Synergistic cancer treatment

Abstract

Conjugates of topoisomerase I inhibitors linked to a macromolecule through a linkage that undergo beta elimination in situ in combination with one or more of an assessed defect in DNA damage response (DDR) in a subject bearing cancer, a cell cycle checkpoint inhibitor and/or a DDR inhibitor provides improved outcomes for cancer-bearing subjects.

Claims

1. A method to treat cancer in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a conjugate of the topoisomerase I inhibitor SN-38 coupled to a macromolecule through a linker, in combination with an effective amount of a poly(ADP-ribose) polymerase (PARP) inhibitor, wherein said conjugate has the following formula: ##STR00003## wherein m=1-6 and n=200-250.

2. The method of claim 1 wherein m is 1 and n is about 225.

3. The method of claim 1, wherein the subject has a genetic defect in a DNA damage response (DDR).

4. The method of claim 2, wherein the subject has a genetic defect in a DNA damage response (DDR).

5. The method of claim 1, wherein the subject is a human.

6. The method of claim 2, wherein the subject is a human.

7. The method of claim 1, wherein the conjugate and the PARP inhibitor are administered simultaneously.

8. The method of claim 2, wherein the conjugate and the PARP inhibitor are administered simultaneously.

9. The method of claim 1, wherein the conjugate and the PARP inhibitor are administered sequentially.

10. The method of claim 2, wherein the conjugate and the PARP inhibitor are administered sequentially.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic outline of the invention approach wherein single treatment with topoisomerase I inhibitor may be offset by various repair or cell cycle checkpoints as in panel A. When the subject has an inherent DDR defect, e.g., a mutation in the BRCA gene as in panel B, the effect of topoisomerase I inhibition is strengthened, and this is further strengthened by an inhibitor of DNA damage repair such as a PARP inhibitor as in panel C (PARP is poly ADP ribose polymerase).

(2) FIG. 2 shows the state of the art with regard to sensitivity to topoisomerase I inhibitors of various DDR defects, both with respect to germline in non-germline DDR associated with various genes.

(3) FIGS. 3A-3C show the synergistic effect of an SN-38 conjugate of the invention with an inhibitor of PARP on tumor growth and on event free survival.

(4) FIGS. 4A-4C show the impact of BRCA 1 or BRCA 2 deficiency on effectiveness of the SN-38 conjugate in treating tumors in mice.

MODES OF CARRYING OUT THE INVENTION

(5) The invention takes advantage of synergistic attacks on the DNA damage response that might be mounted in cancer cells to affect their successful replication. The topoisomerase I inhibitor conjugates that cause DNA damage may be combined with either inhibitors of DDR or other inhibitors that interfere with DNA damage repair or replication. The DDR is an extremely complex process involving various mechanisms of fixing DNA to correct errors that occur either through mutation or through errors in the replication process itself. Part of this response is also a control mechanism involving cell cycle checkpoints that ensures that DNA is properly repaired or replicated before the cell divides or alternatively to effect apoptosis so that error-ridden DNA is not transmitted to daughter cells. The present invention employs a combination of a particular DDR inhibitor—a topoisomerase I inhibitor with other obstacles to successful replication including other inhibitors of DDR and inhibitors of cell cycle checkpoint pathways including instances wherein the cancer cells themselves are defective in their ability to respond to DNA damage.

(6) The invention utilizes a conjugate of a topoisomerase I inhibitor coupled to a macromolecule through a linker that provides decoupling through a beta elimination mechanism. Suitable topoisomerase I inhibitors are typically camptothecin and analogs, including irinotecan, otherwise 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 and GI 149893.

(7) In some embodiments, the macromolecule is a linear or branched or multi-armed, polyethylene glycol.

(8) Particularly preferred is a conjugate of formula (I)

(9) ##STR00001##

(10) wherein

(11) PEG is linear or branched and, when q is 2-8, multi-armed, polyethylene glycol;

(12) X is (CH.sub.2).sub.m, wherein m=1-6;

(13) L is (CH.sub.2CH.sub.2O).sub.p(CH.sub.2).sub.r, wherein r=1-10 and p=0-10;

(14) R.sup.1 is CN or SO.sub.2NR.sup.2.sub.2, wherein each R.sup.2 is independently alkyl, aryl, heteroaryl, alkylalkenyl, alkylaryl, or alkylheteroaryl, or two R.sup.2 taken together can form a ring;

(15) Y is COR.sup.3 or SO.sub.2R.sup.3, wherein R.sup.3=OH, alkoxy, or NR.sup.4.sub.2, wherein each R.sup.4 is independently alkyl, substituted alkyl, or two R.sup.4 taken together can form a ring; and

(16) q is 1-8.

(17) In particular, this conjugate may have a PEG of average molecular weight 30,000-50,000 Da, and/or wherein q=4, and/or wherein R.sup.1=CN or SO.sub.2NR.sup.2.sub.2 wherein each R.sup.2 is alkyl.

(18) The conjugate may be of the formula:

(19) ##STR00002##

(20) wherein m=1-6 and n is 200-250.

(21) In particular, the conjugate may be PLX038, which is of the above formula where m is 1 and n is approximately 225.

(22) The conjugates useful in the invention are generally provided in standard pharmaceutical formulations in combination with one or more pharmaceutically acceptable excipients, in some cases wherein the pH is between 4.0 and 6.0. Standard formulations can be found, for example, in Remington Pharmaceutical Sciences, Latest Edition, Mack Publishing Company, Easton, Pa.

(23) The invention is based on the favorable properties of a conjugate that has suitable pharmacokinetics for combination with either endogenous DDR defects or with coadministered compounds that are cell cycle checkpoint inhibitors or DDR inhibitors.

(24) In some embodiments, the conjugates, when administered to subjects provide a continuous low dose exposure to the topoisomerase I inhibitor wherein the concentration of the free inhibitor can be maintained between 15 and 5 nM between once or twice weekly administrations or over a protocol of administration, for example, of once every two weeks. In any case, the conjugates provide consistent low dose exposure to the active drug.

(25) As to the identity of the coadministered DDR inhibitors and/or cell cycle checkpoint inhibitors, many are known in the art as set forth, for example, in the Background Art discussion above.

(26) Cell cycle checkpoints include G1-S, S, and G2/M. Any of these can be targeted in combination with the topoisomerase I inhibitor conjugate, and/or in combination with additional agents that target components needed for successful checkpoint transition. This may be also against a background of an endogenous defect in cell cycle checkpoint control.

(27) 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. An extensive list of inhibitors of these targets is found in WO2012/074754.

(28) Suitable DDR inhibitors include those that target homologous recombination (HR), e.g. poly(ADP-ribose) polymerase (PARP) inhibitors and/or other DDR pathways, including an HEJ, HR, alt-NHEJ/MMEJ, SSA, ICL, SSB, BER, TLS, NER and MMR. A large number of agents are in development for addressing these targets, and a number of agents known to do so are now used in the clinic.

(29) All documents cited are incorporated herein by reference in their entirety.

(30) The following example is intended to illustrate, but not limit the invention.

Example 1

Synergistic Effect of PLX038A and PARP Inhibitor Talazoparib (Designated BMN673 or TLZ)

(31) Preparation of murine MX-1 xenografts: The MX-1 cell line was obtained from Charles River Labs (Frederick, Md.)..sup.1 Cells were cultured in RPMI-1640, 10% FBS and 1% 2 mM L-glutamine at 37° C. in 95% air/5% CO.sub.2 atmosphere. .sup.1 Ovejera A A et al. Chemotherapy of human tumor xenografts in genetically athymic mice. Ann Clin Lab Sci 8: 50-6, 1978.

(32) Female NCr nude mice (N CrTac:NCr-Foxnt1.sup.nu; ˜6-7 weeks old) from Taconic Bioscience (Cambridge City, Ind.) were housed at the UCSF Preclinical Therapeutics Core vivarium (San Francisco, Calif.). All animal studies were carried out in accordance with UCSF Institutional Animal Care and Use Committee. Tumor xenografts were established by subcutaneous injection with MX-1 tumor cells (2×10.sup.6 cells in 100 μl of serum free medium mixed 1:1 with Matrigel) into the right flank of female NCr nude mice. When tumor xenografts reached 1000-1500 mm.sup.3 in donor mice, they were resected, cut into even-size fragments (˜2.5×2.5×2.5 mm in size), embedded in Matrigel and re-implanted via subcutaneous trocar implantation in receiver mice..sup.2 .sup.2 Morton C L, Houghton P J. Establishment of human tumor xenografts in immunodeficient mice. Nat Protoc. 2007; 2(2):247-50.

(33) Dosing and tumor volume measurements: Solutions of PLX038A (1.02 mM SN38; 0.26 mM PLX038A conjugate) were prepared in pH 5 isotonic acetate and sterile filtered (0.2 μm) before use. Solutions of BMN673 (52 μM) were prepared in 10% dimethylacetamide/5% Solutol HS15/85% 1×PBS and were sterile filtered (0.2 μm) before use.

(34) Groups (N=4-5/group) were dosed when the group average reached 100-200 mm.sup.3 in size. Mice received vehicle, a single dose of PLX038A (14.7 mL/kg i.p., 15 μmol/kg), daily doses of BMN673 (7.72 mL/kg p.o., 0.4 μmol/kg), or a combination of PLX038A and BMN673 at the same doses. For groups receiving the combination, daily BMN673 dosing began on the same day (FIG. 3A) or after a 4-day delay (FIG. 3B) after dosing PLX038A. Tumor volumes (caliper measurement: 0.5×(length×width.sup.2)) and body weights were measured twice weekly. When vehicle control tumors reached ˜3000 mm.sup.3 in size, mice were treated with the combination of a single dose of PLX038A (15 μmol/kg) and daily BMN673 (0.4 μmol/kg) combination with no delay between dosing (FIG. 3A).

(35) As shown in FIGS. 3A and 3B, administration of PLX038A to mice bearing MX-1 tumors at 15 μmol/kg in combination with daily doses of Talazoparib at 0.4 μmol/kg provides a synergistic effect as compared to either of these drugs alone. This was true whether daily dosage with TLZ began at the same time as PLX038A or 4 days later. A single combination administered to control immediately reduced tumor volume (FIG. 3A).

(36) As shown in FIG. 3C, event-free survival was enhanced synergistically with the combination vs PLX038A and TLZ individually.

Example 2

Synergy of PLX038A and Tumor Cell Defect

(37) MX-1 cells are BRCA 1 deficient and CAPAN-1 cells are supplied as either BRCA 2 deficient (−/−) or not deficient (+/+). The general protocol of Example 1 was followed with mice bearing tumors of these cell lines. For mice with MX-1 tumors, dosages were single i.p. injections of 137 μg/kg of irinotecan or 4, 40 or 120 μg/kg of PLX038A. For mice with CAPAN-1 xenografts, dosages were single i.p. injections of 137 μg/kg irinotecan or 15, 40 or 120 μg/kg of PLX038A. FIGS. 4A-4C show the results of these dosages on tumor volumes, which were measured twice weekly.

(38) As shown in FIG. 4A, all dosages of PLX038A were more effective than irinotecan in reducing tumor volume, with 40 or 120 μg/kg essentially stopping tumor growth. Also shown is the dramatic result of a single dose of 120 μg/kg PLX038A administered when the control tumors reached 2000 mm.sup.3.

(39) A comparison of FIGS. 4B and 4C shows the effect of BRCA 2 deficiency on the effectiveness of treatment with irinotecan or PLX038A—only the very highest dose of PLX038A was comparably effective for both deficient and non-deficient cells. The effectiveness of all other dosage levels was enhanced in the BRCA 2 deficient cells.