Methods for sequentially administering tas-102 and bcl-2 inhibitor for cancer treatment

Abstract

Disclosed is a method of treating or alleviating a symptom of a cancer in a subject, comprising sequentially administering to the subject TAS-102 followed by a BCL-2 inhibitor.

Description

METHODS FOR SEQUENTIALLY ADMINISTERING TAS-102 AND BCL-2 INHIBITOR FOR CANCER TREATMENTCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional application no. 63 / 501,310, filed on May 10, 2023, the disclosure of which is incorporated herein by reference.FIELD

[0002] This disclosure generally relates to cancer therapeutics, and use of combinations of and a BCL-2 inhibitor.BACKGROUND OF THE DISCLOSURE

[0003] Genetic alterations in the tumor suppressor p53 gene (TP53) are found in over 50% of human solid tumors, including breast cancer (BC), ovarian cancer (OC), pancreatic cancer (PDAC), colorectal (CRC), and esophagogastric (EGC) cancers. The p53 protein encoded by TP53 controls genome stability during cell cycle and in response to DNA damage. Genetic alterations (mutations) in p53 contribute to cancer progression, metastasis, and resistance to therapy. However, existing treatment options for p53 mutant cancers are rather ineffective. Therefore, improved methods are needed.SUMMARY OF THE DISCLOSURE

[0004] The present disclosure provides a method of treating or alleviating a symptom of a cancer in a subject by sequentially administering to the subject TAS-102 followed by a BCL- 2 inhibitor. By sequentially administering a thymidine analogue with anti-BCL-2 agents, a greater toxicity against p53-deficient cancers can be achieved and treatment of aggressive cancers resistant to current treatment options can be improved.

[0005] According to an example of the present invention, the BCL-2 inhibitor may be Navitoclax or Venetoclax.

[0006] According to an example of the present invention, the BCL-2 inhibitor may be administered to the subject at least one day the after the TAS-102 is administered to the subject.

[0007] According to an example of the present invention, BCL-2 inhibitor may be administered to the subject one, two, three, or four days after the TAS-102 is administered to the subject. In one example, the TAS-102 and the BLCL-2 inhibitor are both administered to the individual within a period of not more than five day. In certain embodiments,administration of the TAS- 102 may be repeated and the administrations may be separated by at least 1 day or 2 days before the BLCL-2 inhibitor is administered.

[0008] According to an example of the present invention, the cancer may be colorectal cancer, colon cancer, cervical cancer, or pancreatic cancer.

[0009] According to an example of the present invention, the cancer may be a solid tumor.

[0010] According to an example of the present invention, treatment may be applied to solid tumors carrying mutant, deleted, or otherwise dysfunctional p53.BRIEF DESCRIPTION OF THE FIGURES

[0011] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0012] Fig. 1 provides a schematic of the therapeutic strategy of sequentially administering TAS- 102 and a BCL-2 inhibitor.

[0013] Fig. 2 provides another schematic of the therapeutic strategy using TAS-102 as an inducer and PARPi as the amplifier.

[0014] Fig. 3 displays potential interactor molecules and treatment options for three thymidine analogues.[0015| Fig. 4 displays the expressions of BCL-2 like genes in breast, colon, and PDAC cancers.

[0016] Fig. 5 displays results (A-D) from a cytotoxicity assay performed in Example 2.

[0017] Fig. 6 displays results from a cytotoxicity assay of Navitoclaz in CRC and PDAC lines.

[0018] Fig. 7 displays results from a cytotoxicity assay of Navitoclax in CRC lines.

[0019] Fig. 8 displays an isobologram analysis to demonstrate the dose-relationship forTAS-102 and Navitoclax for HCT116-p53WT and HCT116-p53KO.

[0020] Fig. 9 displays results from a cytotoxicity assay of ABT-199 (Venetoclax) in CRC and PDAC lines.

[0021] Fig. 10 displays a comparison of cytotoxicity assays of TAS-102 with Navitoclax and TAS-102 with ABT-199 (Venetoclax) in CRC lines.

[0022] Fig. 11 provides graphs summarizing data obtained as follows. HCT116 colon cancer cells were implanted subcutaneously into SCID female mice (2x10A6 cells / injection). At about 100mm3, mice were randomized into groups and treatment is initiated: (1) Vehicle,(2) BCL-2 inhibitor (BCL2i) (illustrated using 50mg / kg Navitoclax), (3) 50mg / kg TAS 102, (4) TAS102+BCL2i (50mg / kg each). On day 15 Navitoclax / BCL2i treatment was stopped, TAS 102 was continued. (A) Tumor volume in mm3 (n=8-9mice / group). (B) Tumor Growth Inhibition was calculated relative to the Vehicle group; TGI (%) = 100*(Vv-Vi) / Vv.DETAILED DESCRIPTION OF THE DISCLOSURE

[0023] Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0024] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0025] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. The steps of the method described in the various examples and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an example, the method consists essentially of a combination of the steps of the methods disclosed herein. In another example, the method consists of such steps.

[0026] As used in the specification and the appended claims, the singular forms “a” "and” and “the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another example includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” or “approximately” it will be understood that the particular value forms another example. The term “about” and “approximately” in relation to a numerical value encompass variations of + / -10%, +7- 5%, or + / - 1%.

[0027] Any result obtained by using a described combination as described herein can be compared to a suitable control. In examples, the control is a result obtained using only one of TAS- 102 or a BCL-2 inhibitor as a monotherapy. In examples, the control comprises use of a BCL-2 inhibitor as an initial agent that is administered to the individual. In examples, sequential administration of TAS- 102 followed by a BCL-2 inhibitor provides an improved result relative to a control. In an example, the improved result comprises a reduction intumor volume using the described combination approach, relative to the control, or an inhibition of tumor growth, relative to the control.

[0028] The present disclosure accordingly provides a method of treating or alleviating symptoms of cancer in a subject by sequentially administering to the subject TAS-102 followed by a BCL-2 inhibitor. In examples a therapeutically effective amount of a described combination of agents is administered. In examples a combination of TAS-102 followed by a BCL-2 inhibitor may exhibit a synergistic effect. The term “therapeutically effective amount” as used herein refers to an amount of an agent sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. The amount desired or required will vary depending on the particular compound or composition used, its mode of administration, patient specifics and the like. Appropriate effective amounts can be determined by one of ordinary skill in the art informed by the instant disclosure using routine experimentation. For example, a therapeutically effective amount, e.g., a dose, can be estimated initially either in cell culture assays or in animal models. An animal model can also be used to determine a suitable concentration range, and route of administration. Such information can then be used to determine useful doses and routes for administration in humans, or to non-human animals. A precise dosage can be selected by in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of components to achieve a desired effect, such as a modification in a threshold number of cells. Additional factors which may be taken into account include the particular gene or other genetic element involved, the type of condition, the age, weight and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance / response to therapy. In certain examples, a therapeutically effective amount is an amount that reduces one or more signs or symptoms of a disease, and / or reduces the severity of the disease. A therapeutically effective amount may also inhibit or prevent the onset of a disease, or a disease relapse.

[0029] In examples a described combination agents is sequentially administered to an individual who has any type of cancer. In an example, the individual has a cancer comprising p53-mutant cancer cells. Thus, in examples, p53 wild type cells may be unaffected.

[0030] TAS-102 is composed of Triflurthymidine (TFT) and tipiracil, a thymidine phosphorylase inhibitor that enhances bioavailability of TFT. TAS-102 is approved for treatment in patients with refractory CRC, although its antitumor effect is marginal. Furthermore, retrospective clinical data indicates that the clinical benefit of TAS-102 in refractory metastatic p53-mutant CRC is limited, if present at all. The structures of thecomponents of TAS-102 are known in the art. In an example, a TAS-102 formulation is the formulation sold under the brand name LON SURF.

[0031] As shown in the examples, BCL-2-like proteins were identified as interacting partners for thymidine analogues. As shown in Fig. 1, thymidine analogue Trifhirthymidine / TFT (a component of TAS-102; tipiracil is a thymidine phosphorylase inhibitor) activates Base-Excision repair (BER) when incorporated into DNA. BER excises TFT resulting in single-strand breaks (SSBs) and double-strand breaks (DSBs) that activate DNA damage response (DDR) that induces the p53-p21 axis and DNA repair. BCL-2 proteins prevent cell death while their inhibition enhances cell death.

[0032] As shown in Fig. 1, the thymidine analogues are incorporated into genomic DNA, followed by their excision by BER machinery of the cell. In p53-deficient cells, this action leads to accumulation of DNA breaks but a limited death. The blockade of BCL-2 protein activity greatly enhances cytotoxicity of TAS-102. A sequential treatment regimen is preferred in order to achieve selective elimination of cancer cells compared to non-tumor human cells, therefore limiting the impact on normal tissues, and reducing potential adverse side effects. This antitumor action is associated with G2-arrest of tumor cell proliferation and 2-fold increase in tumor cell death.

[0033] In a method of the present disclosure, TAS- 102 is administered to a patient first, following the administration of a BCL-2 inhibitor. The BCL-2 inhibitor may be administered on the same day as the TAS-102, or one, two, three, or four days after the administration of TAS-102. In one example, the doses of TAS-102 and BCL-2 inhibitor are both administered within a five-day period. In examples, the TAS-102 is administered as a first dose, and the BCL-2 inhibitor is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours of the TAS-102. In examples, an individual may be treated with a described consecutive treatment for a period, followed by a period wherein neither TAS-102 or the BCL-2 inhibitor is administered, or only one of TAS-102 or the BCL-2 is administered. In an example, such period may be 1-14 days.

[0034] TAS-102 and BCL-2 may be administered orally, intravenously, intramuscularly, intrathecally, subcutaneously, sublingually, buccally, rectally, vaginally, nasally, by inhalation or nebulization, or transdermally.

[0035] The following examples are presented to illustrate the present disclosure. The examples are not intended to be limiting in any matter.EXAMPLE 1[00361 Although the present disclosure has been described with respect to one or more particular examples and / or examples, it will be understood that other examples and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

[0037] Current treatment protocols commonly use DNA-damaging drugs such as cisplatin, carboplatin, gemcitabine. These drugs showed toxicity regardless of p53 status, whereas TAS102-PARPi combination is selective for p53-deficient cancers. Based on this understanding, potential interacting biomolecules (proteins) were studied to determine a way to enhance or confer resistance to TAS 102 or the TAS102-PARPi combination.

[0038] In this example, three thymidine analogues; triflurthymidine, floxuridine, and ethynyl-deoxyuridine, that can act through a common mechanism involving Base Excision Repair (BER) were screened. The screening results, displayed in Fig. 3, identified a set of proteins interacting with these thymidine analogues.

[0039] Preclinical studies were performed with such targets, i.e., BCL-2 -like proteins. The results of this study supported a treatment strategy combining TAS- 102 and anti-BCL-2 agents for treatment of colon and pancreatic cancers (Fig.2). Fig. 4 displays the expressions of BCL-2 like genes in breast, colon, and PDAC cancers.

[0040] This example found the TAS102-PARPi treatment method displayed in Fig. 2, was far superior to either drug alone against p53 -deficient tumors in preclinical models without adverse effects in mice. Further, it was determined that the antitumor action was associated with G2-arrest of tumor cell proliferation and 2-fold increase in tumor cell death.EXAMPLE 2

[0041] In this example, cytotoxicity assays were performed to establish the cytotoxicity of TAS- 102 in combination with a BCL-2 inhibitor.

[0042] The BCL-2 inhibitor may be Venetoclax (ABT-199) or Navitoclax (ABT-263). Venetoclax is a BCL-2 selective inhibitor with Ki of <0.01 nM in cell-free assays, > 4800- fold more selective versus BCL-xL and BCL-w, and no activity to MCL-1. Navitoclax is a potent inhibitor of BCL-xL, BCL-2 and BCL-w with Ki of < 0.5 nM, <1 nM and <1 nM in cell-free assays but binds more weakly to Mcl-1 and AL

[0043] Figure 5D shows an isobologram analysis to demonstrate the dose-response relationship for TAS- 102 (Y-axis, fraction of IC50 TAS 102) and the BCL-2 inhibitor, Navitoclax (X-axis, fraction of IC50 Navitoclax).100441 Cytotoxicity studies were performed in breast and colon cancer cells. In the cytotoxicity assays for TAS-102 in combination with BCL-2 inhibitor, Navitoclax, as displayed in Fig. 5, cells were seeded at 5,000 cells / well in a 96 well-plate. The day after seeding, the cells were treated with TAS-102 at varying doses for 24 hours. The treated cell plates were replenished with cell culture media containing the BCL-2 inhibitor, Navitoclax. The control cell plates were replenished with cell culture medium without a BCL-2 inhibitor. All cells were incubated for 96 hours. The cells were stained with 1% Methylene Blue for 30 minutes, rinsed with water, dried and solubilized in 5% SDS stock solution in phosphate- buffered saline (PBS), and read at 650 nm. The optical density values were normalized to control / DMSO and the IC50 was calculated using three-parameter nonlinear regression. The data is represented as Mean + / - SEM.

[0045] Fig. 6 displays a cytotoxicity assay of Navitoclaz in CRC and PDAC lines performing the methods described above.

[0046] Fig. 7 displays a cytotoxicity assay of Navitoclax in CRC lines performing the methods described above.

[0047] Fig. 8 displays an isobologram analysis to demonstrate the dose-relationship for TAS-102 and Navitoclax for HCT116-p53WT and HCT116-p53KO. As shown, the combination index for HCT116-p53WT is 0.141 and the combination index for HCT116- p53KO is 0.12. An antagonistic reaction exists when the combination index is greater than 1.0, and a synergistic reaction exists when the combination index is less than 0.75.

[0048] Fig. 9 displays a cytotoxicity assay of ABT-199 (Venetoclax) in CRC and PDAC lines performing the methods described above.

[0049] Fig. 10 displays a comparison of cytotoxicity assays of TAS-102 with Navitoclax and TAS-102 with ABT- 199 (Venetoclax) in CRC lines.

[0050] Fig. 1 1 provides an in vivo example. As shown in this figure, HCT116 colon cancer cells were implanted subcutaneously into SCID female mice (2x10A6 cells / injection). At about 100mm3, mice were randomized into groups and treatment is initiated: (1) Vehicle, (2) BCL-2 inhibitor (BCL2i) (illustrated using 50mg / kg Navitoclax), (3) 50mg / kg TAS 102, (4) TAS102+BCL2i (50mg / kg each). On dayl5 Navitoclax / BCL2i treatment was stopped, TAS102 was continued. (Panel A) Tumor volume in mm3 (n=8-9mice / group). (Panel B) Tumor Growth Inhibition was calculated relative to the Vehicle group; TGI (%) = 100*(Vv- Vi) / Vv. Thus, the present illustrates a benefit of the described approach in a relevant animal model of cancer and demonstrates tumor growth inhibition (TGI), as evidenced by inhibitingan increase in tumor volume over time is improved when using the described combination of agents, relative to use of either agent alone.100511 While the disclosure has been described through specific embodiments, routine modifications will be apparent to those skilled in the art and such modifications are intended to be within the scope of the present disclosure.

Claims

CLAIMS:1 . A method of treating or alleviating a symptom of a cancer in a subject, the method comprising administering to the subject TAS- 102 followed by administering a BCL-2 inhibitor.

2. The method of claim 1, wherein the BCL-2 inhibitor is navitoclax or venetoclax.

3. The method of claim 1 or claim 2, wherein the BCL-2 inhibitor is administered to the subject on the same day the TAS- 102 is administered to the subject.

4. The method of claim 1 or claim 2, wherein the BCL-2 inhibitor is administered to the subject one, two, three, or four days after the TAS- 102 is administered to the subject.

5. The method of claim 1 or claim 2, wherein the cancer is colorectal cancer, colon cancer, cervical cancer, or pancreatic cancer.

6. The method of claim 5, wherein the symptom of the cancer is a solid tumor.

7. The method of claim 6, wherein the solid tumor is carrying a mutant or deleted gene encoding p53.

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