Patent Application
20250049795
The present disclosure relates to combination therapies for treating proliferative cell diseases and conditions. In one particular application, the disclosure provides a method which comprises administering to a subject such as a cancer patient, an effective amount of a particular thiazole-pyrimidine anti-proliferative compound(s) as a single agent or in combination with an immune checkpoint inhibitor(s) such as an anti-PD-1 antibody.
The present application claims priority from Australian Provisional Patent Application No. 2022900660 titled “USE OF 5-(2-((5-(4-(DIMETHYLAMINO)PIPERIDIN-1-YL)PYRIDIN-2-YL)AMINO)-5-FLUOROPYRIMIDIN-4-YL)-N,4-DIMETHYLTHIAZOL-2-AMINE IN COMBINATION THERAPIES FOR CANCER” and filed on 17 Mar. 2022, the content of which is hereby incorporated by reference in its entirety.
Methods of cancer immunotherapy that enhance a patient's immune system to target tumours have recently been developed (see, for example, Rizvi N A et al., Science 348:124-8, 2015; and Pardoll D M., Nat Rev Cancer 12:252-64, 2012). One class of such methods has arisen through the discovery of immune checkpoint receptors, such as PD-1 (programmed cell death protein 1; CD279) and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), that repress the activity of anti-tumour (cytotoxic) T cells. In particular, this discovery has led to the development of blocking antibodies directed against certain examples of these receptors (or their ligands), including for example, pembrolizumab (a humanised antibody directed against PD-1), nivolumab (another anti-PD-1 antibody), atezolizumab (a monoclonal antibody directed against the PD-1 ligand, PD-L1, so as to inhibit interaction between PD-1 and PD-L1), durvalumab (another anti-PD-L1 antibody) and ipilimumab (a monoclonal antibody directed against CTLA-4). Some patients treated with checkpoint inhibitors such as these, experience a durable tumour regression (Sharma P et al., Cell 161:205-14, 2015). However, despite promising clinical results, therapies using checkpoint inhibitor(s) (ie termed “checkpoint inhibitor therapy” or “checkpoint blockade therapy”) have, to-date, only proven to be successful in subset(s) of patients (Havel J J et al., Nat Rev Cancer 19(3):133-150, 2019). Moreover, increasingly, evidence is emerging which shows that tumours can acquire resistance against immunotherapies such as checkpoint inhibitor therapy (Koyama S, et al. Nat Commun 7:10501, 2016). Thus, it is desirable to develop new treatment strategies that may broaden the range of tumours and/or patients that respond to immunotherapy, or which may otherwise enhance the specificity and/or efficacy of existing immunotherapies such as those mentioned above.
One potential new strategy is to identify and develop small molecule kinase inhibitors which might enhance or complement an immunotherapy. Small molecule inhibitors have many advantages in comparison with antibody-based biologics including, for example, greater exposure in the tumour microenvironment and access to intracellular targets (Petroni G et al., Nat Review Immunol 20:669-679, 2020). Moreover, there is some evidence to indicate that signalling from oncogenic kinase drivers can alter the tumour microenvironment to promote immunosuppression (Ahn R and J Ursini-Siegel, Int J Mol Sci 22(5):2608-2631, 2021), and the inhibition of these oncogenes using small molecule kinase inhibitors can potentially contribute to immune reactivation. In addition, small molecule kinase inhibitors can directly alter immune cell function and contribute to antitumour immunity (Ahn and Ursini-Siegel, 2021 supra). As such, combining small molecule kinase inhibitors, which can induce dramatic but short-lived tumour regression, with an immunotherapy such as checkpoint inhibitor therapy, which provide slower but potentially more durable responses, is an attractive potential new treatment strategy.
The present Applicant has identified and developed a range of novel small molecule kinase inhibitors, particularly directed to cyclin-dependent kinases (CDKs). CDKs are known to be associated with various cyclin subunits, playing pivotal roles in the regulation of a variety of important regulatory pathways in cells, including cell-cycle control, apoptosis, neuronal physiology, differentiation and transcription. As such, aberrant CDK expression and/or activity can cause or contribute to, inter alia, aberrant cell-cycle control, resulting in unlimited cell cycle re-entry and progression, which is a hallmark of human cancers. To date, at least 20 CDKs and 30 cyclins have been identified. They may be classified into two major groups, reflecting their functions, the cell cycle regulator CDKs and the transcription regulator CDKs (Wang S et al., Trends Pharmacol Sci 29(6):302-313, 2008; Diab S et al., J Med Chem 63(14):7458-7474, 2020). The class of the cell cycle regulator CDKs includes CDK 1, 2, 3, 4, 5, 6 and 7 and these function with their cyclin partners (eg cyclins A, B, C, D1, D2, D3, E and F) to regulate promotion of the cell cycle. The class of the transcription regulator CDKs includes CDK 7, 8, 9 and 11, which work together with cyclins C, H, K, L1, L2, T1 and T2. Given the functions of the CDK classes, it is not surprising that CDKs have been implicated in cell proliferation diseases and conditions, particularly cancer. Cell proliferation is a result of the direct or indirect deregulation of the cell division cycle and the CDKs play a critical role in the regulation of the various phases of this cycle. Therefore, CDK inhibitors are useful targets for cancer therapy, and CDK4/6 inhibitors such as palbociclib, ribociclib and abemaciclib have received US Food and Drug Administration (FDA) approval for the treatment of patients with advanced or metastatic breast cancers as a single agent or in combination. Although best known for their function in the blockade of cell-cycle progression, accumulating evidence indicates that the anti-cancer effects of CDK4/6 inhibitors such as these also result from a range of immunostimulatory effects as they interact with malignant cells as well as with immune cell populations of the tumour microenvironment (Petroni G et al., 2020 supra).
The present Applicant has identified a CDK4 specific inhibitor, namely 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (Bantie L et al., Gynecologic Oncology 159 (3):827-838, 2020), that shows significant potential as an immunotherapeutic agent, or to enhance the specificity and/or efficacy of various agents for cancer immunotherapy such as those used in checkpoint inhibitor therapy (eg an anti-PD-1 blocking antibody), and might therefore form the basis of useful novel combination therapies.
In a first aspect, the present disclosure provides a method of treating a proliferative disease or condition in a subject, comprising co-administering 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof) with an immunotherapeutic agent to the subject.
The immunotherapeutic agent may be selected from, for example, agents that are known to those skilled in the art as being capable of promoting an immunoreaction against cancer cells, such as an immune checkpoint inhibitor.
In a second aspect, the present disclosure provides a pharmaceutical composition for treating a proliferative disease or condition in a subject, comprising 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof) and an immunotherapeutic agent, optionally in combination with a pharmaceutically acceptable carrier, diluent and/or excipient.
In a third aspect, the present disclosure provides the use of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof) and an immunotherapeutic agent for treating a proliferative disease or condition in a subject.
In a fourth aspect, the present disclosure provides the use of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof) and an immunotherapeutic agent in the manufacture of a medicament for treating a proliferative disease or condition in a subject.
In a fifth aspect, the present disclosure provides a kit comprising first and second containers (eg vials), wherein the first container contains 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof), and the second container contains an immunotherapeutic agent; optionally packaged with instructions for the use of the kit in the method of the first aspect.
The present Applicant has identified a CDK4 specific inhibitor, namely 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine, suitable for enhancing the activity and/or efficacy of a cancer immunotherapy (eg a therapy involving the administration of an immunotherapeutic agent that promotes an immunoreaction against cancer cells such as those used in checkpoint inhibitor therapy (eg an anti-PD-1 antibody)). As shown in Example 2 hereinafter, it was found that this CDK4 specific inhibitor compound is capable of an anti-proliferative effect through CDK4/6 inhibition and associated immunotherapeutic mode(s) of action (eg the modulation of regulatory T cells, and/or PD-1/PD-1 ligands).
In a first aspect, the present disclosure provides a method of treating a proliferative disease or condition in a subject, comprising co-administering 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof) with an immunotherapeutic agent to the subject.
5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine has been found to possess anti-proliferative activity (eg anti-cancer effects); it is considered that this inhibitor compound blocks tumour cell proliferation by inhibiting the activity of CDK4. In particular, 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine has been found to, for example, inhibit cell growth of cancer cell lines both in vitro and in vivo and is considered to be useful on its own in the treatment of proliferative cell diseases and conditions.
Further, 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine has been found to possess anti-proliferative activity (eg anti-cancer effects) through previously unrecognised immunotherapeutic effects. In particular, this inhibitor compound may, for example, inhibit of cancer cell growth in vivo through promoting an immunoreaction against the cancer cells.
As such, it is considered that 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine has excellent potential to additionally act as an immunotherapeutic agent, especially when used in combination with another immunotherapeutic agent such as those used in checkpoint inhibitor therapy (eg an anti-PD-1 blocking antibody), for the treatment of proliferative cell diseases and conditions.
In particular, as shown in the Example 2 hereinafter, when mice models bearing syngeneic allografts of cancer cell lines were co-administered with 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and another immunotherapeutic agent (in particular, an immune checkpoint inhibitor), enhanced anti-proliferative effects and increased survival rates were achieved (ie relative to the use of the inhibitor compound or other immunotherapeutic agent alone).
In preferred embodiments, the enhanced anti-proliferative effects represents synergism between 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and the other immunotherapeutic agent, as may be assessed by, for example, determining combination index (CI) values according to any of the methodologies well known to those skilled in the art, including the Chou-Talaly method (Chou T C et al., Trends Pharmacol Sci 4:450-454, 1983), wherein CI values of <1 indicate a synergistic interaction between the inhibitor compound and the other immunotherapeutic agent. In some embodiments, the CI values are determined on the basis of the reduced level of tumour volume achieved by the combination treatment in an in vivo mouse model such as has been described in the Example 2 hereinafter.
In this specification, a number of terms and phrases are used which are well known to those skilled in the art. Nevertheless, for the purposes of clarity, a number of these terms and phrases are hereinafter defined.
As used herein, an anti-proliferative activity or effect within the scope of the present disclosure may be demonstrated by the ability to inhibit cell proliferation in an in vitro whole cell assay and/or the ability to reduce tumour volume in vivo. An example(s) of suitable assays for such activity, including methods for performance, are described in the Examples hereinafter.
The term “treating”, as used herein, includes prophylaxis as well as the alleviation of established symptoms of a disease or condition. As such, the act of “treating” a disease or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the disease or condition developing in a subject afflicted with or predisposed to the disease or condition; (2) inhibiting the disease or condition (ie arresting, reducing or delaying the development of the disease or condition or a relapse thereof (in case of a maintenance treatment)) or at least one clinical or subclinical symptom thereof; and (3) relieving or attenuating the disease or condition (ie causing regression of the disease or condition or at least one of its clinical or subclinical symptoms).
As used herein, the phrase “manufacture of a medicament” includes the use of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and/or the immunotherapeutic agent directly as the medicament or in any stage of the manufacture of a medicament comprising the inhibitor compound and/or the immunotherapeutic agent.
The term “pharmaceutically acceptable salt” as used herein, refers to salts that retain the desired biological activity of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine, and include pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic and arylsulfonic. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co, Easton PA 1995.
The term “solvate” refers to any form of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine, resulting from solvation with an appropriate solvent. Such a form may be, for example, a crystalline solvate or a complex that may be formed between the solvent and the dissolved compound.
The term “prodrug” means a compound that undergoes conversion to 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine within a biological system, usually by metabolic means (eg by hydrolysis, reduction or oxidation). For example, an ester prodrug of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine containing a hydroxyl group may be convertible by hydrolysis in vivo to the inhibitor compound. Suitable esters of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine containing a hydroxyl group may be, for example, acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-P-hydroxynaphthoates, gestisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and quinates. As another example, an ester prodrug of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine containing a carboxy group may be convertible by hydrolysis in vivo to the inhibitor compound. Examples of ester prodrugs include those described by Leinweber F J, Drug Metab Rev 18:379-439 (1987). Similarly, an acyl prodrug of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine containing an amino group may be convertible by hydrolysis in vivo to the inhibitor compound. Examples of prodrugs for these and other functional groups, including amines, are provided in Prodrugs: challenges and rewards, Valentino J Stella (ed), Springer, 2007.
It will be understood by those skilled in the art that 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or pharmaceutically acceptable salts, solvates or prodrugs thereof) may exist in different crystalline or polymorphic forms, all of which are encompassed within the scope of the present disclosure.
The term “therapeutically effective amount” or “effective amount” is an amount sufficient to effect beneficial or desired clinical results. A therapeutically effective amount can be administered in one or more administrations. Typically, a therapeutically effective amount is sufficient for treating a disease or condition or otherwise to palliate, ameliorate, stabilise, reverse, slow or delay the progression of a disease or condition such as, for example, cancer or another proliferative cell disease or condition. By way of example only, a therapeutically effective amount of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine, or a pharmaceutically acceptable salt, solvate or prodrug thereof, may comprise between about 0.1 and about 250 mg/kg body weight per day, more preferably between about 0.1 and about 100 mg/kg body weight per day and, still more preferably between about 0.1 and about 25 mg/kg body weight per day. For the other immunotherapeutic agent, a therapeutically effective amount may be expected to vary considerably depending upon the particular immunotherapeutic agent, but may similarly comprise between about 0.1 and about 250 mg/kg body weight every 1-3 weeks, more preferably between about 0.1 and about 100 mg/kg body weight every 1-3 weeks and, still more preferably between about 0.1 and about 25 mg/kg body weight every 1-3 weeks. However, notwithstanding the above, it will be understood by those skilled in the art that the therapeutically effective amount of the compound/agent may also vary and depend upon a variety of factors including the activity of the compound/agent, the metabolic stability and length of action of the particular compound/agent, the age, body weight, sex, health, route and time of administration, rate of excretion of the particular compound/agent, and the severity of, for example, the cancer or other proliferative cell disease or condition to be treated.
In some preferred embodiments, the 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine exhibits anti-proliferative activity in human cell lines, as measured by a standard cytotoxicity assay. Preferably, the inhibitor compound exhibits an IC50 value of less than 5 M, even more preferably less than 1 M as measured by a standard cell viability assay. More preferably still, the inhibitor compound exhibits an IC50 value of less than 0.5 M.
In some preferred embodiments, the 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine inhibits CDK4, as measured by any standard assay well known to those skilled in the art. Preferably, the inhibitor compound exhibits an IC50 value of less than 1 μM or less than 0.5 M as measured by any standard kinase assay well known to those skilled in the art, more preferably still less than 0.1 μM.
The other immunotherapeutic agent may be selected from, for example, agents that are known to those skilled in the art as being capable of promoting an immunoreaction against cancer cells. For example, the other immunotherapeutic agent may be selected from immune checkpoint inhibitors such as any agent (which may be, for example, a protein, peptide, antibody or antibody fragment or a combination thereof) which is capable of inhibiting interactions between one or more immune checkpoint receptor and their ligand(s) (eg by blocking). As will be appreciated by those skilled in the art, among the immune checkpoint inhibitors suitable for use in the method of the first aspect are agents which are capable of inhibiting PD-1 (eg an anti-PD-1 blocking antibody such as pembrolizumab, lambrolizumab, cemiplimab, spartalizumab and nivolumab; or an anti-PD-L1 antibody such as atezolizumab, avelumab and durvalumab, or an anti-PD-L2 antibody), PD-L1, PD-L2, CTLA-4 (eg an anti-CTLA-4 antibody such as ipilimumab), B and T lymphocyte attenuator (BTLA; CD272) (eg an anti-BTLA antibody), the T cell immunoglobulin and mucin domain-3 (Tim-3; CD366) (eg an anti-Tim-3 antibody) or so-called intracellular checkpoints such as E3 ubiquitin-protein ligase (CBL-B) and CISH (cytokine-inducible SH2-containing protein).
It is to be appreciated that in the method of the first aspect, the method may comprise co-administering 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof) and one or more other immunotherapeutic agent.
In some embodiments, the method of the first aspect may further comprise administering one or more additional compound(s) with anti-proliferative activity, such as a compound(s) of one or more of the following categories:
The method of the first aspect will typically be applied to the treatment of cancer or another proliferative cell disease or condition in a human subject. However, the subject may also be selected from, for example, livestock animals (eg cows, horses, pigs, sheep and goats), companion animals (eg dogs and cats) and exotic animals (eg non-human primates, tigers, elephants etc).
Cancers and other proliferative cell diseases and conditions that may be treated in accordance with the method of the first aspect include biliary tract cancer, brain cancer and other cancers of the central nervous system (CNS) (including glioblastomas and medulloblastomas), neuroblastomas, breast cancer, cervical cancer, ovarian cancer (including those arising from epithelial cells, stromal cells, germ cells, and mesenchymal cells), choriocarcinoma, colorectal cancer, endometrial cancer, liver cancer, lung cancer, oesophageal cancer, gastric cancer, haematological neoplasms (including acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), and acute myeloid leukaemia (AML), multiple myeloma, AIDS-associated leukemias and adult T-cell leukemia lymphoma, lymphomas (including Non-Hodgkin's lymphoma, Hodgkin's disease and lymphocytic lymphomas)), intraepithelial neoplasms (including Bowen's disease and Paget's disease), oral cancer (including squamous cell carcinoma), pancreatic cancer, prostate cancer, sarcomas (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma), skin cancer (including melanoma, Kaposi's sarcoma, basocellular cancer, and squamous cell cancer), testicular cancer (including germinal tumours such as seminoma, non-seminoma teratomas, and choriocarcinomas), stromal tumours, germ cell tumours, thyroid cancer (including thyroid adenocarcinoma and medullar carcinoma), and renal cancer (including adenocarcinoma and Wilms' tumour).
In some embodiments, the cancers and other proliferative cell diseases and conditions treated in accordance with the method of the first aspect are characterised by over-expression of CDK4 and/or cyclin D including, for example, lung cancer (Wu et al., J Transl Med 9:38 (2011)), breast cancer (An et al., Am J Pathol 154(1):113-118 (1999)), brain cancers and other cancers of the CNS (eg glioblastomas) and colorectal cancer (Ikeda et al., Jap J Clin Med 54(4):1054-1059 (1996)). CDK4 and/or cyclin D over-expression may be determined by, for example, assessing the amount of mRNA encoding CDK4 and/or cyclin D in a suitable sample using any of the techniques well known to those skilled in the art (eg quantitative amplification techniques such as qPCR).
As will be appreciated by those skilled in the art, the step of co-administering the 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and the other immunotherapeutic agent may involve administering the inhibitor compound and agent simultaneously to the subject or otherwise sequentially in any order (eg within seconds or minutes (eg 10, 60 or 90 minutes) or even hours (eg within 2 to 48 hours)). As such, the 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and the other immunotherapeutic agent can be administered in, for example, the same pharmaceutical composition (ie for simultaneous administration) or in separate pharmaceutical compositions.
In a second aspect, the present disclosure provides a pharmaceutical composition for treating a proliferative disease or condition in a subject, comprising 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof) and an immunotherapeutic agent, optionally in combination with a pharmaceutically acceptable carrier, diluent and/or excipient.
The 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and the other immunotherapeutic agent may be formulated into a pharmaceutical composition in therapeutically effective amounts (which may be lesser amounts than might be used for treatments where the compounds are used alone) with a pharmaceutically acceptable carrier, diluent and/or excipient. Examples of suitable carriers and diluents are well known to those skilled in the art, and are described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 1995. Examples of suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and P J Weller. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of carrier, diluent and/or excipient may be made with regard to the intended route of administration and standard pharmaceutical practice.
A pharmaceutical composition comprising 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and the other immunotherapeutic agent may further comprise any suitable binders, lubricants, suspending agents, coating agents and solubilising agents. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilising agents, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Anti-oxidants and suspending agents may be also used.
A pharmaceutical composition comprising 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and an immunotherapeutic agent may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration. For oral administration, particular use may be made of compressed tablets, pills, tablets, gellules, drops, and capsules. For other forms of administration, a pharmaceutical composition may comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. A pharmaceutical composition comprising 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and an immunotherapeutic agent may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders. A pharmaceutical composition may be formulated in unit dosage form (ie in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose).
The 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine and/or the immunotherapeutic agent may be provided as a pharmaceutically acceptable salt including, for example, suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al., J Pharm Sci 66:1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids (eg sulfuric acid, phosphoric acid or hydrohalic acids), with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (eg by halogen), such as acetic acid, with saturated or unsaturated dicarboxylic acids (eg oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic acid), with hydroxycarboxylic acids (eg ascorbic, glycolic, lactic, malic, tartaric or citric acid), with amino acids (eg aspartic or glutamic acid), with benzoic acid, or with organic sulfonic acids (eg (C1-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted by, for example, halogen) such as methane- or p-toluene sulfonic acid). In addition, the inhibitor compound and/or the other immunotherapeutic agent may be provided in their various crystalline forms, polymorphic forms and (an)hydrous forms. In this regard, it will be well known to those skilled in the art that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation from the solvents used in the synthetic preparation of such compounds.
In a third aspect, the present disclosure provides the use of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof) and an immunotherapeutic agent for treating a proliferative disease or condition in a subject.
In a fourth aspect, the present disclosure provides the use of 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof) and an immunotherapeutic agent in the manufacture of a medicament for treating a proliferative disease or condition in a subject.
In a fifth aspect, the present disclosure provides a kit comprising first and second containers (eg vials), wherein the first container contains 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (or a pharmaceutically acceptable salt, solvate or prodrug thereof), and the second container contains an immunotherapeutic agent; optionally packaged with instructions for the use of the kit in the method of the first aspect.
Methods for synthesising compounds such as 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine have been previously described (see, for example, WO 2017/020065). In some embodiments, 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine may be synthesised by adapting the following general synthetic scheme:
wherein the general reaction conditions are: (a) DMF-DMA or Bredereck's reagent, reflux; (b) Select Fluor, MeOH; (c) Et3N, HgCl2, DCM; (d) TFA/DCM (1:1), reflux; (e) A, B, NaOH, 2-methoxyethanol, microwave and (f) Pd2dba3, xantphose, t-BuONa, dioxane, microwave.
With regard to the description of the synthetic method of scheme 1 above, it will be understood by those skilled in the art that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be readily selected. Moreover, it will be understood by those skilled in the art that the functionality present at various portions of the molecule must be compatible with the reagents and reaction conditions utilised.
Necessary starting materials may be obtained by standard procedures of organic chemistry. The preparation of such starting materials is described in conjunction with the following representative process variants and within the examples hereinafter. Alternatively, necessary starting materials may be obtainable by analogous procedures to those illustrated which are within the ordinary skill of those skilled in the art. Further, it will be appreciated that during the synthesis of the compounds, or during the synthesis of certain starting materials, it may be desirable to protect certain substituent groups to prevent their undesired reaction. Those skilled in the art will readily recognise when such protection is required, and how such protecting groups may be put in place, and later removed. Examples of protecting groups are described in, for example, Protective Groups in Organic Synthesis by Theodora Green (publisher: John Wiley & Sons). Protecting groups may be removed by any convenient method well known to those skilled in the art as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with the minimum disturbance of groups elsewhere in the molecule. Thus, if reactants include, for example, groups such as amino, carboxyl or hydroxyl, it may be desirable to protect the group in some of the reactions mentioned herein.
In addition, those skilled in the art will be able to select appropriate reaction conditions to use in the coupling reaction of the compound of formula A or formula B shown in scheme 1. However, typically, the reaction will be carried out in anhydrous conditions and in the presence of an inert atmosphere, such as argon or nitrogen. The reaction may also be carried out an elevated temperature, such as, for example, within the range of 80 to 180° C. for a suitable time period of, for example, 20 minutes to 48 hours. Suitably, the reaction is carried out under microwave heating, for example, at 80 to 180° C. for 20 minutes to 1.5 hour.
The resultant compound can be isolated and purified using techniques well known to those skilled in the art.
The methods and uses of the disclosure are hereinafter further described with reference to the following, non-limiting examples and accompanying figures.
1H and 13C NMR spectra were recorded at 300 K on a Bruker AVANCE III 500 spectrometer (1H at 500 MHz). 1H NMR spectra were referenced to 1H signals of residual non-deuterated solvents (or tetramethylsilane). High resolution mass spectra were recorded on an AB SCIEX TripleTOF® 5600 mass spectrometer, and ionisation of all samples was carried out using ESI. The purity of compounds was determined by analytical HPLC, and was greater than 95%. Analytic HPLC was carried out on a Shimadzu Prominence UFLC (UltraFast Liquid Chromatograph) system with a CBM-20A communications bus module, a DGU-20A5R degassing unit, an LC-20AD liquid chromatograph pump, an SIL-20AHT autosampler, an SPD-M20A photo diode array detector, a CTO-20A column oven and a Phenomenex Kinetex 5u C18 100A 250 mm×4.60 mm column using Method A (gradient 5 to 95% MeOH containing 0.1% FA over 7 min, followed by 95% MeOH containing 0.1% FA over 13 min at a flow rate of 1 mL/min), Method B (gradient 5 to 95% MeCN containing 0.1% FA over 7 min followed by 95% MeCN containing 0.1% FA over 13 min, at a flow rate of 1 mL/min).
5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (1): To a solution of crude 1-(5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)guanidine trifluoroacetate (524 mg, 2.00 mmol) in 2-methoxyethanol (3 mL) were added ((E)-3-(dimethylamino)-2-fluoro-1-(4-methyl-2-(methylamino)thiazol-5-yl)prop-2-en-1-one (243 mg, 1.00 mmol) and NaOH (80.0 mg, 2.00 mmol). The reaction mixture was heated at 180° C. for 1h under microwave irradiation, cooled down to room temperature, and then concentrated under reduced pressure. The residue was purified by chromatography (silica gel, DCM ramping to DCM:MeOH=90:10 with constant addition of 0.5 ml of 32% ammonia) to give 1 as a brown solid (76 mg, 17.2%). 1H NMR (DMSO-d6) δ 1.50 (q, 2H, J 11.0), 1.84 (d, 3H, J 11.0), 2.21 (s, 7H), 2.47 (s, 3H, thiazole-CH3), 2.64 (t, 2H, J 11.0), 2.86 (t, 3H, J 3.5), 3.63 (d, 1H, J 11.0), 7.39 (app d, 1H, J 7.0), 7.92 (d, 1H, J 9.0), 7.98 (s, 1H), 8.10 (1H, J 4.0), 8.41 (s, 1H), 9.43 (s, 1H). HRMS (ESI): m/z 443.2136 [M+H]+.
Eurofins Pharma Discovery or Reaction Biology Corporation Kinase Profiler services were used to measure inhibition of CDKs and other kinases by radiometric assay (RIA). Inhibition of CDK4/cyclin D1, CDK6/cyclin D3 and CDK9/cyclin T1 were also determined using ADP Glo Kinase assays (Promega Corporation, Madison WI, United States of America). Briefly, the kinase reaction for CDK4/cyclin D1 and CDK6/cyclin D3 was performed with kinase reaction buffer (40 nM Tris base pH 7.5, 20 mM MgCl2, 0.4 mM DTT), 0.1 mg/ml BSA and RB-CTF substrate (retinoblastoma protein1 C-terminal fraction). For CDK9/cyclin T1, the kinase reaction was performed with standard assay buffer and Kinase Dilution Buffer and RBER-IRStide substrate. Serial dilutions of 1:3 were prepared for test compounds for 10 concentrations (from 10 μM to 0.5 nM). The kinase reactions were started by addition of ATP, incubated for 40 min at 370 C and then stopped by adding 10 μL of ADP Glo reagent. After incubation at room temperature (RT) in the dark for 40 min, 20 μL of kinase detection reagent was added per well and incubated for 40 min. Luminescence was measured using an EnVision Multilabel plate reader (PerkinElmer, Buckinghamshire, United Kingdom). Positive and negative controls were performed in the presence and absence of CDK kinases, respectively. Half-maximal inhibition (IC50) values were calculated using a 4-parameter logistic non-linear regression model with Graphpad prism (Version 6.0). Apparent inhibition constants (Ki) values were calculated from Km (ATP) and IC50 values for the respective kinases. The results are shown in Table 1. 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (compound 1) was found to be a highly specific CDK4 inhibitor with a 140-fold selectivity for CDK4 over CDK6 and no activity against other members of CDK family.
Murine triple-negative breast cancer EMT6 tumour cells (ATCC® CRL-2755™) and colorectal cancer CT26.WT cells (ATCC® CRL-2638™) were cultured and expanded in RPMI-1640 medium (Roswell Park Memorial Institute Medium-1640) with L-glutamine and sodium bicarbonate (Sigma-Aldrich, Macquarie Park, NSW, Australia) supplemented with 10% heat-inactivated foetal bovine serum (FBS). Cells were cultured in an incubator at 5% CO2 37° C.
In Vivo Anti-Tumour Activity in Mice with a Colorectal Cancer Syngeneic Allograft
CT26.WT colorectal cancer cells were harvested and re-suspended in a 1:1 mixture of Matrigel™ (#354234; Corning Life Sciences, Corning, NY, United States of America) and serum-free RPMI-1640 medium, and thereafter injected sub-cutaneously (sc) (7×105 cells in a volume of 100 μL) into the right flank of each female BALB/c mouse. After the tumour volume reached ˜100 mm3, the mice were randomised into two groups (n=8/group) and given vehicle (1% CMC in water), compound 1 (150 mg/kg) once daily by oral administration (QD PO), Anti-PD1 (RMP1-14-derived mouse monoclonal antibody, Assay Matrix, Australia; 150 g/mouse/wk) by intraperitoneal (ip) administration, or compound 1 plus anti-PD-1 antibody (
The contribution of immunity toward the improved anti-cancer effects by compound 1 was demonstrated by the increased level of various immune markers such as CD3+ T-cells and CDK8+ T-cells as shown in
The treatment with compound 1 was also found to significantly prolong the survival of the mice as a single agent (p<0.001) and when administered in combination with the anti-PD-1 antibody (p<0.001,
5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (compound 1) was shown to inhibit the CDK4 kinase (known to be involved in the regulation of the cell cycle) and possess anti-tumour efficacy against mice with an intact immune system such as Balb/C mice bearing CT26.WT colorectal cancer syngeneic allografts. Further, this inhibitor compound was found to be capable of increasing the level of various immune markers, indicating that it has excellent potential to act as an immunotherapeutic agent, especially when used in combination with another immunotherapeutic agent such as those used in checkpoint inhibitor therapy, for the treatment of proliferative cell diseases and conditions.
Throughout the specification and the claims that follow, unless the context requires otherwise, the words “comprise” and “include” and variations such as “comprising” and “including” will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.
It will be appreciated by those skilled in the art that the present disclosure is not restricted in its use to the particular application described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be also appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the disclosure as set forth and defined by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022900660 | Mar 2022 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2023/050185 | 3/16/2023 | WO |
