Synergistic cancer therapy

Abstract

To provide improved methods of treatment with topoisomerase I inhibitors in tumor subjects.SOLUTION: Outcomes for cancer-bearing subjects are improved by conjugates of topoisomerase I inhibitors linked to a macromolecule through a linkage that undergoes β-elimination in situ, in combination with one or more assessed defects in DNA damage response (DDR) in a subject bearing cancer, a cell cycle checkpoint inhibitor and / or a DDR inhibitor.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority from U.S. Provisional Application No. 62 / 617,095, filed on Jan. 12, 2018; U.S. Provisional Application No. 62 / 674,483, filed on May 21, 2018; U.S. Provisional Application 62 / 711,421, filed on Jul. 27, 2018; U.S. Provisional Application No. 62 / 716,788, filed on Aug. 9, 2018; U.S. Provisional Application No. 62 / 716,796, filed on Aug. 9, 2018; U.S. Provisional Application No. 62 / 700,147, filed on Jul. 18, 2018; and U.S. Provisional Application No. 62 / 711,423, filed on Jul. 27, 2018, the disclosures of which are hereby incorporated by reference in their entirety.

[0002] Technical field The present invention relates to the use of a topoisomerase I inhibitor linked to a polymer via a linkage that undergoes beta elimination for treating cancer. More specifically, the present invention relates to the treatment of a cancer-bearing subject when the pharmacokinetics are appropriately controlled by the topoisomerase I inhibitor, where the subject has a genetic defect in the DNA damage response (DDR), and / or the treatment involves administering the topoisomerase I inhibitor in combination with an inhibitor of DDR or in combination with a cell cycle checkpoint inhibitor. In certain embodiments, the present invention necessarily involves the use of synthetic lethal interactions in cancer cells when a defect in a gene required for DDR renders another gene essential for cell survival. carrier, and the treatment involves administering the topoisomerase I inhibitor in combination with an inhibitor of DDR or in combination with a cell cycle checkpoint inhibitor. In certain embodiments, the present invention necessarily involves the use of synthetic lethal interactions in cancer cells when a defect in a gene required for DDR renders another gene essential for cell survival. genetic defect, and / or the treatment involves administering the topoisomerase I inhibitor in combination with an inhibitor of DDR or in combination with a cell cycle checkpoint inhibitor. In certain embodiments, the present invention necessarily involves the use of synthetic lethal interactions in cancer cells when a defect in a gene required for DDR renders another gene essential for cell survival. genetic defect, and / or the treatment involves administering the topoisomerase I inhibitor in combination with an inhibitor of DDR or in combination with a cell cycle checkpoint inhibitor. In certain embodiments, the present invention necessarily involves the use of synthetic lethal interactions in cancer cells when a defect in a gene required for DDR renders another gene essential for cell survival. genetic defect, and / or the treatment involves administering the topoisomerase I inhibitor in combination with an inhibitor of DDR or in combination with a cell cycle checkpoint inhibitor. In certain embodiments, the present invention necessarily involves the use of synthetic lethal interactions in cancer cells when a defect in a gene required for DDR renders another gene essential for cell survival. interactions) in cancer cells when a defect in a gene required for DDR renders another gene essential for cell survival.

Background Art

[0003] Topoisomerase I inhibitors are well known in the treatment of various cancers, and topoisomer It is an inhibitor of the essential ligation step catalyzed by topoisomerase I. Topoisomerase I repairs single-stranded DNA damage caused by the relaxation of tension generated by supercoiling during DNA replication. (Topoisomerase I is required for DNA replication.) Topoisomerase I inhibitors include camptothecin and its analogs. Many of these compounds are approved and are used as chemotherapy in the treatment of a variety of cancers.

[0004] In cancer cells with certain genetic defects, it has been observed that the administration of topoisomerase I inhibitors shows enhanced effects compared to cancer cells without such defects. For example, SN-38, a topoisomerase inhibitor, has been administered as a conjugate with polyethylene glycol and to BRCA1-deficient mice with breast tumors, and the combination of BRCA1 deficiency and inhibition by SN-38 of topoisomerase is not only effective but also overcomes ABCG2-mediated chemoresistance. See, for example, Zander, S.A.L. et al., PLOS One (2012) 7:e45248. Furthermore, various topoisomerase inhibitors have been co-administered in combination with additional anticancer agents that are DDR inhibitors and / or cell cycle checkpoint inhibitors. For example, Abal, M. et al., Oncol. Gene (2004) 23:1737-1744; Wainberg, Z.A.et al., Targ Oncol. (2017) 12:775-785; Verschraegen, C.F. et al., Cancer (2013) See 5:418-429; and Gray, J. et al., Cancer Biol. and Ther. (2012) 13:614-622; Josse, R e.al, Cancer Res (2014) 74:6968-6978; Ma, C.X., et al, Breast Cancer Res Treat (2013) 137:483-492. Also, in vitro studies have shown that inhibiting the expression of Werner Syndrome helicase (WRN), a protein important for DNA replication and repair, enhances the effect of irinotecan on cancer cells. See Futami, K., et al., Biol Pharm Bull (2007) 30:1958-1961. Combinations of cell checkpoint inhibitors and various DNA-damaging agents have also been tested in clinical trials. (See Visconti, R. et al., J. Exp. Clin. Cancer Res. (2016) 35:153.)

[0005] Furthermore, extensive knowledge exists regarding deficiencies in the DNA damage response across various genes and genomic locations. (See Knijnenburg, T.A. et al., Cell Reports (2018) 23:239-254.)

[0006] The conjugation of topoisomerase I inhibitors, such as SN-38, to polymers has been reported in Zhao, H. et al., Bioconjugate Chem. (2008) 19:849-859 and Koizumi, F. et al., Cancer Res. (2006) 66:10048-10056. A specific set of conjugates useful in the present invention is disclosed in Santi, D.V. et al., J. Med. Chem. (2014) 57:2303-2314. An additional conjugate, a PEGylated irinotecan known as NKTR-102, is also known.

[0007] The present invention provides an improved method of treatment with a topoisomerase I inhibitor in a tumor subject. This method either takes advantage of the subject's inherent DDR defect associated with germline mutations or other functional impairments in the subject's cancer cells, or utilizes combination therapy with an additional agent that results in synthetic lethality.

SUMMARY OF THE INVENTION

[0008] Disclosure of the invention As is apparent from the previously cited literature, topoisomerase I is known to be essential for DNA replication and is required for cell growth and replication. Inhibitors of topoisomerase I, such as irinotecan and its active metabolite SN-38, have been used to treat cancer by inhibiting proper DNA replication.

[0009] There are also reports of attempts to combine topoisomerase I inhibitors with either a cell cycle checkpoint inhibitor (which disables the mechanism by which the cell determines whether replication has been successfully achieved) or an inhibitor of the additional DNA damage response (DDR). It is also known to administer topoisomerase I inhibitors to cancers that have already been characterized as having defects in DDR. ​​​​

[0010] Some such attempts involve topoisomerase I inhibitors conjugated to solubilizing agents such as polyethylene glycol (PEG). However, the pharmacokinetics of such inhibitors provided heretofore have not been appropriate for obtaining favorable results; moreover, the toxicity of these inhibitors has also been a problem. The protocol of the present invention is, most importantly, implemented in human subjects, but the present invention is also applicable to other mammalian subjects, including laboratory models for testing disease treatment. These protocols are also useful for livestock and pets. Now, by providing a topoisomerase I inhibitor having a bond to a polymer that is cleaved by β-elimination, it has been found that its pharmacokinetics can be adjusted to provide a more effective and tolerable treatment to subjects with defects in DDR, or in combination with an inhibitor of the cell cycle checkpoint pathway and / or an inhibitor of DDR. The conjugate of the present invention can also be administered in a dose that reduces the synergistic toxicity of the topoisomerase I inhibitor and such additional agents.

[0011] The protocol of the present invention is, most importantly, implemented in human subjects, but the present invention is also applicable to other mammalian subjects, including laboratory models for testing disease treatment. These protocols are also useful for livestock and pets.

[0012] Now, by providing a topoisomerase I inhibitor having a bond to a polymer that is cleaved by β-elimination, it has been found that its pharmacokinetics can be adjusted to provide a more effective and tolerable treatment to subjects with defects in DDR, or in combination with an inhibitor of the cell cycle checkpoint pathway and / or an inhibitor of DDR. The conjugate of the present invention can also be administered in a dose that reduces the synergistic toxicity of the topoisomerase I inhibitor and such additional agents. to subjects with defects in DDR, or in combination with an inhibitor of the cell cycle checkpoint pathway and / or an inhibitor of DDR. The conjugate of the present invention can also be administered in a dose that reduces the synergistic toxicity of the topoisomerase I inhibitor and such additional agents. to subjects with defects in DDR, or in combination with an inhibitor of the cell cycle checkpoint pathway and / or an inhibitor of DDR. The conjugate of the present invention can also be administered in a dose that reduces the synergistic toxicity of the topoisomerase I inhibitor and such additional agents. administered.

[0013] Thus, in a first aspect, the present invention relates to a method for treating cancer in a subject in need of cancer treatment, wherein the subject has been identified as having one or more defects in the DNA damage response (DDR). This method comprises administering to the subject an effective amount of a topoisomerase I inhibitor conjugated to a polymer via a linker capable of undergoing cleavage by a β-elimination mechanism. Thus, in a first aspect, the present invention relates to a method for treating cancer in a subject in need of cancer treatment, wherein the subject has been identified as having one or more defects in the DNA damage response (DDR). This method comprises administering to the subject an effective amount of a topoisomerase I inhibitor conjugated to a polymer via a linker capable of undergoing cleavage by a β-elimination mechanism. Thus, in a first aspect, the present invention relates to a method for treating cancer in a subject in need of cancer treatment, wherein the subject has been identified as having one or more defects in the DNA damage response (DDR). This method comprises administering to the subject an effective amount of a topoisomerase I inhibitor conjugated to a polymer via a linker capable of undergoing cleavage by a β-elimination mechanism. administering.

[0014] In a second aspect, the present invention provides a polymer via a linker capable of undergoing cleavage by a β-elimination mechanism The present invention relates to a method of treating cancer in a subject, comprising administering to the subject an effective amount of a topoisomerase I inhibitor bound to: in combination with an effective amount of an additional DDR inhibitor.

[0015] In a third aspect, the present invention provides a method for attaching a polymer to a linker that is susceptible to cleavage by a β-elimination mechanism. an effective amount of a topoisomerase I inhibitor linked to The present invention relates to a method of treating cancer in a subject, comprising administering to the subject a compound that inhibits the pathway.

[0016] In the first aspect of the invention, the method also comprises diagnosing a subject for the presence of the defect. The method may include administering two or more agents, including a conjugate of the invention. In certain embodiments, the co-administration of two or more agents may be simultaneous or concurrent. The administration of the co-administered drugs may be sequential in any order. The difference in the time of administration of the co-administered drugs may be up to several days. Two or more drugs may also be administered in the same composition.

[0017] Combinations of the above approaches are also included in the present invention; A subject having a topoisomerase I inhibitor conjugate can be provided in combination with either or both of an additional DDR inhibitor or a checkpoint pathway inhibitor via a linker that can be cleaved by a beta-elimination mechanism to a subject having a topoisomerase I inhibitor conjugate of the present invention. Independently, regardless of whether the subject shows an inherent defect in DDR, the combination of the topoisomerase I inhibitor conjugate of the present invention with either or both of an additional DDR inhibitor and a cell cycle checkpoint inhibitor is included within the scope of the present invention. Furthermore, the use of two or more DDR inhibitors and / or two or more cell cycle checkpoint inhibitors in combination with a topoisomerase I inhibitor conjugate is also included within the scope of the present invention. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 shows a schematic diagram of the approach of the present invention. Monotherapy with a topoisomerase I inhibitor can be counteracted by various repair or cell cycle checkpoints, as shown in Panel A. If the subject has an inherent DDR defect, for example, a mutation in the BRCA gene as shown in Panel B, the effect of topoisomerase I inhibition is enhanced, which is further enhanced by an inhibitor of DNA damage repair such as a PARP inhibitor (PARP is poly ADP ribose polymerase), as shown in Panel C. [Figure 2] Figure 2 shows the latest information on the sensitivity of various DDR defects to topoisomerase I inhibitors with respect to DDR in both germline and somatic cell lineages related to various genes. [Figure 3] Figures 3A - 3C show the synergistic effect of the SN-38 conjugate of the present invention and a PARP inhibitor on tumor growth and event free survival. [Figure 4] Figures 4A - 4C show the effect of BRCA 1 or BRCA 2 deficiency on the efficacy of the SN-38 conjugate in treating mouse tumors. **DETAILED DESCRIPTION OF THE INVENTION**

[0019] The present invention relates to DNA that may be present in cancer cells to affect good replication Utilize synergistic attacks against the damage response. Topoisomerase I inhibitor conjugates that cause DNA damage can be combined with inhibitors of DDR or other inhibitors that prevent DNA damage repair or replication. DDR is a very complex process involving diverse mechanisms to return DNA to its original state and correct errors that occur due to mutations or errors in the replication process itself. Part of this response is also a control mechanism including cell cycle checkpoints, which ensure that DNA is properly repaired or replicated before cell division, or cause apoptosis so that DNA with errors is not transmitted to daughter cells. The present invention employs combinations of specific DDR inhibitor-topoisomerase I inhibitors with other impediments to good replication, such as other inhibitors of DDR and inhibitors of cell cycle checkpoint pathways (including cases where cancer cells themselves lack the ability to respond to DNA damage).

[0020] The present invention utilizes conjugates of topoisomerase I inhibitors bound to polymers via linkers capable of undergoing cleavage by a β-elimination mechanism. Suitable topoisomerase I inhibitors generally include camptothecin and analogs, such as irinotecan (also known as CPT-11) and its active metabolite SN-38, as well as topotecan, 9-amino-camptothecin, and water-soluble analogs such as GI 147211, GI 149893, etc.

[0021] In certain embodiments, the polymer is a linear or branched or multi-armed polyethylene glycol.

[0022] Conjugates of formula (I) are particularly preferred:

Chemical formula

[0023] In particular, this conjugate has PEG with an average molecular weight of 30,000 - 50,000 Da, and / or q = 4, and / or R 1 = CN or SO2NR 2 2 (where each R 2 is alkyl).

[0024] The conjugate can be of the following formula: [Chemical formula] where m = 1 - 6 and n = 200 - 250.

[0025] In particular, the conjugate can be PLX038, which is of the above formula with m = 1 and n ≈ 225.

[0026] Conjugates useful in the present invention generally are administered in one or more pharma- ceutically acceptable excipients. The composition is provided as a standard pharmaceutical formulation in combination with an agent, and in some cases, the pH is 4.0 to 6.0. Standard formulations can be found, for example, in Remington Pharmaceutical Sciences (latest edition, Mack Publishing Company, Easton, Pa.).

[0027] The present invention relates to combinations of intrinsic DDR defects or cell cycle checkpoint inhibitors. or in combination with a co-administered compound that is a DDR inhibitor. It is based on the advantageous properties of conjugates with kinetic properties.

[0028] In one embodiment, the conjugate, when administered to a subject, provides sustained low dose exposure to a topoisomerase I inhibitor, where the concentration of free inhibitor can be maintained between 15-5 nM during a once or twice weekly administration protocol, or, for example, once every two weeks, in either case providing a consistent low dose exposure to active drug.

[0029] Regarding the identity of the co-administered DDR inhibitors and / or cell cycle checkpoint inhibitors, many are known in the art, for example as described in the Background section above.

[0030] Cell cycle checkpoints include G1-S, S, and G2 / M. , in combination with topoisomerase I inhibitor conjugates, and / or in combination with additional agents that target components required for successful checkpoint transition. This can also be against the backdrop of intrinsic defects in the control of cell cycle checkpoints.

[0031] Suitable cell cycle checkpoint targets include checkpoint kinase 1 or 2 (CHK1 or CHK2), ataxia telangiectasia mutated (ATM) kinase, ataxia telangiectasia and Rad3 related (ATR) kinase, Wee1 kinase, and p53. A comprehensive list of inhibitors of these targets can be found in WO2012 / 074754.

[0032] Suitable DDR inhibitors include those targeting homologous recombination (HR), such as poly(ADP-ribose) polymerase (PARP) inhibitors, and / or those targeting other DDR pathways, including HEJ, HR, alt-NHEJ / MMEJ, SSA, ICL, SSB, BER, TLS, NER, MMR. A number of agents are under development to address these targets, and several agents that are known to do so are currently in use in the clinical setting. All documents cited are hereby incorporated by reference in their entirety.

[0033] The following examples are intended to illustrate the invention and are not intended to limit the invention.

[0034]

Examples

Example

[0035] Example 1 Synergistic effect of PLX038A and PARP inhibitor Talazoparib (alias BMN673 or TLZ) Preparation of mouse MX-1 xenografts: The MX-1 cell line was obtained from Charles River Labs (Frederick, Maryland). 1 Cells were cultured in RPMI-1640, 10% FBS, and 1% 2 mM L-glutamine at 37°C in an atmosphere of 95% air / 5% CO2. 1Ovejera AA et al. Chemotherapy of human tumor xenografts in genetically athymic mice. Ann Clin Lab Sci 8: 50-6, 1978.

[0036] Female NCr nude mice from Taconic Biosciences (Cambridge City, Indiana) (N CrTac:NCr-Foxn1 nu ; approximately 6 - 7 weeks old) were housed at the UCSF Preclinical Therapeutics Core Vivarium (San Francisco, California). All animal experiments were conducted in accordance with the UCSF Institutional Animal Care and Use Committee. Tumor xenografts were established by subcutaneous injection of MX-1 tumor cells (2 × 10 6 cells in 100 μl of serum-free medium mixed 1:1 with Matrigel) into the right flank of female NCr nude mice. When the tumor xenografts reached 1000 - 1500 mm 3 in the donor mice, they were excised, cut into equal-sized fragments (approximately 2.5 × 2.5 × 2.5 mm in size), embedded in Matrigel, and re-implanted subcutaneously by trocar into recipient mice. 2 . 2 Morton CL, Houghton PJ. Establishment of human tumor xenografts in immunodeficient mice. Nat Protoc. 2007;2(2):247-50.

[0037] Administration and tumor volume measurement: A solution of PLX038A (1.02 mM SN38; 0.26 mM PLX038A conjugate) in isotonic acetic acid at pH 5 was A solution of BMN673 (52 μM) was diluted with 10% dimethylacetone and sterile filtered (0.2 μm) before use. The solution was prepared in 5% ethanolamide / 5% Solutol HS15 / 85% 1X PBS and sterile filtered (0.2 μm) before use.

[0038] The mean tumor size of the group (N = 4–5 / group) was 100–200 mm 3 When it reaches Mice were dosed with vehicle, a single dose of PLX038A (14.7 mL / kg i.p., 15 μmol / kg ), daily doses of BMN673 (7.72 mL / kg po, 0.4 μmol / kg), or the same dose of the combination of PLX038A and BMN673. In the group receiving the combination, daily BMN673 administration was initiated on the same day as PLX038A administration (Figure 3A) or 4 days after PLX038A administration (Figure 3B). Tumor volumes (caliper measurements: 0.5 × (length × width) 2 ) and body weight were measured twice weekly. Vehicle control tumors were approximately 3000 mm in size. 3 When this was reached, mice were treated with a combination of a single dose of PLX038A (15 μmol / kg) and daily BMN673 (0.4 μmol / kg) with no delay between doses (Figure 3A).

[0039] As shown in Figures 3A and 3B, administration of 15 μmol / kg PLX038A in combination with a daily dose of 0.4 μmol / kg talazoparib to mice bearing MX-1 tumors resulted in a synergistic effect compared to either of these agents alone. This was true whether daily administration of TLZ was initiated simultaneously with PLX038A or after a 4-day delay. Administration of the single combination to controls resulted in an immediate reduction in tumor volume (Figure 3A).

[0040] As shown in Figure 3C, event-free survival was synergistically improved with the combination versus individual administration of PLX038A and TLZ.

[0041] Example 2 Synergistic action of PLX038A and tumor cell defects MX-1 cells are BRCA 1 deficient, and CAPAN-1 cells are supplied as BRCA 2 deficient (- / -) or non-deficient (+ / +). Using mice bearing tumors of these cell lines, the general protocol of Example 1 was carried out. In the case of mice with MX-1 tumors, the dosage was a single intraperitoneal injection of 137 μg / kg of irinotecan or 4, 40 or 120 μg / kg of PLX038A. In the case of mice with CAPAN-1 xenografts, the dosage was a single intraperitoneal injection of 137 μg / kg of irinotecan or 15, 40 or 120 μg / kg of PLX038A. Figures 4A - 4C show the results of these dosages on tumor volume measured twice a week.

[0042] As shown in Figure 4A, at all dosages of PLX038A, it was more effective than irinotecan in reducing tumor volume, and when the dosage was 40 or 120 μg / kg, tumor growth essentially stopped. Also shown are the dramatic results when a single dose of 120 μg / kg of PLX038A was administered when the control tumor reached 2000 mm 3 .

[0043] The comparison of Figures 4B and 4C shows the effect of BRCA 2 deficiency on the efficacy of treatment with irinotecan or PLX038A - only the highest dose of PLX038A was equally effective in both BRCA 2 - deficient and non - deficient cells. The efficacy at all other dosage levels was enhanced in BRCA 2 - deficient cells. The present invention includes the following aspects. [Item 1] A method for treating cancer in a subject in need of cancer treatment, wherein the subject is diagnosed with one or more defects in DNA damage response (DDR), and administering to the subject an effective amount of a conjugate of a topoisomerase I inhibitor conjugated to a polymer via a linker capable of undergoing cleavage by a β - elimination mechanism. [Item 2] The method according to item 1, comprising administering to the subject an effective amount of an inhibitor of DDR in combination with the conjugate. [Item 3] The method according to item 1, comprising administering to the subject an effective amount of a cell cycle checkpoint inhibitor in combination with the conjugate. [Item 4] The method according to item 1, further comprising diagnosing the subject as to whether there is such a defect. [Item 5] The method according to item 1, wherein the topoisomerase I inhibitor is SN - 38. [Item 6] The method according to item 5, wherein the polymer is polyethylene glycol (PEG). [Item 7] The method according to item 5, wherein the conjugate is PLX038. ​ [Item 8] The method according to any one of items 1 - 7, wherein the defect in DDR in the subject is a defect in homologous recombination repair (HRR) or a defect in single - strand break repair, or the defect is in a tumor suppressor gene. [Item 9] The method according to item 2, wherein the inhibitor of DDR is an inhibitor of HRR repair or an inhibitor of single - strand break repair. [Item 10] The method according to item 3, wherein the cell cycle checkpoint inhibitor is an inhibitor of CHK1, CHK2 or Wee1 kinase. [Item 11] A method for treating cancer in a subject in need of cancer treatment, comprising administering to the subject an effective amount of a conjugate of a topoisomerase I inhibitor conjugated to a polymer via a linker capable of undergoing cleavage by a β - elimination mechanism in combination with an effective amount of an inhibitor of DDR. [Item 12] The method according to item 11, wherein the inhibitor is an inhibitor of HRR repair or single - strand break repair. [Item 13] A method for treating cancer in a subject in need of cancer treatment, the method comprising administering to the subject a conjugate of a topoisomerase I inhibitor conjugated to a polymer via a linker capable of undergoing cleavage by a β-elimination mechanism, in combination with an effective amount of a cell cycle checkpoint inhibitor. [Item 14] The method according to item 13, wherein the cell cycle checkpoint inhibitor is an inhibitor of CHK1, CHK2 or Wee1 kinase. [Item 15] The method according to any one of items 11 to 14, wherein the topoisomerase I inhibitor is SN-38. [Item 16] The method according to item 15, wherein the polymer is polyethylene glycol (PEG). [Item 17] The method according to item 15, wherein the conjugate is PLX038.

Claims

**Claim 1** A medicament for treating cancer in a subject in need of cancer treatment, comprising a conjugate of a topoisomerase I inhibitor conjugated to a polymer via a linker capable of undergoing cleavage by a β-elimination mechanism and an effective amount of an inhibitor of DDR, wherein the conjugate has the following formula: 【Chemical 1】 [wherein m = 1 to 6 and n = 200 to 250.] and the inhibitor is talazoparib, and the conjugate and the inhibitor are administered simultaneously or sequentially, a medicament. **Claim 2** The medicament according to claim 1, wherein the conjugate is PLX038. **Claim 3** The medicament according to claim 1 or 2, wherein the subject has one or more genetic defects in DNA damage response (DDR).

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