[0104] The features disclosed in the preceding description, or the following claims, or the accompanying drawings, are expressed in their specific form, or in terms of means for performing a disclosed function, or methods or processes for obtaining a disclosed result, and can be utilized, as appropriate, separately or in any combination of such features to realize the invention in various of its forms.

[0105] Although the present invention has been described in conjunction with the example embodiments set forth above, numerous equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the example embodiments of the present invention set forth above are considered to be illustrative and not limiting. Various modifications can be made to the described embodiments without departing from the spirit and scope of the present invention.

[0106] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of improving the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0107] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0108] Throughout this specification, including the appended claims, unless the context requires otherwise, the words "comprise" and "include", as well as variations such as "comprises", "comprising" and "including", are understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0109] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, ranges may be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it is understood that by use of the antecedent "about," the particular value forms another embodiment. The term "about" in connection with numerical values ​​is optional and may mean, for example, + / - 10%. EXAMPLES

[0110] [Example] [Example 1] Combination synergy and activity rates in colon cancer AZD7762 (CHEK1, CHEK2) was screened in combination with the TOP1 inhibitor camptothecin in colon cancer cell lines (n=45). To screen efficiently, we used a 2x7 concentration matrix, or "anchor" approach. We screened each anchor compound at two optimized concentrations and a discrete 1,000-fold (7-point) dose-response curve of library compounds. Viability was read 72h after drug treatment using CellTiter-Glo and drug responses to single agents and combination responses were fitted. Single agent and combination viability measurements were fitted for each cell line to derive multiple parameters, including: 1) the impact of anchor on viability; 2) the maximum library concentration used for the library and combination (Library E max and combination E max ) and 3) the estimated library drug concentration that results in a 50% reduction in viability (IC 50 We compared the observed cellular responses to the combinations with the Bliss Independence predicted responses based on monotherapy activity and measured the intensity (ΔIC 50 , i.e., increased sensitivity) or efficacy (ΔE max We classified drug combinations based on a shift in the IC of the combinations relative to Bliss (i.e., decreased cell viability) at either anchor concentration. 50 Or E max A combination-cell line pair was classified as synergistic if the IL-16 expression level was reduced by 8-fold or 20% viability. Good levels of synergy and activity were observed. See Table 1, Synergy Rates in Colon Cancer Cell Lines.

[0111] [Table 1]

[0112] The synergy and activity rates are also subsetted into MSS (n=30) and MSI (n=15), or KRAS mutated (n=24) and KRAS wild type (wt) (n=21) cell lines. More than 50% of MSS or KRAS mutated colon cell lines show synergy and activity. See Table 2.

[0113] [Table 2]

[0114] Activity in MSI (n=15) and MSS (n=30) cell lines was compared (Figure 1a). KRAS mutant (n=24) cell lines were investigated (n=8 TP53 wild type, n=16 TP53 mutant) (Figure 1b). TP53 mutations in a microsatellite stable (MSS) or KRAS mutant background sensitize colon cancer cells to treatment with AZD7762 and camptothecin. In both populations, we found that potency (ΔIC 50 ) and efficacy (ΔE max ) was observed. Plotted are the averaged combination responses. Box plots (box and whisker plots) show the median and interquartile range. A two-tailed Welch t-test was performed ( * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** =p≦0.0001).

[0115] [Example 2] Comparison with other combinations AZD7762 (CHEK1, CHEK2) was screened in combination with chemotherapy drugs in colon cancer cell lines (n=45). Viability was read 72h after drug treatment using CellTiter-Glo and drug responses to single agents and combination responses were fitted. Synergy was evaluated by comparing efficacy (ΔE max ) or intensity (ΔIC 50 ) was determined based on a significant shift in [calculated by Bliss, ΔE max≥20% survival rate or ΔIC 50 ≥ 3 (= 8-fold concentration shift) is synergy]. Activity was determined based on a significant shift in viability reduction of the combination compared to single agent activity (the combination showed 20% more viability reduction than both single agents). As can be seen, the synergy and activity of the claimed combination in colon cancer cell lines is higher than combinations of the CHEK1 inhibitor AZD7762 with other chemotherapeutic agents (Table 3).

[0116] [Table 3]

[0117] The combination of AZD7762 and 5-FU was further investigated (Fig. 2a,b). TP53 mutations in a microsatellite stable (MSS) or KRAS mutated background only marginally sensitize colon cancer cells to treatment with AZD7762+5-FU. In both populations, we found that the intensity (ΔIC 50 ) and efficacy (ΔE max ) was observed. Plotted are the averaged combination responses. Box plots show median and interquartile range. Welch's two-tailed t-test was performed ( * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** =p≦0.0001).

[0118] [Example 3] CHEK1 specificity To test CHEK specificity, we seeded SW620, SW837, SNU-81 or LS-1034 cells in 96-well plates (770-2,750 cells per well) and treated them with camptothecin (anchor, 0.025 μM) in combination with six CHEK inhibitors with different specificities (library, all administered at a maximum working concentration of 1 μM unless otherwise indicated): AZD7762 (CHEK1, CHEK2), pleksertib (CHEK1, CHEK2), MK-8776 (CHEK1, CHEK2, CDK2), SAR-020106 (CHEK1), ravusertib (CHEK1) and CCT241533 (CHEK2, 2 μM). After 96 h of drug treatment, viability was measured using CellTiter-Glo® 2.0 (CTG, Promega). Fit the drug response curves and calculate the combination response metric, ΔE max and ΔIC 50 was calculated by comparing the observed combination response with the expected (Bliss) combination response. max and ΔIC 50 The higher the IC, the stronger the combination response. SW620, SW837, SNU-81, and LS-1034 are all MSS and KRAS-TP53 double-mutated colon cancer cell lines. The CHEK1-selective inhibitors SAR-020106 and ravusertib showed potency (ΔIC) in combination with camptothecin in the four cell lines. 50 Median: 8.5-10.5-fold shift) and efficacy (ΔE max Both drugs produced a large shift in IL-1 expression (median: 0.22-0.24), whereas the CHEK2-selective inhibitor CCT-241533 did not (Figure 3).

[0119] siRNA experiments were also performed (Figures 4a and 4c). Figure 4a shows A) pooled siRNAs against CHEK1, CHEK2 or PLK1 (cell death positive control) + / - SN-38 (TOP1). Readout: Viability (CTG). Figure 4c shows individual siRNAs against CHEK1 (cell death positive control) + / - SN-38 (TOP1). Readout: Viability (CTG).

[0120] For siRNA experiments, SW837 and SNU-81 cells (8,000 and 16,000 cells per well, respectively) were reverse transfected using Lipofectamine RNAiMax (Thermofisher) with a non-targeting pool of siRNA (siNT, Dharmacon, D-001810-10-05) as a negative control, a polo-like kinase 1 (PLK1) pool (Dharmacon, L-003290-00-0010) as a positive control, a CHEK1 pool (Dharmacon, L-003255-00-0005) or four individual CHEK1 siRNAs (LQ-003255-00-0005) and a CHEK2 pool (Dharmacon, L-003256-00-0005). After 30 h, SN-38 or DMSO at doses ranging from 0.025 μM or 0.001 to 9 μM was added and viability was measured after 72 h using CTG. Signals were normalized to siNT+DMSO control. Statistical significance between conditions was tested using Student's t-test: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** =p≦0.0001. Figures 4a and 4c confirm that the effect of the TOP1+CHEK combination is primarily mediated by inhibition of CHEK1.

[0121] Western blot analysis was also performed to confirm gene knockdown (Figures 4b and 4d). SW837 or SNU-81 cells were reverse transfected with siNT, siCHEK1 or siCHEK2 as described above. 72 h after transfection, cells were harvested and lysed with RIPA buffer (Sigma-Aldrich, R0278). Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes and probed for anti-CHEK1 (Santa Cruz Biotechnology, sc-8408, 1:200, mouse), anti-CHEK2 (Cell Signaling Technologies, D9C6, 1:1000, rabbit) and anti-β-actin (Abcam, ab6276, 1:5000, mouse) (as a loading control). Figure 4b confirms knockdown of CHEK1 and CHEK2. Figure 4d confirms knockdown of CHEK1.

[0122] [Example 4] Combinations compared with single agents Cells (from cell lines LS-1034, SW837 and SNU-81) were seeded at 50,000 cells per well in 6-well plates. Drugs (0.1 nM SN-38, 0.5 μM ravsertib, 0.5 μM CCT241533) or DMSO were added on day 1 and refreshed via medium change on day 8. 14 days after the start of drug treatment, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS (10 min at room temperature) and stained with Giemsa (10%, Sigma-Aldrich) for at least 30 min at room temperature.

[0123] As can be seen from Figure 5, in three separate cell lines tested, the combination of ravusertib (CHEK1) and TOP1i (SN-38) results in less colony formation than either agent alone, or TOP1i in combination with CCT241533 (a selective inhibitor of CHEK2).

[0124] The inventors state that due to their small size it is difficult to quantitate the number of colonies formed, which often depends on the cell line used.

[0125] [Example 5] Rabusertib (CHEK1i) and TOP1i lead to cell death that exceeds additive responses Cells were seeded in 96-well plates (typically 5,000-16,000 cells per well). After 24 h, drugs (0.125 μM staurosporine (positive control), 0.025 μM SN-38, 0.75 μM ravsertib, 0.75 μM CCT241533) or DMSO and real-time fluorescent reagents for detection of cell death (CellTox™ Green, 1:1000, Promega) or caspase 3 / 7 activity (IncuCyte® Caspase 3 / 7 Red, 1:1000, Essen Bioscience) were added. Pictures were recorded using an Incucyte (Essen Bioscience) every 2 h for 96 h. Recorded fluorescent signals were measured as the mean intensity per cell area and normalized to the 0 h time point. Average of 3-4 biological replicates.

[0126] Snapshots of cell death signals recorded at 72 h were converted to the plot shown in FIG. 6. GCU is the abbreviation for Green Calibration Units. The top panel (three rows, Observed-Additive, Additive, and Observed, with the "Observed-Additive (GCU)" scale as key) shows the additive response (sum of SN-38 and CHEKi single agent responses, Additive row) and the response observed in the experiment (Observed row), where delta is the difference between the observed and additive responses (Observed-Additive row). The middle panel (three rows, DMSO, SN-38, and CHEKi, with the "CTOX signal (GCU)" scale as key) shows the effect of individual drugs on cell death, with DMSO as a control in the absence of either drug. The bottom panel (three rows, MSS, KRAS_mutation, and TP53_mutation, with "Feature" color coding as legend) shows the KRAS and TP53 mutation status and microsatellite stability (MSS) status in cell lines (cell lines are identified below).

[0127] As can be seen from FIG. 6, in many colon cancer cell lines, particularly those that are MSS or KRAS-TP53 double mutated, CHEK1i in combination with TOP1i leads to increased cell death that exceeds an additive response, unlike CHEK2i.

[0128] [Example 6] PARP cleavage to determine cell death SW837 (1 million) or SNU-81 (1.5 million) cells were seeded in 10 cm dishes and treated the next day with drugs (SN-38 at 0.025 nM, ravusertib at 1.5 μM, CCT241533 at 1.5 μM, MG-132 at 2 μM (positive control)) or DMSO. After 72 h, live and dead cells were harvested and lysed in RIPA buffer (Sigma-Aldrich) supplemented with 1 mM DTT (Cayman Chemicals) and protease and phosphatase inhibitors (Roche). Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and probed for anti-PARP (Cell Signalling Technologies, 9542, 1:1,000, rabbit) and anti-β-tubulin (Sigma-Aldrich, T4026, 1:5,000, mouse) (as loading control). The results are shown in FIG. 7 and confirm that the combination leads to increased cell death over the individual agents.

[0129] [Example 7] Effect of CHEK1i and TOPi on tumor volume change or survival 4.5 × 10 in 30% Matrigel 6 LS-1034 cells, 5 × 10 6 SW837 cells or 2.5 x 10 6 SNU-81 cells were injected subcutaneously into the right flank of 6-week-old NOD / SCID mice. Tumors grew to an average volume of approximately 300–400 mm. 3Once the NIH score was reached, mice were randomized into treatment groups: LS-1034: n=6 mice to vehicle, n=11 to irinotecan, n=12 to ravusertib and irinotecan + ravusertib, SW837: n=6 mice to vehicle and ravusertib, n=8 to irinotecan, n=4 to irinotecan + ravusertib, SNU-81: n=5 mice to vehicle and irinotecan + ravusertib, n=6 to ravusertib, n=10 to irinotecan.

[0130] Rabusertib was administered orally at 200 mg / kg daily (vehicle: 16.66% Captisol®, CyDex Inc, in 25 mM phosphate buffer, pH 4) and irinotecan was administered intraperitoneally at 25 mg / kg twice weekly (vehicle: saline). Tumor size was assessed weekly by caliper measurement and calculated using the formula 4 / 3π×(d / 2) 2 Approximate mass volumes were calculated using the formula: ×D / 2, where d is the short diameter of the tumor and D is the long diameter of the tumor. Results were based on a minimum of 4 mice per treatment group at a pre-specified endpoint (mean volume of 1500 mm within each treatment group in studies aimed at evaluating drug efficacy). 3 progression of tumors exceeding 750 mm in volume in survival studies aimed at assessing tumor control by therapy for at least 3 weeks; 3 Interpretation was considered when the tumor progression in more than 10 individuals was reached, or at least 3 weeks after treatment discontinuation. Operators were blinded during measurements. In vivo procedures and related biobank data were managed using Laboratory Assistant Suite49. Animal handling was approved by the Italian Ministry of Health (authorization 806 / 2016-PR).

[0131] Statistical significance for tumor volume changes during treatment was calculated using two-way ANOVA. For endpoint comparisons, statistical analysis was performed by unpaired two-tailed Welch t-test. Statistical analysis in survival experiments was performed by log-rank (Mantel-Cox) test. For all tests, the level of statistical significance was set at p<0.05. Graphs were generated and statistical analysis was performed using the GraphPad Prism (v9.0) statistical package.

[0132] Figure 8a shows the change in tumor volume over time. Figure 8b shows the change in tumor volume at the endpoint. Figure 8c shows the survival rate of SNU-81-implanted mice after the end of treatment. As can be seen from the figures, the impact of the combination therapy on tumor volume at the end of treatment and tumor growth inhibition over time was more significant than irinotecan alone (Welch's t-test, p<0.05) (Figures 8a and 8b). For SNU-81, the combination therapy was relatively similar to irinotecan alone, but tumor growth resumption after withdrawal was delayed in mice treated with the combination compared to those treated with irinotecan alone (log-rank Mantel-Cox test, endpoint 750 mm), suggesting a health disadvantage. 3 ) (Figure 8c).

[0133] [Example 8] Effects of CHEK1i and TOPi on DNA double-strand breaks, apoptosis and proliferation in tumors Morphometric quantification of Ki67, active caspase 3 (active cas-3), and phosphorylated H2AX immunoreactivity was performed in mice treated with vehicle (tumors with a mean volume of 1500 mm 3LS-1034 xenografts from mice treated with 100 μg / mL ELISA (until 1 h was reached) or with the indicated compounds (after 72 h). Tumors (n=1–3 per treatment group) were explanted and subjected to histological quality inspection and immunohistochemical analysis using the following antibodies: anti-mouse Ki-67 (MIB-1) (Dako #GA626), anti-rabbit cleaved caspase 3 (Asp175) (Cell Signaling #9661) and anti-rabbit phosphorylated histone H2AX (Ser139) (20E3) (Cell Signaling #9718). After incubation with secondary antibodies, immunoreactivity was revealed by DAB chromogen (Dako). Images were captured by Leica LAS EZ software using a Leica DM LB microscope. Morphometric quantification was performed by ImageJ software using spectral image segmentation. Software output was manually verified by visual inspection of digital images. Each point represents the value measured in one optical field (40x for Ki67 and phosphorylated H2AX, 20x for active caspase-3), with 2-10 optical fields (Ki67 and phosphorylated H2AX) and 3-5 optical fields (active caspase-3) per tumor, depending on the section area (n=12-30 for Ki67 and phosphorylated H2AX, n=10-15 for active caspase-3). Plots show mean ± SD. Mean + / - SD. Statistical analysis by unpaired two-tailed Welch t-test.

[0134] The results are shown in Figure 9, where the combination led to an increase in DNA double strand breaks (phosphorylated H2AX positive cells), more apoptotic cells (active caspase 3 positive cells), and fewer proliferating cells (reduced Ki67 positive cells) in tumors in vivo.

[0135] [Example 9] TOP1i IC 50 Effect of siCHEK1 on SW837 and SNU-81 cells (8,000 and 16,000 cells per well, respectively) were reverse transfected using Lipofectamine RNAiMax (Thermofisher) with a non-targeting pool of siRNA (siNT, Dharmacon, D-001810-10-05) as a negative control, a CHEK1 pool (Dharmacon, L-003255-00-0005) or a CHEK2 pool (Dharmacon, L-003256-00-0005). After 30 h, SN-38 or DMSO was added in the dose range of 0.001-9 μM and viability was measured after 72 h using CTG. Signals were normalized to siNT+DMSO control. Mean + / - SD of three independent replicates.

[0136] The results are shown in Table 4 and Figure 10. siCHEK1 was an IC 50 These results are consistent with CHEK1 inhibition leading to increased sensitivity to TOP1i, suggesting that combination therapy of CHEK1i and TOP1i could be used to reduce the dose of TOP1i administered to patients in the clinic, potentially reducing clinical toxicity associated with TOP1 inhibition.

[0137] [Table 4]

[0138] [Example 10] Combination of other CHEK1 and topoisomerase inhibitors SN-38 in colon cancer cell lines method: Screens were performed in three colon cancer cell lines (CL-11, SNU-81, and SW837) using a 7x7 matrix approach generating 49 wells of data per cell line / drug combination. For each combination, one CHEK1 inhibitor was combined with SN-38 across a dose range of 1,000 discrete fractions (7 points). Viability was measured using CellTiter-Glo reagent 72 h after drug treatment. Single agent and combination viability measurements were fitted for each cell line to derive multiple parameters, including ranges of single agent values ​​and synergy scores.

[0139] For all 49 concentration combination measurements, Bliss excess was calculated by comparing the observed cellular response to the combination with the Bliss-independent predicted response based on monotherapy activity. The "Bliss window" was reported as the maximum mean Bliss excess value measured in 25 possible 3x3 submatrices, or "windows," of the 7x7 dose matrix.

[0140] In addition, HSA (highest single agent) excess was calculated by comparing the observed cellular response to the combination with the highest single agent response of either drug A or drug B for all 49 concentration combination measurements. The "HSA window" was reported as the largest mean HSA excess value measured in 25 possible 3 x 3 sub-matrices or "windows" within the 7 x 7 dose matrix.

[0141] result: The screen consisted of SN-38 (a topoisomerase I inhibitor) in combination with each of the following CHEK1 inhibitors: MK-8776, prexasertib, ravusertib, SAR-020106, and SRA737. All three cell lines are MSS and KRAS / TP53 double mutant. The results show high synergy for SN-38 in combination with any of the CHEK1 inhibitors, as seen by the high scores in the Bliss and HSA windows (Figure 11).

[0142] Consideration The inventors explain that the results show that the combination of a TOP1 inhibitor plus CHEK1 inhibition is synergistic in MSS or KRAS-TP53 double mutant colon cancer cells, leading to apoptosis and growth inhibition of tumor xenografts.

[0143] Camptothecin is an analog of the standard of care chemotherapy irinotecan used for the treatment of colon cancer, and CHEK1 inhibitors, including AZD7762, have been shown to enhance the response of DNA damaging compounds through the abrogation of DNA damage-induced cell cycle arrest. Notably, we observed that this combination produced high synergy rates in MSS colon cancer cell lines (62.1% and 53.3% for both screening configurations), with MSS cell lines showing significantly higher potency and efficacy than MSI cell lines (Welch's two-tailed t-test, p<0.001, Fig. 1a). Furthermore, KRAS mutant colon cancer cells showed high synergy (46% and 48%) for this combination, and we found that KRAS-TP53 double mutant cell lines had significantly stronger combination responses than KRAS single mutant cell lines (Welch's two-tailed t-test, p<0.0001, Fig. 1b). Thus, the inventors have identified two potential molecular contexts, MSS and KRAS-TP53 double mutant colon, in which notable benefit may be derived from the combination of CHEKi and TOP1i.

[0144] To confirm the observed synergy and to explore whether it was dependent on inhibition of CHEK1, CHEK2 or both, we combined camptothecin with six CHEK inhibitors with different specificities. The CHEK1 selective inhibitors SAR-020106 and ravsertib showed potency (ΔIC 50 Median: 8.5-10.5-fold shift) and efficacy (ΔE maxIn contrast to the CHEK2-selective inhibitor CCT241533, which led to a significant shift in IC (median: 0.22–0.24) in both groups, the CHEK2-selective inhibitor CCT241533 did not (Figure 3). Furthermore, combining SN-38, an active metabolite of the TOP1 inhibitor irinotecan, with siRNA against CHEK1 but not CHEK2 resulted in a synergistic decrease in viability (Figures 3–5). Addition of siCHEK1 but not siCHEK2 significantly reduced survival rates compared to the control group (Figures 3–5). 50 This resulted in a shift of at least 7-fold from the original value [SNU-81: 7.2-fold (IC 50 siNT: 611.1 nM, siCHEK1: 84.4 nM, siCHEK2: 714.7 nM), SW837: 1 / 120 (IC 50 (siNT: 84.4 nM, siCHEK1: 0.69 nM, siCHEK2: 66.6 nM)], suggesting that modulation of CHEK1 could be used to reduce the dose of TOP1 inhibitors. The results indicate that the effect of the TOP1i+CHEKi combination is primarily mediated through inhibition of CHEK1.

[0145] We next evaluated the combination using colony formation assays. Combining low concentrations of SN-38 with ravusertib (CHEK1) resulted in fewer colonies formed and increased cell death than either agent alone or SN-38 combined with CCT241533 (CHEK2) (Figures 5 and 6). In many colon cell lines, especially those with weak to moderate responses to SN-38 alone, the effects of SN-38 + ravusertib on cell death were greater than additive, consistent with CHEK1 inhibition enhancing the effects of TOP1 inhibition. The effects of the combination in colon cell lines ranged from less than additive to strongly enhancing, with all MSS cell lines and most KRAS mutant cell lines showing at least additive responses. We further found that the combination was associated with activation of caspase 3 / 7 and cleavage of PARP, both markers of apoptosis (Figure 7).

[0146] To investigate the effect of the combination of TOP1i and CHEK1i on tumor growth in vivo, we implanted three colon cancer cell lines (LS-1034, SW837, SNU-81) into NOD / scid mice and treated them with irinotecan (TOP1), ravusertib (CHEK1), or a combination of the two drugs. In LS-1034 and SW837, the combination of TOP1i and CHEK1i showed more cell death than SNU-81 in vitro (Figure 6), and the effect of the combination on tumor volume at the end of treatment and tumor growth inhibition over time was more pronounced than that of irinotecan alone (Welch's t-test, p<0.05), whereas in SNU-81, the effect of the combination was more similar to that of irinotecan alone (Figures 8a and b). However, mice treated with the combination showed a delayed resumption of SNU-81 tumor growth after withdrawal compared with those treated with irinotecan alone, suggesting a health disadvantage (log-rank Mantel-Cox test, endpoint 750 mm 3 ) (FIG. 8c). Furthermore, combination treatment resulted in increased DNA double-strand breaks (phosphorylated H2AX-positive cells), less proliferative, and more apoptotic tumor cells than were observed in irinotecan-treated LS-1034 tumors 72 h after treatment initiation (FIG. 9).

[0147] Taken together, the inventors believe that these data validate the combination of TOPi and CHEK1i as a potent combination in MSS and KRAS-TP53 double mutant colon cancer cells, driving cell apoptosis and enhancing responses compared to irinotecan alone.

[0148] As a proof of concept, we have tested the combination of irinotecan and CHEK1i in vitro and in vivo. Although the combination of CHEK1i and DDA has been linked to TP53 and KRAS, to the best of our knowledge, this is the first report of significant activity of CHEK1i in combination with chemotherapy in MSS and KRAS-TP53 double mutant colon cancer. Clinical trials combining CHEKi with chemotherapy have demonstrated variable antitumor activity, especially for unselected patients, and have been accompanied by toxicity. As irinotecan is approved for the treatment of colon cancer, and ravusertib is a CHEK1 selective agent with an acceptable safety profile in phase I clinical trials, these data indicate that this combination, if potential toxicity is appropriately considered, provides an effective treatment option for patients with MSS or KRAS-TP53 double mutant colon cancer. These patients represent a population with current unmet clinical needs, and the synergy rates we observed exceed those of combinations previously used in clinical trials or currently being studied in clinical trials. For example, non-selective CHEK inhibitors have been clinically tested in unselected patient populations in combination with chemotherapeutic agents (particularly gemcitabine) and have generally been unsuccessful due to lack of efficacy or toxicity, or both. Our observations indicate that CHEK1 inhibition in combination with TOP1i in patients with MSS or KRAS-TP53 mutated colon cancer may be particularly responsive.

[0149] References In order to more fully describe and disclose the present invention and the state of the art to which it pertains, a number of publications are cited above. Full citations for these references are set forth below. Each of these references is incorporated herein in its entirety. Berge et al., J Pharm Sci, 1977 66(1) p 1 Bliss, Annals of Applied Biology, 1939, 26(3), 585-615 ClinicalTrials.gov Identifier: NCT01341457 EP3411036 Kawakami et al., Curr. Treat. Options Oncol., 2015 Jul;16(7):30 (PMID:26031544) Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins Scagliotti et al. Invest New Drugs,27 Jun 2016, 34(5):625-635 (PMID:27350064) Weiss et al. Invest New Drugs 2013 31(1):136-44 (PMID:22492020) van Ark-Otte et al., Br J Cancer, 1998 Jun; 77(12): 2171-2176 https: / / www.fda.gov / medical-devices / in-vitro-diagnostics / list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools https: / / www.medicines.org.uk / emc / product / 481 / smpc#gref https: / / reference.medscape.com / drug / camptosar-irinotecan-342252

[0150] For standard molecular biology techniques, see Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual. 3rd ed. 2001, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press.

[0151] Prevailing Terms 1. A combination of a CHEK1 inhibitor and a TOP1 inhibitor for use in a method of treating colorectal cancer in a patient, wherein the TOP1 inhibitor is a camptothecin. 2. The combination for use according to clause 1, wherein the colorectal cancer is KRAS-TP53 double mutant colorectal cancer. 3. The combination for use according to clause 1, wherein the colorectal cancer is microsatellite stable. 4. The combination for use according to clause 1, wherein the colorectal cancer is KRAS-TP53 double mutant and microsatellite stable colorectal cancer. 5. The combination for use according to clause 4, wherein the camptothecin is selected from irinotecan, SN-38, topotecan and camptothecin. 6. The combination for use according to clause 4, wherein the camptothecin is irinotecan. 7. The combination for use according to any one of clauses 1 to 6, wherein the CHEK1 inhibitor is selected from ravusertib, SAR-020106, AZD7762, prexasertib, MK-8776, CCT245737, CHIR-124, PF-477736, VX-803, GDC-0575, ESP-01 and BEBT-260. 8. The combination for use according to clause 7, wherein the CHEK1 inhibitor is selected from ravusertib, SAR-020106, AZD7762, prexasertib and MK-8776. 9. The combination for use according to clause 8, wherein the CHEK1 inhibitor is ravusertib. 10. The combination for use according to any one of clauses 1 to 9, wherein the CHEK1 inhibitor and the TOP1 inhibitor are administered separately.

Claims

1. 1. A combination of a CHEK1 inhibitor and a TOP1 inhibitor for use in a method of treating colorectal cancer in a patient, wherein the TOP1 inhibitor is a camptothecin derivative.

2. 1. A CHEK1 inhibitor for use in a method of treating colorectal cancer in a patient, wherein the CHEK1 inhibitor is administered to the patient in combination with a TOP1 inhibitor, wherein the TOP1 inhibitor is a camptothecin derivative.

3. 1. A TOP1 inhibitor for use in a method for treating colorectal cancer in a patient, wherein the TOP1 inhibitor is administered to the patient in combination with a CHEK1 inhibitor, and the TOP1 inhibitor is a camptothecin derivative.

4. 10. The combination for use according to claim 1, the CHEK1 inhibitor for use according to claim 2, or the TOP1 inhibitor for use according to claim 3, wherein the colorectal cancer is KRAS-TP53 double mutant colorectal cancer.

5. 10. The combination for use according to claim 1, the CHEK1 inhibitor for use according to claim 2, or the TOP1 inhibitor for use according to claim 3, wherein the colorectal cancer is microsatellite stable.

6. 4. The combination for use according to claim 1, the CHEK1 inhibitor for use according to claim 2, or the TOP1 inhibitor for use according to claim 3, wherein the colorectal cancer is KRAS-TP53 double mutant and microsatellite stable colorectal cancer.

7. 10. The combination for use according to claim 1, the CHEK1 inhibitor for use according to claim 2, or the TOP1 inhibitor for use according to claim 3, wherein the camptothecin derivative is selected from irinotecan, SN-38, topotecan and camptothecin.

8. 10. The combination for use according to claim 1, the CHEK1 inhibitor for use according to claim 2, or the TOP1 inhibitor for use according to claim 3, wherein the camptothecin derivative is irinotecan.

9. 10. The combination for use according to claim 1, the CHEK1 inhibitor for use according to claim 2, or the TOP1 inhibitor for use according to claim 3, wherein the camptothecin derivative is camptothecin.

10. 10. The combination for use according to claim 1, the CHEK1 inhibitor for use according to claim 2, or the TOP1 inhibitor for use according to claim 3, wherein the camptothecin derivative is SN-38.

11. the CHEK1 inhibitor is selected from ravsertib, SAR-020106, AZD7762, prexasertib, MK-8776, CCT245737, CHIR-124, PF-477736, VX-803, GDC-0575, ESP-01, and BEBT-260; optionally, the CHEK1 inhibitor is selected from ravsertib, SAR-020106, AZD7762, prexasertib, and MK-8776; Optionally, the combination for use according to claim 1, the CHEK1 inhibitor for use according to claim 2, or the TOP1 inhibitor for use according to claim 3, wherein the CHEK1 inhibitor is ravusertib.

12. 10. The combination for use according to claim 1, the CHEK1 inhibitor for use according to claim 2, or the TOP1 inhibitor for use according to claim 3, wherein the CHEK1 inhibitor and the TOP1 inhibitor are administered separately.

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