Combination therapy

Abstract

The present invention relates to a combination therapy regimen of a FAK inhibitor (FAKi) and a FOLFIRINOX chemotherapy regimen, and to the use of this therapy regimen as a means of treating cancer in patients who require cancer treatment, particularly in patients with fibrous cancers / tumors such as pancreatic cancer. Accordingly, the first embodiment provides a method for treating cancer in a subject who requires cancer treatment, comprising administering a combination of a FAK inhibitor (FAKi) and a FOLFIRINOX chemotherapy regimen.

Description

[Technical Field]

[0001] Related applications This application claims priority from Australian Provisional Patent Application No. 2023 / 900354, filed on 14 February 2024, the entire disclosure of which is incorporated herein by reference.

[0002] Field of Invention This invention relates to a combination therapy regimen of a FAK inhibitor (FAKi) and the FOLFIRINOX chemotherapy regimen, and to the use of this therapy regimen as a means of treating cancer in patients who require cancer treatment, particularly in patients with fibrous cancers / tumors such as pancreatic cancer. [Background technology]

[0003] Background of the Invention One of the barriers to the effectiveness of chemotherapy in solid tumors is the complexity of the tumor's cellular and stromal structure. Fibrotic reactions in the tumor microenvironment, known as fibrosis, are characterized by excessive turnover and remodeling of the extracellular matrix (ECM). In fibrous tumors such as pancreatic cancer (and particularly pancreatic ductal adenocarcinoma (PDAC)), increased collagen production and crosslinking can not only promote disease progression and immunosuppression but also negatively impact treatment response and the effectiveness of chemotherapy.

[0004] Standard treatment options for these patients are currently limited to gemcitabine in combination with nab-paclitaxel and FOLFIRINOX. However, the mean 5-year survival rate has remained largely unchanged at around 11% over the past 40 years. Therefore, there remains a need for new combination therapies to improve the effectiveness of chemotherapy agents, particularly FOLFIRINOX, compared to their effectiveness when used alone.

[0005] Focal adhesion kinases (FAKs) are often hyperactivated and overexpressed in invasive cancers, promoting stromal remodeling and inducing tissue stiffness, which can accelerate cancer cell proliferation, survival, and chemotherapy resistance. Therefore, efforts to improve the efficacy of gemcitabine in combination with nab-paclitaxel through stromal manipulation to make tumors more sensitive to chemotherapy include the use of FAK inhibitors (Le Large et al. J Exp Clin Cancer Res (2021) 40(91), and Murphy et al. Biochem. Soc. Trans (2022) 50(4)). However, to the extent that a synergistic effect of FAK inhibition is observed, FAK inhibitors have been shown to synergize with nab-paclitaxel (microtubule inhibitor) rather than gemcitabine (an antimetabolite).

[0006] FOLFIRINOX is a four-drug chemotherapy regimen initially approved for the treatment of advanced pancreatic cancer, but is also being investigated for the treatment of colorectal cancer and other cancers. It consists of the following four drugs, none of which are microtubule inhibitors, but which play a role in interfering with DNA replication in the same way as gemcitabine: ●FOL-folic acid (also known as folinate calcium or leucovorin), ●F-Fluorouracil (5-FU), ●IRIN - Irinotecan (Camptosar), and ●OX-Oxaliplatin (Eloxatin).

[0007] Therefore, previous efforts have focused on the combination of other drugs in an effort to enhance the effectiveness of FOLFIRINOX through interstitial remodeling.

[0008] For example, IPI-926, an oral hedgehog inhibitor capable of depleting tumor-associated stroma, was tested in combination with FOLFIRINOX in patients with advanced pancreatic cancer. Not only did the treatment fail to result in a consistent increase in tumor perfusion, but another phase II trial of IPI-926 + gemcitabine demonstrated adverse effects of the combination (Ko AH, Pancreas. 2016 Mar;45(3)). Hyaluronic acid (HA) is a major component of the extracellular matrix of tumor stroma. However, a study combining FOLFIRINOX with pegylated human recombinant hyaluronidase (which depletes HA in the extracellular matrix of cancer cells) resulted in increased toxicity and reduced treatment duration compared to FOLFIRINOX alone (Ramanathan RK, et al. J Clin Oncol. 2019 May 1;37(13):1062-1069).

[0009] Collectively, previous research has shown that (a) Whether interstitial modification by any means could enhance the effectiveness of FOLFIRINOX, and (b) Interstitial modification raised the serious question of whether it was a worthwhile investigation when chemotherapeutic agents relied on interfering with DNA replication rather than inhibiting microtubules. Any reference to prior art in this specification does not constitute an endorsement or implied that the prior art forms part of the common general knowledge in any jurisdiction, or that the prior art will be understood, considered relevant, and / or combined with other parts of the prior art as reasonably expected by those skilled in the art. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Le Large et al.J Exp Clin Cancer Res(2021)40(91) [Non-Patent Document 2] Murphy et al. Biochem. Soc. Trans (2022) 50(4)

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

[0011] Thus, in a first embodiment, there is provided a method of treating cancer in a subject needing cancer treatment, the method comprising administering in combination a FAK inhibitor (FAKi) and a FOLFIRINOX chemotherapy treatment regimen.

[0012] In an alternative embodiment, there is provided a combination of a FAK inhibitor (FAKi) and a FOLFIRINOX chemotherapy treatment regimen for use in treating cancer in a patient needing cancer treatment.

[0013] In a further embodiment, there is provided the use of a FAK inhibitor (FAKi) and a FOLFIRINOX chemotherapy treatment regimen in the manufacture of a medicament for treating cancer in a patient needing cancer treatment.

[0014] In a further embodiment, there is provided the use of a FAK inhibitor (FAKi) in the manufacture of a medicament for treating cancer in a patient needing cancer treatment by a combination therapy using the FAK inhibitor (FAKi) together with FOLFIRINOX.

[0015] In yet another embodiment,[[]] ● A method of modifying a FOLFIRINOX treatment regimen ● A method of reducing / mitigating the symptoms of a FOLFIRINOX treatment regimen ● A method for improving the therapeutic efficacy of FOLFIRINOX as compared to its efficacy when used alone ●A method for improving survival and quality of life in patients who would otherwise normally receive FOLFIRINOX chemotherapy alone is provided, each method comprising administering in combination a FAK inhibitor (FAKi) and a FOLFIRINOX chemotherapy treatment regimen. This combination achieves better outcomes than the FOLFIRINOX chemotherapy treatment regimen alone.

[0016] The combination of a FAK inhibitor (FAKi) and a FOLFIRINOX chemotherapy treatment regimen may be administered in each embodiment of the invention in the following ways: ●The FAKi may be administered in a pulse dosing regimen that is discontinued before initiating FOLFIRINOX chemotherapy treatment. ●The FAKi may be administered in a pulse dosing regimen before initiating FOLFIRINOX chemotherapy treatment and continued when FOLFIRINOX chemotherapy treatment is initiated. ●The FAKi may be administered concurrently with FOLFIRINOX chemotherapy treatment and may or may not be administered continuously throughout the treatment cycle.

[0017] Administration of the FAKi in a pulse dosing regimen that is discontinued before initiating FOLFIRINOX chemotherapy treatment is preferred, preferably the FAKi is administered for at least 3, 4, 5, 6, 7, or 8 days before initiation of FOLFIRINOX chemotherapy treatment.

[0018] In each of the above-listed treatment options, the subject may be chemotherapy-naive or may have received previous chemotherapy treatment, which may include previous FOLFIRINOX chemotherapy treatment.

[0019] In each embodiment of the invention, the FAK inhibitor (FAKi) is of formula (I) or formula (II):

Chemical formula

[0020] In preferred embodiments, the FAK inhibitors of formulas (I) and (II) are tartrate salts.

[0021] In embodiments of the present invention, cancer is a solid cancer or tumor, preferably a fibrous cancer or tumor such as pancreatic cancer, ovarian cancer, colon cancer, rectal cancer, fibrosarcoma, and solitary fibrous tumor. In embodiments where the cancer is pancreatic cancer, this is more specifically pancreatic ductal adenocarcinoma (PDAC).

[0022] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, which is provided as an example with reference to the accompanying drawings. [Brief explanation of the drawing]

[0023] [Figure 1-1] Exemplary pulsed dosing regimens of FAK inhibitors and FOLFIRINOX for subcutaneous (Figure 1A) and orthotopic (Figure 1B) injection of tumor cell lines in mice. Mice were treated with FAK inhibitors on day 1 (afternoon), day 2 (morning and afternoon), day 3 (morning and afternoon), and day 4 (morning). Mice were further treated with FOLFIRINOX on days 8 and 9, with oxaliplatin (morning) and irinotecan (afternoon) on day 8, and with leucovorin calcium (morning) and fluorouracil (afternoon) on day 9. [Figure 1-2] Exemplary pulsed dosing regimens of FAK inhibitors and FOLFIRINOX for subcutaneous (Figure 1A) and orthotopic (Figure 1B) injection of tumor cell lines in mice. Mice were treated with FAK inhibitors on day 1 (afternoon), day 2 (morning and afternoon), day 3 (morning and afternoon), and day 4 (morning). Mice were further treated with FOLFIRINOX on days 8 and 9, with oxaliplatin (morning) and irinotecan (afternoon) on day 8, and with leucovorin calcium (morning) and fluorouracil (afternoon) on day 9. [Figure 2]Kaplan-Meier analysis of survival in mice with subcutaneous tumors derived from TKCC10lo patients treated with the following: Example FAK inhibitor AMP945 / saline (1st trace), vehicle / saline (2nd trace), vehicle / FOLFIRINOX (3rd trace), and Example FAK inhibitor AMP945 / FOLFIRINOX (4th trace). Kaplan-Meier curves were compared using the log-rank-Mantel-Cox test. ns, P>0.05, *P<0.05, **P<0.01, ***P<0.001 [Figure 3] Kaplan-Meier analysis of survival in mice with orthotopic (intrapancreatic) tumors derived from TKCC10lo patients treated with vehicle / saline (first trace), example FAK inhibitor AMP945 / saline (second trace), vehicle / FOLFIRINOX (third trace), and example FAK inhibitor AMP945 / FOLFIRINOX (fourth trace). Kaplan-Meier curves were compared using the log-rank-Mantel-Cox test: ns, P>0.05, *P<0.05, **P<0.01, ***P<0.001. [Figure 4-1] (Figure 4A) and (B) tumor volume (mm3) at the endpoint and over time in mice treated with FOLFIRINOX monotherapy or mice primed with AMP945 before FOLFIRINOX. Mice treated with FOLFIRINOX monotherapy showed higher tumor volumes. [Figure 4-2] (Figure 4A) and (B) tumor volume (mm3) at the endpoint and over time in mice treated with FOLFIRINOX monotherapy or mice primed with AMP945 before FOLFIRINOX. Mice treated with FOLFIRINOX monotherapy showed higher tumor volumes. [Figure 5]A) Percentage of cleaved caspase-3 positive cells in response to FOLFIRINOX and combination therapy with a FAK inhibitor. B) Percentage of Ki67 positive cells in response to FOLFIRINOX and combination therapy with a FAK inhibitor. N=5 mice / tumor per treatment group, and 6 regions of interest per tumor for each IHC stain. Results are mean ± SEM. P-values ​​determined using unpaired t-tests with Welsh correction for unequal variances. Unless otherwise specified, all significance is compared to FOLFIRINOX. ns, P>0.05, *P<0.05 [Modes for carrying out the invention]

[0024] definition For the purposes of interpreting this specification, any term used in the singular form also includes its plural form, and vice versa.

[0025] As used herein, unless the context requires otherwise, the term “comprise,” and variations such as “comprising,” “comprises,” and “comprised,” are not intended to exclude further additives, components, elements, or steps.

[0026] When used herein in reference to measurable values ​​such as quantity or temporary duration, “about” is intended to include variations of ±5%, in some cases ±1%, and in some cases ±0.1% from the specified value, as such variations are understood by those skilled in the art to be appropriate for carrying out the disclosed method.

[0027] Throughout this disclosure, various aspects of this disclosure may be presented in scope form. It should be understood that scope form is for convenience and brevity only and should not be interpreted as an inflexible limitation on the scope of this disclosure. Therefore, a scope statement should be considered to specifically disclose all possible sub-ranges and individual numbers within that range. For example, a scope statement such as 1–6 should be considered to have specifically disclosed sub-ranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, and individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0028] As used herein, the term "FOLFIRINOX" generally refers to a combination chemotherapy comprising folinic acid (also known as calcium folinate or leucovorin), 5-fluorouracil, irinotecan hydrochloride, and oxaliplatin. Modifications of FOLFIRINOX are intended to be encompassed by the use of this term throughout this specification. For example, NALIRIFOX is FOLFIRINOX in which irinotecan is a liposomal formulation. Other examples include FOLFOX or FOLFIRI.3, as well as combinations of FOLFIRINOX variants such as FIRGEM, NAB-FOLFIRI, NALIRIFOX, and NAB-FOLFOX. A chemotherapy regimen is considered to be FOLFIRINOX for the purposes of this invention, provided that it has those four components.

[0029] FOLFIRINOX is described herein in terms of chemotherapy, chemotherapy treatment, chemotherapy regimen, chemotherapy treatment regimen, and treatment regimen. These terms are interchangeable, as those skilled in the art will be well aware that FOLFIRINOX is not a single drug or formulation.

[0030] As used herein, “combined therapy” or “combination therapy” means a treatment step involving the administration of both FAKi and FOLFIRINOX. The administration may be simultaneous, at the same time, or as a single formulation / drug. Alternatively, FAKi and FOLFIRINOX may be administered sequentially. Sequentially is intended to mean any time frame between the administration of the first and second components, provided that the effect of the first component is still sufficient to positively influence the effect of the second component.

[0031] As will be further understood by those skilled in the art from the context of this specification as a whole, the components of “combined therapy” or “combination therapy” are preferably formulated as two separate formulations or agents.

[0032] A "pulse dosing" regimen refers to the administration of FAKi for a set period of time, starting before the administration of FOLFIRINOX. FAKi may be discontinued before any round of FOLFIRINOX administration, or it may be continued once FOLFIRINOX administration has started. Pulse dosing regimens may also be combined. For example, FAKi may be administered, discontinued before the first round of FOLFIRINOX administration, and then FAKi may be administered again and continued at the start of the second round of FOLFIRINOX.

[0033] As used herein, the term “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for use in contact with the tissue of a subject (e.g., human) without excessive toxicity, irritation, allergic reaction, or other problems or complications, within the bounds of sound medical judgment, and that is commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc., must also be “acceptable” in the sense that it is compatible with the other components of the formulation.

[0034] As used herein, the term “pharmaceutically acceptable salt” refers to a salt that, within the bounds of sound medical judgment, is suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, or allergic reactions, and that is commensurate with a reasonable benefit / risk ratio. pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Examples of pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, and 2-hydroxyethanesulfate. Examples include nitrates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, and valersates.

[0035] As used herein, “pharmaceutically acceptable excipients” means pharmaceutically acceptable materials included in a composition for purposes other than pharmaceutically effective (this is not intended to exclude materials that may have some biological effect).

[0036] As used herein, “preventing” or “prevention” is intended to mean at least reducing the likelihood (or susceptibility) to the risk of contracting a disease or condition (i.e., preventing the development of at least one of the clinical symptoms of a disease in an individual who is potentially exposed to or susceptible to the disease but has not yet experienced or shown any symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known to physicians. Those skilled in the art will understand that “prevention” is not an absolute term. In particularly preferred embodiments, the methods of the present invention may be for preventing or reducing the severity of symptoms of a disease or condition described herein, or for inhibiting or minimizing its progression. Thus, the methods of the present invention have utility as therapeutic and preventive methods.

[0037] The terms “treatment” or “treating” include delaying, slowing, stabilizing, recovering, curing, reducing, alleviating, altering, repairing, suppressing exacerbation, improving, or influencing the disease or condition, the symptoms of the disease or condition, or the risk (or susceptibility) to the disease or condition. “Treatment” refers to any measure of success in treating or improving an injury, pathology, or condition, including any objective or subjective parameters such as relief, remission, reduced exacerbation rate, reduced disease severity, stabilization, symptom reduction, or creating a more tolerable injury, pathology, or condition for the individual, slowing the rate of degeneration or decline, or preventing the final debilitating stage of degeneration. Treatment may not necessarily result in complete clearance of the disease or disability, but it can reduce or minimize infectious complications and side effects, and the progression of the disease or disability. Whether the treatment was successful or not can be monitored, among other things, by physical examination of the individual, CT scan, MRI, or blood biomarkers.

[0038] As used herein, the term “therapeutic dose” refers to the amount of an active compound, or a material, composition, or dosage form containing an active compound, or a treatment and its components, that, when administered according to a desired therapeutic regimen, is effective in producing a certain degree of desired therapeutic effect and is commensurate with a reasonable benefit / risk ratio.

[0039] Detailed description of the embodiment The interaction between tumor and stroma is mediated by bidirectional integrin-mediated signaling, specifically by focal adhesion kinases (FAKs). FAKs are often overactivated and overexpressed in invasive cancers, promoting stromal remodeling and inducing tissue stiffness that can accelerate cancer cell proliferation, survival, and chemotherapy resistance. FAK inhibitors (FAKi), such as the class of 2,4,5-substituted pyrimidines described in WO2012 / 110774, possess their own utility for cell adhesion, cell migration, cell invasion, cell proliferation, and reducing chemotherapy resistance. Furthermore, FAK inhibitors have applicability for inducing apoptosis in cells in an unsuitable extracellular matrix environment and reducing angiogenesis.

[0040] The inventors have surprisingly found that stromal manipulation (or "priming") via short-term FAK inhibition using specific FAK inhibitor compounds can make pancreatic ductal adenocarcinoma (PDAC) cells and other solid tumors more vulnerable and therefore more responsive to subsequent FOLFIRINOX chemotherapy, resulting in unexpectedly improved survival in animal models compared to other FAKi / FOLFIRINOX combinations. This is despite previous research in the field that (a) the synergistic effect between other FAK inhibitors and chemotherapeutic agents was mediated through mechanisms unrelated to FOLFIRINOX, and (b) in many cases, stromal modification was detrimental to the efficacy of FOLFIRINOX and other chemotherapeutic agents (gemcitabine) that rely on interference with DNA replication.

[0041] FOLFIRINOX is a chemotherapy regimen typically used to treat advanced pancreatic cancer. It consists of folinic acid (leucovorin), F-fluorouracil, IRIN-irinotecan, and OX-oxaliplatin. ●Leucovorin is converted to another reduced folate, 5,10-methylenetetrahydrofolate, which stabilizes the binding of FdUMP (the pharmacologically active form of fluorouracil) to thymidylate synthase, thereby enhancing the inhibition of thymidylate synthase by FdUMP. ●Fluorouracil is metabolized to 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP), which is an inhibitor of thymidylate synthase, an enzyme important in DNA repair and replication. ● Irinotecan and its active metabolite SN-38 bind to the topoisomerase I-DNA complex, preventing the religation of double-strand DNA breaks and single-strand DNA breaks that lead to cytotoxicity. ● Oxaliplatin can form interstrand and intrastrand crosslinks in DNA by replacing its oxalate ligand. These crosslinks inhibit DNA replication and transcription, which can cause cytotoxicity.

[0042] In a first embodiment of the present invention, a method for treating cancer in a person requiring cancer treatment is provided, comprising administering a FAK inhibitor (FAKi) in combination with a FOLFIRINOX treatment regimen.

[0043] The present invention further provides a combination of a FAK inhibitor and a FOLFIRINOX chemotherapy regimen for use in the treatment of cancer in patients requiring cancer treatment.

[0044] The present invention further provides the use of an FAK inhibitor in the manufacture of a drug administered to a subject with cancer, after which the subject is administered a FOLFIRINOX treatment regimen.

[0045] In a further embodiment, the use of FAK inhibitors (FAKi) in combination therapy with FOLFIRINOX is provided for the manufacture of drugs for treating cancer in patients who require cancer treatment.

[0046] The combination of a FAK inhibitor (FAKi) and the FOLFIRINOX chemotherapy regimen may be administered in each embodiment of the present invention by the following method: ●FAKi may be administered in a pulse dosing regimen that is discontinued before initiating FOLFIRINOX chemotherapy. ●FAKi may be administered in a pulsed dosing regimen before initiating FOLFIRINOX chemotherapy and may be continued once FOLFIRINOX chemotherapy has started. ●FAKi may be administered concurrently with FOLFIRINOX chemotherapy. In this embodiment, "concurrently" can mean at the same time (e.g., oral administration of FAKi and intravenous infusion of FOLFIRINOX chemotherapy) or concurrently if FAKi is formulated together with the components of FOLFIRINOX chemotherapy.

[0047] The above dosing options can also be combined across multiple rounds of a treatment regimen. For example, for the first treatment round, FAKi may be administered in a pulsed dosing regimen that is discontinued before initiating FOLFIRINOX chemotherapy, and for the second treatment round, FAKi may be administered in a pulsed dosing regimen before initiating FOLFIRINOX chemotherapy and continued once FOLFIRINOX chemotherapy is initiated.

[0048] The administration of FAKi in a pulsed dosing regimen that is discontinued before initiating FOLFIRINOX chemotherapy is preferred, and preferably, FAKi was administered for at least 3, 4, 5, 6, 7, or 8 days prior to the initiation of FOLFIRINOX chemotherapy. Accordingly, a preferred embodiment of the present invention provides a method for treating cancer in a subject requiring cancer treatment, comprising administering a FAK inhibitor (FAKi) in combination with a FOLFIRINOX treatment regimen, wherein the FAKi is administered to the subject for at least 3 days, preferably 4 to 7 days, and discontinued before the initiation of the FOLFIRINOX treatment regimen.

[0049] In these embodiments, as detailed below, the FOLFIRINOX treatment regimen is the U.S. Food and Drug Administration-approved "standard" FOLFIRINOX treatment regimen for use in treating patients with pancreatic cancer.

[0050] The combination therapy FAKi / FOLFIRINOX is expected to be synergistic and improve the therapeutic efficacy of FOLFIRINOX compared to its efficacy when used alone. Improvements in therapeutic efficacy and outcomes can be evaluated or demonstrated by one of the following: ● The probability of survival and improvement in quality of life in patients who would normally have received the FOLFIRINOX treatment regimen itself, and / or ● Modification of the standard FOLFIRINOX treatment regimen, and / or ● Fewer or less serious side effects of the standard FOLFIRINOX treatment regimen, including a reduction in the degree of toxicity.

[0051] When referring to a standard FOLFIRINOX treatment regimen, it refers to the following regimen: [Table 4]

[0052] The treatment regimen is typically repeated in 2-week cycles for up to 6 months, or 12 cycles.

[0053] Therefore, in one embodiment of the present invention, a method for modifying a standard FOLFIRINOX treatment regimen in a patient requiring FOLFIRINOX treatment, (a) A step of administering a FAK inhibitor (FAKi) for at least 3 days, (b) The optional step of discontinuing FAKi administration, (c) Steps to initiate a modified FOLFIRINOX treatment regimen, A method is provided in which the modification is a reduction in the dose of one or more of the four components of FOLFIRINOX, and / or a reduction in the time over which one or more of the four components of FOLFIRINOX are infused, and / or a reduction in the treatment frequency or the total number of treatment cycles.

[0054] Any such modification is assumed to be beneficial to the patient in terms of treatment time and treatment side effects (including lower toxicity), improved patient compliance, and the cost of treatment when used in smaller doses.

[0055] In an alternative embodiment, a method is provided for reducing the incidence or severity of adverse events experienced by a subject compared to those experienced with FOLFIRINOX alone, comprising administering a FAK inhibitor (FAKi) in combination with a FOLFIRINOX treatment regimen, wherein the FAKi is administered to the subject for at least 3 days and discontinued before the initiation of the FOLFIRINOX treatment regimen. The FOLFIRINOX treatment regimen may be standard FOLFIRINOX treatment, or modified FOLFIRINOX, wherein the modification involves a reduction in the dose of one or more of the four components of FOLFIRINOX, and / or a reduction in the time during which one or more of the four components of FOLFIRINOX are infused, and / or a reduction in the treatment frequency or the total number of treatment cycles.

[0056] Side effects that may be reduced or mitigated include, but are not limited to, neutropenia, fatigue, headache, nausea, vomiting, diarrhea, loss of appetite, thrombocytopenia, numbness and tingling, anemia, impaired liver function, acute cholinergic syndrome, thrombosis, and neutropenic fever. While not bound by any theory, it is assumed that the reduction or mitigation of these symptoms is a result of modifications to the FOLFIRINOX treatment regime to have lower doses and / or reduced infusion times and / or lower frequency or fewer treatment cycles. The degree of reduction and mitigation may also be considered as prevention of progression of symptoms and side effects.

[0057] In embodiments in which FAKi is administered as a pulsed dosing regimen for a certain period and then discontinued before the FOLFIRINOX treatment regimen is initiated, FAKi is preferably administered for at least 3, 4, 5, 6, 7, or 8 days, more preferably 4 to 7 days.

[0058] FAKi used in embodiments of the present invention FAK inhibitors are defined by formula (I) or (II): [ka] It may be defined by or a pharmaceutically acceptable derivative thereof.

[0059] In the embodiment, the FAK inhibitors of formulas (I) and (II) are tartrate salts.

[0060] In this embodiment, the tartrate may be D-tartrate or L-tartrate, preferably L-tartrate.

[0061] Therefore, in a preferred embodiment of the present invention, a method for treating cancer in a subject requiring cancer treatment, comprising administering a FAK inhibitor (FAKi) in combination with a FOLFIRINOX treatment regimen, wherein the FAKi is administered to the subject for at least 3 days, preferably 4 to 7 days, and discontinued before the initiation of the FOLFIRINOX treatment regimen, and the FAKi is a compound of formula (I): [ka] A method is provided which is defined by or a pharmaceutically acceptable derivative or salt thereof.

[0062] Preferably, FAKi is a tartrate salt.

[0063] Conditions that should be treated with FAKi / FOLFIRINOX combination therapy The combination therapy of the present invention can be used, specifically, as an anticancer therapy in the treatment of proliferative disorders.

[0064] In embodiments, cancer is selected from solid tumors, including but not limited to bone cancer, brainstem glioma, breast cancer, adrenal cancer, anal cancer, bladder cancer, endocrine cancer, esophageal cancer, head and neck cancer, kidney cancer or ureteral cancer, liver cancer, parathyroid cancer, penile cancer, small intestine cancer, thyroid cancer, urethral cancer, cervical cancer, endometrial cancer, fallopian tube cancer, renal pelvis cancer, vaginal cancer, vulvar cancer, colon cancer, cutaneous melanoma or intraocular melanoma, fibrosarcoma, lung cancer, lymphocytic lymphoma, neoplasms of the central nervous system (CNS), ovarian cancer, pancreatic cancer, pituitary adenoma, primary CNS lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, soft tissue sarcoma, skin cancer, spinal axial tumor, solitary fibrous tumor, gastric cancer, and uterine cancer.

[0065] In preferred embodiments, the cancer is a fibrous cancer or tumor, such as pancreatic cancer, ovarian cancer, colon cancer, rectal cancer, fibrosarcoma, and solitary fibrous tumor. In embodiments where the cancer is pancreatic cancer, this is more specifically pancreatic ductal adenocarcinoma (PDAC).

[0066] In the embodiment, the FAK inhibitor is administered in a dose of 10 mg to 1 g, preferably 50 mg to 600 mg, and most preferably 100 mg to 400 mg.

[0067] Any type of cell may be treated, including but not limited to the lungs, gastrointestinal tract (e.g., intestines, colon, rectum), breasts (mammary glands), ovaries, prostate, liver (hepatic), kidneys (renal), bladder, pancreas, brain, and skin.

[0068] Administration FAKi compounds or pharmaceutical compositions containing the active compound may be administered to the target by any convenient route of administration, regardless of the desired site of action, including but not limited to systemic / peripheral or oral (e.g., by ingestion) and parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous, and intradermal).

[0069] Standard and modified FOLFIRINOX treatment is administered parenterally.

[0070] The subjects may be eukaryotes, animals, vertebrates, mammals, rodents (e.g., guinea pigs, hamsters, rats, mice), murids (e.g., mice), canids (e.g., dogs), felines (e.g., cats), equids (e.g., horses), primates, anthropoids (e.g., monkeys or apes), monkeys (e.g., marmosets, baboons), apes (e.g., gorillas, chimpanzees, orangutans, gibbons), or humans.

[0071] formulation While FAKi can be administered alone in combination therapy, it is preferable to present them as a pharmaceutical composition (e.g., a formulation) containing at least one FAKi as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants, or other materials well known to those skilled in the art, and optionally other therapeutic or prophylactic agents.

[0072] Accordingly, the present invention further provides pharmaceutical compositions for use in the methods of the present invention as defined above, and methods for preparing pharmaceutical compositions, comprising mixing at least one FAKi as defined above with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers, or other materials as described herein.

[0073] FAKi may also be combined in formulations with FOLFIRINOX for parenteral administration.

[0074] Suitable carriers and excipients can be found in standard pharmaceutical textbooks, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0075] Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boron, sulfamic acid, and hydrobromic acid, or salts of pharmaceutically acceptable organic acids such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, malic acid, citric acid, lactic acid, mucoic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0076] General information regarding pharmaceutically acceptable salt types and their formation is known to those skilled in the art and is found in common texts such as “Handbook of Pharmaceutical Salts” PHStahl, CGWermuth, 1st edition, 2002, Wiley-VCH and SMBerge et al., J. Pharmaceutical Sciences, 1977, 66, 1-19.

[0077] The basic nitrogen-containing group may be quaternized with lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides, dialkyl sulfates such as dimethyl sulfate and diethyl sulfate, and other agents.

[0078] The formulations may be conveniently presented in unit dosage forms and may be prepared by any method well known in the field of pharmacy. Such methods include the step of associating FAKi with a carrier constituting one or more minor components. Generally, formulations are prepared by homogeneously and closely associating FAKi with a liquid carrier, a pulverized solid carrier, or both, and then, if necessary, forming the product.

[0079] The formulation may be in the form of a liquid, solution, suspension, emulsion, elixir, syrup, tablet, lozenge, granule, powder, capsule, cachet, pill, ampoule, suppository, pessary, ointment, gel, paste, cream, spray, mist, foam, lotion, oil, bolus, lick, or aerosol.

[0080] Preparations suitable for oral administration (e.g., by ingestion) may be presented as separate units such as capsules, cachetes, or tablets, each containing a predetermined amount of tartrate, as powders or granules, as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, as boluses, as licks, or as pastes.

[0081] Preferably, the FAKi formulation is suitable for oral administration, which can be administered sequentially with the FOLFIRINOX treatment regimen, or for the same duration as the FOLFIRINOX treatment regimen, but as a formulation of its own. However, it is conceivable that FAKi may be formulated together with one of the components of the FOLFIRINOX treatment regimen for co-administration.

[0082] Tablets may be prepared by conventional means, such as compression or molding, using one or more optional components. Compressed tablets can be prepared by compressing a free-flowing form of FAK inhibitor, such as powder or granules, mixed with one or more optional binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropyl methylcellulose), fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica), disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surfactants or dispersants or wetting agents (e.g., sodium lauryl sulfate), and preservatives (e.g., p-methyl hydroxybenzoate, p-propyl hydroxybenzoate, sorbic acid), using a suitable machine. Molded tablets can be prepared by molding a mixture of powder compounds moistened with an inert liquid diluent using a suitable machine. The tablets may optionally be coated or scored, and may be formulated, for example, to provide sustained or controlled release of the FAK inhibitor therein using hydroxypropyl methylcellulose in various proportions to provide a desired release profile. The tablets may optionally be provided with an enteric coating to result in release to a portion of the gastrointestinal tract other than the stomach.

[0083] Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous, and intradermal) include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions that may contain antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners, and liposomes or other particulate systems designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use with such formulations include sodium chloride injection, Ringer's solution, or Ringer's lactate injection. Typically, the concentration of tartrate in the solution is about 1 ng / ml to about 10 μg / ml, for example, about 10 ng / ml to about 1 pg / ml. The formulations may be presented in sealed containers of unit doses or multiple doses, such as ampoules and vials, and may be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier, such as sterile water for injection, immediately before use. Immediate injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. The formulations may also be in the form of liposomes or other particulate systems designed to target the FAK inhibitor to blood components or one or more organs.

[0084] Modification of the FOLFIRINOX regimen The standard FOLFIRINOX treatment regimen consists of the following: [Table 5]

[0085] The treatment regimen is typically repeated in 2-week cycles for up to 6 months, or 12 cycles.

[0086] If the patient is treated with FAKi before administration of the standard FOLFIRINOX treatment regimen, the standard treatment can be modified as follows: ● Use less of one or more of the four components of FOLFIRINOX, and / or ● To shorten the injection time of one or more of the four components of FOLFIRINOX, and / or ● Reduce the frequency of treatment, and / or ● Reduce the total number of treatment cycles. Any such modification is assumed to be beneficial to the patient in terms of treatment time, patient compliance, and treatment side effects (including lower toxicity), and in terms of the cost of treatment when used in smaller doses.

[0087] The fix may include one of the following: ● Oxaliplatin 50-80 mg / m² 2 To reduce the dose to and / or ● Leucovorin 200-350 mg / m² 2 To reduce the dose to and / or ● Irinotecan 130-165 mg / m² 2 To reduce the dose to and / or ● Fluorouracil 1800-2200 mg / m² 2 , 2400 mg / m² 2 Reduce the dosage to

[0088] Preferably, the modification involves reducing the number of cycles from 12 to less than 10. For the patient, this reduction in the number of cycles represents a one-month reduction in treatment duration.

[0089] The treatment involves FOLFOX or FOLFIRINOX-related therapeutic variants such as FOLFIRI.3, as well as combinations of FOLFIRINOX variants such as FIRGEM, NAB-FOLFIRI, NALIRIFOX, and NAB-FOLFOX.

[0090] FAKi administration and dosage The FAK inhibitor is preferably administered in a pulsed dosing regimen, and the FAK inhibitor is provided in a single dose throughout the entire course of treatment, or intermittently (e.g., in doses divided at appropriate intervals), as a high dose, prior to the administration of the FOLFIRINOX treatment regimen.

[0091] Preferably, the pulsed dosing regimen is accompanied by the administration of FAKi for at least 3, 4, 5, 6, 7, or 8 days, most preferably 4 to 7 days, prior to the administration of the FOLFIRINOX treatment regimen.

[0092] Generally, the preferred dose of FAKi is in the range of approximately 100 pg to 250 mg per kilogram of body weight per day.

[0093] It will be understood that the present invention, as disclosed and defined herein, extends to all alternative combinations of two or more of the individual features referred to or revealed in this document or drawings. All of these different combinations constitute various alternative aspects of the present invention. [Examples]

[0094] The present invention will now be described with reference to the following non-limiting embodiments.

[0095] Example 1 - Preparation of FAK inhibitors The FAK inhibitor of the present invention was prepared according to the procedure disclosed in U.S. Patent No. 9,174,946,B2 (the contents of which are incorporated in their entirety).

[0096] Example 2 - Materials and Methods statistical analysis Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc., CA), and significance is indicated as ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. After normality confirmation using the Shapiro-Wilk test, the data were analyzed using an unpaired t-test with Welch correction for normally distributed data, or the Mann-Whitney test for non-normally distributed data. A one-sample t-test was performed for data normalized to 1. The FUCCI cell cycle data analysis was evaluated using two-way analysis of variance (ANOVA) with Tukey correction for multiple comparisons. Kaplan-Meier curves were compared using the Log-Rank-Mantel-Cox test. For all other datasets, p-values ​​were determined by a standard one-way ANOVA with Tukey correction for multiple comparisons for normally distributed data, or the Kruskal-Wallis test with Dunn's multiple comparisons for non-normally distributed data.

[0097] animal Animal experiments were conducted in accordance with the Garvan / St. Vincent's Animal Ethics Committee guidelines (19 / 10, 19 / 13, 22 / 09, 22 / 10) and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Mice were kept in IVC isolation cages with a 12-hour light / dark cycle and allowed to feed ad libitum.

[0098] Drug treatment schedule The stock solution of the FAK inhibitor (AMP945 / nalmafotinib) was prepared at 10 mM in DMSO for in vitro use in cell culture medium at a concentration range of 5–100 nM using DMSO as a vehicle control. For the in vivo study, FAKi was dissolved in 0.5% w / v hydroxypropyl methylcellulose, 0.5% benzyl alcohol, and 0.4% Tween® 80 in sterile water and administered twice daily by forced oral administration for 3 days using a 22-gauge nutrient tube (Instech Laboratories, FTP-22-25) coated with sucrose solution (24%, Sigma-Aldrich, S9378).

[0099] In the subcutaneous model, treatment with FAKi (10 mg / kg) was performed 6 days after KPC cell injection (30 mm 3 (Average tumor volume), and for TKCC10lo cells, 24-28 days later (50 mm 3 Treatment was initiated when the tumor was palpable and detectable by IVIS imaging (mean flux 1 × 10⁹) 4 weeks after intrapancreatic injection of TKCC10 cells. In the pancreatic orthotopic model, FAKi treatment was initiated when the tumor was palpable and detectable by IVIS imaging (mean flux 1 × 10⁹) 4 weeks after intrapancreatic injection of TKCC10 cells.

[0100] FOLFIRINOX was administered intraperitoneally via solution, oxaliplatin (Clifford Hallam Healthcare Pty Ltd, 5 mg / kg), irinotecan (Clifford Hallam Healthcare Pty Ltd, 25 mg / kg), calcium leucovorin (Clifford Hallam Healthcare Pty Ltd, 100 mg / kg), and 5-fluorouracil (Baxter Healthcare Pty Ltd, 25 mg / kg), or via a saline vehicle. All procedures were performed according to the treatment schedule outlined in the corresponding examples, drawings, and captions.

[0101] cell culture Primary KPC cells are terminal Pdx1-Cre and LSL-Kras G12D / +, LSL-Trp53 R172H / + The KPC mouse (previously isolated from J.P. Morton et al., Proc Natl Acad Sci U S A 107, 246 - 251 (2010), J.P. Morton et al., Gastroenterology 139, 292 - 303 (2010), and S.R. Hingorani et al., Cancer Cell 7, 469 - 483 (2005)), while telomerase immortalized fibroblasts (TIF) were also previously generated (C. Vennin et al., Sci Transl Med 9, (2017), K.J. Murphy et al., Sci Adv 7, eabh0363 (2021), Z. Erami et al., Cell Rep 14, 152 - 167 (2016), and J. Munro, K. et al., Oncogene 20, 3541 - 3552 (2001)). KPC cells and TIF were maintained in Dulbecco's modified Eagle's medium (DMEM, high glucose, pyruvate, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 10 mM HEPES (Gibco). TKCC10lo cells were maintained in a 1:1 mixture of M199 medium / Ham's F12 medium (Gibco) supplemented with 7.5% FBS, 15 mM HEPES, 2 mM glutamine, 1×MEM vitamins, 25 ng / ml apo-transferrin, 0.2 IU / ml insulin, 6.5 mM glucose, 40 ng / ml hydrocortisone, 20 ng / ml EGF, 0.5 pg / ml triiodothyronine, and 2 μg / ml O-phosphorylethanolamine. All cells were cultured in the presence of penicillin / streptomycin (100 U / ml and 100 mg / ml respectively) and maintained at 37°C and 5% CO2. Experiments were performed at 20% oxygen for KPC cells and TIF in a HeraCell 150i CO2 / O2 incubator and at 5% oxygen for the TKCC10lo strain. All cell lines were confirmed to be mycoplasma-free.

[0102] Generation of stable cell lines The generation of stable cell lines expressing the pPBDEST-Lyn-FAK biosensor was achieved by co-transfection of the pPBDEST-Lyn-FAK vector and pCMV-hyPBase transposase (obtained from Welcome Trust Sanger Institute, (KJ Murphy et al., Sci Adv 7, eabh0363 (2021), K. Yusa et al. Proc Natl Acad Sci USA 108, 1531-1536 (2011), and J. Seong et al., Nat Commun 2, 406 (2011)) using the Lipofectamine 3000 reagent according to the manufacturer's instructions. KPC-FUCCI cells were also obtained using a third-generation lentiviral packaging system, as previously achieved, with the FUCCI cell cycle reporters (mKO2-hCdt1 and mAG-hGeminin (A. Chou et al., Gut Cells were modified to express either 67,2142-2155(2018) or luciferase-GFP (pLV430G). After transfection and transduction, positive cells were selected by fluorescence-activated cell sorting (FACS), respectively.

[0103] Cell-derived matrix (CDM) assay CDM was established as described above (C. Vennin et al., Sci Transl Med 9, (2017), KJ Murphy et al., Sci Adv 7, eabh0363 (2021), Z. Erami et al., Cell Rep 14, 152-167 (2016), KJ Murphy et al., Bio Protoc 12, (2022), and E. Cukierman, et al. Science 294, 1708-1712 (2001)). To produce CDM on a glass-bottom dish, the surface was coated with 2% gelatin, solidified at 37°C for 2 hours, then rinsed twice with Dulbecco's phosphate-buffered saline (PBS), and fixed in formalin at room temperature for 30 minutes. After rinsing twice with PBS, the fixed gelatin crosslinks were quenched in 1M sterile glycine at room temperature for 30 minutes. Before TIF cell seeding, the coated plates were rinsed twice with PBS and once with DMEM. TIF was grown until confluent. Then, the medium supplemented with ascorbic acid (50 mg / mL) was refreshed every 7 days to remove the TIF (extraction buffer: 0.5% v / v Triton® X-100, 20 mM ammonium hydroxide, 1% w / v sodium deoxycholate). Before seeding cancer cells, the CDM was rinsed with PBS and DMEM.

[0104] Organ-type infiltration assay Collagen Extraction: Collagen I was extracted from rat tails as described above (C. Vennin et al., Sci Transl Med 9, (2017), KJ Murphy et al., Sci Adv 7, eabh0363 (2021), and C. Vennin et al., Nat Commun 10, 3637 (2019)). Briefly, after thawing the rat tails, collagen tendons free of epithelial and skeletal structures were pulled using prong forceps. The tendons extracted from 10–12 tails were then solubilized in 1500 mL of 0.5 M acetic acid on a magnetic stirrer at 4°C for 48–72 hours. The mixture was then filtered through a strainer to remove the sheath, and precipitated with 10% (w / v) sodium chloride on a magnetic stirrer for 6–8 hours. Once an opaque, homogeneous white solution was formed, the mixture was centrifuged at 10,000 rpm at 4°C for 30 minutes. The obtained collagen precipitate was dissolved overnight in 400-600 mL of 0.25 M acetic acid at 4°C on a magnetic stirrer, and the solution was dialyzed in 4 L of 17.4 mM acetic acid for 4 days, refreshing the acid every 12 hours.

[0105] Contraction assay: Organoid matrices were generated as described in (C. Vennin et al., Sci Transl Med 9, (2017), KJ Murphy et al., Sci Adv 7, eabh0363 (2021), and C. Vennin et al., Nat Commun 10, 3637 (2019)). Briefly, 8 × 10 4 Each TIF / matrix was embedded in acid-extracted rat tail collagen (approximately 2.5 mg / mL) in 1×MEM and 8.8% FBS, and neutralized with sodium hydroxide. Before separation, the matrix was coagulated at 37°C and shrunk for 12 days in DMEM containing 10% FBS, 10 mM HEPES, and penicillin / streptomycin (100 U / ml and 100 mg / ml, respectively). For initial treatment (priming), the matrix was treated during shrunk with a vehicle and 5 nM, 10 nM, 20 nM, 50 nM, or 100 nM FAK inhibitors on day 0, and refreshed on day 6.

[0106] Infiltration assay: 12 days after contraction, wash with vehicle or 20 nM FAK inhibitor, 1 × 10 5 KPC cells were seeded into a contracted matrix. 72 hours after cancer cell growth, the seeded matrix was moved to an air-liquid interface on a metal grid, and KPC cancer cells were allowed to invade the matrix for 14 days. For the later treatment regimen, the medium was supplemented with vehicle or 20 nM FAK inhibitor and refreshed three times a week during cell invasion. Longer treatment involved FAK inhibitor treatment both during matrix contraction and invasion. The matrix was then fixed in 10% neutral buffered formalin, embedded in paraffin blocks, sectioned, and processed for histological and immunohistochemical (IHC) analysis (Garvan Histopathology Core Facility). Using cell counts calculated from H&E staining, the cell invasion index was calculated as the total number of invading cells divided by the number of cells at the top of the matrix, and normalized to the mean invasion index of vehicle cells.

number

[0107] ICGC and APGI patient data For IHC analysis of patient TMA from the APGI cohort, tumor cores (3 per patient) from deceased patients, including the complete survival dataset (158 total), were scored for stromal integrity (picrosilius red) and analyzed using TWOMBLI. For pTyr-397-FAK, DAB strength was determined using QuPath(2). Kaplan-Meier curves were generated using GraphPad Prism and log-rank tests performed to determine significance.

[0108] Subcutaneous injection and in vivo imaging 1 × 10⁶ mice in PBS 6Individual KPC-FAK or KPC-FUCCI cells were injected into the posterior flank of BALB / c-Fox1nuAusb mice while they were under anesthesia (3% isoflurane, 1 L / min O2, with continuous vacuum to remove excess isoflurane). The tumors were allowed to develop to an average volume of 30 mm³ for 6 days for KPC tumors before the start of the treatment schedule. For KPC-FAK-derived tumors, mice were treated twice daily for 3 days with narmafotinib (10 mg / kg) or vehicle, with the final treatment performed 4 hours before skin flap surgery and subsequent in vivo imaging (Figure 4A). For KPC-FUCCI-derived tumors, mice were treated with narmafotinib (10 mg / kg), followed by skin flap surgery and imaging 24 hours after treatment. For skin flap surgery, mice were terminally anesthetized using a mixture of 10 mg / kg xylazine and 50 mg / kg zoletil, while maintaining anesthesia (3% gaseous isoflurane, O2 1 L / min, with continuous vacuum to remove excess isoflurane). Subcutaneous tumors were surgically exposed through small incisions around the tumor, which were then enlarged using blunt incisions to separate the epidermis and dermis from the peritoneal wall, thereby creating a skin flap. The skin flap was enlarged to a suitable distance from the body for in vivo imaging. Once the tumor was surgically exposed, the mice were restrained on a 37°C heating stage for up to 40 minutes and imaging was performed using mice maintained under anesthesia. After in vivo imaging, the tumors were fixed in formalin for histological preparation.

[0109] Subcutaneous injection of PDCL 1.5 × 10⁴ 6 Individual TKCC10lo cells were injected into the posterior flank of NOD.Cg-PrkdcscidIL2rgtm1Wjl / SzAusb mice while they were under anesthesia (3% isoflurane, 1 L / min O2, with excess isoflurane removed by continuous vacuum). The tumors were treated for 23–29 days, until the start of the treatment schedule, at a rate of 50 mm. 3The average volume was increased. The treatment schedule was initiated with forced oral administration of narmafotinib (10 mg / kg) or vehicle twice daily (day 1: afternoon, day 4: morning), followed by intraperitoneal injection of oxaliplatin (Clifford Hallam Healthcare Pty Ltd, 5 mg / kg), irinotecan (Clifford Hallam Healthcare Pty Ltd, 25 mg / kg), calcium leucovorin (Clifford Hallam Healthcare Pty Ltd, 100 mg / kg), and 5-fluorouracil (Baxter Healthcare Pty Ltd, 25 mg / kg), or a saline vehicle in solution on days 8 and 9, and the cycle was restarted from day 12 onwards (Figure 6A). For the timed endpoint study, mice were treated with narmafotinib (10 mg / kg) or vehicle twice daily for 3 days over 2 cycles (start day 1: afternoon and end day 4: morning), with the final treatment 4 hours before tumor collection from the timed endpoint mice. Oxaliplatin (Clifford Hallam Healthcare Pty Ltd, 5 mg / kg), irinotecan (Clifford Hallam Healthcare Pty Ltd, 25 mg / kg), calcium leucovorin (Clifford Hallam Healthcare Pty Ltd, 100 mg / kg), and 5-fluorouracil (Baxter Healthcare Pty Ltd, 25 mg / kg), or a saline vehicle were administered intraperitoneally by injection on days 8 and 9 over 2 cycles, and tumors were collected 24 hours after the final treatment.

[0110] Orthotopic injection For orthotopic survival experiments, NOD.Cg-PrkdcscidIL2rgtm1Wjl / SzAusb mice were anesthetized (3% isoflurane, 1 L / min O2, with continuous vacuum to remove excess isoflurane), and TKCC10lo-Luc cells (1 × 10⁶ cells in 50 μl PBS / Matrigel (1:1) per mouse) were injected into the pancreas during laparotomy (7). Briefly, an incision was made below the left ribs through the skin and peritoneum to expose the pancreas, where tumor cells were injected using a 29G needle. The peritoneal wall was then sutured and cut using Vicryl reabsorbable sutures. For analgesia, mice were subcutaneously treated with buprenorphine (0.075 mg / kg) and locally treated with bupivicaine (8 mg / kg). Treatment was initiated when the tumor was both palpable and visible by IVIS monitoring (mean flux 1 × 10⁹). Treatment was initiated with forced oral administration of narmafotinib (10 mg / kg) or vehicle twice daily (day 1: afternoon, day 4: morning), oxaliplatin (Clifford Hallam Healthcare Pty Ltd, 5 mg / kg), irinotecan (Clifford Hallam Healthcare Pty Ltd, 25 mg / kg), calcium leucovorin (Clifford Hallam Healthcare Pty Ltd, 100 mg / kg), and 5-fluorouracil (Baxter Healthcare Pty Ltd, 25 mg / kg), or saline vehicle on days 8 and 9 (Figure 7B). Tumor growth was monitored weekly using IVIS imaging. The experimental endpoint was determined by the presence of ascites, a 10% or greater overnight weight loss, a 20% or greater total weight loss throughout the experiment, a hunched posture, or signs of pain. At the endpoint, the animals were euthanized, the pancreatic tumor, liver, lungs, and spleen were removed, visible metastases were quantified, and the tissues were formalin-fixed for histological treatment. If the endpoint was caused by non-PDAC-related symptoms, the mouse was excluded from the study as a censoring event.

[0111] Immunoblot Cells were rinsed twice in PBS, and lysates were prepared in RIPA protein lysis buffer (50 mM HEPES, 1% Trition X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM EDTA, 50 mM NaF, 10 mM Na3VO4, and 1× protease inhibitor cocktail (Roche)). Protein concentrations were determined by Bradford assay, and the lysate volume was adjusted accordingly to a final concentration of 1 μg / μL. Protein separation was performed by gel electrophoresis using 4–12% or 10% Bis-Tis protein gels. The separated proteins were transferred to a PVDF membrane and blocked overnight at 4°C in Tris-buffered saline and BSA dissolved in 0.1% Tween® 20 (TBST). After rinsing with TBST, the membrane was incubated overnight at 4°C in primary antibody solution (TBS / BSA). The antibodies and their respective dilutions are provided in Table 1. After rinsing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, diluted in 1% skim milk / TBST, GE Healthcare Limited) at room temperature for 2 hours, and then rinsed with TBST. The HRP signal was visualized on Fusion FX (Vilber) using super-enhanced chemiluminescence (ECL) or ECL reagent. Protein signal concentration analysis was performed in Image J (NIH). [Table 1]

[0112] For picrosirius red staining, sections were dewaxed with xylene and rehydrated in graded ethanol washes. The following hematoxylin counterstained sections were stained with 0.02% phosphomolybdic acid and 0.1% picrosirius red (Polysciences) for fibrous collagen. The sections were then rinsed with acidified water and dehydrated in graded ethanol before coverslipping. Slides were scanned using an Aperio slide scanner. For APMA tumor sections, picrosirius red coverage was analyzed using ImageJ (see Macro:Picrosirius red Coverage), while the intensity and coverage of picrosirius red-stained organotype matrix and tumor were analyzed using an in-house MATLAB® (Mathworks, US) script.

[0113] For IHC on Leica Bond RX, organ matrix and tumor sections were dewaxed using Bond Dewax Solution (Leica, AR2992) on Leica Bond RX. Subsequently, heat-induced epitope recovery (HIER) was performed for 30 minutes using epitope recovery solution 2 (pH=9, Leica AR9640) at 93°C for organ matrix sections and 100°C for tumor sections, after removing cleavage caspase-3 which was removed after 20 minutes. Details of primary antibody dilution and incubation time are provided in Table 2. IHC staining was performed on Leica Bond RX autostainers, followed by hematoxylin counterstaining on Leica Autostainer XL and coverslipping on Leica Coverslipper (CV5030). Samples were scanned using an Aperio slide scanner. Organotype matrices and tumor sections stained with caspase-3 and Ki67 were analyzed for positive and negative cells, and DAB intensity for pTyr-397-FAK (pTyr-397) was evaluated using QuPath (P. Bankhead et al., Sci Rep 7, 16878 (2017)). [Table 2]

[0114] In vitro and in vivo imaging techniques and data analysis Polarization imaging of picrosilius red stained tissue: Polarization microscopy was performed on fixed, deparaffinized, and rehydrated 4 μm sections stained with 0.1% picrosilius red (Polysciences, 29401-250). Polarization signals of fibrous collagen were obtained using an Olympus U-Pot polarizer and Olympus U-ANT transmitted light analyzer attached to a DM4000 microscope (Leica). Birefringence signals were quantified and analyzed using Image J. In short, a hue-saturation balance (HSB) threshold was applied (high birefringence / red-orange 0>H<29|0>S<255|70>B<255, moderate birefringence / yellow 30>H<44|0>S<255|70>B<255, low birefringence / green 45>H<245|0>S<255|70>B<255). Then, the relative area of ​​the fibers was calculated as a percentage of the total fibers (0>H<245|0>S<255|70>B<255).

[0115] Second Harmonic Generation (SHG) Imaging: SHG imaging was performed on an inverted Leica DMI6000 SP8 confocal microscope equipped with a titanium-sapphire femtosecond laser (Coherent Chameleon Ultra II) excitation source operating at 80 MHz and tuned to a wavelength of 880 nm. SHG intensity was recorded on RLD-HyD at 440 / 20 nm. For organoid matrices, three representative fields (512px × 512px) were imaged across a 3D z-stack (80 μm depth with a 2.52 μm step size and 30 μm depth with a 1.26 μm step size, respectively). For tissue sections, five regions of interest in deparaffinized, rehydrated 4 μm unstained sections were imaged with a 1.26 μm step size and a 20 μm depth. SHG signal intensity was quantified using MATLAB® (Mathworks, US). Tumor samples from the APMA cohort were tile-scanned using a Leica Stellaris 8 DIVE multiphoton inverted microscope. A Ti:sapphire femtosecond pulsed laser (MaiTai eHP DeepSea, Spectra Physics), operating at 80 MHz and tuned to a wavelength of 880 nm, was used as the excitation source, and SHG intensity was recorded on an RLD-HyD at 440 nm. Individual z-stacks were then exported, and SHG coverage analysis was performed using ImageJ.

[0116] FUCCI Cell Cycle Reporter Imaging: Imaging of the FUCCI cell cycle reporter in biological tissue was performed on an inverted Leica Stellaris 8 DIVE multiphoton inverted microscope. A Ti:sapphire femtosecond pulsed laser (MaiTai eHP DeepSea, Spectra Physics), operating at 80 MHz and tuned to a wavelength of 920 nm, was used as the excitation source, and a 25 × 0.95 NA water immersion objective lens was used for imaging. Signals were recorded using an RLD-HyD detector (bandpass emission filters were used for SHG signals at 460 / 50 nm, mAzami Green at 525 / 50 nm, and mKusabira Orange at 585 / 40 nm). Ten regions of interest (ROIs, 512 px × 512 px) per tumor were imaged over a 20 μm Z-stack with a step size of 2.52 μm. Next, using Leica LASX software, we generated 3D maximum projections and analyzed them using QuPath to quantify the proportion of red, green, and yellow nuclei, respectively, representing G1 / 0, G2 / M, or G1 / S cell cycle phases.

[0117] FLIM-FRET imaging of FAK biosensors: In vitro measurement of FAK activity, 1 × 10⁻⁶ 5KPC-FAK cells were seeded and adhered to CDM, and then treated with a vehicle or FAK inhibitor for 4 hours prior to FLIM-FRET imaging on the CDM. For in vivo measurement of ECFP fluorescence lifetime in subcutaneous xenografts, KPC-FAK cancer cells were injected into the flanks of BALB / c-Fox1nuAusb mice. Four hours after treatment with a vehicle or FAK inhibitor (10 mg / kg), the tumors were surgically exposed using skin flap surgery. Imaging was performed on an inverted Leica DMI6000 SP8 confocal microscope equipped with a titanium-sapphire femtosecond laser resonator (Coherent Chameleon Ultra II) excitation source tuned to a wavelength of 840 nm for ECFP excitation. Signals were recorded using an RLD-HyD detector (using bandpass emission filters of 435 / 40 nm for SHG signals and 483 / 40 nm for FLIM). FLIM data was acquired using the Picoharp300 TCSPC system (Picoquant), and image stabilization was performed using Galene (SCWarren et al., Elife 7, (2018)). Fifty cells per condition were acquired in vivo across multiple ROIs per tumor at a scan rate of 400 Hz, with an acquisition time of 2 minutes 30 seconds and a pixel residence time of 5 μs. ECFP lifetime analysis was performed using FLIMfit by manually selecting the target single-cell membrane region and recording an exponential function that fit the fluorescence decay data. Reference lifetime was calculated using Chroma slides. A lifetime map was generated from the raw data using smoothing with a 2x2 pixel kernel and the application of a standard rainbow lookup table, where blue indicates low ECFP fluorescence lifetime and green to yellow indicates high ECFP fluorescence lifetime. To exclude areas of unspecified signals, the background intensity threshold was set to the average background pixel value for each image, shown as black on the lifetime map.

[0118] IVIS imaging: Orthotopic tumor growth was monitored via luciferase signal imaging on the IVIS spectrum (PerkinElmer). Luciferin (150 mg / kg, Gold Biotechnology) was administered intraperitoneally 3 minutes prior to imaging. Anesthetized mice (2 L isoflurane, 1 L / min O2, with continuous vacuum to remove excess O2 and isoflurane) were placed on an IVIS stage with their left flank exposed, and the signal was acquired with an open filter and small binning. Tumor burden was determined based on total flux.

[0119] Example 3 - Treatment regimen In the in vitro study, the fibroblasts were TIF (telomerase-immortized fibroblasts), while the cancer cells were KPC cells (PDAC mouse cells).

[0120] Cells derived from TKCC10lo patients were injected either subcutaneously into the posterior flank of NOGIL2 mice or orthotopically into the pancreas for orthotopic studies. The tumors were allowed to grow until they were palpable.

[0121] Treatment for subcutaneous or orthotopic studies followed the regimens described in Figures 1A and 1B, respectively. FAKi (10 mg / kg) or the vehicle as described herein was administered over four days according to the described schedule, with the first day of administration counted as day 1. On day 8, oxaliplatin (5 mg / kg) was administered in the morning, followed by irinotecan (25 mg / kg) in the afternoon. On day 9, leucovorin calcium (100 mg / kg) was administered in the morning, followed by fluorouracil (25 mg / kg) two hours after the leucovorin calcium treatment. Mice were monitored until day 12. The regimen was repeated for up to 20 cycles, or until the experimental endpoint was reached. At the experimental endpoint, mice were sacrificed, tumors were excised, and analyzed. Exemplary FAKi (a compound of formula I called AMP945 or narmafotinib) was administered to mouse models in a pulsed dosing regimen in combination with FOLFIRINOX, according to the treatment regimens summarized in Table 3 below. [Table 3-1] [Table 3-2]

[0122] Compared to FOLFIRINOX alone, AMP945 priming increased mouse survival (Figure 2 - subcutaneous study). Post-subcutaneous cell injection treatment showed an approximately 35% increase in patient survival after the combined use of AMP945 and FOLFIRINOX.

[0123] To evaluate the long-term effects of AMP945 in combination with FOLFIRINOX on survival, an orthotopic patient-derived model was used. Luciferase-expressing TKCC10lo cells were orthotopically (intrapancreatically) injected into mice (Figure 1B), and tumor growth was monitored via imaging using a whole-body in vivo imaging system (IVIS). 1 × 10⁶ images were obtained via IVIS imaging. 9 Upon reaching the mean flux, mice were randomly assigned to separate groups receiving pulsed treatment cycles as described, before FOLFIRINOX administration on days 8 and 9, and before 3 days of priming with AMP945, until the study endpoint was reached.

[0124] AMP945 priming prior to FOLFIRINOX resulted in a significant survival benefit compared to FOLFIRINOX alone, demonstrating that AMP945 priming can improve the efficacy of FOLFIRINOX and lead to increased survival in this setting. In mice carrying xenografts derived from pancreatic cancer patients, the median survival time was significantly extended when AMP945 was used in combination with FOLFIRINOX chemotherapy (Figure 3 and the table below). [Table 6]

[0125] Example 4 - Tumor Volume Once control (vehicle or FOLFIRINOX only) mice reached the endpoint, matched AMP945-primed mice were also collected. Before IHC analysis, the tumors were formalin-fixed and paraffin-embedded.

[0126] Combination therapy shows that tumor volume grows at a slower rate compared to FOLFIRINOX monotherapy (Figures 4A and B). (Figure 4A) and (B) tumor volume (mm²) at the endpoint and over time in mice treated with FOLFIRINOX monotherapy or mice primed with AMP945 before FOLFIRINOX. 3 ).

[0127] A reduction in tumor volume typically occurs in two ways: through cell death or a reduction in cell proliferation. From a therapeutic standpoint, a reduction in tumor volume through cell death is preferable to a reduction in proliferation.

[0128] Caspases are a family of enzymes crucial for initiating and executing apoptosis within cells. Caspase-3 is a protein that is cleaved and therefore activated at the initiation of apoptosis. Thus, an increase in levels in the assay indicates an increase in apoptosis and can be used to investigate the cytotoxicity and efficacy of potential therapeutic agents. Figure 5A (cleaved caspase-3 positive cells (%)) demonstrates that combination therapy resulted in a higher increase in caspase-3 cleavage compared to FOLFIRINOX alone. This supports the administration of FAKi AMP945, which enhances the efficacy of FOLFIRINOX.

[0129] Ki67 expression is strongly associated with tumor cell proliferation and growth and is widely used as a proliferation marker. Figure 5B shows that there is no significant difference in cell proliferation between subjects receiving FOLFIRINOX monotherapy and those receiving combination therapy, suggesting that the reduction in tumor volume is most likely due to apoptosis rather than a decrease in cell proliferation.

Claims

1. A method for treating cancer in a patient requiring cancer treatment, comprising administering a FAK inhibitor in combination with a FOLFIRINOX chemotherapy regimen.

2. Combination therapy of FAK inhibitors with the FOLFIRINOX chemotherapy regimen for use in cancer treatment in patients requiring cancer treatment.

3. The use of FAK inhibitors in the manufacture of drugs for treating cancer in patients who require cancer treatment by combination therapy using FAK inhibitors together with the FOLFIRINOX chemotherapy regimen.

4. The method, combination, or use according to any one of claims 1 to 3, wherein the FAK inhibitor is administered before the administration of the FOLFIRINOX chemotherapy regimen.

5. The method, combination, or use according to claim 4, wherein the administration of the FAK inhibitor is discontinued before the administration of the FOLFIRINOX chemotherapy regimen.

6. The method, combination, or use according to any one of claims 1 to 4, wherein the FAK inhibitor is administered in a continuous dosing regimen.

7. Formula (I) or (II): 【Chemistry 4】 The method, combination, or use according to any one of claims 1 to 6, wherein the FAK inhibitor is defined as, or a pharmaceutically acceptable derivative thereof.

8. The method, combination, or use according to claim 7, wherein the FAK inhibitor is a compound of formula (I).

9. The method, combination, or use according to claim 7 or 8, wherein the FAK inhibitor is a tartrate salt.

10. The method, combination, or use of any one of claims 1 to 9, wherein the cancer is selected from solid tumors, including but not limited to bone cancer, brainstem glioma, breast cancer, adrenal cancer, anal cancer, bladder cancer, endocrine cancer, esophageal cancer, head and neck cancer, kidney cancer or ureteral cancer, liver cancer, parathyroid cancer, penile cancer, small intestine cancer, thyroid cancer, urethral cancer, cervical cancer, endometrial cancer, fallopian tube cancer, renal pelvis cancer, vaginal cancer, vulvar cancer, colon cancer, cutaneous melanoma or intraocular melanoma, fibrosarcoma, lung cancer, lymphocytic lymphoma, neoplasm of the central nervous system (CNS), ovarian cancer, pancreatic cancer, pituitary adenoma, primary CNS lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, soft tissue sarcoma, skin cancer, spinal axial tumor, solitary fibrous tumor, gastric cancer, and uterine cancer.

11. The method, combination, or use according to claim 10, wherein the cancer is pancreatic cancer, ovarian cancer, colon cancer, rectal cancer, fibrosarcoma, or solitary fibrous tumor.

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