RU2800951C2 - Ret inhibitor for use in the treatment of cancer with ret alteration - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the methods for treating cancer with an activating alteration rearranged during transfection (RET-altered cancer). Variants of a method of treating cancer having an activating transfection-rearranged alteration (RET-altered cancer) in a subject in need thereof are provided, comprising administering to the subject a therapeutically effective amount, 300 mg or 400 mg, of compound 1 or a pharmaceutically acceptable salt thereof once a day.

EFFECT: achievement of the maximum therapeutic effect while maintaining the safety of the medicinal product.

37 cl, 13 dwg, 2 tbl, 3 ex

Description

The present application claims priority under U.S. Provisional Application No. 62/652284, filed April 3, 2018, U.S. provisional application no. 62/656297, filed April 11, 2018, U.S. provisional application no. 62/657605, filed April 13, 2018, and U.S. Provisional Application No. 62/741683, filed October 5, 2018, the contents of which are hereby incorporated by reference in their entirety.

The present disclosure relates to methods of treating a subject suffering from cancer having an RET activating alteration by administering an effective amount of a selective RET inhibitor, i.e., a compound that is specifically designed to selectively target one or more RET or RET altered kinases. As used in the present description, the term "having cancer" means the presence of cancer. In other words, a subject suffering from cancer has cancer. More specifically, the methods described herein relate to the treatment of a subject suffering from cancer characterized by an activating RET alteration. In some embodiments, the selective RET inhibitor is Compound 1 or pharmaceutically acceptable salts thereof. In some embodiments, the selective RET inhibitor is administered once a day. In some embodiments, the effective amount is 60 mg to 400 mg, 100 mg to 400 mg, 300 mg, or 400 mg. In some embodiments, the effective amount is 60 mg to 400 mg, 100 mg to 400 mg, 300 mg, or 400 mg administered once a day. In some embodiments, the cancer is RET-altered solid tumor, RET-altered non-small cell lung cancer, or RET-altered thyroid cancer. In some embodiments, the cancer is brain cancer, where the brain cancer is associated with non-small cell lung cancer. The present disclosure also relates to methods of treating RET-altered cancer by administering a physiologically effective dose of a selective RET inhibitor that causes robust down-regulation of at least one effect marker.

Receptor tyrosine kinase (RTK) RET, along with glial cell line neurotrophic factors (GDNF) and GDNF-α (GFRα) family receptors, is required for the development, maturation, and maintenance of several types of neural, neuroendocrine, and genitourinary tissues. However, a growing body of evidence points to aberrant activation of RET as a critical factor in tumor growth and proliferation in a large number of solid tumors (Mulligan LM., Nat. Rev. Cancer. 14: 173-186 (2014)). Oncogenic activation of RET occurs through amplification of a functional mutation or rearrangement of the RET gene, resulting in the production of a RET fusion protein with constitutively active RET signaling that promotes ligand-independent tumor growth. Oncogenic activation of RET was first described in hereditary and sporadic thyroid cancer and later in non-small cell lung cancer (NSCLC).

Oncogenic RET rearrangements have been identified in 1-2% of NSCLC cases (Lipson, D. et al., Nat. Med. 18: 382-384 (2012); Takeuchi, K. et al., Nat. Med. 18: 378-381 (2012) Stransky, N. et al., Nat Commun 5: 4846 (2014)). This creates a constitutively active kinase that promotes tumorigenesis. As with anaplastic lymphoma kinase (ALK) and c-ros oncogene (ROS) 1-rearranged NSCLC, RET-rearranged NSCLC usually has adenocarcinoma histology (although sometimes squamous) and occurs in young non-smokers. Because diagnostic testing for RET is not standard of care, RET rearranged patients with advanced NSCLC are treated according to NCCN guidelines for epidermal growth factor receptor (EGFR-) and ALK-negative adenocarcinoma. This usually includes chemotherapy using a platinum doublet or, more recently, a checkpoint inhibitor, but clinical response and overall survival, especially in RET-rearranged NSCLC with these agents, are not well understood. Follow-up therapy other than chemotherapy and checkpoint inhibitors for refractory patients according to NCCN guidelines is the best maintenance therapy or clinical trial.

Initial case reports and uncontrolled studies using the multikinase RET inhibitors (MKIs) cabozantinib, vandetanib, sorafenib, and alectinib in patients with known RET-rearranged NSCLC demonstrated clinical activity, suggesting that RET may be a valid target in NSCLC. Although these early studies have observed encouraging response rates (~12%-60%) (Horiike A et al., Lung Cancer 93: 43-6 (Mar. 2016); Lin JJ et al., J Thorac Oncol. 11 (11 ): 2027-32 (Nov. 2016); Gautshi O et al., J Clin Oncol. 34 (suppl; abstr 9014) (2016)) response time is typically less than a year. Treatment with MKI has been associated with significant toxicity requiring treatment interruption and/or dose modification, which likely limits the effects needed to effectively inhibit RET.

Oncogenic RET activation is also associated with thyroid cancer. Thyroid cancer consists primarily of differentiated thyroid cancer (DTC; ~90% of cases), medullary thyroid cancer (MTC; ~5% of cases), and anaplastic thyroid cancer (<5% of cases). DTC arises sporadically from thyroid follicular cells and consists of papillary thyroid cancer (PTC) (~80% of all thyroid cancers) and follicular thyroid cancer. In contrast, MTS arises from parafollicular C cells and occurs in both hereditary and sporadic forms. Oncogenic activation of RET is the driving force in both MTS and RTS.

Recurrent gene rearrangements involving RET and the gene encoding the dimerization domain have been identified in approximately 5%-20% of adult sporadic papillary tumors. RET kinase-activating mutations occur in almost all cases of hereditary MTS (87%-97%) (Machens A et al., N Engl J Med 349: 1517-25 (2003); Mulligan LM et al., Nature 363 (6428): 458-60 (1993 Jun 3); Mulligan LM et al., J Int Med. 238 (4): 343-346 (1995)) and in about 43%-65% of cases of sporadic MTS (Elisei R. et al., J Clin Endocrinol Metab 93: 682-687 (2008); Moura MM et al., British Journal of Cancer 100: 1777-1783 (2009)). These RET mutations occur in the extracellular domain (mainly at position C634), which promote ligand-independent RET dimerization and activation, and mutations in kinase domains (primarily M918T, A883F, or V804L/M), which promote RET autoactivation and subsequent oncogenic signaling (Romei C et al., Nat Rev Endocrinol. 12(4): 192-202 (2016 Apr.)).

PTC and MTS are treated surgically at localization (Fagin JA & Wells SA Jr., N Engl J Med. 375(11): 1054-67 (2016 Sep 15)). Ablative therapy with radioactive iodine (RAI) is effective in patients with recurrent RTS; however, patients eventually become refractory to RAI. Because MTS originates from follicular C cells, RAI is not effective. With progression, RAI-refractory RTS and MTS respond poorly to chemotherapy, and systemic treatment with small molecule MKI is the standard of care for both. Sorafenib and lenvatinib are MKI-approved for progressive and/or symptomatic RAI-refractory PTK. Cabozantinib and vandetanib are MKI-approved for advanced MTS and are used regardless of RET mutational status. MKIs used to treat thyroid cancer have broad activity against many kinases (eg, RAF, MET, EGFR, VEGFR1-3, PDGFR, RET, and others) and are associated with significant dermatological, cardiovascular, and gastrointestinal side effects . Therefore, the National Clinical Practice Guidelines in Oncology from the National Comprehensive Cancer Network (available at https://www.nccn.org/professionals/physician_gls/f_guidelines.asp) recommend close monitoring and treatment suspension and/or dose modification in case of side effects associated with drugs, with these drugs. For patients with disease progression on MKI therapy or intolerance to MKI, there are no effective treatments, and NCCN guidelines recommend participation in clinical trials.

Given the strong genetic and preclinical evidence that activated RET is the cause of oncogenic disease, the lack of available selective RET inhibitors, and the poor prognosis for many patients with RET-altered tumors, there remains a need to identify dosing ranges and regimens with adequate safety, efficacy, and tolerability of selective RET inhibitors for the treatment of RET-altered cancer.

Small molecule compounds that selectively inhibit RET are desirable agents for the treatment of cancer having an RET-activating alteration. One small molecule is (1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4 -(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexanecarboxamide (Compound 1). Compound 1 has the chemical structure:

In March 2017, compound 1 (also known as BLU-667) entered phase I clinical trials in the US for the treatment of patients with thyroid cancer, non-small cell lung cancer, and other advanced solid tumors (NCT03037385). WO 2017/079140, incorporated herein by reference, describes the synthesis of Compound 1 (Example Compound 130) and also describes the therapeutic activity of this molecule to inhibit, regulate and/or modulate RET kinase (Assays, Example 10 on page 72- 74.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1A, 1B and 1C are a series of histograms showing the effect of Compound 1 on DUSP6 and SPRY4 expression in LC2/ad (FIG. 1A), MZ-CRC-1 (FIG. 1B), and TT (FIG. 1C) cells.

Fig. 2 is a bar graph that shows a sustained decrease in expression of the MAPK target genes DUSP6 and SPRY4 in the KIF5B-RET NSCLC PDX model.

Fig. 3 is a graph that demonstrates the in vivo antitumor activity of Compound 1 in a cabozantinib resistant tumor model derived from the engineered KIF5B-RET V804L cell line.

Fig. 4A is a graph that shows tumor size and reduction in calcitonin and CEA (carcinoembryonic antigen) levels during treatment with Compound 1. A patient with RET mutant MTC (RET L629P, D631_R635DELINSG, V637R MTC) was treated with 60 mg once daily, and then sequentially increase the dose to 300 mg once a day. Fig. 4B are CT scans of the same RET mutant MTC patient shown in FIG. 4A, at baseline (upper) and after 8 weeks of treatment with Compound 1 (lower), showing rapid reduction in tumor growth. Fig. 4C is a graph that shows tumor size and reduction in calcitonin and CEA levels in a patient with RET M918T mutant MTC during treatment with Compound 1 at 300 mg once daily. Fig. 4D are CT scans of the M918T RET mutation patient of FIG. 4C, tumor at baseline (upper) and after 24 weeks of compound 1 treatment (lower). Fig. 4E is a graph that shows ctDNA analysis of M918T RET plasma levels from a patient with MTS during treatment. Tumor biopsy before and after treatment revealed a 93% decrease in DUSP6 and an 86% decrease in SPRY4 mRNA expression after 28 days of Compound 1 treatment.

Fig. 5A is a graph showing the reduction in lung tumor and KIF5B-RET and TP53 cDNA during treatment with 200 mg Compound 1 once daily; fig. 5B are computed tomography images that illustrate the tumor at baseline (upper) and after 32 weeks of Compound 1 treatment (lower).

Fig. 6A is a graph showing mean plasma concentration (ng/mL) versus time (h); fig. 6B is a bar graph that shows the percentage change in mean expression levels of the DUSP6 and SPRY4 genes from baseline.

Fig. 7A is a bar graph that shows the dose-dependent reduction in CEA in patients measured during cycle 2, day 1. FIG. 7B is a bar graph that shows the dose-dependent decrease in calcitonin in patients measured during cycle 2, day 1.

Fig. 8 is a waterfall plot that shows the maximum tumor reduction - the sum of the change in diameter compared to the baseline percentage - in patients in the phase I clinical study. End of data collection: April 6, 2018.

Fig. 9A is a CT scan of the brain at baseline before Compound 1 treatment. FIG. 9B is a CT scan of the brain after 8 weeks of Compound 1 treatment.

Fig. 10 is a chart showing patient response rates in RET-altered NSCLC. End of data collection: April 6, 2018

Fig. 11A is a baseline CT scan before Compound 1 treatment. FIG. 11B is a CT scan after 8 weeks of Compound 1 treatment. FIG. 11C is a baseline CT scan before Compound 1 treatment. FIG. 11D is a CT scan after 8 weeks of Compound 1 treatment.

Fig. 12 is a graph that shows that the response rate in patients with medullary thyroid cancer increases with dose and duration of therapy. In particular, the graph shows the dosing response rate of Compound 1 at 60 to 200 mg once daily and 300/400 mg once daily over a period of 8 to 24+ weeks.

Fig. 13 is a computed tomography image at baseline (BSL) and after 5 months of treatment with compound 1 at a dose of 400 mg once a day.

Abbreviations and definitions

The following abbreviations and terms throughout this document refer to the meanings indicated:

"Compound 1" is (1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy- 4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexanecarboxamide:

As used in the present description, "DOR" means the duration of the response.

As used herein, "PD" means disease progression.

As used herein, "SD" means stable disease.

As used herein, "CR" means complete response.

As used herein, "ORR" refers to the overall response rate.

As used in the present description, "CBR" means the frequency of clinical effectiveness.

As used herein, "PFS" means progression-free survival.

As used herein, a "fusion" is a protein that results from a chromosomal translocation in which two genes are joined by an in-frame coding sequence to form a chimeric protein. In some embodiments, the fusion is a chromosomal translocation in which the kinase domain of one protein is fused to the dimerization domain of another gene.

As used herein, a "RET altered cancer" is a cancer having an activating transfection rearranged (RET) alteration that triggers tumorigenesis. Non-limiting examples of RET-activating alterations include mutations, fusions, and copy number variations.

As used herein, "RET fusion" is a rearrangement of genes. RET rearrangements create a chimeric protein that juxtaposes the RET kinase domain and another protein's dimerization domain, creating a constitutively activated dimer that triggers tumorigenesis.

As used herein, a "chimeric RET protein" is the result of a gene rearrangement.

As used herein, an "activating RET mutation" means a mutation in the RET kinase that promotes ligand-independent constitutive activation of the RET kinase that triggers tumorigenesis. For example, RET mutations may occur in extracellular cysteine residues (eg, C620R or C634R/W) that trigger aberrant receptor dimerization, or RET mutations may occur in the intracellular kinase domain.

As used herein, a "RET inhibitor" is a compound that inhibits RET kinase activity. The RET kinase is a wild type RET kinase and/or one or more RET altered kinases (eg, RET fusion, RET mutation, or RET copy number variation).

Examples of RET inhibitors include, but are not limited to, Compound 1, LOXO-292 (selpercatinib), cabozantinib, vandetanib, alectinib, sorafenib, levatinib, ponatinib, dovitinib, sunitinib, foretinib, sitravatinib, DS-5010 (BOS172738), and RXDX- 105.

In some embodiments, the RET inhibitor may also inhibit other kinases. As used herein, a "multikinase RET inhibitor" is a compound that inhibits wild-type RET kinase and inhibits at least one other kinase to an equal or greater extent than wild-type RET kinase. Examples of multikinase RET inhibitors include: cabozantinib; vandetanib; alectinib; sorafenib; levatinib, ponatinib; dovitinib; sunitinib; foretinib; sitravatinib; DS-5010; and RXDX-105.

As used herein, the term "selective RET inhibitor" means a compound that selectively inhibits RET kinase. The RET kinase may include the wild type RET kinase and/or one or more RET altered kinases (eg, RET fusion, RET mutation, or RET copy number variations). The inhibitory activity of a selective RET inhibitor on RET kinase is more potent in terms of IC ₅₀ value (i.e., IC ₅₀ value is subnanomolar) compared to its inhibitory activity on many other kinases (e.g., KDR, VEGFR-2, ABL, EGFR, FGFR2, HER2, IGFIR, JAKI, KIT, MET, AKTI, MEK1). Efficacy can be measured using known biochemical assays. Examples of selective RET inhibitors include compound 1 and selpercatinib.

As used herein, the term "subject" or "patient" refers to organisms that are to be treated by the methods of this disclosure. Such organisms include, but are not limited to, mammals (eg, mice, monkeys, horses, cattle, pigs, dogs, cats, and the like) and, in some embodiments, humans.

Many cancers are associated with aberrant RET expression (Kato et al., Clin. Cancer Res: 23(8): 1988-97 (2017)). Non-limiting examples of "cancer" as used herein include lung cancer, head and neck cancer, gastrointestinal cancer, breast cancer, skin cancer, genitourinary cancer, gynecological cancer, hematological malignancy, central nervous system cancer ( CNS), peripheral nervous system cancer, endometrial cancer, colorectal cancer, bone cancer, sarcoma, spitzoid tumor, adenosquamous carcinoma, pheochromocytoma (PCC), hepatocellular carcinoma, multiple endocrine neoplasias (MEN2A and MEN2B), and inflammatory myofibroblastic tumor. For other examples, see Nature Reviews Cancer 14: 173-86 (2014).

Additional non-limiting examples of cancers include hemangiopericytoma, differentiated thyroid cancer, anaplastic thyroid cancer, lung carcinosarcoma, urothelial ureteral carcinoma, uterine carcinosarcoma, basal cell carcinoma, Merkel cell carcinoma, atypical lung carcinoma, fallopian tube adenocarcinoma, ovarian epithelial carcinoma, salivary gland adenocarcinoma , meningioma, duodenal adenocarcinoma, cervical adenocarcinoma, adrenal carcinoma, gastroesophageal junction carcinoma, skin squamous cell carcinoma, pancreatic ductal adenocarcinoma, prostate adenocarcinoma, esophageal adenocarcinoma, endometrial adenocarcinoma, serous ovarian carcinoma, unknown primary carcinoma, urothelial (transition nocellular) carcinoma of the urinary bladder, squamous cell lung cancer, colorectal adenocarcinoma, head and neck squamous cell carcinoma, and gastric adenocarcinoma.

In some embodiments, the cancer is liver cholangiocarcinoma. In some embodiments, the cancer is duodenal adenocarcinoma. In some embodiments, the cancer is an endometrioid adenocarcinoma of the uterine endometrium.

In some embodiments, MEN2A is associated with pheochromocytoma and parathyroid hyperplasia.

In some embodiments, MEN2B is associated with mucosal neuromas, pheochromocytomas, intestinal ganglioneuromas, and marfanoid habitus.

In some embodiments, the lung cancer is selected from small cell lung cancer (SCLC), lung adenocarcinoma, non-small cell lung cancer (NSCLC), bronchioloalveolar cancer, and mesothelioma. In some embodiments, the lung cancer is SCLC. In some embodiments, the lung cancer is NSCLC.

In some embodiments, the head and neck cancer is selected from thyroid cancer and salivary gland cancer. In some embodiments, the thyroid cancer is selected from papillary thyroid carcinoma (PTC), metastatic papillary thyroid cancer, medullary thyroid cancer (MTC), diffuse sclerosing variant papillary thyroid cancer, and thyroid cancer. In some embodiments, the cancer is familial medullary thyroid cancer. In some embodiments, the thyroid cancer is RTS. In some embodiments, the thyroid cancer is MTS.

In some embodiments, the gastrointestinal cancer is selected from esophageal cancer, esophageal and gastric cancer, gastrointestinal stromal tumor (e.g., imatinib-resistant gastrointestinal stromal tumor), small intestine cancer, diffuse gastric cancer, and ampullary cancer.

In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the skin cancer is melanoma or non-melanoma.

In some embodiments, the genitourinary cancer is selected from colon cancer, metastatic colon cancer, bladder cancer, renal cell carcinoma (RCC), prostate cancer, hepatobiliary cancer, intrahepatic bile duct cancer, adrenocortical cancer, pancreatic cancer, and adenocarcinoma of the pancreatic ducts.

In some embodiments, the gynecological cancer is selected from uterine sarcoma, germ cell tumor, cervical cancer, rectal cancer, testicular cancer, and ovarian cancer.

In some embodiments, the hematologic cancer is selected from leukemia, primary myelofibrosis with secondary acute myeloid leukemia, myelodysplasia (MDS), non-Hodgkin's lymphoma, chronic myeloid leukemia, Philadelphia chromosome acute lymphoblastic leukemia, and chronic myelomonocytic leukemia (CMML).

In some embodiments, the cancer of the peripheral nervous system is a paraganglioma. In some embodiments, the endometrial cancer is endometrial adenocarcinoma. In some embodiments, the sarcoma is a soft tissue sarcoma.

In some embodiments, the central nervous system (CNS) cancer is selected from brain cancer associated with lung cancer and glioma.

It is known that lung cancer spreads to the brain in about 40% of cases when metastases occur. In lung cancer, this is considered the fourth stage of the disease, and the average life expectancy with brain metastases is usually less than a year. Lung cancer with brain metastases has a relatively poor prognosis, for example, with chemotherapy drugs. Brain metastases are difficult to treat for many reasons. Often, by the time a patient has the first symptoms, they already have multiple lesions. Brain metastases tend to be very aggressive. The brain has many defenses to reduce the penetration of harmful substances. In particular, the blood-brain barrier prevents many drugs, such as compounds, from entering the brain. Treatment options can damage surrounding normal tissues and have a significant impact on quality of life. In particular, there is a need for compounds that can be administered at a safe dose, are well tolerated, and penetrate the brain for the treatment of brain metastases.

In some embodiments, the cancer is a brain metastasis associated with lung cancer.

In some embodiments, the cancer is "RET-altered cancer," which, as used herein, means a cancer having an RET-activating alteration. In some embodiments, the RET-altered cancer has a RET mutation or a rearrangement of the RET gene. In some embodiments, the RET-altered cancer is a RET-altered solid tumor.

As used herein, the term "effective amount" refers to an amount of a selective RET inhibitor (eg Compound 1 or a pharmaceutically acceptable salt thereof) sufficient to achieve beneficial or desired results. Beneficial or desired results may be a therapeutic benefit or outcome, or a physiological benefit or outcome. An effective amount may be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration.

As used herein, the term "therapeutically effective amount" refers to an amount of a selective inhibitor (eg, Compound 1 or a pharmaceutically acceptable salt thereof) sufficient to achieve beneficial or desired therapeutic results in a subject. A therapeutically effective amount may be administered to a subject in need thereof in one or more administrations, uses, or dosages, and is not intended to be limited to a particular formulation or route of administration. In some embodiments, the therapeutically effective amount provides the desired safety, effect, and tolerability. The selection of a therapeutically effective amount, ie the correct dose for administering a compound, is a necessary step in the development of a pharmaceutical preparation for clinical use. Without reliable information about the dosage, doctors cannot prescribe a particular drug to patients. Therefore, determining the correct dosage of a drug is a key question that can only be answered in clinical trials. If a dose and frequency of administration that provides safe and predictable administration cannot be determined, then the compound may not be a medically useful or commercially viable pharmaceutical product.

As used herein, the term "physiologically effective amount" refers to an amount of a selective inhibitor (eg, Compound 1 or a pharmaceutically acceptable salt thereof) sufficient to achieve a favorable or desired physiological outcome in a subject. The physiological result may be a stable suppression of at least one marker of effect in the subject.

As used herein, the term "treatment" includes any effect, such as amelioration, reduction, modulation, amelioration, or elimination, that results in amelioration of a condition, disease, disorder, or the like, or alleviation of their symptoms.

As used herein, an "effect marker" means expression of DUSP6 mRNA, expression of SPRY4 mRNA, CEA, calcitonin, KIF5B ctDNA, or TP53 ctDNA.

Some exemplary embodiments of the invention include the following:

1. A method of treating a subject suffering from cancer having an activating transfection rearranged (RET) alteration comprising administering to the subject a therapeutically effective amount of 300 to 400 mg of Compound 1 or a pharmaceutically acceptable salt thereof once daily.

2. The method according to Embodiment 1 wherein the administration amount is 300 mg.

3. The method according to embodiment 1 or 2, wherein the administration amount is 400 mg.

4. The method according to any one of embodiments 1-3, wherein the cancer is selected from papillary thyroid carcinoma (PTC), medullary thyroid cancer (MTC), pheochromocytoma (PCC), pancreatic ductal adenocarcinoma, multiple endocrine neoplasia (MEN2A and MEN2B) , metastatic breast cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), chronic myelomonocytic leukemia (CMML), colorectal cancer, ovarian cancer, inflammatory myofibroblastic tumor and salivary gland cancer.

5. The method according to any one of embodiments 1-3, wherein the cancer is selected from esophageal cancer, skin cancer (non-melanoma), endometrial cancer, head and neck cancer, bladder cancer, prostate cancer, hematologic malignancy, leukemia, soft tissue sarcoma , renal cell carcinoma (RCC), non-Hodgkin's lymphoma, hepatobiliary cancer, adrenocortical cancer, myelodysplasia (MDS), uterine sarcoma, embryonic cell tumor, cervical cancer, cancer of the central nervous system, bone cancer, ampullary cancer, gastrointestinal stromal tumor intestinal tract, small intestine cancer, mesothelioma, rectal cancer, paraganglioma, and intrahepatic bile duct cancer.

6. The method according to any one of embodiments 1-3, wherein the cancer is selected from adenocarcinoma, spitzoid tumor, lung adenocarcinoma, adenosquamous cancer, colon cancer, metastatic colon cancer, metastatic papillary thyroid cancer, diffuse sclerosing variant papillary thyroid cancer, primary myelofibrosis with secondary acute myeloid leukemia, diffuse gastric cancer, thyroid cancer, and bronchioloalveolar cancer.

7. The method according to any one of embodiments 1-3, wherein the cancer is selected from hepatobiliary cancer, ampullary cancer, small intestine cancer, intrahepatic bile duct cancer, metastatic colon cancer, lung cancer associated brain cancer, brain metastasis associated with lung cancer, and retroperitoneal paraganglioma.

8. The method according to any one of embodiments 1-3, wherein the cancer is selected from medullary thyroid cancer (MTC) and non-small cell lung cancer (NSCLC).

9. The method according to embodiment 8, wherein the cancer is selected from sporadic MTC, metastatic RET-altered NSCLC, tyrosine kinase inhibitor (TKI)-refractory KIF5B-RET NSCLC, and KIF5B-RET NSCLC.

10. The method according to any one of embodiments 1-3, wherein the cancer is selected from brain cancer associated with lung cancer.

11. The method of Embodiment 10 wherein the brain cancer is a brain metastasis.

12. The method according to any one of embodiments 1-11, wherein the RET activating alteration comprises a RET mutation or rearrangement (fusion) of the RET gene.

13. The method according to any one of embodiments 1-11, wherein the RET activating alteration is a RET mutation.

14. The method according to embodiment 12 or 13, wherein the RET mutation is a point mutation.

15. The method according to any one of embodiments 12-14, wherein the RET mutation is a resistance mutation.

16. The method according to any one of embodiments 12-15, wherein the RET alteration is a RET mutation selected from Table 1.

17. The method according to any one of embodiments 12-16, wherein the RET mutation is V804M, M918T, C634R, or C634W.

18. The method according to any one of embodiments 1-4, 8, 9, and 12-16, wherein the cancer is RET-altered medullary thyroid cancer (MTC).

19. The method of Embodiment 18 wherein the cancer is familial MTS.

20. The method of Embodiment 18 wherein the cancer is sporadic MTS.

21. The method according to any one of embodiments 1-3 and 12-19, wherein the cancer is MTC having the M918T mutation.

22. The method according to any one of embodiments 1-3 and 12-19, wherein the cancer is an MTC having a C634R mutation.

23. The method according to any one of embodiments 1-3 and 12-19, wherein the cancer is an MTC having a V804M mutation.

24. The method according to any one of embodiments 1-3, 6, and 12-16, wherein the cancer is a paraganglioma.

25. The method of Embodiment 24 wherein the cancer is a retroperitoneal paraganglioma.

26. The method according to any one of embodiments 1-3, 6, 12-16, 24, and 25, wherein the paraganglioma has the R77H mutation.

27. The method according to any one of embodiments 1-11, wherein the RET activating alteration is a rearrangement (fusion) of a gene.

28. The method of Embodiment 27, wherein the RET activating alteration is a fusion with a RET fusion partner selected from Table 2.

29. The method according to embodiment 27 or 28, wherein the fusion is KIF5B-RET, CCDC6-RET, KIAA1468-RET, or NCOA4-RET.

30. The method according to any one of embodiments 1-4 and 27-29, wherein the cancer is RET-altered NSCLC.

31. The method of embodiment 30 wherein the cancer is an NSCLC having a KIF5B-RET fusion.

32. The method of embodiment 30 wherein the cancer is an NSCLC having a CCDC6-RET fusion.

33. The method of embodiment 30 wherein the cancer is an NSCLC having a KIAA1468-RET fusion.

34. The method of embodiment 30 wherein the cancer is an NSCLC having a RET fusion identified as FISH positive.

35. The method according to embodiment 29 or 30, wherein the RET alteration is KIF5B-RET V804L (cabozantinib resistant).

36. The method according to embodiment 29 or 30, wherein the RET alteration is CCDC6-RET V804M (ponatinib resistant).

37. The method according to any one of embodiments 1-4 and 27-29, wherein the cancer is a RET altered PTC.

38. The method of embodiment 37 wherein the cancer is a PTC having a CCDC6-RET fusion.

39. The method of embodiment 37 wherein the cancer is a PTC having an NCOA4-RET fusion.

40. The method according to any one of embodiments 1-3 and 27-29, wherein the cancer is RET-altered intrahepatic bile duct carcinoma.

41. The method of embodiment 40 wherein the cancer is an intrahepatic bile duct carcinoma having an NCOA4-RET fusion.

42. The method according to any one of embodiments 1-41, wherein the subject has not received prior treatment with a multikinase RET inhibitor.

43. The method according to any one of embodiments 1-41, wherein the subject has received one or more prior treatments with a multikinase RET inhibitor.

44. The method of embodiment 43 wherein the RET multikinase inhibitor is selected from lenvatinib, vandetanib, cabozantinib, and RXDX-105.

45. The method according to any of embodiments 1-41, wherein the subject has not previously received platinum treatment.

46. The method according to any one of embodiments 1-41, wherein the subject has previously received platinum treatment.

47. The method according to any one of embodiments 1-41, wherein the subject has previously received treatment with a selective RET inhibitor.

48. The method according to any one of embodiments 1-47, wherein the subject has not received prior chemotherapy.

49. The method according to any one of embodiments 1-47, wherein the subject has previously received chemotherapy.

50. The method of embodiment 49 wherein the prior chemotherapy is selected from carboplatin, pemetrexed, abraxane, cisplatin, bevacizumab, and combinations thereof.

51. The method according to any of embodiments 1-42, wherein the subject has not received prior immunotherapy.

52. The method according to any one of embodiments 1-42, wherein the subject has previously received immunotherapy.

53. The method of embodiment 52, wherein the prior immunotherapy is selected from ipilimumab, pembrolizumab, nivolumab, MPDL3280A, MEDI4736, and combinations thereof.

54. A method of treating a subject suffering from brain cancer associated with RET-altered lung cancer, comprising administering to the subject a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof.

55. The method of Embodiment 54 wherein the brain cancer is a brain metastasis.

56. A method of treating a subject suffering from cancer having an activating RET mutation, comprising administering to the subject a physiologically effective amount of an RET inhibitor, wherein administration of the RET inhibitor is associated with sustained down-regulation of at least one effect marker in the subject.

57. The method of embodiment 56 wherein the RET inhibitor is administered orally.

58. The method according to embodiment 56 or 57 wherein the RET inhibitor is Compound 1 or a pharmaceutically acceptable salt thereof.

59. The method according to any one of embodiments 56-58, wherein the effect marker is selected from DUSP6 mRNA expression, SPRY4 mRNA expression, carcinoembryonic antigen level, and calcitonin level.

60. The method according to any one of embodiments 56-58, wherein the effect marker is a KIF5B ctDNA level or a TP53 ctDNA level.

61. The method according to any one of embodiments 56-59, wherein the amount administered to the subject causes greater than 95% down regulation of at least one effect marker.

62. The method according to any one of embodiments 56-59, wherein the amount administered to the subject causes more than 94%, more than 93%, more than 92%, more than 91%, more than 90%, more than 89%, more than 88%, more than 87% , more than 86%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55% or more than 50% down-regulation of at least one effect marker.

63. The method according to embodiment 61, wherein the amount administered to the subject causes greater than 89%, greater than 88%, greater than 87%, greater than 86%, greater than 85%, greater than 80%, greater than 75%, or greater than 70% down regulation of at least one effect marker.

64. The method according to any one of embodiments 56-59, wherein at least two effect markers are down-adjusted.

Some of the RET point mutations in Table 1 are described in: U.S. Patent Application Publication No. 2014/0272951; Krampitz et al., Cancer 120: 1920-31 (2014); Latteyer et al., J Clin. Endocrinol. Metab. 101(3): 1016-22 (2016); Silva et al. Endocrine 49.2: 366-72 (2015); Jovanovic et al., Prilozi 36(1): 93-107 (2015); Qi et al., Oncotarget 6 (32): 33993-4003 (2015); Kim et al. ACTA ENDOCRINOLOGICA-BUCHAREST 11.2, 189-194, (2015); Cecchirini et al. Oncogene, 14: 2609-12 (1997); Karrasch et al., Eur. Thyroid J 5(1): 73-77 (2016); Scollo et al., Endocr. J 63: 87-91 (2016); and Wells et al., Thyroid 25: 567-610 (2015).

R525W and A513D may act in combination with S891A to enhance oncogenic activity.

Some of the RET Mergers in Table 2 are described in: Grubbs et al., J Clin Endocrinol Metab, 100: 788-93 (2015); Halkova et al., Human Pathology 46: 1962-69 (2015); US Patent No. 9297011; US Patent No. 9216172; Le Rolle et al., Oncotarget 6 (30): 28929-37 (2015); Antonescu et al., Am J Surg Pathol 39(7): 957-67 (2015); U.S. Patent Application Publication No. 2015/0177246; U.S. Patent Application Publication No. 2015/0057335; Japanese Patent Application Publication No. 2015/109806 A; Publication of Chinese Patent Application No. 105255927 A; Fang, et al., Journal of Thoracic Oncology 11.2 (2016): S21-S22; European patent application publication no. EP 3037547 A1; Lee et al., Oncotarget DOI: 10.18632/oncotarget.9137, e-published ahead of printing, 2016; Saito et al., Cancer Science 107: 713-20 (2016); Pirker et al., Transl Lung Cancer Res, 4(6): 797-800 (2015); and Joung et al., Histopathology 69(1): 45-53 (2016).

One skilled in the art can determine if a subject has a RET altered cell, cancer, gene, or gene product, e.g., having a mutation, e.g., fusion, deletion, insertion, translocation, frameshift, duplication, mutation point, and/or rearrangement, for example, using a method selected from hybridization-based methods, amplification-based methods, microarray analysis, flow cytometry, DNA sequencing, next generation sequencing (NGS), primer extension, PCR, in situ hybridization, fluorescence in situ hybridization, dot- blotting and southern blotting.

Primary tumor samples may be taken from a subject to detect fusion. Samples are processed, nucleic acids are isolated using methods known in the art, then the nucleic acids are sequenced using methods known in the art. The sequences are then mapped to individual exons and transcriptional expression scores (such as RPKM or reads per kilobase per million reads mapped) were quantified. Raw sequences and exon array data are available from sources such as TCGA, ICGC, and NCBI Gene Expression Omnibus (GEO). For a given sample, the coordinates of individual exons are annotated with gene identifier information, and exons belonging to kinase domains are labeled. Exon levels are then normalized by z-score for all tumor samples.

Genes are then identified in which the 5' exons are expressed at significantly different levels than the 3' exons. The sliding frame is used to define the break point in a single pattern. Specifically, at each iteration, an incremental breakpoint divides the gene into 5' and 3' regions, and a t-statistic is used to measure the difference in expression (if any) between the two regions. The breakpoint with the maximum t-statistic is chosen as the probable merge breakpoint. As used herein, a "break point" is the boundary at which two different genes merge. This is sometimes referred to as the "merge point". The location where the difference in exon expression is greatest between 5' and 3' is the putative fusion break point. In this way, thousands of tumor samples can be quickly profiled by generating a list of fusion candidates (ranked by t-statistic). High-ranking candidates can then be tested and fusion partners can be identified by examining the raw RNA-seq datasets and identifying chimeric pairs and/or split reads that support the fusion. Possible mergers can then be experimentally verified as described below.

Alternatively, the methods described in Wang L et al., Genes Chromosomes Cancer 51(2): 127-39 (2012). doi: 10.1002/gcc.20937, Epub 2011 Oct 27; and Suehara Y et al., Clin Cancer Res. 18(24): 6599-608 (2012). doi: 10.1158/1078-0432.CCR-12-0838, Epub 2012 Oct 10, can also be used.

It has been suggested that incorporating pharmacodynamic evaluation of molecularly targeted therapies into clinical trials may simplify the drug development process (Tan DS et al., Cancer J 15(5): 406-20 (2009); Sarker D & Workman P. Adv Cancer Res 96: 213-68 (2007)). Pharmacodynamic biomarkers have been successfully used for the clinical development of kinase inhibitors, including imatinib and gefitinib (Sarker D & Workman P. Adv Cancer Res 96: 213-68 (2007); Baselga J et al., J Clin Oncol 23 (23): 5323-33 (2005); Druker BJ et al., N Engl J Med 344 (14): 1031-7 (2001)). As described herein, Compound 1 dose-dependently inhibited RET and SHC activation, reflecting the inhibition of DUSP6 and SPRY4 transcription in RET-driven preclinical models, indicating that these transcripts may serve as biomarkers of RET inhibitory activity. The translational capacity of these markers was established in this study, in which the reduction in MTC tumor induced by Compound 1 treatment was associated with effective inhibition of DUSP6 and SPRY4 expression in tumor tissue. To the knowledge of the Applicant, this represents the first confirmation of RET targeting by a small molecule inhibitor, multipurpose or selective, in a clinical setting. These effect markers can be used to more accurately determine the optimal dose and regimen needed to effectively inhibit RET.

Although Compound 1 may be administered alone, in some embodiments, Compound 1 may be administered as a pharmaceutical formulation wherein Compound 1 is combined with one or more pharmaceutically acceptable excipients or carriers. Compound 1 may be formulated for administration by any convenient route for human or veterinary use. In some embodiments, the compound included in the pharmaceutical formulation may be active per se or may be a prodrug, eg, capable of being converted to the active compound under physiological conditions.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, substances, compositions and/or dosage forms which, under sound medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problem. or complications commensurate with a reasonable benefit/risk ratio.

Examples of pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; (21) cyclodextrins such as Captisol®; and (22) other non-toxic compatible substances used in pharmaceutical compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Solid dosage forms (eg, capsules, tablets, pills, dragees, powders, granules, etc.) may include one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and/or any of the following: (1) excipients or diluents such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; (3) humectants such as glycerin; (4) disintegrants such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retardants such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (8) scavengers such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) colorants.

Liquid dosage forms may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate , propylene glycol, 1,3-butylene glycol, oils (particularly cottonseed, peanut, corn, germ, olive, castor and sesame), glycerin, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof.

Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide. or mixtures thereof.

Powders and sprays may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

Dosage forms for topical or transdermal administration of compound 1 include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalation dosage forms. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers or propellants that may be required.

When Compound 1 is administered as a pharmaceutical to humans and animals, it may be administered alone or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (for example, 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage forms can be administered topically, orally, transdermally, rectally, vaginally, parenterally, intranasally, intrapulmonary, intraocularly, intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intradermally, intraperitoneally, subcutaneously, intradermally, or by inhalation.

The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited in this application are expressly incorporated into this description by reference.

Examples

Example 1: Expression analysis of DUSP6 and SPRY4

Cells were treated with these compounds for 7 hours before lysis with RLT buffer (QIAGEN, Hilden, Germany) containing 1% β-mercaptoethanol. Total RNA was isolated using the Rneasy Plus Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. First strand cDNA was synthesized using Superscript VILO Master Mix (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Real-time qPCR was performed on a ViiA 7 Real Time PCR System (Thermo Fisher Scientific). For qRT-PCR, expression of the glucuronidase beta (GUSB) reference gene was used to normalize expression of the target genes DUSP6, SPRY4, and glycogen synthase kinase-3 beta (GSK3B). Repeat real-time qPCR reactions were analyzed for each sample, and QuantStudio Real-Time PCR software (Life Technologies, Carlsbad, CA) normalized mean DUSP6, SPRY4, or GSK3B expression to mean GUSB reference gene expression in each sample. FIGS. 1A-1C show the relative transcript expression of the RET pathway targeting DUSP6 and SPRY4 and the AKT pathway targeting GSK3B 7 hours after treatment of L2C/ad cells (FIG. 1A), MZ-CRC-1 cells (FIG. 1B) or MTC TT cells (Fig. 1C) with compound 1 or cabozantinib. Figure 2 shows the relative expression of DUSP6, SPRY4 and GSK3B transcripts from KIF5B-RET NSCLC PDX. Tumors were harvested at the indicated time (hours) after the last dose. Data are mean + SD. *P<0.05, **P<0.01, ***p<0.001, two-tailed Student's t-test. SD, standard deviation.

Example 2 Preparation of KIF5B-RET Ba/F3 Cells and ENU Mutagenesis Assays

DNA encoding the amino acid sequence of human KIF5B-RET variant 1 was placed in a lentiviral vector under a doxycycline-inducible promoter to maximize expression with the carboxy-terminal FLAG epitope to facilitate immunodetection of the fusion with anti-FLAG antibodies. Lentivirus-mediated gene transduction was used to express KIF5B-RET in Ba/F3 cells, KIFSB-RET-dependent cells were selected by removing IL-3, and expression of the KIF5B-RET fusion protein was confirmed by immunoblot analysis. To generate Ba/F3 cells bearing V804 substitutions, WT KIF5B-RET Ba/F3 cells were mutagenized overnight with ENU and plated in 96-well plates for a period of 2 weeks in the presence of 6 concentrations of MKIs (ponatinib, regorafenib, cabozantinib, or vandetanib). Selected concentrations ranged from 2×-64×IC ₅₀ proliferation for each compound: 125 nM to 4 µmol/L cabozantinib, 20-640 nM ponatinib, and 250 nM to 8 µmol/L vandetanib. Genomic DNA was isolated from resistant clones and Sanger sequencing was used to identify those containing substitutions. In FIG. 3 shows the antitumor activity of Compound 1 versus cabozantinib in KIF5B-RET V804L Ba/F3 allografts.

Example 3: Phase I study

A phase I, first human study (NCT03037385) was initiated to determine the maximum tolerated dose, safety profile, pharmacokinetics, and provisional antitumor activity of Compound 1 in advanced, RET-altered NSCLC, MTS, and other solid tumors. Prior to enrollment in the study, written informed consent was obtained from all patients for Compound 1 treatment and collection of blood and tumor samples for diagnostic biomarker analysis to characterize potential predictive biomarkers of safety and efficacy. Adult patients (≥18 years of age) were required to have advanced, inoperable solid tumors, with an Eastern Joint Oncology Group General Condition score of 0 to 2, and acceptable bone marrow, liver, kidney, and heart function. Compound 1 was administered orally once a day for a 4 week cycle using a Bayesian optimal interval schedule. At dose levels ≥120 mg, a documented RET alteration was additionally required for inclusion in the study. Adverse events were assessed according to the Criteria for assessing the severity of the most common adverse events (CTAE). The radiological response by computed tomography was assessed using RECIST version 1.1 (European Journal of Cancer 45: 228-247 (2009)). Plasma ctDNA levels were assessed using a PlasmaSELECT™-R64 NGS panel (Personal Genome Diagnostics, Baltimore, MD). Serum calcitonin levels in MTS patients were measured by ELISA (Medpace, Cincinnati, OH). Tumor levels of DUSP6/SPRY4 were analyzed by real-time qPCR (Molecular MD, Portland, OR).

Specific examples

Patient 1 was a 27 year old with sporadic MTS carrying multiple RET mutations (L629P, D631_R635DELINSG, and V637R). The patient was a patient who had not received tyrosine kinase inhibitor treatment prior to treatment with Compound 1 with highly invasive disease that required emergency tracheostomy and extensive surgery, including total thyroidectomy, central cervical dissection, bilateral cervical dissection at levels 1-4, total thymectomy, and median sternotomy. The postoperative course was complicated by chylothorax. The interdisciplinary medical consensus was against radiotherapy to the neck, and rescanning showed disease in the left paratracheal region with invasion of the trachea and esophagus, and metastasis of the disease to the lungs and liver. The two FDA-approved multikinase drugs for the treatment of MTS (vandetanib and cabozantinib) were not considered appropriate for this patient given their associated risk of VEGFR-related toxicity, which may include impaired wound healing and increased risk of fistula formation and bleeding (CAPRELSA (vandetanib) [ package leaflet] Cambridge, MA: Sanofi Genzyme; 2016; COMETRIQ (cabozantinib) [package leaflet]. South San Francisco, CA: Exelixix, Inc.; 2018). Therefore, the patient was enrolled in the Compound 1 clinical trial and started treatment at the second dose level (60 mg, QD (once a day)). Notably, after 28 days of compound 1 treatment, a >90% decrease in serum tumor marker calcitonin was observed (FIG. 4A). After 8 weeks, target lesions were reduced by 19%. Following a sequential dose increase of Compound 1 to 200 mg QD, the patient achieved a partial response with >30% tumor reduction according to the Solid Tumor Response Evaluation Criteria (RECIST) version 1.1 (FIG. 4B). This patient subsequently increased the dose to 300 mg once daily and achieved a confirmed partial response (maximum reduction of 47%) at 10 months. Overall, carcinoembryonic antigen (CEA) levels decreased by 57% over this period. Improvement in health with Compound 1 treatment allowed the patient's tracheostomy tube to be removed and return to baseline weight after losing several kilograms of pre-treatment weight. Compound 1 was well tolerated during 11 months of continuous treatment, with the only drug-related adverse effect being a transient decrease in grade 1 white blood cells, which resolved without drug interruption or dose changes. As of April 13, 2018, the patient continued treatment.

Patient 2 was a 56 year old patient with sporadic RET M918T mutant MTC who responded to treatment and then progressed on vandetanib starting therapy with Compound 1, 300 mg QD. Early signals of clinical activity appeared within the first few weeks of Compound 1 treatment: serum calcitonin decreased >90% and CEA decreased by 75% after 28 days (FIG. 4C). Circulating tumor DNA (cDNA) RET M918T decreased by 47% after 28 days and was not detected after 56 days. Paired tumor biopsies collected before treatment and 28 days after treatment demonstrated a 93% reduction in DUSP6 and an 86% reduction in SPRY4 mRNA expression, confirming inhibition of the RET pathway in the tumor (Fig. 4E). Importantly, these signs of activity were confirmed by radiographic response (-35%) according to RECIST 1.1 at 8 weeks (Fig. 4D). The patient tolerated Compound 1 treatment well with no dose interruption; Medication-related side effects included grade 1 nausea and hyperphosphatemia. The patient continued therapy after 8 months with a confirmed partial response (maximum reduction of 47%) as of April 13, 2018.

Patient 3 was a 37-year-old patient with metastatic RET-altered NSCLC that progressed on cisplatin, pemetrexed, and bevacizumab, had a positive tumor tissue test for RET fusion by FISH analysis. The patient started treatment with 200 mg QD Compound 1 and baseline ctDNA analysis revealed a canonical KIF5B-RET fusion and a concomitant TP53 mutation. Tumor reduction (-25%) was noted at the first radiographic evaluation after 8 weeks of treatment and correlated with a concomitant decrease in KIF5B-RET and TP53 ctDNA levels (FIG. 5A). The patient achieved a partial response at the second radiographic evaluation at 16 weeks (Figure 5B) and continued treatment for 10 months with a confirmed partial response as of April 13, 2018. As observed in patients with MTS described above, Compound 1 is well tolerated. all drug-related side effects are grade 1, including constipation (resolved), dry skin, rash, and leukopenia.

Patient 4 was a 69-year-old NSCLC patient who underwent lung resection, nephrectomy, and pleural drainage. The patient started treatment with 400 mg compound 1 once a day. Tumor reduction was noted in relation to KIF5B-RET NSCLC brain metastases (FIG. 9). In particular, the patient showed signs of intracranial antitumor activity. At baseline, the patient had an approximately 6 mm brain metastasis that appeared to resolve after 8 weeks of treatment. At the time of the 8-week evaluation, the patient was found to have stable disease.

Patient 5 was a 74 year old ex-smoker with locally advanced KIF5B-RET NSCLC. The CT images of the patient are shown in Fig. 11A-11D. The patient received concurrent chemoradiotherapy with cisplatin and pemetrexed followed by treatment with carboplatin and nab-paclitaxel and eventually progressed. Next-generation sequencing of tumor tissue, along with FISH, revealed a KIF5B-RET fusion, and the patient was included in a clinical trial testing the combination regimen of vandetanib and everolimus (NCT01582191). The patient achieved a partial response, but rescanning after 11 cycles showed progression of the disease, which was associated with clinical symptoms of increased dyspnea and worsening general condition. The patient was then enrolled in the phase 1 study of compound 1. After 16 weeks of treatment with compound 1 (300 mg QD), the patient had a partial response with a 34% reduction in tumor volume (FIGS. 11C and 11D) and improvement in dyspnea and general condition. Compound 1 was well tolerated throughout treatment, and as of April 13, 2018, the patient had experienced no drug-related side effects.

Patient 6 was a 23-year-old female with RTS, a sclerosing variant (CCDC6-RET fusion) who was diagnosed with symptomatic diffuse lung metastases 6 years ago requiring supplemental oxygen since diagnosis. She progressed on sorafenib and lenvatinib. She started treatment with compound 1 at a dose of 400 mg once a day. In FIG. 13 shows tumor shrinkage after 5 months of compound 1 treatment. She was accustomed to room air for 5 months.

Measurement of ctDNA levels

Plasma levels of one example of an effect marker, ctDNA (eg, KIF5B or TP53 ctDNA), can be assessed using a PlasmaSELECT™-R64 NGS panel (Personal Genome Diagnostics, Baltimore, MD). PlasmaSELECT™ 64 analyzes circulating tumor DNA for genetic alterations in cancer. In particular, PlasmaSELECT™ 64 evaluates a target panel of 64 well-studied cancer genes. Cell-free DNA is extracted from plasma and obtained using proprietary methods that are suitable for low copy number DNA samples. The samples were then processed using a proprietary capture and high coverage next-generation sequencing process.

Steady-state plasma concentration, RET IC ₉₀ and brain IC ₉₀ (predictable)

Blood samples were collected at predetermined time points from patients receiving 30 to 600 mg of compound 1 orally once a day. Plasma samples were analyzed for compound 1 using a validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method. Compound 1 plasma concentration versus time data were plotted using Phoenix WinNonlin© (Version 6.4, Certara LP) or Graphpad Prism (Version 7.02). In FIG. 6A shows a plot of plasma concentration versus time for Compound 1 at steady state. RET IC ₉₀ and IC ₉₀ brain (predicted) are based on predictions and extrapolations based on PK and PD data in animals.

The twice-daily (BID) dosing regimen has also been studied in a phase I clinical trial. The BID dosing regimen was started with a total daily dose of 300 mg (200 mg in the morning, 100 mg in the evening). A total of 9 patients were enrolled in the BID escalation program: 4 patients with a total daily dose of 300 mg (200 mg in the morning, 100 mg in the evening) and 5 patients with a total daily dose of 200 mg (100 mg BID). Of the first 4 patients included in a total daily dose of 300 mg, 2 patients experienced dose-limiting toxicity (DLT) of grade 3 hypertension and the dose was subsequently reduced to 100 mg BID. Two of 5 patients taking 100 mg BID experienced DLT, including 1 patient with grade 3 hypertension and 1 patient with grade 3 tumor lysis syndrome. Based on overall safety, impact, and tolerability, QD was the best dosing regimen and was chosen to increase the dose.

All publications and patents mentioned in this document are incorporated herein by reference in their entirety.

Claims (43)

1. A method of treating a cancer having an activating transfection-rearranged alteration (RET-altered cancer) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount, 300 mg or 400 mg, of Compound 1 or a pharmaceutically acceptable salt thereof once every day, where compound 1 is 2. The method of claim 1 wherein the cancer is a solid tumor. 3. The method of claim 2 wherein the cancer is selected from colorectal cancer, head and neck cancer, hepatobiliary cancer, ovarian cancer, pancreatic cancer, sarcoma, and lung cancer. 4. The method of claim 3 wherein the cancer is selected from small cell lung cancer (SCLC), lung adenocarcinoma, non-small cell lung cancer (NSCLC), bronchioloalveolar cancer and mesothelioma. 5. The method of claim 3 wherein the head and neck cancer is selected from salivary gland cancer and thyroid cancer. 6. The method of claim 5 wherein the thyroid cancer is selected from differentiated thyroid cancer (DTC), medullary thyroid cancer (MTC), and anaplastic thyroid cancer. 7. The method of claim 6 wherein the differentiated thyroid cancer is papillary thyroid cancer (PTC). 8. The method of claim 3 wherein the hepatobiliary cancer is cholangiocarcinoma. 9. The method according to any one of paragraphs. 1-8, where the RET-altered cancer contains a RET fusion. 10. The method of claim 1 wherein the RET-altered cancer is non-small cell lung cancer (NSCLC) having RET fusion. 11. The method of claim 1 wherein the RET-altered cancer is a thyroid cancer having RET fusion. 12. The method of claim 11 wherein the cancer is differentiated thyroid cancer. 13. The compound of claim 11 wherein the cancer is papillary thyroid cancer. 14. The method of claim 11 wherein the cancer is anaplastic thyroid cancer. 15. The method according to any one of paragraphs. 9-14 where the RET fusion is selected from CLIP 1, PIBF1, BCR, FGFRIOP, CEP55, CUX1, MYH13, PTClex9, MPRIP, CCDC6, KIF5B, TRIM33, MBD1, RAB61P2, PRKAR1A, TRIM24, KTN1, HOOK3, TRIM27, AKAP13, FKBP15, ERC1, KIAA1468, KIAA1217, NCOA4, GOLGA5, SPECC1L, TBL1XR1 and ACBD5. 16. The method of claim 9, wherein the RET fusion is selected from CCDC6, KIF5B, TRIM33, TRIM24, and NCOA4. 17. The method of claim 10, wherein the RET fusion is selected from MPRIP, KIF5B, CCDC6, NCOA4, TRIM33, KIAA1468 and KIAA1217. 18. The method according to any one of paragraphs. 11-14 where RET fusion is selected from ERC1, GOLGA5, MYH13, PTClex9, MBD1, RAB61P2, PRKAR1A, TRIM24, KTN1, HOOK3, TRIM27, AKAP13, FKBP15, SPECC1L, TBL1XR1, ACBD5, CCDC6, NCOA4, TRIM33, KIAA1468 and KIAA1217 . 19. The method according to any one of paragraphs. 1-8, where the RET-altered cancer contains the RET mutation. 20. The method of claim 1 wherein the RET altered cancer is a RET mutant thyroid cancer. 21. The method according to claim 19 or 20, where the RET mutation or RET mutant is V804L, V804M, V804E, M918T, C609Y, C609S, C609G, C609R, C609F, C609W, C611R, C611S, C611G, C611Y, C611F, C611W, C618S, C618Y, C618R, C618G, C618F, C618W, C620S, C620W, C620R, C620G, C620L, C620Y, C620F, C630A, C630R, C630S, C630Y, C630F, C634W, C6 34Y, C634S, C634F, C634G, C634L, C634A, C634T, L790F, R844W, R844Q, R844L, A883F, A883S, A883T, K666E, K666M or K666N. 22. The method according to any one of paragraphs. 19-21, where the cancer is medullary thyroid cancer (MTC). 23. The method of claim 22, wherein the RET mutation or RET mutant is sporadic. 24. The method according to any one of paragraphs. 1-23 where the cancer is metastatic. 25. The method according to any one of paragraphs. 1-24, where the subject has previously received treatment with platinum. 26. The method of claim 25 wherein the platinum is selected from cisplatin and carboplatin. 27. The method according to any one of paragraphs. 1-26, where the subject has previously received treatment with cabozantinib or vandetanib. 28. The method according to any one of paragraphs. 1-27, where the subject is administered 300 mg of compound 1 once a day. 29. The method according to any one of paragraphs. 1-27, where the subject is administered 400 mg of compound 1 once a day. 30. The method according to claim 1, where RET-altered cancer is a RET-mutant thyroid cancer; and the subject is administered orally 300 mg or 400 mg of compound 1 once a day. 31. The method according to claim 1, where RET-altered cancer is non-small cell lung cancer (NSCLC) having RET fusion; and the subject is administered orally 300 mg or 400 mg of compound 1 once a day. 32. The method according to claim 1, where RET-altered cancer is a thyroid cancer having RET fusion; and the subject is administered orally 300 mg or 400 mg of compound 1 once a day. 33. The method according to any one of paragraphs. 1-32 wherein the compound is administered as one or more solid dosage forms. 34. The compound of claim 33, wherein the one or more solid dosage forms are tablets. 35. The method of claim 33 wherein the one or more solid dosage forms are capsules. 36. A method of treating cancer having an activating transfection-rearranged alteration (RET-altered cancer) in a subject in need thereof, comprising administering to the subject 300 mg of Compound 1 or a pharmaceutically acceptable salt thereof once daily, wherein Compound 1 is 37. A method of treating a cancer having an activating transfection-rearranged alteration (RET-altered cancer) in a subject in need thereof, comprising administering to the subject 400 mg of Compound 1 or a pharmaceutically acceptable salt thereof once daily, wherein Compound 1 is