Use of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-D]pyrimidine-5-carboxamide for tumor treatment

Abstract

This invention provides a novel RET inhibitor that exhibits excellent efficacy in suppressing tumor metastasis to the central nervous system (CNS) and / or tumor recurrence in the CNS, in the treatment of human patients with solid tumors afflicted with RET gene abnormalities. [Solution] A composition for treating human patients with solid tumors having RET gene abnormalities, comprising 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide, represented by the following formula, or a salt thereof. JPEG2026143696000015.jpg56167

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority to U.S. Provisional Application No. 63 / 116,282 filed on 20 November 2020 and U.S. Provisional Application No. 63 / 229,626 filed on 5 August 2021, and each of these in whole forms part of this specification by reference for all purposes.

[0002] This application relates to a composition and method for treating patients with cancer having a RET gene abnormality, including the administration of HM06. [Background technology]

[0003] Receptor tyrosine kinases (RTKs) play crucial roles in various cellular processes, including proliferation, motility, differentiation, and metabolism. Therefore, dysregulation of RTK signaling can lead to certain human diseases, such as cancer. Mutations in RTK-encoding genes, such as EGFR, HER2 / ErbB2, MET, and RET (REarranged during transfection), are diverse. RET is a single-pass transmembrane receptor tyrosine kinase required for the normal development, maturation, and maintenance of multiple tissues and cell types (Non-Patent Literature 1; Non-Patent Literature 2). RET activation occurs through oncogenic mutations in familial and sporadic cancers, such as thyroid cancer (papillary / medullary thyroid carcinoma) and lung cancers like non-small cell lung cancer (NSCLC). Recently, RET has also been associated with the progression of mammary gland tumors and pancreatic tumors, in particular (Non-Patent Literature 3). RET gene abnormalities have also occurred in brain metastases and / or leptomeningeal metastases. Patients with progressive NSCLC and RET rearrangement may have central nervous system (CNS) metastases.

[0004] The RET gene has been found to be an oncogenic driver when activated by genetic abnormalities, such as chromosomal rearrangement (RET gene fusion), point mutations, copy number increase, overexpression, or ligand-induced activation. Downstream pathways that lead to cell proliferation, migration, and differentiation include the RAS / MEK / ERK pathway, P13K / AKT pathway, JAK / STAT pathway, p38 pathway, MAPK pathway, and protein kinase C pathway. The RET kinase domain is conserved during fusion, preserving downstream intracellular kinase activity intact. RET gene fusion can occur in NSCLC (incidence 1-2%), papillary thyroid carcinoma (PTC), colorectal cancer (e.g., CCDC6-RET fusion), and breast cancer (e.g., ERC1-RET fusion). RET gene point mutations occur in multiple endocrine neoplasia 2A (MEN2A), familial medullary thyroid carcinoma (FMTC), and many hereditary forms of medullary thyroid carcinoma (MTC), including MEN2B. In sporadic MTC, RET mutations are identified in up to 50% of patients. RET gene copy number increases occur in NSCLC, breast cancer, pancreatic cancer, and glioblastoma (Non-Patent Literature 4; Non-Patent Literature 3; Non-Patent Literature 5).

[0005] RET gene fusions are a novel oncogenic driver in NSCLC. RET fusions in NSCLC are present in a large number of cases and are mutually exclusive with EGFR, KRAS, ALK, HER2, and BRAF mutations. This suggests that RET fusions are an independent oncogenic driver in NSCLC. These repeat gene fusions were first discovered in late 2011 and have since been confirmed by multiple independent researchers (Non-Patent Literature 6; Non-Patent Literature 7). The fusions are oncogenic when expressed in, for example, Ba / F3 cells and NIH-3T3 cells, and these cells respond to sorafenib, sunitinib, and vandetanib. It may acquire sensitivity to various RET inhibitors (Non-Patent Literature 8; Non-Patent Literature 9).

[0006] Multi-kinase inhibitors (MKIs) with activity against RET receptor tyrosine kinases, such as cabozantinib, vandetanib, and lenvatinib, have demonstrated limited efficacy in a small number of patients with medullary thyroid carcinoma and RET-fusion NSCLC (Non-Patent Literature 10; Non-Patent Literature 11; Non-Patent Literature 12). The degree of overall clinical utility achieved with these MKIs may be lower than that of targeted therapies in patients with different molecular subtypes of NSCLC. Furthermore, the risk / benefit profile may be hindered by the observation of serious toxicity resulting from more potent inhibition of non-RET kinases such as VEGFR2 (Non-Patent Literature 4).

[0007] Two selective RET inhibitors, selpercatinib (LOXO-292) (NCT03157128) and pralcetinib (BLU-667) (NCT03037385), were approved by the U.S. FDA in 2020 for the treatment of adult patients with metastatic RET fusion-positive non-small cell lung cancer (Non-Patent Literature 13; Non-Patent Literature 14). In December 2018 and November 2019, the selective RET inhibitors BOS-172738 and TPX-0046 entered clinical trials (NCT03780517 and NCT04161391, respectively). These RET-specific drugs had not been extensively tested in samples or in animal models resistant to multi-kinase inhibitors, such as cabozantinib, vandetanib, and RXDX-105.

[0008] It remains unclear how effective these RET-specific inhibitors are against lung cancer that has metastasized to the brain. The duration of the CNS response and their effect in delaying the appearance of brain metastases are still unknown. In general, the CNS, including the brain, is protected by the blood-brain barrier (BBB), a protective endothelial tissue surrounding the CNS, which is a major obstacle to the systemic delivery of high molecular weight therapeutic and diagnostic agents to the CNS. The brain permeability of drugs used to treat neurological disorders, such as large biopharmaceuticals or even low molecular weight drugs with low brain permeability, is limited in part due to the large and impermeable BBB.

[0009] Patients with RET abnormalities have a rare disease with high unmet medical needs. Despite clinical improvements with the introduction of novel targeted therapies, many patients eventually relapse. Patients with progressing disease have limited treatment options. Therefore, there is still a need for novel agents to treat patients who have relapsed. Furthermore, information is limited regarding the frequency, responsiveness, and overall treatment outcomes of RET rearrangement CNS metastases in patients with advanced NSCLC. The frequency of CNS involvement in these patients is 25% at diagnosis, but the lifetime prevalence can reach almost half. Poor intracranial response has been reported in patients treated with various multi-kinase inhibitors (Non-Patent Literature 15; Non-Patent Literature 16).

[0010] Therefore, there is a need for novel RET inhibitors that can overcome CNS relapse and have a desirable tolerability profile. The availability of potent and selective RET inhibitors could offer clinical benefits not only to patients with resistance mutations but also to those who are naive to RET-targeted agonists. High CNS penetration could also represent a substantial clinical improvement in NSCLC patients with brain metastases or leptomeningeal disease.

[0011] In this specification, RET-specific inhibitors referred to as "HM06," "TAS0953 / HM06," or "HM06 / TAS0953" are in clinical development. HM06 / TAS0953 is a potent and highly selective inhibitor of RET phosphorylation. The antitumor efficacy of HM06 / TAS0953 in preclinical models supports therapeutic interest for this selective RET inhibitor for clinical use. HM06 / TAS0953 has been shown to be effective against patients treated with RET inhibitors. It inhibited the proliferation of RET rearrangement-positive lung cancer cell lines derived from patient samples without [amplification]. These results indicate that HM06 / TAS0953 was more effective than RET multi-kinase inhibitors in inhibiting the proliferation of cell lines harboring RET fusions. HM06 / TAS0953 was also tested against cell lines derived from patient samples that are resistant to different RET multi-kinase compounds. These results suggest that HM06 / TAS0953 is effective against cell lines that are resistant to different RET multi-kinase inhibitors and resistant to the inhibitory effects of cabozantinib, RXDX-105, and vandetanib.

[0012] Additional preclinical data presented herein show that HM06 / TAS0953 exhibits inhibitory activity against solid tumor xenografts, including an orthotopic xenograft model of brain metastasis of lung cancer. These data indicate that HM06 / TAS0953 penetrates the brain and is effective against tumors with RET gene abnormalities in the brain.

[0013] HM06 / TAS0953 has the potential to provide clinical utility without potentially causing adverse reactions resulting from inhibition of non-RET kinases. For example, HM06 / TAS0953 can provide improved treatment and disease management options for NSCLC patients with brain metastasis and / or leptomeningeal disease. Furthermore, HM06 / TAS0953 may be beneficial for patients who are resistant to other RET kinase or multi-kinase inhibitors (e.g., patients who have progressed or developed intolerance to other medicaments). In view of the limited number of tumor patients with RET gene abnormalities and the rarity of diseases with high unmet medical needs, patients may benefit from treatment with HM06 / TAS0953.

Prior Art Literature

Non-Patent Literature

[0014]

Non-Patent Literature 1

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

[0015] The present invention provides a composition and method for treating patients with cancer having a RET gene abnormality, comprising the administration of HM06 / TAS0953.

[0016] This disclosure relates to a method for treating a human patient with non-small cell lung cancer (NSCLC) having a RET gene abnormality, comprising administering to the human patient a composition comprising 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide. The present invention provides a method in which a human patient is administered a dosage equivalent to approximately 40 mg to approximately 3000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.

[0017] The disclosure also provides a method for treating a human patient with locally advanced or metastatic non-small cell lung cancer (NSCLC) having a RET gene abnormality, comprising administering to the human patient a composition comprising 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide, wherein the human patient is administered a dose equivalent to approximately 40 mg to approximately 3000 mg of free base of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide per day.

[0018] The disclosure further provides a method for treating a human patient with metastatic non-small cell lung cancer (NSCLC) having a RET gene abnormality with brain and / or leptomeningeal metastases, comprising administering to the human patient a composition comprising 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide, wherein the human patient is administered an effective amount of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide.

[0019] This disclosure provides a method for treating a human patient with a solid tumor having a RET gene abnormality, comprising administering to the human patient a composition comprising 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide, wherein the human patient is administered a dosage equivalent to the free base of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide at a rate of approximately 40 mg to approximately 3000 mg per day.

[0020] The disclosure also provides a method for treating a human patient with a solid tumor having a RET gene abnormality, comprising administering to the human patient a composition comprising 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide, wherein the RET gene abnormality comprises a solvent front mutation of the RET protein.

[0021] The disclosure also provides a method for treating a human patient with a solid tumor having a RET gene abnormality with brain and / or leptomeningeal metastasis, comprising administering to the human patient a composition comprising 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide, wherein the human patient is administered an effective amount of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide.

[0022] In some embodiments, the effective dose is approximately 40 mg to approximately 3000 mg per day. This is an equivalent dose to the free base of -amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide.

[0023] In some embodiments, brain and / or leptomeningeal metastases are asymptomatic.

[0024] In some embodiments, human patients are administered a dose equivalent to approximately 150 mg to approximately 640 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 160 mg to approximately 640 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 320 mg to approximately 640 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 480 mg to approximately 640 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 640 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.

[0025] In some embodiments, human patients are administered a dose equivalent to approximately 480 mg to approximately 3000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 480 mg to approximately 2000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 480 mg to approximately 1500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 480 mg to approximately 1000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 640 mg to approximately 3000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 640 mg to approximately 20 A dose equivalent to 00 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base is administered. In some embodiments, human patients are administered a dose equivalent to approximately 640 mg to approximately 1500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 640 mg to approximately 1280 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 640 mg to approximately 1000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 3000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 2000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.In some embodiments, human patients are administered a dose equivalent to approximately 1500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 1280 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 1000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.

[0026] In some embodiments, human patients are administered a dose equivalent to approximately 150 mg to approximately 500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 160 mg to approximately 500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day. In some embodiments, human patients are administered a dose equivalent to approximately 150 mg or approximately 160 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.

[0027] In some embodiments, the composition is administered orally. In some embodiments, the composition is administered orally as a single tablet or as a group of tablets. In some embodiments, each tablet contains about 10 mg or about 50 mg of 4-amino-N-[4-(methoxymethylcellulose). Contains a dose equal to the free base of [(Cymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide.

[0028] In some embodiments, the composition comprises the dihydrochloride salt of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide.

[0029] In some embodiments, the composition further comprises citric acid, crystalline cellulose, lactose, polyvinyl N-pyrrolidone, sodium lauryl sulfate, and / or glyceryl behenate.

[0030] In some embodiments, the composition is administered once daily (QD) or twice daily (BID). In some embodiments, the composition is administered twice daily (BID).

[0031] In some embodiments, human patients are administered a dose equivalent to approximately 160 mg to approximately 1500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID). In some embodiments, human patients are administered a dose equivalent to approximately 160 mg to approximately 1000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID). In some embodiments, human patients are administered a dose equivalent to approximately 160 mg to approximately 750 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID). In some embodiments, human patients are administered a dose equivalent to approximately 160 mg to approximately 640 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID). In some embodiments, human patients are administered a dose equivalent to approximately 160 mg to approximately 500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID). In some embodiments, human patients are administered a dose equivalent to approximately 160 mg to approximately 320 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID).

[0032] In some embodiments, human patients are administered a dose equivalent to approximately 320 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID). In some embodiments, human patients are administered a dose equivalent to approximately 500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID). In several embodiments, human patients receive approximately 640 mg of 4-amino-N-[4-(methoxymethyl) twice daily (BID). A dose equivalent to the free base of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide is administered. In some embodiments, human patients are administered a dose equivalent to approximately 750 mg of the free base of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide twice daily (BID). In some embodiments, human patients are administered a dose equivalent to approximately 1000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID). In some embodiments, human patients are administered a dose equivalent to approximately 1500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropa-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID).

[0033] In some embodiments, the dosage is the same for patients weighing more than 50 kg and patients weighing less than 50 kg.

[0034] In some embodiments, the composition is administered in at least one 21-day treatment cycle.

[0035] In some embodiments, a RET gene abnormality includes at least one of the following: RET gene fusion, point mutation, deletion mutation, copy number increase of the RET gene, overexpression of any one or more of these, and overexpression of the RET gene. In some embodiments, a RET gene abnormality includes a RET gene fusion. In some embodiments, a RET gene abnormality includes a RET gene fusion with CCDC6, KIF5B, or TRIM33.

[0036] In some embodiments, the RET gene abnormality includes resistance mutations in the RET protein. In some embodiments, the RET gene abnormality includes solvent front mutations and / or mutations in the hinge region of the RET protein. In some embodiments, the RET gene abnormality includes mutations in the RET protein at amino acid residues 730, 736, 760, 772, 804, 806, 807, 808, 809, 810, and / or 883. In some embodiments, the RET gene abnormality includes mutations in the RET protein at amino acid residues 804, 806, 807, 808, 809, and / or 810. In some embodiments, the RET gene abnormality includes mutations in the RET protein at amino acid residue 810. In some embodiments, the RET gene abnormalities include mutations in the RET protein, including: a) V804X mutation (where X is any amino acid other than valine or glutamic acid), b) Y806X mutation (where X is any amino acid other than tyrosine), c) A807X mutation (where X is any amino acid other than alanine), d) K808X mutation (where X is any amino acid other than alanine), e) Y809X mutation (where X is any amino acid other than tyrosine), and / or f) G810X mutation (where X is any amino acid other than glycine). In some embodiments, RET gene abnormalities include RET protein mutations, including: a) L730Q or L730R mutations, b) G736A mutations, c) L760Q mutations, d) L772M mutations, e) V804L or V804M mutations, f) Y806C, Y806S, Y806H, or Y806N mutations, g) G810R, G810S, G810C, G810V, G810D, or G810A, and / or A883V mutations. In some embodiments, the RET gene abnormalities are: a) V804L or V804M mutations, b) Y806C, Y806S, Y806H, or Y806N mutations, and / or c) G810R, G810S, G810C, G810V, G810D, or G810A mutations. This includes mutations in the RET protein. In some embodiments, the RET gene abnormality includes G810R, G810S, G810C, G810V, G810D, or G810A mutations in the RET protein. In some embodiments, the RET gene abnormality includes the G810R mutation in the RET protein.

[0037] In some embodiments, the cancer or tumor is resistant to at least one multikinase inhibitor. In some embodiments, the cancer or tumor is resistant to at least one RET selective inhibitor. In some embodiments, the cancer or tumor is not resistant to 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide, but is resistant to at least one other RET selective inhibitor. In some embodiments, the cancer or tumor is resistant to serpercatinib and / or pralcetinib. In some embodiments, the cancer or tumor includes cells that are resistant to serpercatinib and / or pralcetinib.

[0038] In some embodiments, the human patient has previously received prior treatment for cancer or tumor. In some embodiments, the cancer or tumor being treated has progressed after prior treatment for cancer or tumor. In some embodiments, the human patient has developed intolerance to prior treatment for cancer or tumor. In some embodiments, the human patient has been previously administered a multikinase inhibitor. In some embodiments, the human patient has been previously administered cabozantinib, vandetanib, lenvatinib, and / or RXDX-105. In some embodiments, the human patient has been previously administered a RET selective inhibitor. In some embodiments, the human patient has been previously administered serpercatinib and / or pralcetinib. In some embodiments, the human patient has never been previously administered a RET selective inhibitor.

[0039] In some embodiments, the human patient has at least one of the following: salivary gland cancer, lung cancer, colorectal cancer, thyroid cancer, breast cancer, pancreatic cancer, ovarian cancer, skin cancer, and brain tumor. In some embodiments, the human patient has at least one of the following: medullary thyroid carcinoma or anaplastic thyroid carcinoma, metastatic breast cancer, and metastatic pancreatic adenocarcinoma.

[0040] Further objectives and benefits will be described in part in the following explanation, and some will be understood from the explanation, i.e., learned through practice. The objectives and benefits will be understood and achieved by the elements and combinations specifically indicated in the attached claims.

[0041] Naturally, the general description above and the detailed description below are illustrative and descriptive, and do not limit the scope of the claims.

[0042] The attached drawings are incorporated into this specification and constitute part of it, illustrating one or more embodiments and, together with the descriptions, illustrating the principles set forth herein. [Brief explanation of the drawing]

[0043] [Figure 1A] This figure shows the efficacy of HM06 / TAS0953 in a KIF5B-RET Luc fusion-positive brain metastasis model. Figure 1A shows the antitumor efficacy of HM06 / TAS0953 administered at 50 mg / kg BID compared to a vehicle control. [Figure 1B] Figure 1B shows the percentage change in body weight of KIF5B-RET Luc-fused brain metastasis mice treated with HM06 / TAS0953 compared to vehicle control mice. [Figure 1C] Figure 1C shows that the survival rate of the HM06 / TAS0953 group was higher than that of the vehicle control group. [Figure 1D] Figure 1D shows IVIS images and pathological conditions of mouse brains treated with HM06 / TAS0953, compared to the control group. [Figure 2]This figure shows the effect of HM06 / TAS0953 on KIF5B-RET fusion tumor volume in a vandetanib-resistant mouse model compared to serial vandetanib treatment. [Figure 3A] This figure shows the efficacy of HM06 / TAS0953 in inhibiting the proliferation of RET fusion-positive cell lines, compared to three RET multikinase inhibitors: cabozantinib, RXDX-105, and vandetanib. Figure 3A shows treatment with a treatment-naive cell line (KIF5B-RET) derived from a sample obtained from a patient who had never been treated with anticancer therapy. [Figure 3B] Figure 3B shows the treatment in the CCDC6-RET fusion cell line. [Figure 3C] Figure 3C shows the effect on an isogenic counterpart in the same cell line expressing a blank control plasmid. [Figure 3D] Figure 3D shows the treatment in the TRIM33-RET fusion cell line. [Figure 3E] Figure 3E shows treatment with a cell line (CCDC6-RET) derived from a sample obtained from a patient who was resistant to cabozantinib. [Figure 3F] Figure 3F shows the treatment of cell lines obtained from patients resistant to RXDX-105. [Figure 4A] This figure shows the efficacy of HM06 / TAS0953 against the proliferation of 3T3-CCDC6-RET xenograft tumors. Figure 4A shows the volume change of each individual tumor from the start to the end of treatment. The results are the mean ± SE of five individual tumors at each time point. [Figure 4B] Figure 4B shows the volume changes of each tumor. *p<0.05, compared to the vehicle treatment group. [Figure 4C] Figure 4C shows animal body weight as a function of time. The results are the mean ± SE of 5 animals per group. [Figure 5A] This figure shows the efficacy of HM06 / TAS0953 against the proliferation of xenograft tumors (TRIM33-RET fusion). Figure 5A shows tumor volume as a function of time. The results are the mean ± SE of five individual tumors at each time point. [Figure 5B] Figure 5B shows the volume changes of each individual tumor from the start to the end of the treatment. In all groups, there was a significant reduction in mean tumor volume compared to the vehicle treatment group (p<0.05). [Figure 5C] Figure 5C shows animal body weight as a function of time. Results are the mean ± SE of 5 animals per group. At all time points after the start of treatment, there was a significant decrease in animal body weight in the vandetanib-treated group (p<0.05). [Figure 6A] This figure shows the efficacy of HM06 / TAS0953 against the proliferation of PDX tumors (CCDC6-RET) derived from tumor samples obtained from patients who had no longer responded to cabozantinib. Figure 6A shows tumor volume as a function of time. Results are the mean ± SE of five individual tumors at each time point. [Figure 6B] Figure 6B shows the volume changes of each individual tumor from the start to the end of the treatment. In all groups, there was a significant reduction in tumor volume compared to the vehicle treatment group (p<0.05). [Figure 6C] Figure 6C shows animal body weight as a function of time. The results are the mean ± SE of 8 animals per group. [Figure 7A] This figure shows the efficacy of HM06 / TAS0953 against the proliferation of PDX tumors (CCDC6-RET) originating from patients resistant to RXDX-105 treatment. Figure 7A shows tumor volume as a function of time. Results are the mean ± SE of five individual tumors at each time point. [Figure 7B] Figure 7B shows the volume changes of each individual tumor from the start to the end of the treatment. In all groups, there was a significant reduction in tumor volume compared to the vehicle treatment group (p<0.05). [Figure 7C] Figure 7C shows animal body weight as a function of time. The results are the mean ± SE of 5 animals per group. [Figure 8A]This figure shows the efficacy of HM06 / TAS0953 against the proliferation of PDX tumors (CCDC6-RET) derived from patients with poor response to RXDX-105. Figure 8A shows tumor volume as a function of time. Results are the mean ± SE of five individual tumors at each time point. In all groups, there was a significant reduction in tumor volume compared to the vehicle treatment group (p<0.05). [Figure 8B] Figure 8B shows the volume changes of individual tumors from the start to the end of treatment. In the QD group, one tumor increased by only 20.7% with HM06 / TAS0953 at 100 mg / kg. [Figure 8C] Figure 8C shows animal body weight as a function of time. The results are the mean ± SE of 5 animals per group. [Figure 9A] This figure shows the efficacy of HM06 / TAS0953 against the proliferation of tumors (TRIM33-RET fusion) transplanted into the brains of mice. Figure 9A shows images of bioluminescence signals from the start to the end of treatment. [Figure 9B] Figure 9B shows the quantitative analysis of luminescence (left) and animal weight measurement (right). There was a significant reduction in tumor volume compared to the vehicle treatment group (p<0.05). Results represent the mean ± SE of 5 animals (vehicle) or 4 animals (HM06). [Figure 9C] Figure 9C shows a Kaplan-Meier plot illustrating the survival rate of tumor-bearing mice throughout the entire study. [Figure 10A] This figure shows the efficacy of HM06 / TAS0953, vandetanib, and LOXO-292 against the proliferation of tumors (TRIM33-RET fusion) transplanted into the brains of mice. Figure 10A shows representative images of bioluminescence 33 and 93 days after cell transplantation. [Figure 10B] Figure 10B shows the quantification of the luminescence signal. The results are the mean ± SE of 6 animals per group. [Figure 10C] Figure 10C shows a Kaplan-Meier plot illustrating the survival rate of tumor-bearing mice throughout the study. The p-values ​​adjusted to compare the HM06-treated group with the LOXO-292-treated group are shown below the graph. [Figure 10D]Figure 10D shows the animal's body weight over time. [Figure 11A] This figure shows the pharmacokinetic profile of HM06 / TAS0953 in rats. Figure 11A shows the plasma concentrations over time after a single oral administration of HM06 / TAS0953 at 3 mg / kg, 10 mg / kg, 30 mg / kg, and 50 mg / kg. [Figure 11B] Figure 11B shows the plasma concentrations over time after a single intravenous administration of HM06 / TAS0953 at a dose of 3 mg / kg. [Figure 11C] Figure 11C shows the pharmacokinetic profiles of HM06 / TAS0953 in PFC, CSF, and plasma (total and free fractions) after oral administration of 10 mg / kg to freely moving adult male Han®Wistar rats. The 1:1 ratio of free plasma concentration to free brain concentration indicates that HM06 / TAS0953 freely crosses the blood-brain barrier. [Figure 12A] This figure shows the X-ray crystal structure of the region of RET amino acid residues 806-810 in complexes of wild-type RET with TAS compound 1, BLU-667, and LOXO-292. Figure 12A shows, based on cocrystal structure data, that BLU-667 and LOXO-292 bind to the same pocket (pocket B) of RET, while TAS compound 1 binds to different pockets (pocket A) of RET in different binding modes. [Figure 12B] Figure 12B shows the cocrystal complex of RET with TAS compound 1, BLU-667, and LOXO-292. [Figure 12C] Figure 12C shows the cocrystal complex of RET and TAS compound 1. [Figure 13A] This figure shows the effects of HM06 / TAS0953, LOXO-292, and BLU-667 in a Ba / F3 KIF5B-RETG810R cell-supported nude mouse model. Figure 13A shows the effect on tumor volume, with HM06 / TAS0953, LOXO-292, and BLU-667 administered twice daily at a dose of 10 mg / kg, respectively. [Figure 13B] Figure 13B shows the effect of a dose of 30 mg / kg administered twice daily. [Figure 13C] Figure 13C shows the change in body weight during the treatment period in nude mice carrying Ba / F3 KIF5B-RETG810R cells. [Figure 14A] This figure shows the effects of HM06 / TAS0953, LOXO-292, and BLU-667 in a Ba / F3 KIF5B-RETG810R cell-supported nude mouse model. Figure 14A shows the effect on tumor volume, with HM06 / TAS0953 administered twice daily at a dose of 50 mg / kg, and LOXO-292 and BLU-667 administered twice daily at a dose of 30 mg / kg, respectively. [Figure 14B] Figure 14B shows the change in body weight during the treatment period in nude mice carrying Ba / F3 KIF5B-RETG810R cells. [Figure 15A] This figure shows the phosphorylation of RET in Ba / F3 KIF5B-RETG810R tumors 1 hour after administration of HM06 / TAS0953, BLU-667, and LOXO-292. Ba / F3 KIF5B-RETG810R-carrying mice were orally administered once at doses of 10 mg / kg, 30 mg / kg, or 50 mg / kg of HM06 / TAS0953, or 10 mg / kg or 30 mg / kg of LOXO292 and BLU667. One hour after administration, the tumors were collected and lysed. The cell lysates were immunoblotted to detect the indicated proteins. [Figure 15B] This figure shows the phosphorylation of RET in Ba / F3 KIF5B-RETG810R tumors 1 hour after administration of HM06 / TAS0953, BLU-667, and LOXO-292. Ba / F3 KIF5B-RETG810R-carrying mice were orally administered once at doses of 10 mg / kg, 30 mg / kg, or 50 mg / kg of HM06 / TAS0953, or 10 mg / kg or 30 mg / kg of LOXO292 and BLU667. One hour after administration, the tumors were collected and lysed. The cell lysates were immunoblotted to detect the indicated proteins. [Modes for carrying out the invention]

[0044] Description of specific sequences Table 1 lists the specific sequences referenced herein.

[0045] [Table 1] JPEG2026143696000002.jpg233170JPEG2026143696000003.jpg233170JPEG20261436960 00004.jpg233170JPEG2026143696000005.jpg233170JPEG2026143696000006.jpg154170

[0046] Description of specific embodiments As used herein, "TAS0953 / HM06," "HM06 / TAS0953," or "HM06" are synonymous and, unless otherwise specified, refer to the free base form of 4-amino-N-(4-(methoxymethyl)phenyl)-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide, as well as any salt forms thereof, including the dihydrochloride salt. The dihydrochloride salt of 4-amino-N-(4-(methoxymethyl)phenyl)-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide is also synonymously referred to as "TAS0953-01 / HM06-01", "HM06-01", or "HM06-01 / TAS0953-01". The molecular formula of the free base form HM06 / TAS0953 is C26H30N6O3, and its molecular weight is 474.57. The structural formula of the free base is as follows: [ka]

[0047] The molecular formula of HM06-01 / TAS0953-01 is C26H32O3N6Cl2, and its molecular weight is 547.54. The chemical structure of the dihydrochloride salt is as follows: [ka]

[0048] In some embodiments, the free base form is the active pharmaceutical ingredient (API). In some embodiments, the dihydrochloride salt is the active pharmaceutical ingredient (API). In some embodiments, the API is a white to off-white solid. In some embodiments, the API is freely soluble in water.

[0049] The dosage form of a composition containing HM06 / TAS0953 can be any form, oral or parenteral. Such formulations are not particularly limited and include, for example, oral compositions such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions, and parenteral compositions such as injections, suppositories, and inhalants. Injections can be administered intravenously alone or in mixtures with common adjuvants, such as glucose or amino acids, or, as needed, alone, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal. Suppositories are administered rectally.

[0050] In some embodiments, HM06 / TAS0953 is prepared as a dihydrochloride (HM06-01 / TAS0953-01) and formulated into tablets. In some embodiments, the tablets are for oral administration. In some embodiments, the tablets are formulated in doses of about 10 mg / unit or about 50 mg / unit (expressed as free base), or in other dosages as described in the specification. In some embodiments, the tablets are administered orally as a single tablet or as a group of tablets.

[0051] In some embodiments, the composition contains a standard, widely used excipient. In some embodiments, the composition contains at least one excipient. In some embodiments, The composition comprises at least one antioxidant, at least one filler, at least one disintegrant, at least one surfactant, and / or at least one lubricant. In some embodiments, the composition comprises citric acid, crystalline cellulose (e.g., Avicel PH200 LM), lactose (e.g., lactose 316 Fast Flo), polyvinyl N-pyrrolidone (e.g., crospovidone), sodium lauryl sulfate, and / or glyceryl behenate (e.g., Compritol ATO 888).

[0052] Generally, human patients are administered a composition containing HM06 / TAS0953 in an effective dose. In some embodiments, human patients are administered a composition containing HM06 / TAS0953 in a dose equivalent to approximately 40 mg to approximately 3000 mg of free base of HM06 / TAS0953 per day. In some embodiments, human patients are administered a composition containing HM06 / TAS0953 in a dose equivalent to approximately 40 mg to approximately 1000 mg of free base of HM06 / TAS0953 per day. In some embodiments, the dose of HM06 / TAS0953 is administered once daily (QD) or multiple times daily (e.g., BID or TID). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to approximately 20 mg to approximately 1500 mg of free base of HM06 / TAS0953 twice daily (BID). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to approximately 20 mg to approximately 500 mg of free base of HM06 / TAS0953 twice daily (BID).

[0053] In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 150 mg to 640 mg of free base of HM06 / TAS0953 per day (for example, approximately 75 mg to 320 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 160 mg to 640 mg of free base of HM06 / TAS0953 per day (for example, approximately 80 mg to 320 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 150 mg to 500 mg of free base of HM06 / TAS0953 per day (for example, approximately 75 mg to 250 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 160 mg to 500 mg of free base of HM06 / TAS0953 per day (for example, approximately 80 mg to 250 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 320 mg to 640 mg of free base of HM06 / TAS0953 per day (for example, approximately 160 mg to 320 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 480 mg to 640 mg of free base of HM06 / TAS0953 per day (for example, approximately 240 mg to 320 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a daily dose of approximately 480 mg to 3000 mg of free base of HM06 / TAS0953 (for example, approximately 240 mg to 1500 mg of free base of HM06 / TAS0953 twice daily).In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 480 mg to 2000 mg of free base of HM06 / TAS0953 per day (for example, approximately 240 mg to 1000 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 480 mg to 1500 mg of free base of HM06 / TAS0953 per day (for example, approximately 240 mg to 750 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, HM06 / T is administered. The dosage of AS0953 is equivalent to approximately 480 mg to 1280 mg of free base of HM06 / TAS0953 per day (for example, approximately 240 mg to 640 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to approximately 480 mg to 1000 mg of free base of HM06 / TAS0953 per day (for example, approximately 240 mg to 500 mg of free base of HM06 / TAS0953 twice daily).

[0054] In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 640 mg to 3000 mg of free base of HM06 / TAS0953 per day (for example, approximately 320 mg to 1500 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 640 mg to 2000 mg of free base of HM06 / TAS0953 per day (for example, approximately 320 mg to 1000 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 640 mg to 1500 mg of free base of HM06 / TAS0953 per day (for example, approximately 320 mg to 750 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to a range of approximately 640 mg to 1280 mg of free base of HM06 / TAS0953 per day (for example, approximately 320 mg to 640 mg of free base of HM06 / TAS0953 twice daily). In some embodiments, the dosage of HM06 / TAS0953 administered is equivalent to approximately 640 mg to 1000 mg of free base of HM06 / TAS0953 per day (for example, approximately 320 mg to 500 mg of free base of HM06 / TAS0953 twice daily).

[0055] In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 40 mg per day (e.g., approximately 20 mg twice a day), approximately 60 mg per day (e.g., approximately 30 mg twice a day), approximately 80 mg per day (e.g., approximately 40 mg twice a day), approximately 100 mg per day (e.g., approximately 50 mg twice a day), approximately 120 mg per day (e.g., approximately 60 mg twice a day), approximately 140 mg per day (e.g., approximately 70 mg twice a day), and approximately 160 mg per day (e.g., approximately 80 mg (mg twice a day), approximately 180 mg per day (for example, approximately 90 mg twice a day), approximately 200 mg per day (for example, approximately 100 mg twice a day), approximately 220 mg per day (for example, approximately 110 mg twice a day), approximately 240 mg per day (for example, approximately 120 mg twice a day), approximately 260 mg per day (for example, approximately 130 mg twice a day), approximately 280 mg per day (for example, approximately 140 mg twice a day), approximately 300 mg per day (for example, approximately 150 mg twice a day), approximately 320 mg per day (for example, approximately 1 60 mg twice a day), approximately 340 mg per day (for example, approximately 170 mg twice a day), approximately 360 mg per day (for example, approximately 180 mg twice a day), approximately 380 mg per day (for example, approximately 190 mg twice a day), approximately 400 mg per day (for example, approximately 200 mg twice a day), approximately 420 mg per day (for example, approximately 210 mg twice a day), approximately 440 mg per day (for example, approximately 220 mg twice a day), approximately 460 mg per day (for example, approximately 230 mg twice a day), approximately 480 mg per day (for example) Contains approximately 240 mg twice daily, approximately 500 mg per day (for example, approximately 250 mg twice daily), approximately 750 mg per day (for example, approximately 375 mg twice daily), approximately 1000 mg per day (for example, approximately 500 mg twice daily), approximately 1280 mg per day (for example, approximately 640 mg twice daily), approximately 1500 mg per day (for example, approximately 750 mg twice daily), approximately 2000 mg per day (for example, approximately 1000 mg twice daily), or approximately 3000 mg per day (for example, approximately 1500 mg twice daily).

[0056] In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 40 mg or more per day (for example, approximately 20 mg twice a day), approximately 60 mg or more (for example, about 30 mg twice a day), about 80 mg or more per day (for example, about 40 mg twice a day), about 100 mg or more per day (for example, about 50 mg twice a day), about 120 mg or more per day (for example, about 60 mg twice a day), about 140 mg or more per day (for example, about 70 mg twice a day), about 160 mg or more per day (for example, about 80 mg twice a day), about 180 mg or more per day (for example, about 90 mg twice a day), about 200 mg or more per day (for example, about 100 mg twice a day) ), approximately 220 mg or more per day (for example, approximately 110 mg twice a day), approximately 240 mg or more per day (for example, approximately 120 mg twice a day), approximately 260 mg or more per day (for example, approximately 130 mg twice a day), approximately 280 mg or more per day (for example, approximately 140 mg twice a day), approximately 300 mg or more per day (for example, approximately 150 mg twice a day), approximately 320 mg or more per day (for example, approximately 160 mg twice a day), approximately 340 mg or more per day (for example, approximately 170 mg twice a day), approximately 360 mg or more per day (For example, about 180 mg twice a day), about 380 mg or more per day (for example, about 190 mg twice a day), about 400 mg or more per day (for example, about 200 mg twice a day), about 420 mg or more per day (for example, about 210 mg twice a day), about 440 mg or more per day (for example, about 220 mg twice a day), about 460 mg or more per day (for example, about 230 mg twice a day), about 480 mg or more per day (for example, about 240 mg twice a day), about 500 mg or more per day (for example, about 250 mg once a day) The dosages are approximately 640 mg or more per day (for example, approximately 320 mg twice a day), approximately 750 mg or more per day (for example, approximately 375 mg twice a day), approximately 1000 mg or more per day (for example, approximately 500 mg twice a day), approximately 1280 mg or more per day (for example, approximately 640 mg twice a day), approximately 1500 mg or more per day (for example, approximately 750 mg twice a day), approximately 2000 mg or more per day (for example, approximately 2000 mg twice a day), or approximately 3000 mg or more per day (for example, approximately 1500 mg twice a day).

[0057] In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 640 mg or more per day (for example, approximately 320 mg twice a day). In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 1000 mg or more per day (for example, approximately 500 mg twice a day). In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 1280 mg or more per day (for example, approximately 640 mg twice a day). In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 1500 mg or more per day (for example, approximately 750 mg twice a day). In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free base) is approximately 2000 mg or more per day (for example, approximately 1000 mg twice a day).

[0058] In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 3000 mg or less per day (for example, approximately 1500 mg or less twice a day). In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 2000 mg or less per day (for example, approximately 1000 mg or less twice a day). In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 1500 mg or less per day (for example, approximately 750 mg or less twice a day). In some embodiments, the dosage of HM06 / TAS0953 (expressed in terms of free bases) is approximately 1280 mg or less per day (for example, approximately 640 mg or less twice a day). In some embodiments, the dosage of HM06 / TAS0953 (expressed as free base) is approximately 1000 mg or less per day (for example, approximately 500 mg or less twice a day). In some embodiments, the dosage of HM06 / TAS0953 is approximately 640 mg or less per day (for example, approximately 320 mg or less twice a day). In some embodiments, the dosage of HM06 / TAS0953 is approximately 400 mg or less per day (for example, approximately 200 mg or less twice a day).

[0059] In some embodiments, the dosage of HM06 / TAS0953 (expressed as free base) is approximately 150 mg per day (e.g., approximately 75 mg twice a day). In some embodiments, the dosage of HM06 / TAS0953 is approximately 160 mg per day (e.g., approximately 80 mg twice a day). In some embodiments, the dosage of HM06 / TAS0953 is approximately 320 mg per day (e.g., approximately 160 mg twice a day). In some embodiments, the dosage of HM06 / TAS0953 is approximately 640 mg per day (e.g., approximately 320 mg twice a day). In some embodiments, the dosage of HM06 / TAS0953 is approximately 1000 mg per day (e.g., approximately 500 mg twice a day). In some embodiments, the dosage of HM06 / TAS0953 is approximately 1280 mg per day (e.g., approximately 640 mg twice daily). In some embodiments, the dosage of HM06 / TAS0953 is approximately 1500 mg per day (e.g., approximately 750 mg twice daily). In some embodiments, the dosage of HM06 / TAS0953 is approximately 2000 mg per day (e.g., approximately 1000 mg twice daily). In some embodiments, the dosage of HM06 / TAS0953 is approximately 3000 mg per day (e.g., approximately 1500 mg twice daily). In some embodiments, dosage changes may occur during treatment.

[0060] In some embodiments, the human patient is 12 years of age or older. In some embodiments, the human patient is 12 years of age or older and weighs less than 50 kg. In some embodiments, the dosage administered is the same for patients weighing more than 50 kg and patients weighing less than 50 kg.

[0061] In some embodiments, for patients weighing less than 50 kg, the dosage of HM06 / TAS0953 is approximately 3000 mg or less per day (e.g., 1500 mg twice a day), approximately 2000 mg or less per day (e.g., 1000 mg twice a day), approximately 1500 mg or less per day (e.g., 750 mg twice a day), approximately 1280 mg or less per day (e.g., 640 mg twice a day), and approximately 1000 mg per day. The following dosages are possible (for example, 500 mg twice a day), approximately 640 mg or less per day (for example, 320 mg twice a day), approximately 320 mg or less per day (for example, 160 mg twice a day), approximately 160 mg or less per day (for example, 80 mg twice a day), approximately 120 mg or less per day (for example, 60 mg twice a day), approximately 80 mg or less per day (for example, 40 mg twice a day), or approximately 40 mg or less once a day. For patients weighing over 50 kg, the dosage of HM06 / TAS0953 is approximately 3000 mg or less per day (e.g., 1500 mg twice a day), approximately 2000 mg or less per day (e.g., 1000 mg twice a day), approximately 1500 mg or less per day (e.g., 750 mg twice a day), approximately 1280 mg or less per day (e.g., 640 mg twice a day), and approximately 1000 mg or less per day (e.g., 500 mg twice a day). Dosages may be approximately 640 mg or less once daily (e.g., 320 mg twice daily), approximately 480 mg or less per day (e.g., 240 mg twice daily), approximately 320 mg or less per day (e.g., 160 mg twice daily), approximately 240 mg or less per day (e.g., 120 mg twice daily), approximately 160 mg or less per day (e.g., 80 mg twice daily), or approximately 80 mg or less per day (e.g., 40 mg twice daily). In some embodiments, dose changes may occur during treatment.

[0062] In some embodiments, a human patient has or has been diagnosed with salivary gland cancer, lung cancer, colorectal cancer, thyroid cancer, breast cancer, pancreatic cancer, ovarian cancer, skin cancer, or brain cancer. In some embodiments, a human patient has or has been diagnosed with medullary thyroid carcinoma or anaplastic thyroid carcinoma, metastatic breast cancer, or metastatic pancreatic adenocarcinoma. In some embodiments, a human patient has or has been diagnosed with non-small cell lung cancer (NSCLC). Examples of NSCLC include adenocarcinoma and large cell carcinoma (non-squamous cell carcinoma), and Squamous cell carcinoma is one example. In some embodiments, human patients have locally advanced or metastatic NSCLC, or have been diagnosed with such cancer.

[0063] In some embodiments, a primary tumor (or multiple tumors) in a human patient can metastasize to the central nervous system (CNS). For example, a human patient may have, or be diagnosed with, a primary tumor and CNS metastases. In some embodiments, a human may have, or be diagnosed with, metastatic NSCLC with brain and / or leptomeningeal metastases.

[0064] As used herein, “brain and / or leptomeningeal metastases” also means brain metastases or leptomeningeal diseases, and include asymptomatic and symptomatic conditions, which may be measurable or unmeasurable.

[0065] Where used herein, unless otherwise specified, "RET" refers to the gene encoding the RET protein and / or the RET protein, or a variant, polymorphism, or part of the RET gene or protein.

[0066] As used herein, "RET protein" or "RET polypeptide" refers to the tyrosine kinase receptor encoded by the RET gene (also referred to as the RET proto-oncogene), and may include the whole or a portion of the RET protein.

[0067] In some embodiments, the RET protein comprises the amino acid sequence of SEQ ID NO: 2 or a portion thereof.

[0068] In some embodiments, the RET protein is a mutant RET protein. In some embodiments, the RET protein contains the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a portion thereof.

[0069] In some embodiments, the RET protein is encoded by a RET gene containing a RET gene mutation.

[0070] As used herein, “RET gene abnormality” refers to the difference between a mutant RET gene sequence and a wild-type RET gene sequence. For example, RET gene abnormalities can be chromosomal rearrangements, point mutations, copy number increases, overexpression, and / or ligand-induced activation.

[0071] The presence or absence of RET gene abnormalities can be determined by any one of the many methods understood in the field, including, but not limited to, DNA or RNA sequencing or FISH analysis (e.g., Subbiah, V et al., Ann Oncol. 2021, 32(2):261-268; Lin JJ et al., Ann Oncol. 2020, 31(12):1725-1733; Solomon See BJ et al., J Thorac Oncol. 2020, 15(4):541-549. In some embodiments RET gene abnormalities are detected by sequencing of circulating tumor DNA. In some embodiments, RET gene abnormalities are detected by targeted single amplicon sequencing.

[0072] In some embodiments, the RET gene containing the RET gene abnormality encodes a mutant RET protein, such as a RET protein fusion and / or a solvent front mutation.

[0073] In some embodiments, cancer or tumors include RET gene abnormalities. In some embodiments, patients have RET gene abnormalities selected from chromosomal rearrangement (RET gene fusion), point mutations, copy number increase, overexpression, and ligand-induced activation. Overexpression may result from copy number increase or transcriptional upregulation, which may lead to increased local receptor concentrations and abnormal activation. Copy number increase or overexpression may refer to wild-type RET or mutant RET. For example, overexpression of mutant RET has been found in MEN2-associated tumors. Stable overexpression of both mutant M918T and wild-type RET has been found in two SCLC cell lines. Ligand-induced activation of RET may result in stimulation of multiple signaling pathways, including the MAP kinase / Erk pathway and the PI3 kinase / Akt pathway. This activation may occur in both wild-type RET and mutant RET.

[0074] In some embodiments, RET gene fusion occurs in NSCLC, papillary thyroid carcinoma (PTC), colorectal cancer (CCDC6-RET fusion), or breast cancer (ERC1-RET fusion). In some embodiments, RET gene point mutations occur in medullary thyroid carcinoma (MTC), which includes multiple endocrine neoplasia 2A (MEN2A), MEN2B, or familial medullary thyroid carcinoma (FMTC). In some embodiments, RET gene copy number increase occurs in NSCLC, breast cancer, pancreatic cancer, or glioblastoma.

[0075] In some embodiments, cancer or tumors include fusions of the C-terminal kinase domain of RET with the N-terminal sequence of kinesin family member 5B (KIF5B-RET), CCDC6-RET (RET-PTC1), NCOA4-RET (RET-PTC3), or TRIM33-RET (RET-PTC7). KIF5B-RET gene fusions can result in a 2- to 30-fold increase in RET transcription, suggesting that RET kinase activity drives tumorigenesis in these cases. In some embodiments, RET fusions include fusions of RET with PRKAR1A, TRIM24, GOLGA5, KTN1, MBD1, or TRIM27. In some embodiments, RET fusions include fusions of RET with TRIM33, KIF5B, CCDC6, or KIF5B.

[0076] In some embodiments, human patients do not have EGFR, KRAS, ALK, HER2, ROS1, BRAF, and / or METex14 activating mutations. RET fusions in NSCLCs are present in the majority of cases and are mutually exclusive with EGFR, KRAS, ALK, HER2, and BRAF mutations.

[0077] Examples of multikinase inhibitors (MKIs) with activity against RET receptor tyrosine kinase (RTK) include cabozantinib, vandetanib, alectinib, sunitinib, sorafenib, pazopanib, ponatinib, regorafenib, apatinib, citravatinib, RXDX-105, and lenvatinib. In some embodiments, human patients have been previously treated with cabozantinib, vandetanib, lenvatinib, RXDX-105, or another multikinase inhibitor. In some embodiments, human patients experienced disease progression after prior treatment. In some embodiments, human patients developed intolerance to prior treatment. In some embodiments, human patients have not been previously treated with a multikinase inhibitor.

[0078] In some embodiments, human patients have previously been treated with selpercatinib (LOXO-292), pralcetinib (BLU-667), BOS-172738, or another RET-selective inhibitor. In some embodiments, human patients experienced disease progression after prior treatment. In some embodiments, human patients developed intolerance to prior treatment. In some embodiments, human patients have not previously been treated with a RET-selective inhibitor.

[0079] Despite clinical improvement observed with targeted agonists, patients with RET abnormalities frequently experience relapses. In some embodiments, the cancer or tumor has resistance mutations or has developed such resistance mutations. In some embodiments, the cancer or tumor is resistant to one or more multikinase inhibitors. In some embodiments, the cancer or tumor is resistant to one or more RET selective inhibitors. In some embodiments, the cancer or tumor is resistant to serpercatinib and / or pralcetinib. In some embodiments, the cancer or tumor contains cells that are resistant to serpercatinib and / or pralcetinib.

[0080] In some embodiments, HM06 / TAS0953 is effective in treating cancer or tumors that have or have developed resistance mutations in the RET protein. In some embodiments, the RET gene abnormality includes a solvent front mutation and / or a hinge region mutation of the RET protein. In some embodiments, the RET gene abnormality includes a solvent front mutation of the RET protein.

[0081] In some embodiments, the RET gene abnormality includes mutations in the RET protein at amino acid residues 730, 736, 760, 772, 804, 806, 807, 808, 809, 810, and / or 883. In some embodiments, the RET gene abnormality includes mutations in the RET protein at amino acid residues 804, 806, 807, 808, 809, and / or 810. In some embodiments, the RET gene abnormality includes mutations in the RET protein at amino acid residue 810.

[0082] In some embodiments, the RET gene abnormality includes RET protein mutations, including the following mutations: a) V804X mutation (where X is any amino acid other than valine or glutamic acid); b) Y806X mutation (where X is any amino acid other than tyrosine); c) A807X mutation (where X is any amino acid other than alanine); d) K808X mutation (where X is any amino acid other than alanine); e) Y809X mutation (where X is any amino acid other than tyrosine); and / or f) G810X mutation (where X is any amino acid other than glycine).

[0083] In some embodiments, the RET gene abnormality includes a RET protein mutation containing the V804X mutation, where X is any amino acid other than valine or glutamic acid. In some embodiments, the RET gene abnormality includes a RET protein mutation containing the Y806X mutation, where X is any amino acid other than tyrosine. In some embodiments, the RET gene abnormality includes a RET protein mutation containing the A807X mutation, where X is any amino acid other than alanine. In some embodiments, the RET gene abnormality includes a RET protein mutation containing the K808X mutation, where X is any amino acid other than alanine. In some embodiments, the RET gene abnormality includes a RET protein mutation containing the Y809X mutation, where X is any amino acid other than tyrosine. In some embodiments, the RET gene abnormality includes a RET protein mutation containing the G810X mutation, where X is any amino acid other than glycine.

[0084] In some embodiments, the RET gene abnormality includes RET protein mutations, including: a) V804L or V804M mutations; b) Y806C, Y806S, Y806H, or Y806N mutations; and / or c) G810R, G810S, G810C, G810V, G810D, or G810A mutations. In some embodiments, the RET gene abnormality includes V804L or V804M mutations in the RET protein. In some embodiments, the RET gene abnormality includes Y806C, Y806S, Y806H, or Y806N mutations in the RET protein. In some embodiments, the RET gene abnormality includes G810R, G810S, G810C, G810V, or G810D mutations in the RET protein. , or including the G810A mutation. In some embodiments, the RET gene abnormality includes the G810R mutation in the RET protein.

[0085] In some embodiments, the RET gene abnormality includes RET protein mutations, including: a) L730Q or L730R mutations; b) G736A mutations; c) L760Q mutations; d) L772M mutations; and / or e) A883V mutations. In some embodiments, the RET gene abnormality includes L730W or L730R mutations in the RET protein. In some embodiments, the RET gene abnormality includes G736A mutations in the RET protein. In some embodiments, the RET gene abnormality includes L760Q mutations in the RET protein. In some embodiments, the RET gene abnormality includes L772M mutations in the RET protein. In some embodiments, the RET gene abnormality includes A883V mutations in the RET protein. [Examples]

[0086] Example 1. Brain transfer characteristics of HM06 / TAS0953 In the initial study, the ability of HM06 / TAS0953 to cross the blood-brain barrier was evaluated. HM06 / TAS0953 was dissolved in 0.5% HPMC and 0.1N hydrochloric acid and administered orally as a single dose to BALB / cAJcl-nu / nu mice (CREA Japan Co., Ltd.). The mice were subcutaneously transplanted with the BaF3 / KIF5B-RET_RFP cell line (i.e., a KIF5B RET fusion-positive cell line). One hour after administration, blood was collected from the inferior vena cava under isoflurane anesthesia, and then the whole brain was removed. The blood sample was centrifuged to obtain a plasma sample. The brain was homogenized with three times the volume of water using an ultrasonic homogenizer to obtain brain homogenate.

[0087] The compound concentrations of HM06 / TAS0953 in plasma and brain homogenate were measured by LC-MS / MS, and the compound concentration in brain was calculated by multiplying the compound concentration in brain homogenate by a coefficient of 4. See Table 2 below. The Kp value (= compound concentration in brain homogenate / compound concentration in plasma) was calculated from the brain / plasma compound concentration ratio. The unbound compound concentrations in plasma and brain were calculated from the plasma protein unbound ratio and the brain protein unbound ratio, and the Kp,uu value was calculated from the brain / plasma unbound compound concentration ratio. Brain penetration characteristics were evaluated based on the calculated Kp value and Kp,uu value. Compounds showing a Kp value of 0.1 or higher were considered to have brain penetration characteristics, and compounds showing a Kp,uu value of 0.3 or higher were considered to have excellent brain penetration characteristics (Varadharajan S, et al. J Pharm Sci. 2015, 104:1197-1206).

[0088] [Table 2]

[0089] HM06 / TAS0953 exhibits high Kp and Kp,uu values, demonstrating excellent brain penetration characteristics. This data suggests that HM06 / TAS0953 may be effective in treating brain metastatic lesions or other CNS diseases.

[0090] Example 2. HM06 / TAS0953 exhibits strong efficacy in a brain metastasis model. The efficacy of HM06 / TAS0953 in treating RET abnormality-positive tumors was tested in a KIF5B-RET fusion-positive brain metastasis model. HM06 / TAS0953 showed a potent and stable effect.

[0091] In this model, KIF5B-RET Luc fusion-positive NIH3T3 cells (2.5 × 10⁶) 5 Cells (from a mouse) were transplanted into the brains of athymoid nude mice (Charles River Co., Ltd., Japan) at a depth of 3 mm, 3 mm anterior and 2 mm right-lateral to the lambdoid suture. Six days after transplantation, the mice were intravenously injected with luciferin (Fujifilm Wako Pure Chemical Corporation), and the luminescence of the mice was measured using an in vivo imaging system (Lumina II, PerkinElmer). The total flux (p / s) of the dorsal and lateral regions of interest of the mouse was quantified using Living Image software (PerkinElmer), and the sum of both fluxes was calculated. The total photons obtained by adding up the X values ​​were used as the luminescence signal. Mice were randomly assigned to three groups of 10 mice each, so that the average total photons in each group were equal. Then, they were orally administered either Vehicle (0.1 mol / L hydrochloric acid-containing 0.5% hydroxypropyl methylcellulose) or TAS0953 (12.5 mg / kg or 50 mg / kg) twice daily (bid) starting from day 7. The luminescence signal of the mice was measured once a week until the end of the study.

[0092] HM06 / TAS0953 demonstrated potent and stable efficacy. Figure 1A shows the antitumor efficacy of HM06 / TAS0953 administered at 50 mg / kg via BID compared to the vehicle control. Figure 1B shows the percentage change in body weight and indicates that mice treated with HM06 / TAS0953 were well-tolerated at the tested dose, while the vehicle control group showed suppressed animal mortality and weight gain due to tumor burden. Figure 1C shows that the survival rate in the HM06 / TAS0953 group was higher than in the vehicle control group. Figure 1D shows in vivo imaging system (IVIS) images and brain pathology of treated mice compared to the vehicle control.

[0093] These data establish that HM06 / TAS0953 may have a potent antitumor effect on brain metastases in RET mutation-positive patients and demonstrate long-term survival. These data also show that HM06 / TAS0953 exhibits good brain penetration in mice.

[0094] Example 3. HM06 / TAS0953 is effective in a vandetanib-resistant tumor model. To evaluate whether switching to HM06 / TAS0953 has a significant effect on tumor size, the efficacy of HM06 / TAS0953 after vandetanib treatment was compared to continued vandetanib treatment. The results suggest that HM06 / TAS0953 may be effective against treatment-resistant tumors.

[0095] A mouse fibroblast cell line transfected with the fusion kinase KIF5B-RET (NIH3T3 KIF5B-RET) was placed in the right thorax of a 6-week-old male BALB / cA Jcl-nu mouse in a 5x10⁶ cell transfected cell line. 6 Cells were transplanted into mice. After tumor transplantation, the long axis (mm) and short axis (mm) of the tumor were measured using calipers, and the tumor volume (TV) was calculated according to the following formula (A). The mice were then assigned to groups so that the average TV of each group was uniform. The day on which this group assignment (n=5 or 6 / group) was performed was designated as day 1.

[0096] Vandetanib was administered orally at a dose of 100 mg / kg / day daily to vandetanib-treated mice until the average tumor volume exceeded three times the average tumor volume on day 1. On day 16, The vandetanib-treated group was divided into two groups: Group 1: Vandetanib continuation group; Group 2: HM06 / TAS0953-treated group. In the HM06 / TAS0953-treated group, after switching from vandetanib to HM06 / TAS0953, HM06 / TAS0953 was administered orally at a dose of 100 mg / kg / day (50 mg / kg, BID) daily.

[0097] As an indicator of antitumor effect, the TV on day 31 was measured for each group, and the relative tumor volume (RTV) relative to day 1 was calculated using the following formula (B) to evaluate the antitumor effect.

[0098] The results are shown in Figure 2. (In the figure, symbols indicate...) * This shows a significant difference between the HM06 / TAS0953 treatment group and the continuous vandetanib treatment group. (A): TV (mm 3 )=(long axis x short axis 2 ) / 2 (B): RTV = (TV on day n) / (TV on day 1) n: Displayed measurement date

[0099] Statistically, the mean RTV value in the HM06 / TAS0953 treatment group was significantly lower than that of the group continuing vandetanib treatment (Student's t-test, p<0.05). These data indicate that HM06 / TAS0953 was effective against vandetanib-resistant tumors.

[0100] Example 4. Preclinical evaluation of HM06 / TAS0953 in a lung cancer cell line driven by RET rearrangement. Next, the efficacy of HM06 / TAS0953 in inhibiting the proliferation of RET fusion-positive cell lines was examined in comparison with three RET multikinase inhibitors (cabozantinib, RXDX-105, and vandetanib). The efficacy of HM06 / TAS0953 was tested in two cell lines derived from treatment-naive samples. One cell line was derived from a sample obtained from a patient who had never received any anticancer therapy, and this sample was KIF5B-RET fusion-positive. Treatment with HM06 / TAS0953 inhibited cell proliferation with an IC50 of 0.03 μM (Figure 3A). This was 7 to 24 times more potent than the multikinase inhibitors, cabozantinib, RXDX-105, and vandetanib.

[0101] HM06 / TAS0953 was tested in isogeneic cell lines to investigate its efficacy against CCDC6-RET fusion-driven cell lines and its nonspecific effects in non-tumor-forming control cell lines. HM06 / TAS0953 inhibited the proliferation of CCDC6-RET fusion cell lines with an IC50 of 0.1 μM (Figure 3B). The isogeneic counterpart expressing the blank control plasmid of this cell line showed significantly lower sensitivity to HM06 / TAS0953 (17-fold lower) (Figure 3C). CCDC6-RET fusion cell lines were equally sensitive to cabozantinib, RXDX-105, and vandetanib.

[0102] HM06 / TAS0953 was tested in TRIM33-RET fusion-positive cell lines. Cell proliferation was inhibited by HM06 / TAS0953 with an IC50 of 0.006 μM, which was 8 to 20 times lower than the IC50 of proliferation inhibition by three different multi-kinase RET inhibitors (Figure 3D).

[0103] The inhibitory effect of HM06 / TAS0953 was tested in RET fusion-positive cell lines resistant to RET multikinase inhibitors. HM06 / TAS0953 inhibited the proliferation of cell lines (CCDC6-RET fusion) derived from samples of patients resistant to cabozantinib, with an IC50 of 0.06 μM. This was 11.5 times lower than the IC50 of cabozantinib-induced proliferation inhibition of these cells. HM06 / TAS0953 was more potent in inhibiting the proliferation of cabozantinib-resistant cells compared to RXDX-105 and vandetanib (Figure 3E).

[0104] HM06 / TAS0953 inhibited the proliferation of cell lines (KIF5B-RET fusion) derived from samples obtained from patients who had acquired resistance to RXDX-105, with an IC50 of 0.07 μM (Figure 3F). This was significantly lower than the IC50 for proliferation inhibition by cabozantinib (0.34 μM, 95% CI: 0.21-0.54), RXDX-105 (0.49 μM, 95% CI: 0.31-0.76), or vandetanib (0.38 μM, 95% CI: 0.29-0.49).

[0105] These results suggest that HM06 / TAS0953 is more effective than other RET multikinase inhibitors in inhibiting the proliferation of cell lines with RET fusions. These results also suggest that HM06 / TAS0953 is effective against cell lines that are resistant to the inhibitory activity of other RET multikinase inhibitors. Furthermore, HM06 / TAS0953 was effective against cell lines with RET fusions consisting of three different N-terminal fusion partners (CCDC6, KIF5B, and TRIM33).

[0106] Example 5. Evaluation of the efficacy of HM06 / TAS0953 in a preclinical animal model of lung cancer driven by RET rearrangement. The efficacy of HM06 / TAS0953 in reducing the growth of xenograft tumors was confirmed, and its efficacy was further investigated in comparison with vandetanib, a known multi-kinase inhibitor with anti-RET activity. Efficacy was observed in preclinical animal models of RET fusion-positive cancer. These preclinical animal models were sensitive to RET inhibitors by inducing xenograft tumors in immunodeficient mice by transplanting either isogenic cells (NIH-3T3-CCDC6-RET) or patient-derived cells (TRIM33-RET fusion) (Examples 5A and 5B). Efficacy was also observed in multiple patient-derived xenograft (PDX) models generated from samples of patients who had developed resistance to either RXDX-105 or cabozantinib (Example 5C). As described in detail below, in all models, HM06 / TAS0953 was superior to or as effective as vandetanib in inhibiting tumor growth, and in some cases, including tumor regression. Treatment of mice carrying RET-dependent xenograft tumors with HM06 / TAS0953 resulted in a significant reduction in tumor growth at a low dose of only 12.5 mg / kg BID. At doses of 50 mg / kg BID or 100 mg / kg QD, significant reductions in tumor growth were observed, including approximately 100% regression in TRIM33-RET fusion xenograft tumors. Similarly, treatment with HM06 / TAS0953 at 50 mg / kg BID or 100 mg / kg QD significantly reduced growth in cabozantinib-resistant PDX tumors, resulting in approximately 50% tumor regression. HM06 / TAS0953 treatment also induced significant reductions in growth in two PDX models derived from RXDX-105-resistant tumors. Mice treated with vandetanib experienced a significant loss of body weight, while mice treated with HM06 / TAS0953 were well tolerable to all doses tested in this study.

[0107] The effect of HM06 / TAS0953 on the proliferation of xenograft tumors was further observed in a preclinical orthotopic brain model (TRIM33-RET fusion) (Example 5D). Treatment with HM06 / TAS0953 at a BID of 50 mg / kg completely blocked tumor growth in the brain and resulted in a significant increase in the survival rate of tumor-bearing mice. These results suggest that HM06 / TAS0953 is an effective anti-RET inhibitor that can block the proliferation of RET fusion-positive xenograft tumors transplanted subcutaneously or intracranially into mice.

[0108] Methods: Six-week-old female NSG (NOD / SCID gamma) mice (Envigo, Madison, WI) were used for PDX model generation and all efficacy tests, with the exception of NI. H-3T3 xenograft studies use athymic nude mice (Envigo, Madison, WI). Twenty-two days after cell transplantation, a firm signal was detected, and after initiating treatment with Vehicle or 50 mg / kg of HM06 / TAS0953 in BID, mice were randomly assigned to groups of five. Luciferase signals were recorded weekly, and animal body weight was measured twice a week. Mice were sacrificed when signs of pathological conditions, such as impaired coordination or excessive weight loss and fatigue, were detected. In the treatment group, one animal was found to have died early in the study, and therefore the treatment group was reduced to only four animals.

[0109] Vehicles and compounds: Cabozantinib was mixed with 30% propylene glycol, 5% Tween 80, and 65% D5W (water containing 5% glucose) to prepare a suspension. Vandetanib was mixed with 1% sodium carboxymethylcellulose (CMC-Na) to prepare a suspension. HM06 / TAS0953 was mixed with 0.1N HCl and 0.5% hypromellose (HPMC) to prepare a suspension. RXDX-105 was mixed with 15% captisol to prepare a suspension. NIH-3T3 models, TRIM33-RET fusion models, PDX models (CCDC6-RET) obtained from patient samples acquired after RXDX-105 resistance, and models (CCDC6-RET) obtained from patients resistant to cabozantinib were treated with HM06 / TAS0953 free base. All other models were treated with HM06 dihydrochloride.

[0110] Statistical Analysis: Datasets were compared using two-way ANOVA, and significance was identified using Tukey or Sidax multiple comparison tests. A p < 0.05 value was considered statistically significant for the difference between two values ​​or between datasets. Survival curves were compared using the log-rank (Mantel-Cox) test. 95% confidence intervals and all statistical analyses were performed using Graphpad Prism v7 software.

[0111] Example 5A: HM06 / TAS0953 is effective in inhibiting the growth of RET inhibitor-sensitive xenograft tumors. The ability of HM06 / TAS0953 to inhibit the growth of xenograft tumors derived from cells that have not been treated with RET inhibitors was examined. NIH-3T3 cells stably expressing the CCDC6-RET fusion protein were transplanted into the subcutaneous flank of athymic nude mice. The tumors were approximately 100 mm in size. 3Upon reaching this stage, mice were randomly assigned to groups of 5 and treatment was initiated (day 6). The NIH-3T3-CCDC6-RET cell line was generated by stable expression of CCDC6-RET fusion cDNA. Athymic immunodeficient nude mice carrying NIH-3T3-CCDC6-RET xenograft tumors were treated with HM06 / TAS0953 at a dose of 12.5 mg / kg to 100 mg / kg once daily (QD) or twice daily (BID). Vandetanib (100 mg / kg QD) was used for comparison.

[0112] Treatment with HM06 / TAS0953 resulted in a significant reduction in tumor growth at all doses tested (Figures 4A and 4B). Vandetanib treatment also resulted in a significant reduction in tumor volume compared to the vehicle treatment group. However, HM06 / TAS0953 was more effective than vandetanib when administered at 50 mg / kg as BID or 100 mg / kg as QD. In these experiments, much higher doses of vandetanib (100 mg / kg) were used than those shown to inhibit RET fusion-dependent tumor growth (50 mg / kg, QD) (Suzuki M et al., Cancer Sci. 2013, 104(7):896-903 doi 10.1111 / cas.12175). These results suggest that HM06 / TAS0953 inhibits RET fusion-driven tumor growth. This suggests that it is more effective than vandetanib in reducing tumor growth. No significant weight loss occurred in animals at any of the HM06 / TAS0953 doses used (Figure 4C).

[0113] Example 5B: HM06 / TAS0953 is a patient-derived cell line driven by RET fusion. It is effective in inhibiting the proliferation of xenografts. The efficacy study was expanded to a patient-derived cell line xenograft model. TRIM33-RET fusion-positive xenograft tumors were transplanted into the subcutaneous flank of NSG mice. The tumors were approximately 100 mm in size. 3Upon reaching this stage, the mice were randomly assigned to groups of 5 and treatment was initiated (day 22) (Somwar R et al., J Clin Oncol. 2016, 34(15_suppl):9068).

[0114] Treatment with HM06 / TAS0953 resulted in a significant reduction in tumor growth at the three different doses tested (Figure 5A). In all groups, each xenograft tumor regressed. Tumors shrank by 60.7±5% when treated with 50 mg / kg of HM06 / TAS0953 once daily (Figure 5B). No palpable tumors remained in mice treated with 50 mg / kg of BID, and tumor growth was reduced by 90±4% when the animals were treated with 100 mg / kg of QD of HM06 / TAS0953 (Figure 5B). In all HM06 treatment groups, there was no significant change in animal body weight when comparing animal body weight at the start and end of the study. Although vandetanib treatment resulted in complete tumor regression, the animals lost a considerable amount of body weight, and all animals in this group had to be sacrificed on day 47 (Figure 5C). Each animal in the vandetanib-treated group began to lose weight on the third day after the start of treatment.

[0115] Example 5C: HM06 / TAS0953 is effective in inhibiting the growth of PDX tumors that are resistant to RET multikinase inhibitors. To further explore the efficacy of HM06 / TAS0953 in inhibiting tumor growth, the efficacy of inhibitors against tumor growth was examined in three PDX models. These models were resistant to RXDX-105, and one was resistant to cabozantinib.

[0116] PDX tumors (CCDC6-RET) derived from tumor samples obtained from patients who had no longer responded to cabozantinib were minced, mixed with Matrigel, and then transplanted into the subcutaneous flanks of NSG mice. The tumors were approximately 100 mm in size. 3Once reached, mice were randomly assigned to groups of 8 animals per group and treatment was initiated (Day 12). Treatment of tumor-bearing mice with vandetanib caused a significant reduction in tumor growth (Figure 6A), with no palpable tumors remaining at the end of the study (Figure 6B). All tumors in this group regressed 100%. However, the animals lost a substantial amount of body weight (Figure 6C). Although treatment with cabozantinib (30 mg / kg QD) significantly reduced tumor growth when compared to the vehicle-treated group (p<0.05), no tumor regression was observed, and all tumors in this group showed some degree of growth (Figure 6B). Treatment with HM06 / TAS0953 at 50 mg / kg BID or 100 mg / kg QD caused significant reduction in tumor growth (Figure 6A), with tumors regressing by 43.7±3.8% and 47.7±0.9%, respectively. One animal was found dead on Day 25 in the HM06 50 mg / kg BID group, therefore only 7 animals remained in this group at the end of the experiment. The cause of death of the animal was unknown.

[0117] PDX tumors (CCDC6-RET) derived from patients who were resistant to RXDX-105 treatment at the time of tumor sample collection were minced, mixed with Matrigel, and then implanted subcutaneously into the flank of NSG mice. When tumors reached approximately 100 mm 3 Once reached, mice were randomly assigned to groups of 5 animals per group and treatment was initiated (Day 14). Treatment with RXDX-105 (30 mg / kg BID) did not cause a significant change in tumor volume compared to the vehicle-treated group (p>0.05), demonstrating that this model is resistant to RXDX-105 (Figure 7A). Treatment with vandetanib (50 mg / kg QD) caused a significant reduction in tumor volume, with tumors regressing by 27.2±5.2% (p<0.05) (Figures 7A and 7B). Based on the observation of substantial weight loss in Figure 5C, 50 mg / kg QD vandetanib was used in this study and all subsequent studies. Similarly, treatment with HM06 / TAS0953 (50 mg / kg BID or 100 mg / kg QD) caused a significant reduction in tumor volume compared to the vehicle-treated group, with tumors reducing by 8.6± The body mass decreased by 11.8% and 30±7.7% respectively (Figures 7A and 7B). Treatment with either dose of HM06 / TAS0953 did not have a significant effect on animal body mass in these groups (p>0.05) (Figure 7C).

[0118] PDX tumors (CCDC6-RET) derived from patients with poor response to RXDX-105 were minced, mixed with Matrigel, and then transplanted into the subcutaneous flank of NSG mice. The tumors were approximately 100 mm in size. 3 Upon reaching this stage, mice were randomly assigned to groups of 8 and treatment was initiated (day 12). The efficacy of HM06 / TAS0953 at doses of 50 mg / kg BID and 100 mg / kg QD was tested in this model. Treatment with HM06 / TAS0953 at a BID dose of 50 mg / kg resulted in a small but significant reduction in tumor volume compared to the vehicle treatment group (Figure 8A). Treatment with HM06 / TAS0953 at a QD dose of 100 mg / kg was more effective in slowing tumor growth. As shown in Figure 8A, there was no tumor reduction (Figure 8B). HM06 / TAS0953 did not cause any significant changes in animal body weight in either group (Figure 8C).

[0119] These results suggest that HM06 / TAS0953 is effective in reducing the growth of PDX tumors that were resistant to cabozantinib and RXDX-105.

[0120] Example 5D: HM06 / TAS0953 is effective in inhibiting the growth of an orthotopic xenograft tumor model with RET fusion. To evaluate the effect of HM06 on RET fusion-positive tumors in the brain, TRIM33-RET fusion-positive xenograft tumors were transplanted into the brains of NSG mice. These cells were modified to express firefly luciferase to facilitate in vivo bioluminescence imaging. The xenograft tumors were digested, and then single cells were transplanted into the brains of NSG mice. Bioluminescence imaging was performed weekly, and treatment was initiated 22 days after tumor cell transplantation when a robust signal was detected (Day 0 in Panels A and B). Mice were imaged weekly, and after a robust signal was detected, treatment with 50 mg / kg of HM06 / TAS0953 was initiated using BID or vehicle (22 days after transplantation). Bioluminescence signals were quantified as described in the Materials and Methods section. Vehicle-treated mice developed tumors rapidly, as evidenced by a strong bioluminescence signal exceeding the mean onset signal (p<0.05) (Figures 9A and 9B). However, on day 22, a significant difference existed between the vehicle-treated group and the HM06-treated group (p<0.05). Mice treated with HM06 survived significantly longer than mice treated with vehicle (p<0.05), and 28 days after the last animal in the vehicle-treated group had to be sacrificed due to tumor burden, three mice were still alive at the end of the experiment (Figure 9C). The HM06 / TAS0953 treatment had no adverse effects on the body weight of the experimental animals (Figure 9B, right panel). These results confirm that HM06 / TAS0953 is effective against RET-reconstructed tumors that cross the brain and reside there.

[0121] The RET-specific kinase inhibitor HM06 / TAS0953 was observed to block the growth of preclinical models of RET-rearranged tumors that had not been treated with RET inhibitors, as well as tumors resistant to the multi-kinase RET inhibitors cabozantinib and RXDX-105. The efficacy of HM06 / TAS0953 was comparable to that of vandetanib. However, treatment with vandetanib resulted in significant weight loss in the animals, a finding not observed with HM06. Importantly, HM06 / TAS0953 suppressed the growth of preclinical models of RET-fusion-positive lung cancer in the brain and extended overall survival.

[0122] Example 6. An orthotopic xenograft model of lung cancer driven by RET reconstruction in the brain. Evaluation of the effectiveness of HM06 / TAS0953 The efficacy of HM06 / TAS0953 in a brain orthotopic model of TRIM33-RET fusion-positive lung cancer was compared with LOXO-292 and vandetanib. Treatment of mice with brain tumors with HM06 / TAS0953 (50 mg / kg BID) more effectively blocked tumor growth than LOXO-292 (10 mg / kg and 25 mg / kg BID doses). HM06 / TAS0953 significantly increased the survival time of tumor-bearing mice compared to both doses of LOXO-292. Vandetanib (50 mg / kg QD) did not reduce tumor growth or survival in tumor-bearing animals. These results suggest that HM06 / TAS0953 is more effective than LOXO-292 in reducing tumor growth in the brain.

[0123] Preparation of cells for injection into animal brains and quantification of bioluminescence images: TRIM33-RET fusion cells were transduced with a retrovirus containing a GFP luciferase construct, and GFP-positive cells were isolated by FACS. These cells were injected subcutaneously into the flanks of NSG mice to induce xenograft tumors. The tumors were collected and digested for 60 minutes in a GentleMACS tissue processor (Miltenyi Biotech) using a tumor isolation enzyme set (Miltenyi Biotech), then the digestive enzymes were neutralized by adding growth medium containing 10% FBS, and the cells were pelleted by centrifugation. The cells were then resuspended in fresh growth medium, passed through a 75 μm filter, counted, washed once with PBS, and resuspended in PBS at a density of 100,000 cells / μL. Isolated tumor cells were injected into the brains of anesthetized mice using a Hamilton syringe with a 26G needle (1 μL) at the following coordinates: anterior (X): 0.5, posterior (Y): 1.5, dorsal (Z): 2.5. The wounds were sealed, and the mice were allowed to recover. Bioluminescence imaging was performed weekly to monitor tumor growth, and the images were analyzed using ImageJ software. Pixels in a region of interest (ROI) encompassing the entire luminescent area of ​​each individual mouse were identified using a threshold function, and their area (A) and mean intensity (I) were measured. The mean background pixel intensity (B) was measured from the non-luminescent area. The total luminescence (L) of each individual mouse was quantified using the following formula: L = (IB) × A. This was adjusted for background intensity.

[0124] After a robust signal was detected, mice were randomly assigned to groups of six. Ten days after cell transplantation, treatment was initiated with Vehicle, 50 mg / kg of BID HM06, 100 mg / kg of QD HM06 / TAS0953, and 10 mg / kg of BID LOXO-292. Treatment of mice with vandetanib (50 mg / kg QD) and 25 mg / kg of BID LOXO-292 was initiated 28 days after transplantation because it took a longer time to obtain a robust bioluminescence signal from these mice. Bioluminescence was recorded weekly, and animal body weight was measured twice a week. Mice were sacrificed when signs of pathological conditions, such as impaired coordination or excessive weight loss and fatigue, were detected.

[0125] Vehicles and compounds: Vandetanib was mixed with 1% sodium carboxymethylcellulose (CMC-Na) to prepare a suspension. HM06 / TAS0953 was mixed with 0.1N HCl and 0.5% hypromellose (HPMC) to prepare a suspension.

[0126] Statistical Analysis: Datasets were compared using two-way ANOVA, and significance was identified using Tukey or Sidax multiple comparison tests. A p<0.05 value was considered statistically significant for the difference between two values ​​or between datasets. Survival curves were compared using the log-rank (Mantel-Cox) test. All graphs and statistical analyses were performed using Graphpad Prism 8 software.

[0127] HM06 / TAS0953 is more effective than LOXO-292 in inhibiting the growth of orthotopic xenograft tumor models with RET fusion: Vehicle-treated mice showed average openness As evidenced by the strong bioluminescence signal exceeding the initial signal (Figures 10A and 10B), tumors developed rapidly. By day 33 of treatment, the tumor-bearing mice in the vehicle group became ill and had to be sacrificed. In contrast, five days after the start of treatment, there was a significant decrease in luciferase signaling obtained from mice treated with 50 mg / kg BID (p=0.0012) or 100 mg / kg QD HM06 / TAS0953 (p=0.0008). Administration of 50 mg / kg HM06 / TAS0953 in BID blocked tumor growth throughout the entire duration of the study (131 days of treatment). Treatment with LOXO-292 in 10 mg / kg BID did not cause any decrease in bioluminescence signaling, and tumors continued to grow while mice were treated with LOXO-292 (Figure 10B). High doses of LOXO-292 (25 mg / kg BID) reduced tumor growth during the first three weeks of treatment, but thereafter, tumors began to expand 64 days after the start of treatment and continued to expand until mice had to be sacrificed due to high tumor burden (Figure 10B). Treatment with HM06 / TAS0953 resulted in a significant increase in survival time of tumor-bearing mice compared to LOXO-292 at 10 mg / kg BID (p=0.0012) and LOXO-292 at 25 mg / kg BID (p=0.001). At the end of the study, six mice in the HM06 / TAS0953 50 mg / kg BID group had low / undetectable luciferase signals, and all mice were still alive at the end of the study. There was no difference in survival rates between the two LOXO-292 groups. No treatments had any adverse effects on the body weight of the experimental animals (Figure 10D). These results confirm that HM06 / TAS0953 transversely migrates to the brain and is effective against RET-reconstructed tumors present there.

[0128] The RET-specific kinase inhibitor HM06 / TAS0953 blocked the proliferation of RET-fused lung cancer cells transplanted into the brains of mice, resulting in increased survival time for the tumor-bearing animals. Considering that LOXO-292 at a BID dose of 10 mg / kg was sufficient to induce regression of RET-fusion-positive tumors transplanted into the subcutaneous flanks of mice, these data suggest that delivery of LOXO-292 to tumor sites in the brain is poor, even at high doses of 25 mg / kg BID.

[0129] Example 7. Safety pharmacology test Safety pharmacology studies were conducted on the cardiovascular, respiratory, and central nervous systems. HM06-01 / TAS0953-01 did not produce any convulsive activity or biologically relevant changes in the respiratory system.

[0130] Central nervous system Because HM06-01 / TAS0953-01 readily crosses the blood-brain barrier, its effects on the CNS were evaluated in multiple non-GLP and GLP safety pharmacology studies.

[0131] In a 2-week toxicological GLP study in rats, the Irwin test was performed 1.5 hours after the second dose on day 1 for rats administered HM06-01 / TAS0953-01 daily at doses of 25 mg / kg, 50 mg / kg, and 125 mg / kg in BID. Each group consisted of 10 male rats and 10 female rats. Animals administered 50 mg / kg and 125 mg / kg in BID showed decreased exploratory behavior and arousal. Female rats treated with 125 mg / kg in BID also had a higher mean urine pool size (0.8 compared to 0.0 for controls) and lower rectal temperature (37.1°C compared to 37.7°C for controls). However, other Irwin test parameters were normal, and the animals' health status did not change macroscopically; therefore, these changes were judged not to be harmful.

[0132] To evaluate the potential pro-convulsant and anti-convulsant effects, HM06-01 / TAS0953-01 was administered to rats via forced oral infusion at SID dose levels of 0 mg / kg, 30 mg / kg, 100 mg / kg, and 200 mg / kg. Each group consisted of male rats. The study consisted of 10 rats. To evaluate potential convulsive activity, the rats were administered HM06-01 / TAS0953-01 or 35 mg / kg of d-amphetamine at these oral dose levels, followed one hour later by a subthreshold dose of 25 mg / kg of PTZ to assess the convulsive effect. HM06-01 / TAS0953-01 showed no convulsive effect. None of the rats in the control group or the HM06-01 / TAS0953-01 treatment group exhibited stage 5, tonic / clonic seizures. On the other hand, the positive control group, which had rats pre-treated with d-amphetamine, showed recurrent behavior typical of dopamine agonist treatment in rats, with 3 out of 10 rats exhibiting full tonic / clonic seizures. To evaluate the relative anticonvulsant effect, Wistar rats were orally administered either HM06-01 / TAS0953-01 or diazepam. HM06-01 / TAS0953-01, at doses of 100 mg / kg and 200 mg / kg, resulted in a reduction in the total number of seizures induced by the pentylenetetrazole preload test. These data suggest that HM06-01 / TAS0953-01 does not exhibit convulsive activity in rats and, notably, may possess anticonvulsant properties.

[0133] In 2-week and 4-week toxicity studies in dogs (further described in Example 8), CNS symptoms were observed in dogs after the initial dose at a daily dose of 15 mg / kg or higher of BID during routine clinical observation, and after the initial dose at a BID dose of 5 mg / kg or higher in the PK study. To evaluate the potential neurotoxic effects of HM06-01, Fluoro-Jade C staining was performed on nine different brain regions (i.e., frontal pole, optic chiasm, infundibulum, mammillary body, base of the third cranial nerve, anterior and occipital pole of the pons, cerebellum, and medulla oblongata) of control dogs and dogs treated with 45 mg / kg of BID HM06-01 / TAS0953-01 (samples were taken from the 2-week toxicity studies in dogs). Furthermore, for verification using an alternative method, ATF3 immunostaining was performed on only one control dog and one high-dose dog treated with 45 mg / kg of BID, and no signs of neuronal degeneration or distress were observed. No differences were observed between the brains of control dogs and the brains of dogs treated with HM06-01 / TAS0953-01. This means that no evidence of neuronal degeneration and / or stressed / damaged neurons was found anywhere in the range of canine brains tested.

[0134] Cardiovascular system In the two-week GLP toxicity study in dogs described above, the effect of HM06-01 / TAS0953-01 on ECG was examined on day 12. No evidence of QT prolongation was observed after repeated administration of HM06-01 / TAS0953-01 at BID levels of 15 mg / kg, 30 mg / kg, and 45 mg / kg. Heart rate and ECG were also unaffected at all tested dose levels. In contrast, female dogs treated with high doses showed a decrease in arterial blood pressure 5 hours after the first daily dose and 1 hour after the second daily dose. The mean arterial blood pressure was 91 mmHg and 93 mmHg compared to 127 mmHg and 115 mmHg in the vehicle group, and 119 mmHg compared to baseline. After 8 hours, arterial blood pressure returned to control / baseline levels. This decrease was consistent and significant enough to be considered related to the test product, although it was not statistically significant compared to the control. Nevertheless, given that the degree of change was limited, occurred only in high-dose females, and this blood pressure reduction did not have a negative impact on the animals' health, it was considered not harmful.

[0135] The effect of HM06-01 / TAS0953-01 on ECG was investigated at week 1 (day 2), week 4 (day 23), and at the end of the 2-week recovery period in dogs as described above (and further described in Example 8) of a 4-week toxicity GLP study. On day 2, 2 hours after the second daily dose, no effect of HM06-01 / TAS0953-01 was observed at the BID dose levels of 15 mg / kg, 30 mg / kg, and 45 mg / kg. On the other hand, on day 23, in males with BID doses of 15 mg / kg and 30 mg / kg, electrocardiogram parameters were improved. No test-related effects were observed. Conversely, in males at a dose level of 45 mg / kg in the BID group, a minor, potentially test-related increase in heart rate was observed, and in females at dose levels of 15 mg / kg, 30 mg / kg, and 45 mg / kg, minor (up to 8% compared to control) test-and-dose-related fridericia and Van der Water-corrected QTc interval prolongation were observed. These electrocardiographic effects were no longer observed at the end of the two-week recovery period. Furthermore, there were no abnormalities in electrocardiographic rhythm or waveform on days 2 and 23, and at the end of the recovery period.

[0136] respiratory system The effects of HM06-01 / TAS0953-01 on the respiratory system were also investigated in a 4-week GLP study in rats after repeated administration of HM06-01 / TAS0953-01 at BIDs of 25 mg / kg, 50 mg / kg, and 90 mg / kg. This GLP study is further described in Example 8. Each group consisted of 5 animals / groups / sex. No toxicologically relevant effects were observed on respiratory parameters at week 4, and no delayed chronic effects were observed at week 6, 2 weeks after the end of the administration period.

[0137] Example 8. 4-week repeated-dose toxicity study in rats and dogs. The adverse effects of HM06 / TAS0953 were evaluated in a 4-week GLP toxicity study in rats and dogs using the HM06 / TAS0953 dihydrochloride (HM06-01 / TAS0953-01) mentioned above.

[0138] 4-week toxicity study in rats In a 4-week GLP toxicity study in rats, animals were administered BID HM06-01 / TAS0953-01 at doses of 0 mg / kg, 25 mg / kg, 50 mg / kg, and 90 mg / kg via forced oral administration. In the high-dose 90 mg / kg BID group, test-related deaths were observed: three female rats died between days 10 and 14, or were sacrificed prematurely for ethical reasons (due to severe clinical signs such as decreased activity, abnormal respiratory rate, and piloerection). At autopsy, lung discoloration was generally observed, histologically correlated with harmful moderate to significant inflammatory changes in the lungs (alveolar / perivascular inflammation, macrophage aggregation, alveolar edema / hemorrhage). Additionally, one female rat (satellite) was found dead on day 7, prior to administration. However, autopsy revealed no clinical symptoms or abnormalities, and the association with test-agent administration was deemed unlikely.

[0139] Clinical signs related to the test product were observed in both sexes and in a dose-dependent manner. These signs included, for example, abnormal respiratory rate, abnormal respiratory sounds, decreased / increased activity, locomotor stereotypy, trunk flexion posture, low carriage, gait disturbance, pedaling, eye closure, chewing motion, pupillary abnormalities, and coldness to the touch. These clinical observations were temporary and had a high incidence 30 minutes after administration. All of these signs did not persist in recovered animals after the end of administration.

[0140] At BIDs of 25 mg / kg and 50 mg / kg, there was no effect on body weight. However, at BID of 90 mg / kg, a 10% decrease in body weight was observed in both sexes, associated with minimal weight gain and decreased food consumption. These effects reversed at the end of the recovery period.

[0141] There were no ophthalmic changes, and no toxicologically relevant acute or chronic effects were observed in respiratory function assessments. There were also no effects on coagulation parameters.

[0142] Hematologically, dose-dependent increases in reticulocytes were observed in both sexes. Additionally, an increase in neutrophils was observed only in males, and increases in platelets were observed in males receiving medium and high doses, as well as in females receiving high doses. A significant increase in white blood cell count was observed only in males at high doses. All of these effects were reversed at the end of the recovery period.

[0143] Compared to the control group, the following dose-dependent changes in mean clinical chemistry parameters were observed and were determined to be test-related: Dose-dependent increases in AST and ALT were observed in both sexes. At all doses, AP increased in males. Elevated cholesterol and decreased triglycerides were observed in males receiving moderate and high doses. All of these effects were reversed at the end of the recovery period.

[0144] A dose-dependent increase in urinary protein concentration was observed in both sexes, with a higher incidence in high-dose males (7 out of 10 males at 1 g / L) and higher incidence in medium and high-dose females (4 out of 10 females and 3 out of 7 females at 1 g / L or higher, respectively). This effect reversed at the end of the recovery period. max This was generally observed 7 hours after the first daily dose on day 1 (1 hour after the second daily dose) and 1 hour after the first daily dose on day 28, and increased with increasing dose levels. max The levels increased. T1 / 2 was observed after the second daily dose, where it could be estimated, and this ranged from 2.03 hours to 3.67 hours. Systemic exposure to HM06-01 / TAS0953-01 increased in a manner roughly proportional to the dose, with increasing dose levels, and was similar between sexes. No systemic accumulation was observed on day 28.

[0145] At the end of the administration period, microscopic findings of HM06-01 / TAS0953-01-related inflammation were observed in the lungs, pancreas (vacuole formation / apoptotic acinar cells), femur (epiphyseal cartilage hypertrophy), and testes / epididymis (Sertoli cell vacuolation, tubular degeneration, increased intratubular cell debris, decreased sperm concentration). At the end of the recovery period, in end-of-period sacrifice and early death, test-related findings in the pancreas were completely reversible, with sporadic occurrences of minimal severity mainly in the lungs and femur at a BID of 90 mg / kg. This indicates that recovery is progressing. The presence of test-related changes in the testes and epididymis during the recovery period, with increased incidence and severity at a BID of 90 mg / kg, indicates incomplete reversibility.

[0146] Based on these results, the severe toxicity dose 10 (STD10) was set at a BID of 50 mg / kg. This corresponds to the mean AUC. (0-t) This corresponds to 50,300 ng·h / mL in males and 37,200 ng·h / mL in females on day 1, and 73,000 ng·h / mL in males and 74,300 ng·h / mL in females on day 28.

[0147] 4-week toxicity study in dogs In a 4-week GLP toxicity study in dogs, animals were administered BID HM06-01 / TAS0953-01 at doses of 0 mg / kg, 15 mg / kg, 30 mg / kg, and 45 mg / kg by forced oral administration for four consecutive weeks, followed by a two-week recovery period. In the high-dose 45 mg / kg BID group, test-related deaths were observed: two males were euthanized prematurely on days 17 and 18 for ethical reasons (severe clinical signs associated with significant weight loss). The primary cause of death was mild to moderate multiple subacute inflammation with alveolar or bronchoalveolar epithelial circulation, and one male had alveolar foreign body granuloma. The other male showed minimal pancreatic acinar vacuolar formation / apoptosis with prostatic apoptosis, and a correlation with the test product cannot be ruled out.

[0148] Clinical signs related to the test product were dose-dependent and were observed to begin from day 1 at BID doses of 15 mg / kg or higher. These observations included decreased activity and tremors at all doses. Symptoms include rubbing, lying down, soiling of fur / skin, closed eyes, gait abnormalities, and coldness to the touch; with medium and high doses, vomiting and salivation, discolored urine / red stools, suppressed / exhausted, and apparent muscle atrophy (hind limbs). However, these clinical signs were transient and their incidence increased 30 minutes after administration.

[0149] Low doses had no effect on body weight, however, weight loss was observed in females given 30 mg / kg and 45 mg / kg in BID between day 1 and day 29, and in males given 45 mg / kg in BID who were slaughtered before the scheduled date (-25% in male #644 and -12% in male #642, on days 15 and 17, respectively). At the end of the recovery period, females given medium and high doses had gained weight, but this weight gain was less than that of the control animals.

[0150] There were no changes in ophthalmological, urinalysis, blood coagulation, or clinical chemistry parameters.

[0151] Hematologically, both sexes showed an increase in total white blood cell count (dose-dependent at all doses) due to an increase in monocyte count, and an increase in neutrophil count (only at high doses in females, and at medium and high doses in males); reticulocyte count increased only at high doses in males, and platelet count increased at high and / or medium doses in both sexes. The decrease in eosinophil count was deemed suspicious, considering that it was observed only in females and was not dose-dependent. All of these effects were reversible at the end of the recovery period, with the exception of high-dose monocytes in males and eosinophils in females.

[0152] t max This was observed between 1 hour and 8 hours after the first daily dose (1 hour after the first daily dose and 2 hours after the second daily dose). AUC (0-t)Based on this, systemic exposure to HM06 / TAS0953 increased slightly more than dose-proportional, with the exception of males and females at day 28 between the 30 mg / kg and 45 mg / kg BIDs, where this increase was less than dose-proportional. Similar exposure to HM06 / TAS0953 was observed in males and females at day 1 and day 28 for all administered doses. Compared to day 1, exposure to HM06 / TAS0953 at day 28 was similar in the 30 mg / kg BID, while in some individuals at the 15 mg / kg and 45 mg / kg BIDs it was decreased or remained similar.

[0153] At the end of the administration period, a decrease in mean thymus weight was observed in all females, as well as a decrease in mean prostate weight in surviving males receiving 30 mg / kg BID and 45 mg / kg BID. Histopathological examination revealed that HM06-01 / TAS0953-01 induced microscopic inflammatory findings in the lungs (subacute inflammation with alveolar or bronchoalveolar epithelial circulation), pancreas (acinate cell vacuolation / apoptosis), and thymus (atrophy). At the end of the recovery period, test-related findings in the lungs indicated progress in recovery. Histological findings observed in the pancreas and thymus at the end of the recovery period showed similar severity and / or sporadic occurrences in the control and treatment groups, and there were no signs of spontaneous toxicity or functional modification in the tested beagle dogs.

[0154] Based on these results, the maximum dose without serious toxicity (HNSTD) was established in this study as a BID of 30 mg / kg. This dose corresponds to a mean AUC(0-t) of 21,400 ng·h / mL in males and 29,200 ng·h / mL in females on day 1, and 22,300 ng·h / mL in males and 29,100 ng·h / mL in females on day 28.

[0155] Example 9. Correlation between free plasma concentration of HM06 / TAS0953 and free concentration in the brain and cerebrospinal fluid. Free-roaming adult male Han(trademark)Wistar rats were administered HM06 / TAS0953 dihydrochloride in single doses of 3 mg / kg, 10 mg / kg, and 50 mg / kg (the dose is measured by the amount of administration). The pharmacokinetics of HM06 / TAS0953 in the prefrontal cortex, cerebrospinal fluid (CSF), and plasma after oral administration (showing free bases) were evaluated according to Table 3 below.

[0156] [Table 3]

[0157] HM06 / TAS0953 exposure in the prefrontal cortex (PFC), plasma, and cerebrospinal fluid (CSF) increased broadly in a dose-proportional manner with increasing dose across the dose range used from 3 mg / kg to 50 mg / kg. A Tmax of 0.5 to 1 hour indicates that HM06 / TAS0953 is rapidly absorbed, with a short half-life, good F% (46% to 51%), moderate plasma clearance, large volume of distribution, and limiting renal clearance. Figure 11A shows the time-course plasma concentrations after single oral administration of HM06 / TAS0953 at 3 mg / kg, 10 mg / kg, 30 mg / kg, and 50 mg / kg. Figure 11B shows the time-course plasma concentrations after single oral and intravenous administration of 3 mg / kg of HM06 / TAS0953.

[0158] Once equilibrium was reached between compartments, the ratio of HM06 / TAS0953 concentrations observed in MetaQuant microdialysis fluid from the PFC, CSF, and free plasma fractions was close to 1:1:1. This concentration ratio was maintained from 2 to 6.5 hours after HM06 / TAS0953 administration (up to 8 hours in the CSF). Figure 11C shows the superimposed pharmacokinetic profiles in the free plasma fraction, PFC, and CSF from 2 to 6.5 hours after administration.

[0159] The 1:1 ratio of free plasma concentration to free brain concentration indicated that HM06 / TAS0953 readily crosses the blood-brain barrier. The free plasma concentration of HM06 / TAS0953 can be used to estimate a good approximation of free concentrations in the brain and CSF. High brain permeability may improve CNS outcomes (e.g., control of CNS metastases, response duration, and / or protection from CNS metastases).

[0160] Example 10. Evaluation of target selectivity of HM06 / TAS0953 for RET The RET selectivity of HM06 / TAS0953 was compared with that of LOXO-292 and BLU-667. As shown in Table 4, HM06 / TAS0953 is an IC 50 Based on the values, it exhibits higher RET kinase selectivity compared to LOXO-292 and BLU-667.

[0161] [Table 4]

[0162] From a safety perspective, the high target selectivity of HM06 / TAS0953 for RET may minimize adverse effects due to potential off-target kinase inhibition, which represents a potential clinical benefit.

[0163] Example 11. Determination of the starting dose for human clinical trials The calculation of the starting dose for human clinical trials is based on GLP-compliant toxicity studies of 4-week repeated doses, and is based on one-tenth of the dose at which serious toxicity occurs in 10% of treated rats (STD10) and one-sixth of the maximum dose at which serious toxicity does not occur in dogs (HNSTD), as per ICH S9 guidelines.

[0164] In a 4-week rat toxicity study, STD10 was set at 50 mg / kg as the BID (Beginning Intake). A BID of 50 mg / kg (equivalent to a daily dose of 100 mg / kg) corresponds to a human equivalent dose (HED) of 8 mg / kg as the BID. Applying a safety factor of 10, a high starting dose of 0.8 mg / kg, or 48 mg as the BID, is possible for a patient weighing 60 kg (BW). An additional safety factor of 2.5 is added because dose-dependent alveolar inflammation and testicular and epididymal changes were observed in 2-week and 4-week rat toxicity studies, with progressive recovery and incomplete reversibility observed at the end of the 2-week recovery period, and CNS effects were observed in rat FOB after oral administration of doses of 50 mg / kg or higher as the SID (Single Intake). This leads to a proposed human starting dose of 20 mg as the BID.

[0165] The maximum dose without serious toxicity in dogs (HNSTD) is the high dose (30 mg / kg BID) tested in a 4-week toxicity study in dogs. A dose of 30 mg / kg BID (equivalent to a daily dose of 60 mg / kg) corresponds to a human equivalent dose (HED) of 16.2 mg / kg BID, or 972 mg BID, for a 60 kg BW patient. Applying a safety factor of 6 as specified in the ICH S9 guidelines, a high starting dose of 2.7 mg / kg, or 162 mg BID, is possible for a 60 kg patient. Consistent with rat studies, an additional safety factor of 2.5 is applied because dose-dependent alveolar inflammation was observed in dogs as well, with progressive recovery at the end of the 2-week recovery period. This is because QTcF prolongation was observed in a 4-week study in dogs, and CNS-related clinical signs were observed from day 1 after the initial administration at doses ≥ 15 BID mg / kg / day during the overall toxicity study. This leads to a proposed human starting dose of 64.8 mg as the BID.

[0166] The starting dose for human clinical trials was determined to be 20 mg BID (i.e., 40 mg / day per patient) based on data from a 4-week repeated-dose GLP-compliant toxicity study. This is based on one-tenth of the dose at which serious toxicity occurred in 10% of treated rats (STD10) and one-sixth of the maximum dose at which serious toxicity did not occur in dogs (HNSTD). The accelerated escalation design (ATD) allows for administering the investigational drug within an acceptable tolerable range while avoiding exposing too many patients to potentially ineffective doses.

[0167] Example 12. Evaluation of the safety, tolerability, pharmacokinetics (PK), and antitumor activity of HM06 / TAS0953 in patients with progressive solid tumors and RET gene abnormalities. A Phase I / II open-label, single-arm, first-in-human trial will be conducted to evaluate the safety, tolerability, pharmacokinetics (PK), and antitumor activity of HM06 / TAS0953. The trial will consist of two parts: a Phase I part using dose-escalation and dose-expansion cohorts, and a Phase II part using three cohorts. Patients with RET gene mutation-positive advanced solid tumors will receive treatment. In both parts, treatment cycles will consist of 21 consecutive days of administration with no interruption between cycles. Administration will continue until disease progression, loss of clinical benefit, occurrence of an unacceptable adverse event, initiation of new anticancer therapy, withdrawal of consent, physician's decision, death, or loss of follow-up. The Phase I / II trial will also include a safety evaluation, which will include assessment of the frequency, severity, and relevance of TEAEs and serious adverse events (SAEs), changes in hematological and blood chemical levels, physical examination evaluation, vital signs, and electrocardiogram (ECG).

[0168] The HM06 / TAS0953 tablets should be administered orally on a BID (approximately every 12 hours) while fasting (i.e., no food should be consumed 2 hours before and 1 hour after drug administration).

[0169] The Phase I trial will involve oral treatment with HM06 / TAS0953, starting with a dose of 20 mg twice daily, followed by continuous daily dosing until the maximum tolerated dose (MTD) is reached, with each cycle lasting 21 days.

[0170] Preclinical efficacy data (ED in mouse models) 50 Based on safety data (oral doses of 3 mg / kg / day in rats in a 4-week toxicology study were 25 mg / kg and 50 mg / kg as BIDs), and a human clearance of 6.4 mL / min / kg predicted by a well-stirred model from recombinant CYP data, assuming a 50% oral bioavailability in humans, HM06 / TAS0953 shows signs of antitumor efficacy in cancer patients starting at approximately 40 mg as a BID, and the daily exposure AUC of HM06 / TAS0953 0-24,ss The expected dose is ≥1650 ng·h / mL. The maximum dose can range from 500 mg BID to 1500 mg BID, and the daily exposure AUC is also possible. 0-24,ss This could result in exposures ranging from ≥22000 ng·h / mL to ≤63000 ng·h / mL. These exposure levels are equivalent to the safe dose of 25 mg / kg BID, which is lower than the STD10 BID of 50 mg / kg in rats, and the exposure achieved at STD10, respectively (adjusted for humans by multiplying the AUC values ​​measured in animals by the animal-to-human unbound fraction ratio). Based on the above assumptions, the maximum dose to be administered in this trial could range from 500 mg BID to 1500 mg BID.

[0171] The Phase II trial involved oral treatment with the recommended dose twice daily, in consecutive daily doses, and the cycle was This will include a 21-day continuation.

[0172] Phase I dose escalation The Phase I dose escalation trial will follow an accelerated dose escalation design (ATD), starting with a BID of 20 mg and gradually increasing the dose in 100% increments in an initial acceleration phase (one patient per dose level). After a cohort of 80 mg BID, the acceleration phase will be converted to a 3+3 design. During the acceleration phase, if no clinically significant Grade 2 or higher drug-related toxicity or dose-limiting toxicity (DLT) is observed by the Safety Review Committee (SRC) during the first cycle of treatment (i.e., the first 21 days of treatment, Cycle 1), and if less than 0 / 3 or 2 / 6 of patients experience DLT in the standard phase, dose escalation will continue with new patients until the maximum tolerated dose (MTD) is reached.

[0173] The first cohort will consist of one patient who will receive HM06 / TAS0953 for 21 consecutive days in a 21-day cycle. If no relevant toxicity is observed in this patient during cycle 1, the next cohort will be initiated. Based on the study protocol, there will be one to a maximum of three cohorts in the dose escalation phase, while the number of cohorts in the standard phase is unpredictable as dose escalation will continue until the MTD is reached.

[0174] MTD is defined as follows: the highest dose level in which 33% or less of patients experience DLT in cycle 1.

[0175] DLT is defined as follows: Toxicity that occurs during the first cycle of treatment, as detailed in the study protocol (excluding adverse events determined by the principal investigator to be clearly related to disease progression or a co-occurring disease and unrelated to the investigational drug).

[0176] The Phase II Recommended Dose (RP2D) is defined as follows: the dose tested in Phase II based on overall safety, tolerability, PK data, and estimates of effective exposure extrapolated from nonclinical data, derived from patients treated at dose escalation and expansion levels. The RP2D may be less than or equal to the MTD, but will not be higher than the MTD. If the MTD is not reached, the RP2D cannot be higher than the highest dose tested.

[0177] The primary objectives of the Phase I dose escalation study are to determine the maximum tolerated dose (MTD) within the first 21 days of treatment (Cycle 1) and to identify the recommended Phase II dose (RP2D).

[0178] The primary secondary objectives of the Phase I dose escalation study are to evaluate: individual PK profiles of HM06 / TAS0953 and its metabolites in plasma after single and multiple doses (high-density sampling); urinary excretion after single dose; safety and tolerability; antitumor activity; and changes in RET gene status in circulating free nucleic acids obtained by fluid biopsy during treatment. Pharmacokinetic evaluation of HM06 / TAS0953 and its metabolites can be assessed by measuring plasma concentrations and PK parameters of HM06 / TAS0953 and its major metabolites, including, but not limited to, the area under the curve (AUC0-24) from time 0 to 24 hours, maximum drug concentration (Cmax), time to reach maximum plasma concentration (Tmax), and degree of accumulation. The concentration of HM06 / TAS0953 in cerebrospinal fluid (CSF) can be determined. Plasma samples can be collected simultaneously to estimate the CSF / plasma ratio.

[0179] The trial may include up to 36 evaluable patients for DLT evaluation. The count will depend on the number of patient substitutions required and possible during dose escalation. The targeted trial population may include patients with RET gene mutation-positive advanced solid tumors.

[0180] Phase I dose expansion: In the Phase I dose expansion trial, the RP2D dose level will be expanded with the enrollment of additional patients. In a subset of patients, the effect of diet on HM06 / TAS0953 bioavailability will be evaluated according to a randomized crossover design.

[0181] The primary objective of the Phase I dose escalation trial is to determine the recommended Phase II dose (RP2D) in the target population, which will be used in three Phase II cohorts. The dose escalation timeframe will be the first 21 days (Cycle 1) and each cycle (21 days) of treatment over approximately 10 months (or earlier if the patient discontinues the trial).

[0182] The primary secondary objectives of the Phase I dose expansion trial are to evaluate: individual PK profiles of HM06 / TAS0953 and its metabolites in steady-state plasma by high-density sampling from a subset of patients for individual PK characterization and by sparse sampling from all other patients for population PK analysis; the effect of diet on the bioavailability of HM06 / TAS0953 in a subset of patients; safety and tolerability; antitumor activity; and changes in RET gene status in circulating free nucleic acids obtained by liquid biopsy during treatment.

[0183] This trial may include 20 to 30 patients, including at least 10 patients with measurable CNS metastases at baseline (as recommended by the RANO Working Group). A preliminary assessment of the effect of diet on HM06 / TAS0953 bioavailability during dose escalation can be performed in 10 patients included in the PK assessment.

[0184] The targeted study population includes patients with locally advanced or metastatic NSCLC who have a primary RET gene fusion (with or without resistance mutations) and have been previously exposed to a RET-selective inhibitor. These patients are those whose disease progression has been recorded after receiving existing treatments that the investigator considered to have demonstrated clinical benefit, or who are unable to receive such treatments.

[0185] Phase II: In the Phase II trial, patients will be treated at the RP2D level in three cohorts: Cohort 1 and Cohort 2 (pivotal), and Cohort 3 (exploratory).

[0186] The primary objective of the Phase II trial is to evaluate the antitumor activity (overall and, where appropriate, intracranially) of selected RP2D in three distinct populations. The response rate will be evaluated approximately every 6 weeks (±1 week) for the first 6 months, and thereafter every 9 weeks (±1 week) until disease progression or completion of the trial in patients who do not experience disease progression.

[0187] The primary secondary objectives of the Phase II trial are to evaluate: safety and tolerability in three cohorts; individual PK profiles of HM06 / TAS0953 and its metabolites in steady-state plasma by sparse sampling in all patients for population PK analysis; and changes in RET gene status in circulating free nucleic acids obtained by fluid biopsy during treatment. HM06 / TAS0953 concentrations in cerebrospinal fluid (CSF) can be determined. Plasma samples can be collected simultaneously to estimate the CSF / plasma ratio.

[0188] Additional secondary outcome measures may include any of the above, as determined by the principal investigator: ORR (objective response rate); disease control rate; time to tumor response; duration of response; progression-free survival; progression-free survival; overall survival; and any of the above, particularly concerning the central nervous system.

[0189] The Phase II trial can include 55 patients in a one-stage design within Cohort 1. This trial can include up to 61 patients in Cohort 2 according to the Simon two-stage design (24 patients continuing from Stage 1 and 37 in Stage 2). This trial can include 3 patients per tumor type within Cohort 3. More patients may be enrolled if clinical benefit is observed.

[0190] The target study population for Cohort 1 (pivotal) may include: patients with locally advanced or metastatic NSCLC who have a primary RET gene fusion (with or without resistance mutations) and have been previously exposed to a RET selective inhibitor; patients whose disease progression has been recorded after receiving existing treatments that the investigator considered to have demonstrated clinical benefit, or who are unable to receive such treatments; with or without measurable brain and / or leptomeningeal metastases.

[0191] The target trial population for Cohort 2 (pivotal) may include: patients with locally advanced or metastatic NSCLC who have a RET gene fusion and are naive to RET selective inhibitors; patients whose disease progression has been recorded after receiving existing treatments that investigators consider to have demonstrated clinical benefit, or who are unable to receive such treatments; regardless of the presence or absence of measurable brain and / or leptomeningeal metastases.

[0192] The target trial population for Cohort 3 (exploratory) may include: patients with RET gene mutation-positive advanced solid tumors (excluding NSCLC patients with primary RET gene fusion) who have rendered all available treatment options ineffective, or whose investigators have determined that other treatments would not provide substantial benefit and / or have refused them.

[0193] The selection criteria for a Phase I / II trial may include the following: Phase I - Common selection criteria for dose escalation / dose expansion: Male or female patients aged 18 or older. The US East Coast Cancer Clinical Group (ECOG) performance score is 0 or 1. RET gene abnormalities can be identified at baseline through tissue biopsy or fluid biopsy. Appropriate hematopoietic function is defined as follows: Platelet count of 100,000 / μL or higher Absolute neutrophil count of 1500 / μL or higher Hemoglobin level of 9.0 g / dL or higher (red blood cell transfusion and erythropoietin may be used to reach 9.0 g / dL, but must be administered at least two weeks prior to the first dose of the investigational drug). Appropriate liver function is defined as follows: serum total bilirubin level ≤ 1.5 times the upper limit of normal (ULN), serum albumin ≥ 2 g / dL, and AST and ALT levels ≤ 2.5 times ULN if liver metastases are absent, and ≤ 5 times ULN if liver metastases are present. Appropriate renal function is defined as follows: serum creatinine ≤ ULN × 2 or estimated creatinine clearance ≥ 60 mL / min. Male and female patients who may have children or are at risk of pregnancy must agree to use a highly effective method of contraception throughout the entire study, from the date they sign the informed consent form, and to continue using it for 180 days after the final dose of their assigned treatment. A patient is defined as fertile if the investigator determines that they are biologically capable of having children and are sexually active. Phase I dose escalation - specific selection criteria: Patients with progressive solid tumors who have evidence of RET gene abnormalities. Measurable and / or unmeasurable diseases as defined by RECIST 1.1 The patient had at least one or more previous lines of treatment for an advanced solid tumor. Patients whose disease progression has been documented, or who are unable to receive such treatment. If a patient has brain and / or leptomeningeal metastases, the male / female patient must be asymptomatic. Both measurable and unmeasurable lesions are acceptable in accordance with RANO Working Group (WG) recommendations. If a patient has previously been treated with a RET-selective inhibitor, at least five times the half-life of HM06 / TAS0953 must have elapsed between the previous RET-selective inhibitor treatment and the first dose of HM06 / TAS0953. Phase I dose expansion - specific selection criteria: Patients with locally advanced or metastatic NSCLC who have a primary RET gene fusion (regardless of the presence of resistance mutations) and have been previously exposed to RET-selective inhibitors: It is stated that the disease progresses after existing therapy, and that the therapy has demonstrated clinical benefit or is not available to the patient, according to the judgment of the principal investigator; Regardless of the presence or absence of brain and / or leptomeningeal metastases at baseline. If a patient has brain and / or leptomeningeal metastases, the male / female patient must have the following: Asymptomatic, untreated brain / leapinging membrane metastases that have not received steroids and anticonvulsants for at least 7 days (these should be considered target lesions if dimensionally appropriate). or Asymptomatic brain metastases that have already been treated with local therapy (WBRT / SRT / surgery) and are clinically stable with at least 7 days of steroid and anticonvulsant use prior to administration of the investigational drug. Measurable diseases as defined by RECIST 1.1 At least five times the half-life of HM06 / TAS0953 must have elapsed between previous RET selective inhibitor treatment and the first dose of HM06 / TAS0953. Prioritizing either a site-controlled NGS trial that has either verbalized the RET condition or demonstrated substantial and long-term (more than 6 months) clinical benefit from previous RET inhibitor treatment. Phase II - Common selection criteria for cohorts 1-3: Male or female patients aged 18 or older. The US East Coast Cancer Clinical Group (ECOG) performance score is between 0 and 2. RET gene abnormalities can be identified at baseline through tissue biopsy or fluid biopsy. Measurable diseases as defined by RECIST 1.1. If a patient has brain and / or leptomeningeal metastases, the male / female patient must have the following: Asymptomatic, untreated brain / leapinging membrane metastases that have not received steroids and anticonvulsants for at least 7 days (these should be considered target lesions if dimensionally appropriate). or Asymptomatic brain metastases that have already been treated with local therapy (WBRT / SRT / surgery) and are clinically stable with at least 7 days of steroid and anticonvulsant use prior to administration of the investigational drug. Appropriate hematopoietic function is defined as follows: Platelet count of 100,000 / μL or higher Absolute neutrophil count of 1500 / μL or higher Hemoglobin level of 9.0 g / dL or higher (red blood cell transfusion and erythropoietin may be used to reach 9.0 g / dL, but must be administered at least two weeks prior to the first dose of the investigational drug). Appropriate liver function is defined as follows: serum total bilirubin level ≤ ULN × 1.5, serum albumin ≥ 2 g / dL, and AST and ALT levels ≤ ULN × 2.5 if liver metastases are absent, and ≤ ULN × 5 if liver metastases are present. Appropriate renal function is defined as follows: serum creatinine ≤ ULN × 2 or estimated creatinine clearance ≥ 60 mL / min. Male and female patients who may have children and are at risk of pregnancy must agree to use highly effective contraception from the time of their first negative pregnancy test at screening, throughout the entire study, and for 180 days after the final dose of their assigned treatment. A patient is defined as fertile if the investigator determines that they are biologically capable of having children and are sexually active. • Phase II Cohort 1 - Specific Inclusion Criteria: Patients with locally advanced or metastatic NSCLC who have a primary RET gene fusion (regardless of the presence of resistance mutations) and have been previously exposed to a RET selective inhibitor. Patients who have experienced disease progression after receiving existing treatments that the investigators considered to have demonstrated clinical benefit, or who are unable to receive such treatments. At least 5 half-lives must have elapsed from the previous RET selective inhibitor therapy to the first administration of HM06 / TAS0953. Either the RET status has been preferentially documented by an NGS test managed at a specific site, or substantial and long-term (more than 6 months) clinical benefit from previous RET inhibitor therapy has been demonstrated. Phase II Cohort 2 - Specific eligibility criteria: Patients with locally advanced or metastatic NSCLC harboring RET gene fusion and no previous exposure to RET selective inhibitors. Patients in whom disease progression has been documented after receiving existing treatment that the investigator considers has demonstrated clinical benefit, or who are unable to receive such treatment. Phase II Cohort 3 - Specific eligibility criteria: Patients with advanced solid tumors harboring RET gene aberrations (other than NSCLC patients with primary RET gene fusion), in whom all available treatment options have become ineffective, or the investigator judges that other treatments provide no substantial clinical benefit, and / or the patient refuses such treatment: this may include (but is not limited to): Medullary thyroid cancer or anaplastic thyroid cancer that has progressed or developed intolerance to selective RET inhibitors; Metastatic breast cancer; Metastatic pancreatic adenocarcinoma; NSCLC with emergence of the RET pathway after therapies including but not limited to EGFR / ALK / ROS1 / BRAF targeted therapies. If the patient has previously been treated with a RET selective inhibitor, at least 5 half-lives must have elapsed from the previous RET selective inhibitor therapy to the first dose of HM06 / TAS0953.

[0194] Exclusion criteria for the Phase I / II trial may include the following: Phase I - Common exclusion criteria for dose escalation / dose expansion: Lactating women. Prior to the first administration of the investigational drug, the agonist or anticancer therapy used in the clinical trial may be used for a period equal to five times the half-life (or, provided there is no residual toxicity, one half-life in the case of long-acting drugs such as anticancer antibodies and other biopharmaceuticals). Patients who have undergone major surgery (excluding the creation of vascular access) within four weeks prior to the first dose of the investigational drug, or who are scheduled to undergo major surgery during the course of the investigational treatment. Patients who have received WBRT within 14 days prior to the first dose of the investigational drug, or who have received other palliative radiotherapy within 7 days, or who have not recovered from side effects of such treatment, and for whom this is clinically significant in the opinion of the principal investigator. A patient with a primary CNS tumor. In accordance with the principal investigator's opinion, clinically significant and uncontrolled cardiovascular disease, including, This includes myocardial infarction, unstable angina, significant valvular or pericardial disease, a history of ventricular tachycardia, symptomatic congestive heart failure (CHF) of New York Heart Association (NYHA) class III-IV, and severe, uncontrolled arterial hypertension within three months prior to the first day of Class I. A duration of overtime QT interval (QTcF) greater than 470 msec, corrected using Fridericia's formula; a personal or family history of long QT syndrome or a history of torsades de pointes (TdP); or a history of uncontrolled and persistent TdP risk factors (e.g., heart failure, hypokalemia, or use of concomitant medications that prolong the QT / QTc interval despite optimal treatment). Currently, there is an active infection of HIV, hepatitis B virus, or hepatitis C virus. There is a documented history of interstitial lung disease, or evidence of current interstitial lung disease requiring steroid medication. Clinically significant gastrointestinal abnormalities that appear to affect drug absorption, as determined by the principal investigator. Clinically significant diseases affecting digestive function (including chronic diarrhea) that are likely to affect the investigational drug therapy, in accordance with the opinion of the principal investigator. Treatment with a potent CYP3A4 inhibitor within one week (7 days) prior to the first dose of the investigational drug. Repeated treatment with a potent CYP3A4 inducer within 3 weeks (21 days) prior to the first dose of the investigational drug. Known hypersensitivity to additives in HM06 / TAS0953. Any disease or medical condition that, in the opinion of the principal investigator, could confound the trial results or pose an undesirable risk to the patient from administering the investigational drug. Phase I dose escalation - specific exclusion criteria: Patients with symptomatic CNS metastases at baseline. Phase I dose expansion - specific exclusion criteria: Patients with symptomatic brain and / or leptomeningeal metastases at baseline that are uncontrolled by local and / or systemic therapy. Known EGFR, KRAS, ALK, HER2, ROS1, BRAF, and METex14 activating mutations exist. Phase II - Common Exclusion Criteria for Cohorts 1-3: A woman who is breastfeeding. Before the first dose of the investigational drug, use of an active agent or anticancer therapy used in the clinical trial for up to five times its half-life (or, provided there is no residual toxicity, up to one half-life in the case of long-acting drugs such as anticancer antibodies and other biopharmaceuticals). Patients who have undergone major surgery (excluding the creation of vascular access) within four weeks prior to the first dose of the investigational drug, or who are scheduled to undergo major surgery during the course of the investigational treatment. Patients who have received WBRT within 14 days prior to the first dose of the investigational drug, or who have received other palliative radiotherapy within 7 days, or who have not recovered from side effects of such treatment, and for whom this is clinically significant in the opinion of the principal investigator. A patient with a primary CNS tumor. Patients with symptomatic brain and / or leptomeningeal metastases at baseline that are uncontrolled by local and / or systemic therapy. In accordance with the principal investigator's opinion, clinically significant uncontrolled cardiovascular disease includes myocardial infarction, unstable angina, significant valvular or pericardial disease, a history of ventricular tachycardia, symptomatic congestive heart failure (CHF) of New York Heart Association (NYHA) class III-IV, and severe uncontrolled arterial hypertension within three months prior to day 1 of cycle 1. A duration of overtime QT interval (QTcF) greater than 470 msec, corrected using Fridericia's formula; a personal or family history of long QT syndrome or a history of torsades de pointes (TdP); or a history of uncontrolled and persistent TdP risk factors (e.g., heart failure, hypokalemia, or use of concomitant medications that prolong the QT / QTc interval despite optimal treatment). Currently, there is an active infection of HIV, hepatitis B virus, or hepatitis C virus. There is a documented history of interstitial lung disease, or evidence of current interstitial lung disease requiring steroid medication. Clinically significant gastrointestinal abnormalities that appear to affect drug absorption, as determined by the principal investigator. Clinically significant diseases affecting digestive function (including chronic diarrhea) that are likely to affect the investigational drug therapy, in accordance with the opinion of the principal investigator. Treatment with a potent CYP3A4 inhibitor within one week (7 days) prior to the first dose of the investigational drug. Repeated treatment with a potent CYP3A4 inducer within 3 weeks (21 days) prior to the first dose of the investigational drug. Known hypersensitivity to additives in HM06 / TAS0953. Any disease or medical condition that, in the opinion of the principal investigator, could confound the trial results or pose an undesirable risk to the patient from administering the investigational drug. Phase II Cohort 1 - Specific Exclusion Criteria: Known EGFR, KRAS, ALK, HER2, ROS1, BRAF, and METex14 activating mutations exist. Phase II Cohort 2 - Specific Exclusion Criteria: Known EGFR, KRAS, ALK, HER2, ROS1, BRAF, and METex14 activating mutations exist. Phase II Cohort 3 - Specific Exclusion Criteria: none.

[0195] Drug administration: HM06 / TAS0953 is prepared as the dihydrochloride salt (HM06-01 / TAS0953-01) for clinical use. The product is formulated into tablets for oral use. The study is conducted using tablets at doses of 10 mg / unit and 50 mg / unit (expressed as free base). The intended storage condition is refrigerated storage (temperature controlled at 2°C to 8°C (36°F to 46°F)).

[0196] HM06 / TAS0953 tablets are to be orally administered BID (approximately every 12 hours) at 20 mg (starting dose) BID in the fasted state (i.e., no food shall be consumed during the period from 2 hours before to 1 hour after drug administration).

[0197] Example 13. Escalation of the dose-escalation part of the Phase I study to a BID dose level of 320 mg Although the dose-escalation part of the Phase I study is still ongoing, safety data obtained from the first treatment cycle of patients dosed with 160 mg BID has enabled dose escalation to a BID dose level of 320 mg. This process complies with the study protocol outlined in Example 12 above, that is, when safety data obtained from the first treatment cycle of enrolled patients is available in accordance with the protocol, a Safety Review Committee (SRC) meeting for dose escalation will be held. As discussed in Example 12 above, the maximum dose to be administered in this study may range from 500 mg BID to 1500 mg BID.

[0198] Example 14. Cellular potency of HM06 / TAS0953 against RET solvent front mutations Although selective RET inhibitors LOXO-292 (selpercatinib) and BLU-667 (pralsetinib) show clinical antitumor activity in non-small cell lung cancer, acquired resistance driven by RET solvent front mutations (e.g., RET G810R / S / C ) has emerged (Subbiah, V et al., Ann Oncol. 2021, 32(2):261-268; Lin JJ et See al., Ann Oncol. 2020, 31(12):1725-1733; Solomon BJ et al., J Thorac Oncol. 2020, 15(4):541-549; Fancelli S et al., Cancers (Basel). 2021, 13(5):1091. doi: 10.3390 / cancers13051091). In such cases, see serpercatinib or prarlsetinib. There are currently no other treatment options available for patients who have experienced a relapse using this method.

[0199] The cytotoxic efficacy against RET wild-type fusions and RET mutations, including the gatekeeper mutation V804L / M and the selpercatinib or pralcetinib resistance mutation G810R / S, was investigated using modified Ba / F3 cells. Approximately 1000 cells were cultured per well in a 96-well plate and treated with HM06 / TAS0953, vandetanib, BLU-667, or LOXO-292 for 72 hours (3 days at 37°C). Cell viability was assessed by luminescence using CellTiter-Glo 2.0 Assay (Promega Corporation). XLfit Using the sigmoid dose-response model in the software (ID Business Solutions), GI5 The zero value (the concentration that results in 50% growth inhibition compared to the untreated control) was calculated. Data are expressed as the mean ± SD of data obtained from three independent experiments.

[0200] The IC50 data is shown in Table 5. Ba / F3 KIF5B-RET G810R / S The cells were found to be resistant not only to BLU-667 but also to LOXO-292. However, Ba / F3 KIF5B-RET G810R / S The cells were sensitive to HM06 / TAS0953.

[0201] Also, Ba / F3 KIF5B-RET V804L / MThe cells were sensitive to HM06 / TAS0953, BLU-667, and LOXO-292, but not to vandetanib.

[0202] [Table 5]

[0203] RET G810R / S To help elucidate the mechanism by which the mutant remains sensitive to HM06 / TAS0953, we examined the crystal structures of the RET kinase domains that complex with TAS compound 1 (which has a structure similar to HM06 / TAS0953), BLU-667, and LOXO-292. From the X-ray crystal structure of the complex, it became clear that TAS compound 1 has a unique binding mode to RET compared to BLU-667 and LOXO-292. Based on the cocrystal structure data, as shown in Figure 12A, BLU-667 and LOXO-292 bind to the same pocket of RET (pocket B), while TAS compound 1 binds to a different pocket of RET (pocket A) with a different binding mode. TAS compound 1 does not completely fill the space in the direction of the G810 side chain, which suggests that HM06 / TAS0953 can effectively avoid steric hindrance caused by solvent front substitution. This characteristic likely contributes to HM06 / TAS0953's ability to maintain its biological efficacy in the G810 mutation.

[0204] The structural formula of TAS compound 1 is as follows: [ka]

[0205] Figures 12B and 12C show magnified structural analyses of the RET cocrystal complex, focusing on the region of RET amino acid residues 806–810. Figure 12B shows the cocrystal complex of RET with TAS compound 1, BLU-667, and LOXO-292, while Figure 12C shows the cocrystal complex of RET with TAS compound 1. Glycine 810 is close to both BLU-667 and LOXO-292, suggesting that substitution at this position could affect steric hindrance to these inhibitors. In contrast, TAS compound 1 is not inserted into the pocket consisting of amino acid residues 806–810.

[0206] This data is consistent with biological data showing that the inhibition patterns against RET mutations (e.g., G810R / S) differ between HM06 / TAS0953 and BLU-667 / LOXO-292.

[0207] Example 15. Pharmacological effects of HM06 / TAS0953 on RET mutants Solvent Front KIF5B-RET G810R / A / C / D / S The sensitivity of various RET mutations, including variants, to HM06 / TAS0953, BLU-667, and LOXO-292 was examined in transiently transfected HEK293 cells using In-Cell Western® (ICW) analysis of KIF5B-RET phosphorylation. IC50 values ​​were calculated based on three independent experiments. The data are summarized in Table 6 below.

[0208] The efficacy of HM06 / TAS0953 against wild-type KIF5B-RET was equivalent to that of BLU-667 and LOXO-292. The pattern of RET mutations inhibited by HM06 / TAS0953 differed from that inhibited by LOXO-292 and BLU-667. HM06 / TAS0953 inhibited the G810X mutation and RET G810XThe IC50 values ​​of HM06 / TAS0953, which inhibits phosphorylation, ranged from 35.4 nmol / L to 282 nmol / L. The efficacy of HM06 / TAS0953 against the G810X mutation was higher than that of BLU-667 and LOXO-292. Furthermore, the efficacy of HM06 / TAS0953 against L730X, G736A, L760Q, L772M, Y806C, and A883V was also higher than that of BLU-667 and LOXO-292, but the efficacy of HM06 / TAS0953 against I788N and L865V was lower than that of BLU-667 and LOXO-292. These data suggest that HM06 / TAS0953 may be effective against point mutations in KIF5B-RET that are resistant to other RET inhibitors, such as LOXO-292 and / or BLU-667.

[0209] Generation of expression vectors An expression vector was generated using gateway technology. First, KIF5B-RET Using PCR products, pDONR 221 vector (Invitrogen Corporation), and Gateway BP Clonase enzyme mix (Invitrogen Corporation), A Tory vector (pENTR / KIF5B-RET) was constructed. Subsequently, a KIF5B-RET expression vector was constructed using an entry vector, a pJTI FAST KO2-PuroR expression vector (which was modified by Taiho Pharmaceutical Co., Ltd. using a pJTI-FAST DEST expression vector (Thermo Fisher Scientific)), and a Gateway LR Clonase enzyme mix (Invitrogen Corporation).

[0210] In-Cell Western Method Jump-In GripTite HEK293 cells were transiently transfected with a KIF5B-RET expression vector using TransIT-X2 (Mirus Bio LLC.). After treating each test compound at various concentrations for 1 hour, the cells were fixed with 20% formalin neutral buffer. The microplates were then blocked at room temperature for 1 hour with Intercept® (PBS) block buffer (LI-COR Inc.). Primary antibodies diluted in (PBS) block buffer (antiphosphorylated RET (Tyr905) antibody (catalog number 3221, Cell Signaling Technology, Inc.) and anti-RET antibody) The animals (catalog number sc-101422, Santa Cruz Biotechnology, Inc.) were incubated overnight at 4°C. The microplate was then washed, and secondary antibodies (goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680RD) (LI-COR Inc.) were added. It was incubated together with [the other organism].

[0211] After washing the microplate, fluorescence in each well was quantified using the Odyssey CLx Imaging System (LI-COR Inc.). The T / C (%) ratio was as follows: Identified: (Average signal of test compound) / (Average signal of control) × 100 Average signal: (fluorescence of phosphorylated RET - background) / (fluorescence of RET - background)

[0212] IC50 values ​​were calculated by curve fitting from concentration vs. T / C (%) curves using XLFit. The IC50 values ​​were determined through three independent experiments.

[0213] [Table 6]

[0214] Example 16. Ba / F3 KIF5B-RETG810R Evaluation of the antitumor effect of HM06 / TAS0953 in nude mice with subcutaneous cell transplantation. HM06 / TAS0953 is KIF5B-RET G810R In animal tumor models, HM06 / TAS0953 demonstrated significant antitumor effects and prominent target inhibition. This suggests that HM06 / TAS0953 may be effective in treating KIF5B-RET gene-positive tumors with the G810R mutation. At low doses of approximately 10 mg / kg twice daily, HM06 / TAS0953 significantly inhibited tumor growth and phosphorylated RET. In addition to its selective and high efficacy against wild-type RET, HM06 / TAS0953 also shows remarkable efficacy against the G810R solventfront mutation. The G810R solventfront mutation is highly resistant to serpercatinib and pralcetinib both in vitro and in vivo.

[0215] In the first test, Ba / F3 KIF5B-RET G810R cells (5×10 6 Cells (from mice) were suspended in 50% Matrigel (Corning Incorporated) / PBS and subcutaneously transplanted into male athymoid nude mice (BALB / cAJcl-nu / nu, CREA Nippon Co., Ltd.). HM06 / TAS0953, LOXO-292, and BLU-667 were mixed in 0.5 w / v% HPMC (Shin-Etsu Chemical Co., Ltd.) containing 0.1 mol / L HCl. One week after transplantation, the mice were randomly divided into different treatment groups so that the mean tumor volume of each group was equal, and each group was orally administered either Vehicle (bid), HM06 / TAS0953 (10 mg / kg, 30 mg / kg, bid), LOXO-292 (10 mg / kg, 30 mg / kg, bid), or BLU-667 (10 mg / kg, 30 mg / kg, bid) for 14 days. The control group received 0.5 w / v% HPMC containing 0.1 mol / L HCl as the vehicle. The length and width of the tumor were measured using a digital caliper (Mitutoyo Corporation), and the tumor volume was calculated as follows: [length × (width)] 2 ] / 2. The tumor volume and body weight of the mice were measured twice a week until the end of the study.

[0216] Dunnett's test was used to determine statistical significance and compare the tumor volume of the treatment group with that of the control group. Statistical analysis was performed using SAS version 9.4 (SAS Institute Japan Co., Ltd.) via EXSUS version 10.0 (CAC Exicare Co., Ltd.). A p-value of less than 0.05 was considered to indicate statistical significance.

[0217] Figures 13A and 13B show the effects of HM06 / TAS0953, LOXO-292, and BLU-667 on tumor volume. These were administered twice daily at low doses of 10 mg / kg (Figure 13A) and high doses of 30 mg / kg (Figure 13B), respectively. Data are expressed as mean ± SE (n=5 for each group). As reflected in Figure 13A, among the low-dose treatment groups, only the group treated with 10 mg / kg of HM06 / TAS0953 showed a significant reduction in tumor volume compared to the control group at the end of the study. * This reflects p<0.05; Dunnett's test). As shown in Figure 13B, at the end of the study, tumor volume in each of the high-dose treatment groups was reduced compared to the control group. * This reflects p<0.05 (Dunnett's test). Furthermore, as shown in Figure 13B, at the end of the study, only the HM06 / TAS0953 group treated with 30 mg / kg did not show tumor regrowth, while the LOXO-292 and BLU-667 groups showed a tendency for tumor regrowth in parallel with the control group. Figure 13C shows the effect of dosage on body weight during the treatment period. There were no significant differences in body weight change between groups. Data are expressed as mean ± SE (n=5 for each group). One mouse in the BLU-667 30 mg / kg group died accidentally.

[0218] In the second test, Ba / F3 KIF5B-RET G810R cells (5×10 6 Cells (mouse) were suspended in 50% Matrigel (Corning Incorporated) / PBS, and then applied to 6-week-old male athymoid nude mice (BALB / cAJcl-nu / nu, CREA Japan Co., Ltd.) in skin cells. The tumors were transplanted. HM06 / TAS0953, LOXO-292, and BLU-667 were mixed with 0.5 w / v% HPMC (Shin-Etsu Chemical Co., Ltd.) containing 0.1 mol / L HCl. Five days after transplantation, mice were randomly divided into different treatment groups so that the average tumor volume of each group was equal. They were orally administered either Vehicle (bid), HM06 / TAS0953 (50 mg / kg, bid), LOXO-292 (30 mg / kg, bid), or BLU-667 (30 mg / kg, bid) for 14 days. The group receiving 0.5 w / v% HPMC containing 0.1 mol / L HCl as Vehicle served as the control group. The length and width of the tumors were measured using a digital caliper (Mitutoyo Corporation), and the tumor volume was calculated as follows: [Length × (Width)] 2 ] / 2. The tumor volume and body weight of the mice were measured twice a week until the end of the study.

[0219] Dunnett's test was used to identify statistical significance and compare the TV of the treatment group with that of the control group. Statistical analysis was performed using SAS version 9.4 (SAS Institute Japan Co., Ltd.) via EXSUS version 10.0 (CAC Exicare Co., Ltd.). A p-value of less than 0.05 was considered to indicate statistical significance.

[0220] Figure 14A shows the effects of HM06 / TAS0953, LOXO-292, and BLU-667 on tumor volume. HM06 / TAS0953 was administered twice daily at a dose of 50 mg / kg, while LOXO-292 and BLU-667 were administered twice daily at a dose of 30 mg / kg, respectively. Data are expressed as mean ± SE (n=5 for each group). The mean tumor volume on day 15 was significantly smaller in the agonist-treated groups than in the control group (p<0.05, Dunnett's test). Furthermore, the mean tumor volume on day 15 was significantly smaller in the HM06 / TAS0953-treated group than in the LOXO-292 and BLU-667 groups (p<0.05, Tukey's test, respectively). While LOXO-292 and BLU-667 showed a tendency toward tumor regrowth on day 15, HM06 / TAS0953 exhibited consistent tumor regression. Figure 14B shows the effect of drug dosage on body weight during the treatment period. There were no significant differences in body weight change between the groups. Data are expressed as mean ± SE (n=5 for each group).

[0221] Ba / F3 KIF5B-RET 1 hour after administration G810R The inhibitory effects of HM06 / TAS0953, LOXO-292, and BLU-667 on RET phosphorylation in tumors were evaluated by Western blotting. (Ba / F3 KIF5B-RET) G810R Mice carrying TAS0953 were orally administered once at doses of 10 mg / kg, 30 mg / kg, or 50 mg / kg, or 10 mg / kg or 30 mg / kg, respectively. One hour after administration, the tumors were collected and lysed. The cell lysates were immunoblotted to detect phosphorylated RET (pRET), RET, and GAPDH (control). As shown in the Western blots of Figure 15A (first trial) and Figure 15B (second trial), a significant decrease in RET phosphorylation was observed in the HM06 / TAS0953 group compared to the control group, while only a slight decrease was observed in the LOXO-292 and BLU-667 groups.

[0222] Western blotting was performed in each of the two studies described above. Tumors were collected one hour after administration. Tumor tissue was lysed using Sample Diluent Concentrate 2 (R&D Systems) with cOmplete®, mini, protease inhibitor cocktail (Roche Applied Science), and PhosSTOP® phosphatase inhibitor cocktail (Roche Applied Science). The lysate was then fused to SDS. -The film was subjected to PAGE and transferred to a PVDF membrane (Trans-Blot turbo Blotting System; Bio-rad). Then, the film was subjected to Blocking One-P Blocked with (Nacalai Tesque Co., Ltd.), and incubated overnight at 4°C with the primary antibody. Phosphorylated Ret(Tyr905) antibody (#3221, Cell Signaling Technology), Ret(C31B4) rabbit mAb (#3223, Cell Signaling Technology), And GAPDH(D16H11)XP(trademark) rabbit mAb (#5174, Cell Signaling Technology) was used as the primary antibody. Subsequently, the membrane was washed, and the secondary antibody (#7074) was used. (with Cell Signaling Technology), incubated at room temperature for 1 hour, and then washed again. Chemiluminescence images were obtained using a chemiluminescence image analyzer (Amersham® Imager 600QC, GE Healthcare Japan Corporation). GAPDH was used as an internal control.

[0223] Equivalents The description above is deemed sufficient to enable those skilled in the art to carry out the embodiments. The above description and examples illustrate the details of a particular embodiment and describe the optimal form intended by the inventors. However, naturally, regardless of how much detail is described above in text, the embodiments can be carried out in many ways and should be interpreted in accordance with the appended claims and any equivalents thereof.

[0224] Where used herein, the term "about" refers to a numerical value, whether explicitly stated or not, which includes, for example, integers, fractions, and percentages. Generally, the term "about" refers to a range of numerical values ​​that a person skilled in the art would consider equivalent to (for example, having the same function or result as) the stated value (e.g., ±5% to 10% of the stated range). Where terms such as "at least" and "about" precede an enumeration of numerical values ​​or ranges, those terms modify all of the numerical values ​​or ranges presented in that enumeration. In some examples, the term "about" may include numerical values ​​rounded to the nearest significant figure.

Claims

1. A composition for treating a human patient with a solid tumor having a RET gene abnormality, comprising 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide or a salt thereof, wherein an effective amount of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide or a salt thereof is used to administer to the human patient, wherein the RET gene abnormality comprises a solvent front mutation of the RET protein.

2. The composition according to claim 1, wherein the RET gene abnormality further comprises at least one of RET gene fusion, point mutation, deletion mutation, copy number increase of the RET gene, overexpression of any one or more of these, and overexpression of the RET gene.

3. The composition according to claim 1 or 2, wherein the RET gene abnormality further comprises a RET gene fusion.

4. The composition according to any one of claims 1 to 3, wherein the RET gene abnormality further comprises a RET gene fusion with a gene encoding CCDC6, KIF5B, or TRIM33.

5. The composition according to any one of claims 1 to 4, wherein the RET gene abnormality further comprises a resistance mutation of the RET protein.

6. The composition according to any one of claims 1 to 5, wherein the RET gene abnormality further comprises a mutation in the hinge region of the RET protein.

7. The composition according to any one of claims 1 to 6, wherein the RET gene abnormality comprises a mutation in the RET protein at least one selected from the group consisting of amino acid residues 730, 736, 760, 772, 804, 806, 807, 808, 809, 810, and 883.

8. The aforementioned RET gene abnormalities are as follows: a) V804X mutation (where X is any amino acid other than valine or glutamic acid), b) Y806X mutation (where X is any amino acid other than tyrosine), c) A807X mutation (where X is any amino acid other than alanine), d) K808X mutation (where X is any amino acid other than lysine), e) Y809X mutation (where X is any amino acid other than tyrosine), and f) G810X mutation (where X is any amino acid other than glycine), The composition according to any one of claims 1 to 7, comprising a mutation of the RET protein including at least one selected from the group consisting of the following.

9. The aforementioned RET gene abnormalities are as follows: a) L730Q or L730R mutation, b) G736A mutation, c) L760Q mutation, d) L772M mutation, e) V804L or V804M mutation, f) Y806C, Y806S, Y806H, or Y806N mutations, g) G810R, G810S, G810C, G810V, G810D, or G810A mutations, and h) A883V mutation, The composition according to any one of claims 1 to 8, comprising a mutation of the RET protein including at least one selected from the group consisting of the following.

10. A composition for treating a human patient with a solid tumor having a RET gene abnormality with brain and / or leptomeningeal metastasis, comprising 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide or a salt thereof, wherein an effective amount of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide or a salt thereof is used to administer the human patient.

11. The composition according to claim 10, wherein the RET gene abnormality includes at least one of RET gene fusion, point mutation, deletion mutation, copy number increase of the RET gene, overexpression of any one or more of these, and overexpression of the RET gene.

12. The composition according to claim 10 or 11, wherein the RET gene abnormality includes a RET gene fusion.

13. The composition according to any one of claims 11 to 12, wherein the RET gene abnormality includes a RET gene fusion with a gene encoding CCDC6, KIF5B, or TRIM33.

14. The composition according to any one of claims 11 to 13, wherein the RET gene abnormality includes a resistance mutation in the RET protein.

15. The composition according to any one of claims 11 to 14, wherein the RET gene abnormality comprises at least one selected from the group consisting of a solvent front mutation of the RET protein and a mutation in the hinge region of the RET protein.

16. The composition according to any one of claims 11 to 15, wherein the RET gene abnormality comprises a mutation in the RET protein at least one selected from the group consisting of amino acid residues 730, 736, 760, 772, 804, 806, 807, 808, 809, 810, and 883.

17. The aforementioned RET gene abnormalities are as follows: a) V804X mutation (where X is any amino acid other than valine or glutamic acid), b) Y806X mutation (where X is any amino acid other than tyrosine), c) A807X mutation (where X is any amino acid other than alanine), d) K808X mutation (where X is any amino acid other than lysine), e) Y809X mutation (where X is any amino acid other than tyrosine), and f) G810X mutation (where X is any amino acid other than glycine), The composition according to any one of claims 1 to 16, comprising a mutation of the RET protein including at least one selected from the group consisting of the following.

18. The aforementioned RET gene abnormalities are as follows: a) L730Q or L730R mutation, b) G736A mutation, c) L760Q mutation, d) L772M mutation, e) V804L or V804M mutation, f) Y806C, Y806S, Y806H, or Y806N mutations, g) G810R, G810S, G810C, G810V, G810D, or G810A mutations, and h) A883V mutation, The composition according to any one of claims 1 to 17, comprising a mutation of the RET protein including at least one selected from the group consisting of the following.

19. The composition according to any one of claims 1 to 18, wherein the effective amount is a dosage equivalent to about 40 mg to about 3000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.

20. The composition according to any one of claims 10 to 19, wherein the brain and / or leptomeningeal metastases are asymptomatic.

21. The composition according to any one of claims 1 to 20, wherein the human patient is administered a dosage equivalent to approximately 150 mg to approximately 640 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.

22. The composition according to any one of claims 1 to 20, wherein the human patient is administered a dosage equivalent to approximately 480 mg to approximately 3000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.

23. The composition according to any one of claims 1 to 20 and 22, wherein the human patient is administered a dosage equivalent to approximately 640 mg to approximately 3000 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.

24. The composition according to any one of claims 1 to 21, wherein the human patient is administered a dosage equivalent to approximately 150 mg to approximately 500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base per day.

25. A composition according to any one of claims 1 to 24, which is administered orally.

26. The composition according to any one of claims 1 to 25, comprising the dihydrochloride salt of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide.

27. The composition according to any one of claims 1 to 26, administered once daily (QD) or twice daily (BID).

28. The composition according to any one of claims 1 to 20 and 25 to 27, wherein the human patient is administered a dose equivalent to approximately 160 mg to approximately 1500 mg of 4-amino-N-[4-(methoxymethyl)phenyl]-7-(1-methylcyclopropyl)-6-(3-morpholinopropane-1-in-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide free base twice daily (BID).

29. The composition according to any one of claims 1 to 28, administered in at least one 21-day treatment cycle.

30. The composition according to any one of claims 1 to 29, wherein the cancer or tumor is resistant to at least one multikinase inhibitor.

31. The composition according to any one of claims 1 to 30, wherein the cancer or tumor is resistant to at least one RET-selective inhibitor.

32. The composition according to any one of claims 1 to 31, wherein the human patient has previously received prior treatment for the cancer or tumor.

33. The composition according to any one of claims 1 to 32, wherein the cancer or tumor to be treated has progressed after prior treatment for the cancer or tumor.

34. The composition according to any one of claims 1 to 33, wherein the human patient developed intolerance to prior treatment for the cancer or tumor.

35. The composition according to any one of claims 1 to 34, wherein the human patient has previously been administered a multikinase inhibitor.

36. The composition according to any one of claims 1 to 35, wherein the human patient has previously been administered at least one selected from the group consisting of cabozantinib, vandetanib, lenvatinib, and RXDX-105.

37. The composition according to any one of claims 1 to 36, wherein the human patient has previously been administered a RET-selective inhibitor.

38. The composition according to any one of claims 1 to 37, wherein the human patient has at least one selected from the group consisting of salivary gland cancer, lung cancer, colorectal cancer, thyroid cancer, breast cancer, pancreatic cancer, ovarian cancer, skin cancer, and brain cancer.

39. The composition according to any one of claims 1 to 38, wherein the human patient has at least one selected from the group consisting of medullary thyroid carcinoma or anaplastic thyroid carcinoma, metastatic breast cancer, and metastatic pancreatic adenocarcinoma.

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