WO2025030049A1 - Sequential treatment method for p53-deficient cancer - Google Patents

Abstract

Disclosed is a method of treating or alleviating a symptom of a cancer in a subject, comprising sequentially administering to the subject a combination of a thymidine analogue and a PARP inhibitor, followed by administration of a G2-kinase inhibiting drug.

Description

SEQUENTIAL TREATMENT METHOD FOR P53-DEFICIENT CANCER

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/517,247, filed August 2, 2023, the entire disclosure of which is incorporated herein by reference.

FIELD OF FIELD OF THE DISCLOSURE

[0002] This disclosure generally relates to cancer therapeutics.

BACKGROUND OF THE DISCLOSURE

[0003] Genetic alterations in the tumor suppressor TP53 gene (p53) are present in most solid malignancies including Colorectal (CRC), Breast (BC), and Pancreatic (PANC) Cancers. Inactivation of p53 (mutation or deletion) drives cancer progression and metastasis [Sabapathy and Lane, 2018], Existing approved therapeutic options for p53-deficient cancer are ineffective and cause toxic side effects stressing the need for better therapeutics [Muller and Vousden, 2014; Sabapathy and Lane, 2018], We previously developed a two-drug combination strategy for treatment of p53-deficient cancers. This method for treatment of p53-deficient cancers combines a DNA damage inducer TAS102 (a fluorinated thymidine analogue) and inhibitors of poly (ADP) ribose polymerase (PARPi) acting as DNA damage amplifier, as described in US 2022/0249531; Pub. Date: Aug. 11, 2022, the disclosure of which is incorporated herein by reference). G2-checkpoint kinases as potential targets for treatment of p53-deficient cancers have been described [Leijen et al., 2010], However, the G2-kinase targeting drugs alone showed a limited efficacy in clinical trials, while their combination with DNA-damaging drugs (cisplatin, carboplatin, gemcitabine) showed toxicity regardless of p53 status [Leijen et al., 2010], There remains on ongoing need for improved combination therapies for treating p53 deficient cancers. The present disclosure is related to this need.

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 a combination of agents. This described strategy involves a sequential procedure aimed to target p53 mutant cancer while protecting p53 wild-type normal tissues. The first step leads to induction of DNA damage selectively in p53-deficient cells, based on our recent discovery that fluorinated thymidine analogues in combination with PARP inhibitors induce DNA damage (double-strand breaks) in p53-deficient cells, while p53 wild-type cells are arrested in cell cycle (proliferation) and can repair the DNA damage (Zonneville et al., Communications Biology, 2021). In the second step, patients are treated with drugs inhibiting molecules (kinases) that control cellcycle transition from G2 to M-phase (separation of chromosomes). Inhibition of these kinases (WEE1 or CHK1) allows cells to proceed to cell division (forming two cells) prior completion of DNA repair. In this case, unrepaired DNA will induce death in newly divided cells. The disclosure includes separating a two-step treatment procedure by 2-3 days gap to provide time for DNA repair in normal p53 wild-type tissues. Thus, the described sequential treatment method is expected to selectively eliminate p53-deficient tumors, while preserving normal tissues and may reduce overall toxicity of the treatments. The described strategy is expected to expand the utility of PARP inhibitors, as the majority of human cancers (>90%) are not currently sensitive to these drugs.

BRIEF DESCRIPTION OF THE FIGURES

[0005] 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.

[0006] FIG. 1 provides a schematic of the therapeutic strategy of sequentially administering TAS- 102 and a PAPR inhibitor. The currently described approach adds administration of a kinase (WEE1 or CHK1) inhibitor.

[0007] FIG. 2 provides a flow cytometry analysis of cells after treatments. (A) propidium iodine (PI) stained human non-tumor MCF10A cells and breast cancer MDA-MB- 231 cells. (B) Staining with PI and annexin V.

[0008] FIG. 3. Cytotoxicity data in cells pretreated with 500nM TAS102+50nM talazoparib followed by wash and incubation with 200nM MK1775 for 72 hrs.

[0009] FIG. 4. Sequential treatment with triple-drug combination of mice bearing colon cancer tumor cell xenografts. HT29 colon cancer cells were implanted sc into SCID mice. At 50mm3 (day 1), mice were randomized, and treatment was initiated as follows: vehicle on schedule 5 days-On, 2days-Off; TAS-Tal: 50mg/kg TAS102 and 0.15mg/kg talazoparib on schedule 5 days-On, 2days-Off; WEE1 inhibitor (WEEli, MK1775) at 30mg/kg for first 2 weeks (cycle 1), and 60mg/kg for cycle 2 on schedule 5days-On/2days- Off; Sequential regimen for each cycle: weekl - TAS-Tal 5days-On/2days-Off, week2 - WEEli; drugs are given as described for TAS-Tal and WEEli. (Panel A) Tumor sizes were measured by calipers 2 times per week. (Panel B) Mouse body weights were measured twice per week. (Panel C) Tumor growth inhibition at day 29 was calculated using TGI (%) = 100 x [Vvehicie - Vtreatment]/Vvehicie. (Panel D) Tumor volume at day 29.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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%, +/- 5%, or +/- 1%.

[0014] Any result obtained by using a described combination as described herein can be compared to a suitable control. In examples, a control comprises use of a thymidine analogue and a PARP inhibitor, without any administration of a G2-kinase inhibiting drug. In examples, use of a described triple combination has a more effective anti-cancer effect than using either of the thymidine analogue or the PARP inhibitor as a monotherapy. In examples, use of a described triple combination is more effective than using a combination consisting of a thymidine analogue and a PARP inhibitor. [0015] The present disclosure provides a method of treating or alleviating symptoms of cancer in a subject by sequentially administering to the subject a two-drug combination of a thymidine analogue (i.e., TAS102) and a Poly(ADP-ribose) Polymerase (PARP) inhibitor followed by G2-kinase inhibiting drug. Administration of the thymidine analog and PARP inhibitor is performed first. After the described combination is administered, the G2 -kinase inhibiting drug is administered.

[0016] In examples a therapeutically effective amount of a described combination of agents is administered. In examples a combination of TAS-102 and PARPi inhibitor, followed by a G2-kinase inhibiting drug, 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. In an example, a symptom of cancer is a tumor. In examples, performing a described method inhibits the growth of a tumor.

[0017] 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. In examples, a described method sensitizes in p53-deficient tumor cells to a described treatment. In examples, non-tumor p53 wild type cells are spared from a growth inhibiting effect of the treatment

[0018] In a method of the present disclosure, a thymidine analogue and a PARP inhibitor administered to a patient first, following the administration of a G2-kinase inhibiting drug. The G2-kinase inhibiting drug may be administered or one, two, three, or four days after the administration of the thymidine analogue and a PARP inhibitor. In one example, a combination of thymidine analogue and a PARP inhibitor are administered first, and the administration of the G2-kinase inhibiting drug is subsequently administered within a five- day period of administering the combination of the thymidine analogue and a PARP inhibitor. [0019] In one example, the thymidine analogue is TAS- 102. This agent 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 the components of TAS-102 are known in the art. In an example, a TAS-102 formulation is the formulation sold under the brand name LONSURF. [0020] In non-limiting examples, the PARP inhibitor comprises a PARP inhibitor sold under the name TALAZOPARIB, OLAPARIB, RUBRACA or ZEJULA. Other PARP inhibitors are known in the art and may be used in the method of this disclosure.

[0021] In examples, the PARP inhibitor and/or the thymidine analogue may be any such agents as described in US patent application publication no. 2022024953 from which the disclosure is incorporated herein by reference. In an example a thymidine analogue may be a referred to as a deoxyuridine analogue.

[0022] In non-limiting examples, the G2-kinase inhibiting drug is an inhibitor of WEE1 kinase or CHK1 kinase. In one example, the WEE1 inhibitor is MK1775, which may also be referred to as AZD1775. In an example, the WEE1 inhibitor is Adavosertib. In an example, the WEE1 inhibitor is Atorvastatin.

[0023] The described agents may be administered orally, intravenously, intramuscularly, intrathecally, subcutaneously, sublingually, buccally, rectally, vaginally, nasally, by inhalation or nebulization, or transdermally.

[0024] In examples, an interval between administration of any described agent or combination of agents may comprise 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. In examples, a 3, 4, 5, 6, 7, days, or longer interval may be used. In examples, an individual may be treated with a described consecutive treatment for a period, followed by a period wherein no drug or no combination of drugs is administered , or a period wherein only two described drugs are administered. In an example, such period may be 1-14 days.

[0025] The following examples are presented to illustrate the present disclosure. The examples are not intended to be limiting in any matter.

EXAMPLE 1

[0026] We showed that the antitumor action is associated with G2-arrest of tumor cell proliferation and 2-fold increase in tumor cell death [Zonneville et al., 2021] and [Alruwaili et al., 2024], We also showed that TAS102-PARPi treatment method was superior to either drug alone against p53-deficient tumors in preclinical models without adverse effects in mice [Alruwaili et al., 2024], To improve the efficacy of a TAS102-Talazoparib regimen we screened for G2-checkpoint inhibitors that could further enhance cell death after a two-drug treatment method. We identified G2-checkpoint WEE1 kinase as a target. We found that the presently described sequential treatment method markedly enhances death of p53-deficient tumor cells (p53-mutant), while sparing non-tumor p53-wild type cells (Fig. 2A). This finding was further confirmed by staining of apoptotic cells with PI and antibody to annexin V (Fig. 2B). Next, we performed cytotoxicity studies in breast and colon cancer cells. Cytotoxicity studies showed that application of WEE 1 inhibitor after pretreatment with TAS102-PARPi (Talazoparib) enhances sensitivity to the two-drug treatment and increases overall toxicity of the new treatment method in p53-deficient cells compared to non-tumor p53 wild type cells (Fig. 3). A sequential treatment regimen was examined in animal models using colon cancer cell xenografts (shown in the panels of Fig. 4). We demonstrated that WEE1 inhibitor monotherapy had a low impact on tumor growth dynamics (Fig. 4 panel A) and at day 29 (Fig. 4, panels C-D). Two-drug combination (TAS102-Talazoparib) showed about 41% tumor growth inhibition (Fig. 4, panels A, C, D). A sequential regimen (2 cycles) showed a reduction in tumor growth dynamics with tumor growth inhibition over 60% at day 29 (Fig. 4, panels A, C, D). Notably, a sequential treatment regimen did not affect mouse body weights and none of the mice showed signs of side toxic effects. Together, these results demonstrate that a sequential treatment method using TAS102-PARPi plus a WEE1 inhibitor increases cell death and enhances sensitivity to drugs in p53-deficient tumor cells, whereas non-tumor p53 wild type cells are spared from the treatment. The in vivo data indicate that a sequential treatment method is safe and effective in preclinical animal models. [0027] 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.

[0028] The following reference listing is not an indication that any particular reference is material to patentability.

Alruwaili MM, Zonneville J, Naranjo MN, Serio H, Melendy T, Straubinger RM, Gillard B, Foster BA, Rajan P, Attwood K, Chatley S, Iyer R, Fountzilas C, Bakin AV: A synergistic two-drug therapy specifically targets a DNA repair dysregulation that occurs in p53-deficient colorectal and pancreatic cancers. Cell Reports Medicine 2024;5: 101434.

Leijen S, Beijnen JH, Schellens JH: Abrogation of the G2 checkpoint by inhibition of Wee-1 kinase results in sensitization of p53-deficient tumor cells to DNA-damaging agents. Current clinical pharmacology 2010;5: 186-191.

Muller PAJ, Vousden KH: Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell 2014;25:304-317.

Sabapathy K, Lane DP: Therapeutic targeting of p53: all mutants are equal, but some mutants are more equal than others. Nature reviews Clinical oncology 2018;15: 13-30.

Zonneville J, Wang M, Alruwaili MM, Smith B, Melnick M, Eng KH, Melendy T, Park BH, Iyer R, Fountzilas C, Bakin AV: Selective therapeutic strategy for p53-deficient cancer by targeting dysregulation in DNA repair. Communications biology 2021;4:862.

Claims

What is claimed is:

1. A method of treating or alleviating a symptom of a cancer in a subject, the method comprising sequentially administering to the individual a combination of a thymidine analogue and a PARP inhibitor, followed by administration of a G2 -kinase inhibiting drug.

2. The method of claim 1, wherein the G2-kinase inhibiting drug is administered to the subject one, two, three, or four days after the combination of thymidine analogue and PARP inhibitor is administered to the subject.

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

4. The method of claim 3, wherein the symptom of the cancer is a solid tumor.

5. The method of claim 4, wherein the solid tumor comprises a mutated p53 gene.

6. The method of claim 5, wherein the thymidine analogue is comprised by TAS102.

7. The method of claim 5, wherein the G2 -kinase inhibiting drug is a WEE1 inhibitor.

8. The method of claim 7, wherein the WEE1 inhibitor comprises MK1775.

9. The method of claim 5, wherein the PARP inhibitor is Talazoparib.

10. The method of claim 1, wherein the thymidine analogue is comprised by TAS 102, the

PARP inhibitor is Talazoparib, and the G2-kinase inhibiting drug is MK1775.