# Patent

## Title
Compositions of combinations of non-covalent DNA binding agents and anti-cancer and/or anti-inflammatory agents and their use in disease treatment

## Abstract

The invention provides for compositions for treating a cancer or an inflammatory disorder comprising a combination of agents in a pharmaceutically acceptable carrier, wherein said agents comprise: (i) a non-covalent DNA binding agent; and (ii) an anti-cancer or anti-inflammatory agent.

## Background
This application is a 371 application of PCT application No. PCT/US2013/028358, filed Feb. 28, 2013, which claims the priority of U.S. Ser. No. 61/621,149, filed Apr. 6, 2012, the contents of all of which are hereby incorporated by reference in their entirety into the present application.


Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
FIELD OF THE INVENTION
The invention relates to non-covalent DNA binding agents, alone or in combination with anti-cancer agents and/or anti-inflammatory agents that can be used to treat cancer and inflammation.
BACKGROUND OF THE INVENTION
Cancers are caused by multiple genetic changes that drive tumorigenesis. Over the past several years, overexpressed oncogenic targets such as receptor tyrosine kinases (RTKs) have been targeted for treatment of cancers. Cancers can also arise from the loss of tumor suppressor gene functions such as through the loss of p53, BRCA1, BRCA2, PTEN and other tumor suppressor genes. Currently no therapeutic approaches have been designed to target cancers that are due to the loss of tumor suppressor gene functions.
The concept of synthetic lethality was introduced, recently, into the field of cancer therapeutics. Initial research in the field of synthetic lethality indicated that two genes are synthetic lethal if mutation of either gene alone is compatible with viability but a mutation of both genes results in cell death. There have been recent examples of treatment of cancers that have a BRCA1 gene deficiency by administration of a DNA crosslinking agent, such as a platinum drug, in combination with an inhibitor of an overexpressed gene, such as PARP, to produce a synthetic lethal outcome in such BRCA1 deficient tumor cells (A. Ashworth: A synthetic lethal therapeutic approach: Poly(ADP) Ribose Polymerase Inhibitors for the Treatment of Cancers Deficient in DNA Double-Strand Break Repair. J Clinical Oncology 26:3785-3790, 2008; Rehman, F. L., Lord, C. J. and Ashworth, A. Synthetic lethal approaches to breast cancer therapy. Nat Rev Clin Oncol 7: 718-724, 2010; O'Shaughnessy, J., Osborne, C., Pippen, J. E., Yoffe, M, Patt, D., Rocha, C., Koo, I. C., Sherman, B. M. and Bradley, C. Iniparib plus chemotherapy in metastatic triple-negative breast cancer. N Engl J Med 364: 205-214, 2011.
Currently, labor intensive bioinformatic analysis and small molecule or RNAi screens are needed to identify synthetic lethal relationships between well-established therapeutic targets and/or lesser-known components of cancer cells' signaling networks.
At present, the only clinical application of synthetic lethality is the use of DNA crosslinking platinum drugs such as carboplatin, together with an antimetabolite such as gemcitabine, in combination with poly (ADP-ribose) polymerase (PARP) inhibitor, such as iniparib in patients with triple-negative breast cancer that have BRCA1 and/or BRCA2 mutations (O'Shaughnessy et al., N Engl J Med 364: 205-214, 2011). Preclinical studies were required to establish synthetic lethal relationships among the combination of a DNA crosslinking agent (platinum), and antimetabolites (gemcitabine) and the inhibition of the DNA repair enzyme PARP, together with the genetic inactivation of tumor suppressor genes BRCA1 or BRCA2.
A clear advantage of cancer treatments based on synthetic lethality is that they have minimal toxicity, because only cells with the impairments that comprise the synthetic lethal relationship (e.g., a mutated gene and a therapeutically inhibited enzyme) should be affected. Those cells should almost exclusively be cancer cells. Treatments based on synthetic lethality offers the advantage of overcoming the problem of targets that, either due to underlying biology or the targets' actual physical make up, are “undruggable” with small molecule and biologic drugs. As much as 75% of the identified molecular targets for cancer may be “undruggable”.
A key obstacle to appropriate treatment of cancers and other inflammatory diseases is the resistance or refractory responses to available therapies. For example, it is well known that tumor cells develop mutations in various genes and/or their expressed proteins. Such mutations allow the tumor cells to become refractory to currently available anticancer agents and thus the patients do not have therapeutic options. The novel invention described in this application shows the benefit of using non-covalent DNA binding agents that show synthetic lethality in tumors that carry mutations, particularly in DNA repair or tumor suppressor genes, that result in a “loss of function” in the cell's ability to either repair itself or go into apoptosis or programmed cell death. Since such mutations in DNA repair or tumor suppressor genes also render the tumor cells refractory to available treatments, the novel combinations of one or more non-covalent DNA binding agents with one or more anticancer or anti-inflammatory agents, represents a novel and unique way to treat tumor cells that have “loss of function” in tumor suppression and/or DNA repair functions.
Furthermore, in view of the fact that a) it is difficult to identify and/or predict synthetic lethal relationships, and b) the importance of cancer treatments based on synthetic lethality, there is a real and immediate need for methods of disease treatment based on combinations of agents that can leverage synthetic lethality and to develop such novel combinations in a rapid time frame, so that it does not involve time consuming identification of synthetic lethal relationships amongst genes. Moreover, such novel compositions of agents should result in treatment methods that are non-toxic. This application describes unique and novel compositions of combinations or one or more non-covalent binding DNA agents with one or more available anticancer agents, including but not limited to, those agents that have become refractory due to mutations in such cells and provide novel methods of therapies for treatment of highly unmet clinical need in cancer and inflammatory diseases, while leveraging, the concept of synthetic lethality.
SUMMARY OF THE INVENTION
The invention relates to novel compositions and methods of disease treatment comprising using one or more non-covalent DNA binding agents to create synthetic lethal combinations in cells that have “loss of function” in tumor suppressor and/or DNA repair pathways. The invention provides for the use of one or more non-covalent DNA binding agents as a monotherapy, that is, they function in the absence of other active agents, to, e.g., create synthetic lethality in tumors that exhibit loss of tumor suppressor gene function, thereby treating disease. In one embodiment of the invention, one or more non-covalent DNA binding agents may be used in combination with one or more anti-cancer agents and/or anti-inflammatory agents to, e.g., create synthetic lethality in tumors that exhibit loss of tumor suppressor gene function, so as to treat disease.
The invention also relates to novel compositions and methods of disease treatment comprising using one or more non-covalent DNA binding agents to treat a subject with at least one of a DNA repair deficiency, dysregulated apoptosis, a replication deficiency, loss of function of a tumor suppressor gene, deficiencies in DNA recombination, a ubiquitin disorder, cell cycle dysregulation and/or dysregulated translesion synthesis. In a further embodiment, one or more non-covalent DNA binding agents may be used with one or more anti-cancer agents in novel compositions and methods of disease.
The invention provides for novel compositions and methods of treating a subject with at least one of a gene deficiency, a protein deficiency, a DNA repair deficiency, dysregulated apoptosis, a recombination deficiency, a replication deficiency, a cell proliferation disorder, dysregulated transcription, loss of function of a tumor suppressor gene, a ubiquitin disorder, cell cycle dysregulation and/or dysregulation of translesion synthesis, comprising administering to the subject a therapeutically effective amount of one or more non-covalent DNA binding agents, as the only active agents, or in combination with one or more anti-cancer and/or anti-inflammatory active agents.
In one embodiment, the DNA repair deficiency is at least one of: DNA mismatch repair (MMR) deficiency, base excision repair (BER) deficiency, nucleotide excision repair (NER) deficiency, recombinational repair deficiency, homologous recombination repair (HRR) deficiency, non-homologous end joining (NHEJ) deficiency, a deficiency in the repair of double stranded breaks, and a deficiency in the repair of chromosomal damage.
The invention also provides for novel compositions and methods of treating a subject with cancer or inflammation, comprising: identifying a subject in need of treatment; administering to the subject a therapeutically effective amount of one or more non-covalent DNA binding agents, as the only active agents, or in combination with one or more anti-cancer and/or anti-inflammatory active agents; wherein following the administration, there is inhibition of inflammation or growth of a cancer cell.
In one embodiment the identification step comprises determining whether the patient has a mutation in at least one of a gene selected from the group consisting of: PTEN, p53, BRCA1, BRCA2, MLH1, PMS1, PMS2, MSH2, MSH6, REVS, XRCC1, XRCC2, XRCC3, RAD51, RAD52, REV, ATM, ATR, and the MRE1/RPA1/RAD51 complex.
The invention also provides for novel compositions and methods of treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of one or more non-covalent DNA binding agents, as the only agent agents, or in combination with one or more anti-cancer active agents, wherein following the administration, there is inhibition of growth of a cancer cell.
In one embodiment, the subject has a loss of function of at least one tumor suppressor gene.
In another embodiment, at least one tumor suppressor gene and/or the gene pathway is selected from the group consisting of: PTEN, p53, BRCA1, BRCA2, MLH1, PMS1, PMS2, MSH2, MSH6, REV3, XRCC1, XRCC2, XRCC3, RAD51, RAD52, REV, ATM, ATR, K-Ras, BRAF and the MRE1/RPA1/RAD51 complex.
In another embodiment, the subject has a DNA mismatch repair gene or pathway deficiency.
In another embodiment, the subject does not have a DNA mismatch repair gene or gene pathway deficiency i.e. the subject has no loss of function in DNA mismatch repair.
In another embodiment, the cancer is mutant K-ras positive or has mutations in the K-Ras pathway.
In another embodiment the cancer is has wild-type K-ras and no mutations in the K-Ras signaling pathway.
In another embodiment, the identification step comprises determining the response of a patient to a therapy for treating cancer.
In another embodiment, the identification step is reported to the subject and/or a health care professional.
In another embodiment, the non-covalent DNA binding agent binds to the minor groove of DNA.
In another embodiment, the non-covalent DNA binding agent binds to a “G-C rich” region of the minor groove.
In another embodiment, the subject has a mutation in at least one of a gene or gene pathway selected from the group consisting of: PTEN, p53, BRCA1, BRCA2, MLH1, PMS1, PMS2, MSH2, MSH6, REV3, XRCC1, XRCC2, XRCC3, RAD51, RAD52, REV, ATM, ATR, K-Ras, BRAF and the MRE1/RPA1/RAD51 complex.
In another embodiment the patient cannot be treated by other therapies i.e. the tumor is refractory or resistant to available therapies.
In another embodiment, the cancer is selected from the group consisting of: breast cancer, colorectal cancer, leukemia, non-small cell lung cancer, ovarian cancer, renal cancer, melanoma, prostate cancer and CNS-cancers. The cancer may be a primary cancer or a metastatic cancer.
In another embodiment, the cancer is triple negative breast cancer.
In another embodiment, the cancer is MMR-deficient colorectal cancer.
In another embodiment, the cancer is glioblastoma.
In another embodiment, the novel composition comprises the non-covalent DNA binding agent or the pharmaceutically acceptable salt or prodrug thereof.
In another embodiment, the subject is a mammal.
In another embodiment, the subject is a human.
In another embodiment, the therapeutically effective amount of one or more non-covalent DNA binding agent is in the range of 0.001 mg to 1000 mg per subject.
In another embodiment, the administration step comprises administering one or more non-covalent DNA binding agent to the subject in accordance with a daily treatment regimen.
In another embodiment the administration step comprises administering one or more non-covalent DNA binding agent as a pharmaceutical formulation.
In another embodiment, the pharmaceutical formulation is a bioequivalent formulation of one or more non-covalent DNA binding agent.
In another embodiment, the pharmaceutical formulation is a pharmaceutically equivalent formulation.
In another embodiment, the pharmaceutical formulation is a therapeutically equivalent formulation.
The invention also provides for a novel, composition of packaged pharmaceutical comprising one or more non-covalent DNA binding agents or pharmaceutically acceptable salt or prodrug thereof, which, upon administration to a subject, inhibits the growth of a cancer cell.
The invention also provides for a novel composition of packaged pharmaceutical comprising: one or more non-covalent DNA binding agents or pharmaceutically acceptable salt or prodrug thereof; and associated instructions for using the non-covalent DNA binding agent(s) to treat cancer.
In one embodiment, one or more of the non-covalent DNA binding agent is present as a pharmaceutical composition comprising a therapeutically effective salt or prodrug thereof and a pharmaceutically acceptable carrier.
In another embodiment, the packaged pharmaceutical further comprises in the instructions a step of identifying a subject in need of such pharmaceutical.
In another embodiment, the packaged pharmaceutical further comprises in the instructions a step of identifying one or more non-covalent. DNA binding agent and one or more anticancer agent as capable of inhibiting the growth of a cancer cell.
In another embodiment, the invention provides for a novel composition of packaged pharmaceutical for administration to a subject comprising: one or more non-covalent DNA binding agents, as the only active agents, or in combination with one or more anti-cancer and/or anti-inflammatory active agents; a test for determining if the subject has a mutation in at least one of a gene; associated instructions for performing the test; and associated instructions for using the non-covalent DNA binding agent to treat cancer and/or inhibit inflammation.
In one embodiment, the gene or gene pathway is selected from the group consisting of: PTEN, p53, BRCA1, BRCA2, MLH1, PMS1, PMS2, MSH2, MSH6, REV3, XRCC1, XRCC2, XRCC3, RAD51, RAD52, REV, ATM, ATR, K-Ras, BRAF and the MRE1/RPA1/RAD51 complex.
The invention provides for novel compositions and methods of inhibiting the growth of a cancer cell comprising administering to the subject a non-covalent DNA binding agent.
In one embodiment, the cancer cell comprises a mutation in at least one of a gene or gene pathway selected from the group consisting of: PTEN, p53, BRCA1, BRCA2, MLH1, PMS1, PMS2, MSH2, MSH6, REV3, XRCC1, XRCC2, XRCC3, RAD51, RAD52, REV, ATM, ATR, K-Ras, BRAF and the MRE1/RPA1/RAD51 complex.
In another embodiment, the non-covalent DNA binding agent binds to the minor groove.
In another embodiment, the non-covalent DNA binding agent binds to a GC rich region of the minor groove.
In another embodiment the subject has a mutation in at least one of a gene or gene pathway selected from the group consisting of: PTEN, p53, BRCA1, BRCA2, MLH1, PMS1, PMS2, MSH2, MSH6, REV3, XRCC1, XRCC2, XRCC3, RAD51, RAD52, REV, ATM, ATR, K-Ras, BRAF and the MRE1/RPA1/RAD51 complex.
Methods are provided for the synthesis of poly(ethylene glycol) (“PEG”) conjugates of non-covalent DNA binding agents of the invention, which conjugates retain unusually high biological potency. Also provides are novel poly(ethylene glycol) (“PEG”) conjugates of non-covalent DNA binding agents of the invention and compositions thereof. Preparation of the pegylated conjugates according to the methods of the present invention reduces or avoids steric inhibition of receptor-ligand interactions that may result from the attachment of PEG to a polypeptide of small molecule of interest. The conjugates of the present invention retain a high level of biological potency compared to those produced by traditional PEG coupling methods that are not targeted to avoid receptor-binding domains of cytokines. The biological potency of the PEG conjugates of non-covalent DNA binding agents of the invention may be higher than that of unconjugated non-covalent DNA binding agents of the invention. The conjugates of the present invention may have an extended half-life in vivo compared to the corresponding unconjugated agents of the invention. The present invention also provides kits comprising such conjugates and/or compositions, and methods of use of such conjugates and compositions in a variety of diagnostic, prophylactic and therapeutic applications.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 presents the effects of non-covalent DNA binding agents in osteosarcoma U20S cells.
FIG. 2 presents the effects of non-covalent DNA binding agents in PTEN-deficient lymphoblastoid CEM cells.
FIG. 3 presents the effects of non-covalent DNA binding agents in leukemia (CEM) cells with PTEN (homologous recombination deficiency).
FIG. 4 presents the effects of non-covalent DNA binding agents in genetically resistant breast cancer cells (MDA-MB-468) cells with deficiencies in PTEN and epigenetic DNA mismatch repair mutations.
FIG. 5 presents the effects of non-covalent DNA binding agents in p53-deficient H1299 cells.
FIG. 6 presents the effects of non-covalent DNA binding agents in colorectals cells with (A) normal (SW403) or (B) mutated (SW480) kras.
FIG. 7 presents the effects of non-covalent DNA binding agents in colorectal cancer cells with (A) mutated kras or (B) mutated kras and having a mismatch repair (MMR) deficiency.
FIG. 8 shows that non-covalent DNA binding agents ((A) 723734 and (B) 726260), are synthetic lethal with homologous recombination repair deficiencies.
FIG. 9 presents the results of a comparison of the activity of non-covalent DNA binding agents in U20S cells wherein MMR, p53 and REV functions have been inhibited using RNAi methods (A) NSC 718813; (B) NSC 723734; (C) NSC 726260.
FIG. 10 presents the results of a comparison of the activity of non-covalent DNA binding agents in isogenic p53-deficient HI299 cells wherein MMR functions have been inhibited using RNAi methods (A) NSC 718813; (B) NSC 723734.
FIG. 11 presents the results of a comparison of the activity of non-covalent DNA binding agents in isogenic MMR-deficient HCTI 16 cells wherein p53 and REV functions have been inhibited using RNAi methods (A) NSC 718813; (B) NSC 723734; (C) NSC 726260; (D) camptothecin.
FIG. 12 presents a comparison of the activity of non-covalent DNA binding agents in p53, mlh1 and rev deficient U20S cells.
FIG. 13 presents the amino acid sequence (A) (SEQ ID NO:1) and the nucleic acid sequence (B) (SEQ ID NO:2) of TP53.
FIG. 14 presents the amino acid sequence (A) (SEQ ID NO:3) and the nucleic acid sequence (B) (SEQ ID NO:4) of MLH1.
FIG. 15 presents the amino acid sequence (A) (SEQ ID NO:5) and the nucleic acid sequence (B) (SEQ ID NO:6) of MSH2.
FIG. 16 presents the amino acid sequence (A) (SEQ ID NO:7) and the nucleic acid sequence (B) (SEQ ID NO:8) of BRCA1.
FIG. 17 presents the amino acid sequence (A) (SEQ ID NO:9) and the nucleic acid sequence (B) SEQ ID NO:10) of REV3L.
FIG. 18 presents the amino acid sequence (A) (SEQ ID NO:11) and the nucleic acid sequence (B) (SEQ ID NO:12) of PARP1.
FIG. 19 presents the amino acid sequence (A) (SEQ ID NO:13) and the nucleic acid sequence (B) (SEQ ID NO:14) of RAD51.
FIG. 20 presents the amino acid sequence (A) (SEQ ID NO:15) and the nucleic acid sequence (B) (SEQ ID NO:16) of MRE11A.
FIG. 21 presents the amino acid sequence (A) (SEQ ID NO:17) and the nucleic acid sequence (B) (SEQ ID NO:18) of ATM.
FIG. 22 presents the amino acid sequence (A) (SEQ ID NO:19) and the nucleic acid sequence (B) (SEQ ID NO:20) of ATR.
FIG. 23 presents the amino acid sequence (A) (SEQ ID NO:21) and the nucleic acid sequence (B) (SEQ ID NO:22) of PTEN.
FIG. 24 presents the amino acid sequence (A) (SEQ ID NO:23) and the nucleic acid sequence (B) (SEQ ID NO:24) of ERCC1.
FIG. 25 presents the amino acid sequence (A) (SEQ ID NO:25) and the nucleic acid sequence (B) (SEQ ID NO:26) of BRCA2.
FIG. 26 presents the amino acid sequence (A) (SEQ ID NO:27) and the nucleic acid sequence (B) (SEQ ID NO:28) of XRCC1.
FIG. 27 presents the amino acid sequence (A) (SEQ ID NO:29) and the nucleic acid sequence (B) (SEQ ID NO:30) of KRAS.
FIG. 28 presents the amino acid sequence (A) (SEQ ID NO:31) and the nucleic acid sequence (B) (SEQ ID NO:32) of BRAF.
FIG. 29 presents the amino acid sequence (A) (SEQ ID NO:33) and the nucleic acid sequence (B) (SEQ ID NO:34) of RAD50.
FIG. 30 presents the amino acid sequence (A) (SEQ ID NO:35) and the nucleic acid sequence (B) (SEQ ID NO:36) of RAD51.
FIG. 31 shows a line graph of the combination effect of NSC 718813 and Vinblastin in MDA-MB-231.
FIG. 32 shows a line graph of the combination effect of NSC 718813 and 5-fluorouracil (5-FU) in MDA-MB-231.
FIG. 33 shows a line graph of the combination effect of NSC718813 with Vinblastin in MDA-MB-468.
FIG. 34 shows a line graph of the combination effect of NSC718813 with Trichostatin in MDA-MB-468.
FIG. 35 shows a line graph of the combination effect of NSC718813 with Camptothecin in MDA-MB-468.
FIG. 36 shows a line graph of the combination effect of NSC718813 with Cyclohexamide in MDA-MB-468.
FIG. 37 shows a line graph of the combination effect of NSC718813 with Mitomycin in MDA-MB-468.
FIG. 38 shows a line graph of the combination effect of NSC718813 with Doxorubicin in MDA-MB-468.
FIG. 39 shows a line graph of the combination effect of NSC718813 with Gefitinib in MDA-MB-468.
FIG. 40 shows a line graph of the combination effect of NSC718813 with 5FU in MDA-MB-468.
FIG. 41 shows a line graph of Trichostatin in CEM cells.
FIG. 42 shows a line graph of the combination effect of NSC 718813 with Cyclohexamide in CEM cells.
FIG. 43 shows a line graph of the combination effect of NSC 718813 with Vinblastin in CEM cells.
FIG. 44 shows a line graph of the combination effect of NSC 718813 with Mitomycin in CEM cells.
FIG. 45 shows a line graph of the combination effect of NSC 718813 with Doxorubicin in CEM cells.
FIG. 46 shows line graphs of 172Tag. FIG. 46A shows NSC 718813 with Paclitaxel in 172Tag. FIG. 46B shows NSC 718813 with Camptothecin in 172Tag. FIG. 46C shows the effect of NSC 718813 with Doxorubicin in MMR deficient cells (172Tag). FIG. 46D shows the combination effect of NSC 718813 with Trichostatin in MMR deficient cell line (172Tag).
FIG. 47 shows line graphs of 172Tag. FIG. 47A shows the effect of NSC 718813 with Mitomycin C in MMR deficient cell line (172Tag). FIG. 47B shows the combination with NSC 718813 and Actinomycin D in MMR deficient cells (172Tag).
FIG. 48 shows line graphs of HeLa. FIG. 48A shows the effect of NSC 718813 with Camptothecin in MMR proficient cells (HeLa). FIG. 48B shows the effect of NSC 718813 with Cyclohexamide in MMR proficient cells (HeLa). FIG. 48C shows the effect of NSC 718813 with Mitomycin C in MMR proficient cells (HeLa). FIG. 48D shows the effect of NSC 718813 with Vinblastine in MMR deficient cells (HEK293T).
FIG. 49 shows line graphs of 293T. FIG. 49A shows the combination effect of NSC 718813 with Mitomycin C in MMR deficient cells (HEK293T). FIG. 49B shows the combination effect of NSC 718813 with Paclitaxel in MMR deficient cells (HEK293T). FIG. 49C shows the combination effect of NSC 718813 with Vincristine in MMR deficient cells (HEK293T). FIG. 49D shows the combination effect of NSC 718813 with Actinomycin in MMR deficient cells (HEK293T).
FIG. 50 shows line graphs of MCF7. FIG. 50A shows NSC 718813 with Doxorubicin in MCF7. FIG. 50B shows NSC 718813 with Paclitaxel in MCF7.
FIG. 51 shows line graphs of CEM. FIG. 51A shows the combination effect in CEM cells NSC 718813 with Vinblastin. FIG. 51B shows cyclohexamide. FIG. 51C shows Trichostatin. FIG. 51D shows Mitomycin C.
FIG. 52 shows line graphs of SW403. FIG. 52A shows NSC 718813 with Vinblastin in SW403. FIG. 52B shows NSC 718813 with camptothecin in SW403. FIG. 52C shows NSC 718813 with Trichostatin in SW403. FIG. 52D shows NSC 718813 with cyclohexamide in SW403.
FIG. 53 shows line graphs of SW403. FIG. 53A shows NSC 718813 with Mitomycin in SW403. FIG. 53B shows NSC 718813 with Doxorubicin in SW403. FIG. 53C shows NSC 718813 with Paclitaxel in SW403. FIG. 53D shows NSC 718813 with actinomycin in SW403.
FIG. 54 shows line graphs of SW403. FIG. 54A shows NSC 718813 with olaparib in SW403. FIG. 54B shows NSC 718813 with Oxaliplatin in SW403. FIG. 54C shows NSC 718813 with Gefitinib in SW403. FIG. 54D shows NSC 718813 with 5FU in SW403.
FIG. 55 shows line graphs of MDA 231. FIG. 55A shows NSC 718813 with Vinblastin in MDA 231. FIG. 55B shows NSC 718813 with Cyclohexamide in MDA-MB-231. FIG. 55C shows NSC 718813 with Trichostatin in MDA-MB-231. FIG. 55D shows NSC 718813 with Mitomycin in MDA-MB-231.
FIG. 56 shows line graphs of MDA-MB-231. FIG. 56A shows NSC 718813 with Paclitaxel in MDA-MB-231. FIG. 56B shows NSC 718813 with Vincristin in MDA-MB-231. FIG. 56C shows NSC 718813 with Doxorubicin in MDA-MB-231. FIG. 56D shows NSC 718813 with 6TG in MDA-MB-231.
FIG. 57 shows line graphs of MDA-MB-231. FIG. 57A shows NSC 718813 in Olaparib in MDA 231. FIG. 57B shows NSC 718813 with Oxaliplatin in MDA-MB-231. FIG. 57C shows NSC 718813 with Gefitinib in MDA-MB-231.
FIG. 58 shows line graphs of MDA-MB-468. FIG. 58A shows NSC 718813 with Vinblastin in MDA-MB-468. FIG. 58B shows NSC 718813 with Camptothecin in MDA-MB-468. FIG. 58C shows NSC 718813 with Trichostatin in MDA-MB-468. FIG. 58D shows NSC 718813 with Cyclohexamide in MDA-MB-468.
FIG. 59 shows line graphs of MDA-MB-468. FIG. 59A shows NSC 718813 with Mitomycin in MDA-MB-231. FIG. 59B shows NSC 718813 with Doxorubicin in MDA-MB-468. FIG. 59C shows NSC 718813 with Paclitaxel in MDA-MB-468. FIG. 59D shows NSC 718813 with Olaparib in MDA-MB-468.
FIG. 60 shows line graphs of MDA-MB-468-468. FIG. 60A shows NSC 718813 with Gefitinib in MDA-MB-468. FIG. 60B shows NSC 718813 with Oxaliplatin in MDA-MB-468. FIG. 60C shows NSC 718813 with Erlonitib in MDA-MB-468.
FIG. 61 shows line graphs of U2OS. FIG. 61A shows NSC 718813 with Olaparib in U2OS. FIG. 61B shows NSC 718813 with Erlonitib in U2OS. FIG. 61C shows NSC 718813 with Gefitinib in U2OS. FIG. 61D shows NSC 718813 with Oxaliplatin in U2OS. FIG. 61E shows NSC 718813 with 5FU in U2OS.
FIG. 62 shows line graphs of SW620. FIG. 62A shows NSC 718813 with Olaparib in SW620. FIG. 62B shows effects of NSC 718813 with Oxaliplatin. FIG. 62C shows NSC 718813 with Gefitinib in SW620. FIG. 62D shows combination SW620 (NSC 718813 with 5FU).
FIG. 63 shows line graphs of representative NSC 718813 (A) effects in tumor cells in the NCI-60 in vitro evaluation.
FIG. 64 shows line graphs of representative NSC 723734 (B) effects in tumor cells in the NCI-60 in vitro evaluation.
FIG. 65 shows line graphs of representative NSC 723732 (C) effects in tumor cells in the NCI-60 in vitro evaluation.
FIG. 66 shows line graphs of representative NSC 726260 (D) effects in tumor cells in the NCI-60 in vitro evaluation.
FIG. 67 shows line graphs of colorectal cancer cells with competent DNA mismatch repair (MMR) are more sensitive to novel PBDs if they also carry mutant K-ras.
FIG. 68 shows line graphs of PBDs that show more potent growth inhibition in K-ras mutant colorectal cancer cells that are DNA mismatch repair (MMR) deficient.
FIG. 69 shows line graphs of breast cancer cells with BRCA/p53 deficiency (MCF-7) that have similar susceptibility to novel PBDs to those breast cancer cells with DNA MMR deficiency (MDA-MB-231).
FIG. 70 shows a line graph of breast cancer cells (MDA-MB-468) with loss of function in PTEN and mlh1 hypermethylation (deficient DNA mismatch repair) that are more susceptible to novel IndUS PBDs.
FIG. 71 shows line graphs of novel IndUS PBDs that are very potent in leukemia cells (CEM) that have loss of function in DNA MMR and PTEN compared to that in MSH2 deficient Jurkat lymphoma cells.
FIG. 72 shows line graphs of novel PBDs that show better potency in growth inhibition of p53-deficient H1299 compared to MMR competent A549 lung cancer cells.
FIG. 73 shows a table and line graphs of comparison of activity of IndUS PBDs in Isogenic U2OS with RNAi knockdowns of MMR, p53 and REV3 functions.
FIG. 74 shows bar graphs of IndUS PBDs showing synthetic lethality as monotherapy in U2OS cells using RNAi knockdown of DNA mismatch repair (MMR), apoptosis (p53) and homologous recombination/translesional synthesis (REV3) genes.
FIG. 75 is a table showing novel PBDs showing synthetic lethality in tumor cells that have loss of DNA mismatch repair (MMR) and/or apoptosis (p53).
FIG. 76 shows line graphs showing lead IndUS PBD compounds having excellent PK with long half-life in rats.
FIG. 77 shows a line graph of intravenous and intraperitoneally administered NSC723734 showing dose-dependent reduction in SW620 colon tumor xenograft.
FIG. 78 shows a line graph of intraperitoneal NSC723734 showing superior activity to NSC718813 in SW620 colon tumor xenograft model following once daily administration for 7 days.
FIG. 79 shows a line graph of NSC718813 that reduces tumor burden in SW620 colon tumor xenograft model following a Q1 Dx5 IV followed by Q4Dx3 IP administration.
FIG. 80 shows a line graph of NSC726260 showing limited pharmacological activity in SW620 colon tumor xenograft model following combined IV and IP administration.
FIG. 81 shows a line graph of NSC723734 showing excellent synergy with cisplatin following intermittent IP administration of the two drugs in SW620 colon tumor xenograft mouse model.
FIG. 82 shows a line graph of NSC723734 that is synergistic with cisplatin and restores antitumor activity of cisplatin at a lower (minimally active) cisplatin dose following intermittent IP administration in SW620 colon tumor xenograft model in mice.
FIG. 83 shows line graphs of quantitative analysis of in vivo SW620 colon tumor xenograft data showing that NSC723734 is synergistic with cisplatin at combination doses achieving >50% efficacy.
FIG. 84 shows line graphs of quantitative analysis of in vivo effects of NSC723734 and cisplatin results in significant dose-reduction index (DRI) supporting the mutual synergism in SW620 colon tumor xenograft mouse model.
FIG. 85 shows a table of novel IndUS anticancer PBDs that are significantly different compared to previously described DNA minor groove binders.



DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
As used herein, “at least one” is intended to mean “one or more” of the listed elements.
Singular word forms are intended to include plural word forms and are likewise used herein interchangeably where appropriate and fall within each meaning, unless expressly stated otherwise.
Except where noted otherwise, capitalized and non-capitalized forms of all terms fall within each meaning.
Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth used in the specification and claims are contemplated to be able to be modified in all instances by the term “about”.
All parts, percentages, ratios, etc. herein are by weight unless indicated otherwise.
As used herein, a “non-covalent DNA binding agent” means an agent that reacts with one or more different positions in a DNA molecule, wherein binding can result in the formation of crosslinkages, either in the same strand (intrastrand crosslink) or in the opposite strands of the DNA (interstrand crosslink). Non-covalent DNA binding agents can also cause interactions between DNA and proteins that are recruited by the DNA. For example, DNA replication is blocked by non-covalent DNA binding agents of the invention that modulate interactions between DNA and genes or proteins which subsequently cause replication arrest, cell cycle arrest and/or cell death if the crosslink is not repaired.
A non-covalent DNA binding agent reacts with DNA via non-covalent interactions, for example, hydrogen bonds, Coulombic interactions, ionic bonds, van der Waals forces, and/or hydrophobic interactions. Non-covalent DNA binding agents of the invention include, but are not limited to, the agents presented herein below. The invention provides for a non-covalent DNA binding agent that binds to the minor groove of DNA. A DNA molecule has two types of grooves, the major groove which has the nitrogen and oxygen atoms of the nucleotide base pairs pointing inward toward the helical axis, and the minor groove, wherein the nitrogen and oxygen atoms of the nucleotides point outwards. The major groove is 22 Å wide and the minor groove is 12 Å wide. The majority of currently available DNA damaging chemotherapeutic agents target the major groove of the DNA.
Most of the currently studied DNA minor groove binding agents target “AT rich” regions of DNA. The current invention provides novel non-covalently linked, DNA minor groove binding agents that target “G-C” rich” regions of the DNA. As used herein, “GC rich region” means between 25% and 80% of the human genome and regions of hundreds of kilobases, often referred to as the isochores, that have relatively homogenous base compositions (Fullerton, S. M., Carvalho, A. B. and Clark, A. G. Local rates of recombination are positively correlated with GC content in human genome. Mol Biol Evol 18(6): 1139-1142, 2001). “GC rich regions” are preferably between 35% and 75% GC, and more preferably between 45% and 75% GC and most preferably, between 60% and 70% GC. There is evidence that the longest eukaryotic exons and the longest prokaryotic genes are the most “GC-rich” Furthermore, the expected length for random reading frames is a function of the sequence GC content, i.e. the higher the GC content, the higher the probability for longer reading frames. On the other hand, the most GC-rich introns are the shorter ones and GC content has a greater effect on the reduction of intron length (Oliver, J. L. and Marin, A. A relationship between GC content and Coding-sequence length. J Mol Evol 43: 216-223, 1996).
As used herein, “DNA repair deficiency” refers to a decrease in the ability of a cell to repair DNA as compared to a wild type or control cell. A “DNA repair deficiency” can be genetic and/or epigenetic in nature (Loeb, L. A., Loeb, K. R. and Anderson, J. P. Multiple mutations and cancer. Proc Nat Acad Sci 100(3): 776-781, 2003; Jones, P. A. and Baylin, S. B. The fundamental role of epigenetic events in cancer. Nat Rev Genetics 3: 415-428, 2002). For instance, DNA repair deficiencies can result in “microsatellite instability”, a key feature of several cancers that are collectively referred to as Lynch tumors (Hewish, M., Lord, C. J., Martin, S. A., Cunningham, D. and Ashworth, A. Mismatch repair deficient colorectal cancer in the era of personalized treatment. Nat Rev Clin Oncol 7: 197-208, 2010). Further, a well defined subtype of colorectal cancer (CRC) is characterized by a deficiency in the mismatch repair (MMR) pathway. MMR deficiency not only contributes to the pathogenesis of a large proportion ( ̃70%) of colorectal cancer, but also determines the response of that subtype of colorectal cancer to many of the drugs that are frequently used to treat colorectal cancer.
A DNA repair deficiency can be determined by methods known in the art including but not limited to assays for microsatellite instability, for example by using a microsatellite instability test distributed by Roche (Cat. No. 12 041 901 00).
Assays for DNA mismatch repair tumors include but are not limited to those presented in Marcus et al., 1999 Am J Surg Pathol Oct: 23(10): 1248-55.
Although there are typical clinical and pathological features associated with MMR-deficiency phenotype in Lynch syndrome cancers, approximately 40% of the Lynch syndrome cases cannot be reliably diagnosed by morphological characteristics alone. A strong relationship exists between sporadic MMR deficiency colorectal cancer (dMMR CRC) and the CpG island methylator phenotype (CIMP) subtype of CRC. CIMP is characterized by regional hypermethylation of CpG islands in the DNA and thus results in the loss of functional MLH1 expression (Hewish et al., Nat Rev Clin Oncol 7: 197-208, 2010). The relationship of CpG island methylation to microsatellite instability can be used to describe the clinical and pathological features of CRC. Hypermethylation (epigenetic) changes of p16 and MLH1 can be determined by methylation-specific polymerase chain reaction (PCR). Methylation of MINT 1, 2, 12 and 31 loci can be assessed by bisulfite PCR. Microsatellite instability and K-ras and p53 status of patient cancer tissues can be assessed by microsatellite PCR, restriction enzyme-mediated PCR and/or immunohistochemistry (IHC) (Hawkins, N., Norrie, M, Cheong, K., Mokany, E., Ku, S-L., Meagher, A., O'Connor, T. and Ward, R. CpG island methylation in sporadic colorectal cancers and its relationship to microsatellite instability. Gastroenterology 122(5): 1376-1387, 2002).
As used herein, a “decrease” in the ability of a cell to repair DNA means that the cell repairs damaged DNA, either due to genetic or epigenetic mutations, such that the repaired DNA is less than 100% error free (for example, 99%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less). A cell that has a DNA repair deficiency also refers to a cell that cannot perform any DNA repair.
As used herein, a “decrease” in the ability of a cell to repair DNA means that the cell repairs damaged DNA at a rate that is less than the rate at which a wild type or control cell repairs DNA.
As used herein, “less than” as it refers to the rate of repair of DNA damage, means that the rate of repair of DNA damage is 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or more, lower than the rate of repair of DNA damage in a wild type or control cell. As used herein, “less than” as it refers to the rate of repair of DNA damage also means that the rate of repair of DNA damage in a cell is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less, lower than the rate of repair of DNA damage in a control or wild type cell.
As used herein, a “DNA repair deficiency” includes but is not limited to: base excision repair deficiency, a deficiency in the repair of double stranded breaks and a deficiency in the repair of chromosomal damage. DNA repair deficiencies can result from genetic changes such as mutated DNA mismatch repair genes like MSH2, Furthermore, DNA repair deficiencies can also include epigenetic changes such as hypermethylation of genes involved in DNA mismatch repair, recombination, replication and/or apoptosis. (Helleday, T., Petermann, E., Lundin, C., Hodgson, B and Sharma, R. A. DNA repair pathways as targets for cancer therapy. Nat Rev. Cancer 8: 193-204, 2008).
As used herein, “apoptosis” or “programmed cell death” refers to a mechanism whereby a cell undergoes death or destruction, for example, to control cell number and proliferation or in response to DNA damage. Many cancer cells do not undergo apoptosis and certain cancers involve an alteration in the apoptotic pathway.
As used herein, “dysregulated apoptosis” refers to a decrease in the ability of a cell to undergo apoptosis or a decrease in the number of cells that undergo apoptosis as compared to a wild type or control cell, for example apoptosis in response to DNA damage. For example, mutations in the p53 gene are a feature of 50% of all reported cancer cases. In the other 50% of cancer cases, the p53 gene is not itself mutated, but the p53-directed apoptosis pathway is partially inactivated (Check, C. F., Verma, C. S., Baselga, J. and Lane, D. P. Translating p53 into the clinic. Nat Rev Clin Oncol 8: 25-37, 2011). P53 protein is a transcription factor that controls the cellular response to stress signals through the induction of cell-cycle arrest, apoptosis and senescence. Apoptosis is detected by any one of the following assays including but not limited to DNA laddering, COMET assays and/or TUNEL staining.
As used herein, a “decrease” in the ability of a cell to undergo apoptosis means that within a population of cells, less than 100% (for example, 99%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less) of the cells undergo apoptosis, as compared to a wild type or control population of cells, for example, wherein 100% of the cells undergo apoptosis
A cell that has dysregulated apoptosis also refers to a cell that does not undergo apoptosis
As used herein, “dysregulated apoptosis” also means that a cell or population of cells undergoes apoptosis at a rate that is less than that of a wild type or control cell or a population thereof.
As used herein, “less than” as it refers to the rate of apoptosis, means 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or more, less than the rate at which a wild type or control cell or a population thereof, undergoes apoptosis. As used herein, “less than” as it refers to the rate of apoptosis also means that the rate of apoptosis is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less, than the rate of apoptosis of a control or wild type cell or a population thereof.
As used herein, a “recombination deficiency” refers to an abnormality in homologous recombination repair in a cell, as compared to a wild type or control cell. While DNA repair is essential for cells to maintain genomic stability, there is increasing evidence that defects in homologous recombination repair (HRR) underlie hereditary and sporadic tumorigenesis (Evers, B. Helleday, T. and Jonkers, J. Targeting homologous recombination repair defects in cancer. Trends Pharmacol Sci 31: 372-380, 2010). Deficiencies in HRR may determine the sensitivity of tumors to many currently available DNA-damaging anti-cancer agents. Furthermore, HRR-deficient tumors are also more susceptible to synthetic lethal interactions. More importantly, HRR-deficient tumors may also have an increased dependence on cell-cycle checkpoints, which could be exploited.
As used herein, a “replication deficiency” refers to an abnormality in DNA replication in a cell, as compared to a wild type or control cell.
A “replication deficiency” includes replication of damaged DNA as determined by, for example, a BrdU assay wherein the thymidine analog, 5-Bromo-2-deoxyuridine (BrdU), is added to the cell growth medium just prior to fixing and the cells are stained with an antibody to BrdU, which detects the thymidine analog in DNA.
A “replication deficiency” also includes replication of DNA prior to cell division.
As used herein, a “cell proliferation disorder” refers to an increase in the number of divisions that a cell undergoes as compared to a wild type or control cell.
A “cell proliferation disorder” also refers to an increase in the rate of cellular division as compared to a wild type or control cell.
A “cell proliferation disorder” also refers to an increase in the frequency of cell division as compared to a wild type or control cell.
A “cell proliferation disorder” also refers to unregulated cell division, for example, the inability of a cell to respond to signals that cause a wild type or control cell to stop dividing or start dividing.
A “cell proliferation disorder” also refers to the inability of a cell to enter senescence.
As used herein, “senescence” refers to a state wherein diploid cells lose the ability to divide.
A “cell proliferation disorder is detected by methods known in the art including but not limited to alamar blue assay, as described herein below.
As used herein, “dysregulated transcription” means transcription of damaged DNA as determined by, for example, real-time reverse transcription polymerase chain reaction (PCR), in vitro transcription methods well known in the art, S1 nuclease assays.
As used herein, a “tumor suppressor gene” includes but is not limited to p53, RB1, WT1, NF1, NF2, APC, TSC1, TSC2, DPC4, DCC, BRCA1, BRCA2, PTEN, STK11, MSH2, MLH1, CDH1, VHL, CDKN2A, PTCH and MEN1.
As used herein, “mutation” refers to a genetic or epigenetic change in phenotype or gene expression.
A “mutation” refers to a change in the genetic sequence, for example a substitution (transition or transversion), a deletion, an insertion (including a duplication) and a translocation.
A “mutation” also refers to a chromosomal rearrangement or a chromosomal translocation.
A “mutation” also refers to an epigenetic mutation or a heritable change in phenotype and or gene expression that occurs via a mechanism that does not require a change in the genetic sequence.
An epigenetic mutation can occur by a variety of mechanisms including but not limited to post-translational modification of amino acids encoding a histone protein, thereby resulting in chromatin remodelling, DNA methylation (hypermethylation or hypomethylation), production of alternate splice forms of RNA and formation of double stranded RNA.
A “mutation” according to the invention can result in a gain in function, a loss of function, an increase or decrease in expression, an increase or decrease in the rate of expression, expression of a defective mRNA and/or expression or translation of a defective protein.
A “function” as used herein includes but is not limited to DNA repair, apoptosis, recombination, replication, cell proliferation, transcription, ubiquitination, cell cycle regulation and translesion synthesis.
“Loss of function” refers to the inability of any cell to perform any of these functions due to any reasons including, but not limited to, mutations, gene silencing and post-translational modifications, that result in a reduction of these functions.
“Gain of function” refers to the increased activity of any cell to perform any of these functions due to any reasons including but not limited to, mutations, gene amplification, overexpression of gene product or proteins and post-translational modifications resulting in amplified activity of such functions.
As used herein, “dysregulation of translesion synthesis” means a decrease in the ability of a cell to undergo translesion synthesis as compared to a wild type or control cell.
As used herein, “translesion synthesis” refers to a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites. Translesion synthesis involves replacing the DNA polymerases that mediate DNA synthesis in the absence of DNA damage with specialized, translesion polymerase (i.e. DNA polymerase IV or V). In addition to replication functions, translesion synthesis is also involved in the homologous recombination repair pathways.
As used herein, “decrease” as it refers to translesion synthesis means that the level of translesion synthesis is 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or more, less than the level of translesion synthesis as compared to a wild type or control cell. As used herein, “decrease” as it refers to translesion synthesis also means that the level of translesion synthesis is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less lower than the level of translesion synthesis in a control or wild type cell.
A “decrease” in translesion synthesis also refers to a decrease in the rate of translesion synthesis as compared to a wild type or control cell.
As used herein, “decrease” as it refers to the rate of translesion synthesis, means 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or more, less than the rate of translesion synthesis in a wild type or control cell. As used herein, “decrease” as it refers to the rate of translesion synthesis also means that the rate is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less, than the rate of translesion synthesis in a control or wild type cell.
As used herein, a “control cell” or “wild type cell” means a cell that is derived from a subject that does not have at least one of a DNA repair deficiency, dysregulated apoptosis, a recombination deficiency, a replication deficiency, a cell proliferation disorder, dysregulated transcription, loss of function of a tumor suppressor gene, a ubiquitin disorder, cell cycle dysregulation and dysregulation of translesion synthesis.
A “control cell” or “wild type cell” also means a cell that is derived from a subject that does not have cancer or an inflammatory disease, and/or does not exhibit any detectable symptoms associated with the disease.
In certain embodiments, a “control cell” means a cell from a subject that has at least one of a DNA repair deficiency, an apoptosis deficiency, a recombination deficiency, a replication deficiency, a cell proliferation disorder, dysregulated transcription, loss of function of a tumor suppressor gene, a ubiquitin disorder, cell cycle dysregulation and dysregulation of translesion synthesis, prior to administration of a DNA binding agent of the invention.
In certain embodiments, a “control cell” means a cell from a subject that has been diagnosed with cancer, prior to administration of a non-covalent DNA binding agent of the invention.
In certain embodiments, a “control cell” means a cell from a subject that has been diagnosed with an inflammatory disease, prior to administration of a non-covalent DNA binding agent of the invention.
In certain embodiments, “patient” or “subject” refers to a mammal that is diagnosed with a disease, e.g., a cancer (including but not limited to cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary, and lymphatic organs; melanomas) an inflammatory disease (including but not limited to autoimmune diseases, such as systemic lupus, rheumatoid arthritis, and multiple sclerosis; graft rejections, such as renal transplant rejection, liver transplant rejection, lung transplant rejection, cardiac transplant rejection, and bone marrow transplant rejection; graft versus host disease) or an infection (including but not limited to bacterial infections, parasitic infections or viral infections. The term “patient” or “subject” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
As used herein, “mammal” refers to any mammal including but not limited to human, mouse, rat, sheep, monkey, dog, cat, goat, rabbit, hamster, horse, cow or pig.
A “non-human mammal”, as used herein, refers to any mammal that is not a human.
As used herein, “control subject” means a subject that does not have a disease, and/or does not exhibit any detectable symptoms associated with that disease, for example cancer or an inflammatory disease.
A “control subject” also means a subject that has a disease, prior to administration of a non-covalent DNA binding agent of the invention.
A “control subject” also means a subject that does not have at least one of a DNA repair deficiency, dysregulated apoptosis, a recombination deficiency, a replication deficiency, a cell proliferation disorder, dysregulated transcription, loss of function of a tumor suppressor gene, a ubiquitin disorder, cell cycle dysregulation and dysregulation of translesion synthesis.
A “control subject” also means a subject that has at least one of a DNA repair deficiency, dysregulated apoptosis, a recombination deficiency, a replication deficiency, a cell proliferation disorder, dysregulated transcription, loss of function of a tumor suppressor gene, a ubiquitin disorder, cell cycle dysregulation and dysregulation of translesion synthesis, prior to administration of a non-covalent DNA binding agent of the invention.
A “control subject” also means a subject that does not have a mutation in at least one of a gene or gene pathway selected from the group consisting of: PTEN, p53, BRCA1, BRCA2, MLH1, PMS1, PMS2, MSH2, MSH6, REV3, XRCC1, XRCC2, XRCC3, RAD51, RAD52, REV, ATM, ATR, K-Ras, BRAF and the MRE1/RPA1/RAD51 complex.
A “control subject” also means a subject has a mutation in at least one of a gene or gene pathway selected from the group consisting of: PTEN, p53, BRCA1, BRCA2, MLH1, PMS1, PMS2, MSH2, MSH6, REV3, XRCC1, XRCC2, XRCC3, RAD51, RAD52, REV, ATM, ATR, K-Ras, BRAF and the MRE1/RPA1/RAD51 complex, prior to administration or a non-covalent DNA binding agent of the invention.
“Treatment”, or “treating” as used herein, is defined as the application or administration of one or more non-covalent DNA binding agent and one or more anticancer or anti-inflammatory agent of the invention, for example, one or more non-covalent DNA minor groove binding agent of the invention, to a subject or patient, or application or administration of one or more non-covalent DNA binding agent and one or more anticancer or anti-inflammatory agent of the invention to an isolated tissue or cell line from a subject or patient, who has a disease, e.g., cancer or an inflammatory disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder. The term “treatment” or “treating” is also used herein in the context of administering agents prophylactically. The term “effective dose” or “effective amount” or “effective dosage” or “therapeutic dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The terms “therapeutically effective dose” and “therapeutically effective amount” are defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.
As used herein, “treating” a disease refers to preventing the onset of disease and/or reducing, delaying, or eliminating disease symptoms, such as an increase in the rate of growth or number of cancer cells. By “treating” is meant restoring the patient or subject to the basal state as defined herein, and/or to prevent a disease in a subject at risk thereof. Alternatively, “treating” means arresting or otherwise ameliorating symptoms of a disease.
“Treatment,” as used herein, includes any drug, drug product, method, procedure, lifestyle change, or other adjustment introduced in an attempt to effect a change in a particular aspect of a subject's health (i.e., directed to a particular disease, disorder, or condition).
As used herein, “inhibition” as it refers to growth of a cancer cell means a decrease in the rate of growth, or a decrease in the amount of growth.
For example, an inhibition of growth of a cancer cell means that the rate of growth of a cancer cell that has been treated with a non-covalent DNA binding agent of the invention is 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or more, less than that of a cancer cell that has not been treated with a non-covalent DNA binding agent of the invention. As used herein, “inhibition” as it refers to the rate of growth of a cancer cell that has been treated with a non-covalent DNA binding agent of the invention also means that the rate is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less, lower than the rate of growth of a cancer cell that has not been treated with a non-covalent DNA binding agent of the invention.
An inhibition of growth of a cancer cell also means that the number or growth of cancer cells that have been treated with a non-covalent DNA binding agent of the invention is 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or more, less than the number or growth of cancer cells that have not been treated with a non-covalent DNA binding agent of the invention. As used herein, “inhibition” as it refers to the rate of growth of a cancer cell also means that the number or growth of cancer cells that have been treated with a non-covalent DNA binding agent of the invention is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less, lower than the growth or number of cancer cells that have not been treated with a non-covalent DNA binding agent of the invention.
As used herein, “K-ras positive” means activating mutations including but not limited to, in the RAS oncogene (KRAS, HRAS and NRAS), PI3K, BRAF, MEK, ERK and MAPK pathways, that are frequent in human cancers. For example, KRAS mutations occur in 60% of pancreatic cancers, 32% of cancers of the large intestine and 17% of lung cancers (Karnoub, A. E. and Weinberg, R. A. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 9: 517-531, 2008). RAS family members signal through numerous effector molecules with diverse functions such as RAF/MAPK, PI3K and RAL proteins (Bommi-Reddy, A. and Kaelin, W. G. Slaying RAS with a synthetic lethal weapon. Cell Res 20: 119-121, 2010).
As used herein, “K-ras negative tumors” means tumors presenting with wild type K-ras. Similarly, “BRAF negative tumors” refers to tumors presenting with wild-type BRAF.
As used herein, a cancer that is “genetically resistant” means those cancers that have developed genetic and/or epigenetic mutations in oncogenes as well as tumor suppressor and DNA repair genes; thereby leading to the genesis of various cancers. Furthermore, those tumors that have loss of tumor suppressor gene function, resulting in dysregulation of DNA repair, recombination, replication, cell cycle regulation and/or apoptosis pathways, are also considered “genetically resistant”.
More specifically, “genetically-resistant” cancers are defined to include all those cancers that either have “functional loss of tumor suppressor genes”, and subtypes of cancers that are resistant to currently available anti-cancer agents. For example, such subtypes of “genetically resistant” cancers include, but are not limited to, metastatic colorectal cancer (mCRC) and other Lynch syndrome tumors, such as endometrial and bladder cancers, that have deficiencies in DNA mismatch repair pathways (dMMR tumors); p53-deficient and/or p53-pathway-deficient tumors; BRCA1 and/or BRCA2-mutated (i.e. homologous recombination repair deficient (dHRR)) tumors such as triple-negative breast cancer and basal-like breast cancer; and PTEN-deficient mCRC subtypes.
Furthermore, “genetically resistant” cancers are also defined to include ‘gain of function’ cancers with KRAS-mutator phenotype, such as mCRC and pancreatic cancers.
As used herein, “determining the response to a therapy for cancer” means comparing a parameter that is indicative of a response to treatment, for example tumor size, rate or growth or number of cancer cells, in a subject before receiving a particular therapy for cancer and after receiving a particular therapy for cancer. “Determining the response to a therapy for cancer” also means comparing a parameter that is indicative of a response to treatment, for example tumor size, rate of growth or number of cancer cells, in a subject that has received a therapy for cancer as compared to a subject that has not received a therapy for cancer. “Determining the response to a therapy for cancer” also means comparing a parameter that is indicative of a response to treatment, for example tumor size, rate of growth or number of cancer cells, in a subject that has received a therapy for cancer as compared to a control subject that has not been diagnosed with cancer and is not in need of cancer treatment.
As used herein, “cannot be treated” means that following receipt of a therapy for cancer there is no change in a parameter that is indicative of a response to treatment, for example tumor size, rate or growth or number of cancer cells, in a subject, as compared to the parameter before receiving the therapy for cancer. “Cannot be treated” also means that following receipt of a particular therapy for cancer, there is no change in a parameter that is indicative of a response to treatment, for example tumor size, rate of growth or number of cancer cells, in a subject that has received a therapy for cancer as compared to a subject that has not received a therapy for cancer. “Cannot be treated” also means that an individual cannot receive a therapy for cancer, for example due to an adverse reaction to the therapy or because they are receiving another treatment that makes it medically unadvisable, for example, due to a negative drug interaction.
“Gene,” as used herein, means a segment of DNA that contains information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other noncoding or untranslated regions that control gene expression.
The invention contemplates novel compositions and methods of treating a subject who has either failed to respond to prior therapy or has been diagnosed with mutations that would render the treatment regimens ineffective based on existing knowledge among those skilled in treatment of cancers. Both cases would result in “refractory” tumors. Such ‘refractory’ tumors would be candidates to receive treatment comprising administering to the subject, a therapeutically effective amount of one or more non-covalent DNA binding agent and one or more available anticancer or anti-inflammatory agents of the invention, for example, one or more DNA minor groove binding agent, either alone or in combination with one or more anti-cancer agents.
As used herein, prior treatment or therapy as it applies to cancer treatment includes but is not limited to surgery, radiotherapy (for example, gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes), endocrine therapy, biologic response modifiers (for example, interferons, interleukins, antibodies, aptamers, siRNAs, oligonucleotides, enzyme, ion channel and receptor inhibitors or activators), hyperthermia and cryotherapy, agents to attenuate any adverse effects (e.g., antiemetics), and other approved chemotherapeutic drugs, including, but not limited to, alkylating drugs (e.g., mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites (e.g., Methotrexate), purine antagonists and pyrimidine antagonists (e.g., 6-Mercaptopurine, 5-Fluorouracil, Cytarabile, Gemcitabine), spindle poisons (e.g., Vinblastine, Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (e.g., Etoposide, Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (e.g., Carmustine, Lomustine), inorganic ions (e.g., Cisplatin, Carboplatin), enzymes (e.g., Asparaginase), and hormones (e.g., Tamoxifen, Leuprolide, Flutamide, and Megestrol).
A method of “administration” useful according to the invention includes but is not limited to intravenous, subcutaneous, intramuscular, intraperitoneal, intracranial and spinal injection, ingestion via the oral route, inhalation, trans-epithelial diffusion (such as via a drug-impregnated, adhesive patch), by the use of an implantable, time-release drug delivery device, which may comprise a reservoir of exogenously-produced agent or may, instead, comprise cells that produce and secrete the therapeutic agent or topical application or administration directly to a blood vessel, including artery, vein or capillary, intravenous drip or injection. Additional methods of administration are provided herein below in the section entitled “Dosage and Administration.”
A “therapeutically effective amount” of a non-covalent DNA binding agent, according to the invention is in the range of 0.001 mg-1000 mg per subject. In another embodiment, a “therapeutically effective amount” of a non-covalent DNA binding agent according to the invention is in the range of 0.01 mg to 100 mg per subject. In another embodiment, a “therapeutically effective amount” of a non-covalent DNA binding agent according to the invention is in the range of 0.1 mg to 10 mg per subject.
As used herein, “basal state” refers to an individual who does not have a disease, e.g., cancer or an inflammatory disorder.
A subject who “does not have a disease” has no detectable symptoms of the disease.
As used herein, “diagnosing” or “identifying a patient or subject having” refers to a process of determining if an individual is afflicted with a disease or ailment, for example cancer as defined herein. Methods well known and accepted in the art are used to diagnose any of the cancers recited herein.
“Cancer” refers to any one of cancer, tumor growth, cancer of the colon, breast, bone, brain and others (e.g., osteosarcoma, neuroblastoma, colon adenocarcinoma), chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), cardiac cancer (e.g., sarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma); lung cancer (e.g., bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma); various gastrointestinal cancers (e.g., cancers of esophagus, stomach, pancreas, small bowel, and large bowel); genitourinary tract cancer (e.g., kidney, bladder and urethra, prostate, testis; liver cancer (e.g., hepatoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bone cancer (e.g., osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma, multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma, benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors); cancers of the nervous system (e.g., of the skull, meninges, brain, and spinal cord); gynecological cancers (e.g., uterus, cervix, ovaries, vulva, vagina); hematologic cancer (e.g., cancers relating to blood, Hodgkin's disease, non-Hodgkin's lymphoma); skin cancer (e.g., malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis); and cancers of the adrenal glands (e.g., neuroblastoma).
An “inflammatory disorder” includes any one or more of the following: autoimmune diseases or disorders: diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis; systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, including keratoconjunctivitis sicca secondary to Sjögren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens Johnson syndrome, idiopathic sprue, lichen planus, Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.
“Inflammatory disorder” also includes any one of rheumatoid spondylitis; post ischemic perfusion injury; inflammatory bowel disease; chronic inflammatory pulmonary disease, eczema, asthma, ischemia/reperfusion injury, acute respiratory distress syndrome, infectious arthritis, progressive chronic arthritis, deforming arthritis, traumatic arthritis, gouty arthritis, Reiter's syndrome, acute synovitis and spondylitis, glomerulonephritis, hemolytic anemia, aplastic anemia, neutropenia, host versus graft disease, allograft rejection, chronic thyroiditis, Graves' disease, primary binary cirrhosis, contact dermatitis, skin sunburns, chronic renal insufficiency, Guillain-Barre syndrome, uveitis, otitis media, periodontal disease, pulmonary interstitial fibrosis, bronchitis, rhinitis, sinusitis, pneumoconiosis, pulmonary insufficiency syndrome, pulmonary emphysema, pulmonary fibrosis, silicosis, or chronic inflammatory pulmonary disease.
As used herein, the term “pharmaceutically acceptable salt” refers to those salts of the compounds formed by the process of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Examples of pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
As used herein, “bioequivalence” or “bioequivalent”, refers to non-covalent DNA binding agents or drug products of the agents of the invention, which are pharmaceutically equivalent, and their bioavailabilities (rate and extent of absorption) after administration in the same molar dosage or amount are similar to such a degree that their therapeutic effects, as to safety and efficacy, are essentially the same. In other words, bioequivalence or bioequivalent means the absence of a significant difference in the rate and extent to which the non-covalent DNA binding agent becomes available from such formulations at the site of action when administered at the same molar dose under similar conditions, e.g., the rate at which a non-covalent DNA binding agent can leave such a formulation and the rate at which it can be absorbed and/or become available at the site of action to affect cancer. In other words, there is a high degree of similarity in the bioavailabilities of two non-covalent DNA binding agent pharmaceutical products (of the same galenic form) from the same molar dose, that are unlikely to produce clinically relevant differences in therapeutic effects, or adverse reactions, or both. The terms “bioequivalence”, as well as “pharmaceutical equivalence” and “therapeutic equivalence” are also used herein as defined and/or used by (a) the FDA, (b) the Code of Federal Regulations (“C.F.R.”), Title 21, (c) Health Canada, (d) European Medicines Agency (EMEA), and/or (e) the Japanese Ministry of Health and Welfare.
Thus, it should be understood that the present invention contemplates novel compositions of one or more non-covalent DNA binding agent formulations, as the only active agents, or in combination with one or more anti-cancer or anti-inflammatory active agents or drug products that may be bioequivalent to other non-covalent DNA binding agent and anti-cancer or anti-inflammatory formulations or drug products of the present invention. By way of example, a first non-covalent DNA binding agent formulation or drug product is bioequivalent to a second non-covalent DNA binding agent formulation or drug product, in accordance with the present invention, when the measurement of at least one pharmacokinetic parameter(s), such as a Cmax, Tmax, AUC, etc., of the first non-covalent DNA binding agent formulation or drug product varies by no more than about ±25%, when compared to the measurement of the same pharmacokinetic parameter for the second non-covalent DNA binding agent formulation or drug product.
As used herein, “bioavailability” or “bioavailable”, means generally the rate and extent of absorption of a non-covalent DNA binding agent into the systemic circulation and, more specifically, the rate or measurements intended to reflect the rate and extent to which a non-covalent DNA binding agent becomes available at the site of action or is absorbed from a drug product and becomes available at the site of action. In other words, and by way of example, the extent and rate of absorption of a non-covalent DNA binding agent from a formulation of the present invention as reflected by a time-concentration curve of the non-covalent DNA binding agent in systemic circulation.
With respect to absolute bioavailability, absolute bioavailability compares the bioavailability (estimated as area under the curve, or AUC) of the active drug in systemic circulation following non-intravenous administration (i.e., after oral, rectal, transdermal, subcutaneous administration), with the bioavailability of the same drug following intravenous administration. It is the fraction of the drug absorbed through non-intravenous administration compared with the corresponding intravenous administration of the same drug. The comparison must be dose normalized if different doses are used; consequently, each AUC is corrected by dividing the corresponding dose administered.
As used herein, the terms “pharmaceutical equivalence” or “pharmaceutically equivalent”, refer to non-covalent DNA binding agent formulations or drug products of these agents that contain the same amount of non-covalent DNA binding agent, in the same dosage forms, but not necessarily containing the same inactive ingredients, for the same route of administration and meeting the same or comparable compendial or other applicable standards of identity, strength, quality, and purity, including potency and, where applicable, content uniformity and for stability. Thus, it should be understood that the present invention contemplates non-covalent DNA binding agent formulations or drug products that may be pharmaceutically equivalent to other non-covalent DNA binding agent formulations or drug products used in accordance with the present invention.
As used herein, the terms “therapeutic equivalence or therapeutically equivalent”, mean those non-covalent DNA binding agent formulations or drug products which (a) will produce the same clinical effect and safety profile when utilizing a non-covalent DNA binding agent drug product to treat a disease, for example cancer, in accordance with the present invention and (b) are pharmaceutical equivalents, e.g., they contain the non-covalent DNA binding agent in the same dosage form, they have the same route of administration; and they have the same non-covalent DNA binding agent strength. In other words, therapeutic equivalence means that a chemical equivalent of a non-covalent DNA binding agent formulation of the present invention (i.e., containing the same amount of the non-covalent DNA binding agent in the same dosage form when administered to the same individuals in the same dosage regimen) will provide essentially the same efficacy and toxicity.
“Biological sample,” as used herein, refers to a material containing, for example, a nucleic acid or other biological or chemical material of interest. Biological samples containing DNA include hair, skin, cheek swab, and biological fluids such as blood, serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus, feces, urine, spinal fluid, and the like. Isolation of DNA from such samples is well known to those skilled in the art.
“Drug” or “drug substance,” as used herein, refers to an active ingredient, such as a chemical entity or biological entity, or combinations of chemical entities and/or biological entities, suitable to be administered to a subject to treat a disease, e.g., cancer or an inflammatory disease. In accordance with the present invention, the drug or drug substance is a non-covalent DNA binding agent or a pharmaceutically acceptable salt thereof.
The term “drug product,” as used herein, is synonymous with the terms “medicine,” “medicament,” “therapeutic intervention,” or “pharmaceutical product.” Most preferably, a drug product is approved by a government agency for use in accordance with the methods of the present invention. A drug product, in accordance with the present invention, contains a non-covalent DNA binding agent.
II. Non-Covalent DNA Binding Agents
The invention provides for novel compositions of one or more non-covalent DNA binding agents, for example one or more non-covalent DNA minor groove binding agents, alone or in combination with one or more available anticancer or anti-inflammatory agent, and their use in treating a disease, for example cancer or an inflammatory disease, according to the methods defined herein.
The invention provides for a library of pyrrolobenzodiazepine dimers (PBDs) (for example as described in U.S. Pat. Nos. 6,362,331, 6,800,622, 6,683,073, 6,884,799 and 7,015,215 the contents of which are incorporated herein by reference in their entirety). Non-covalent DNA binding agents of the invention that are PBDs are non-anthramycin DNA minor groove binding agents that exhibit improved properties, for example, water solubility, and decreased cardiotoxicity and metabolic inactivation as compared to natural anti-cancer antibiotics, for example anthramycin, tomaymycin, sibiromycin and neothramycin. The invention provides for PBDs that demonstrate unique S-phase cell cycle specificity resulting in the stalling of the DNA replication fork.
The invention provides for non-covalent DNA binding agents that are pyrrolobenzodiazepine dimers.
The non-covalent DNA binding agents of the invention are distinct from anti-tumor antibiotics because of the following:

    
    
        They are potent minor groove binders of the DNA with specificity for G-C rich sequences;
        These non-covalent DNA binding agents or intercalators are distinct from previously described DNA minor groove binding agents;
        They exhibit excellent pharmacokinetics in rats;
        They exhibit excellent potency in tumor cells that are deficient in DNA mismatch repair genes and/or pathways, such as those involved in the development of Lynch tumors, that have DNA mismatch repair gene deficiencies-either through genetic or epigenetic mutations;
        These non-covalent DNA binding agents have excellent potency in tumors that exhibit ‘loss of tumor suppressor gene’ function of apoptotic genes such as p53 and PTEN;
        The non-covalent DNA binding agents of the invention show excellent cytotoxic potency in tumor cells that have loss of function in multiple gene targets that regulate DNA repair, replication and/or apoptosis.
    
    


Non-covalent DNA binding agents useful according to the invention include but are not limited to the PBDs presented below:







III. Non-Covalent DNA Binding Agents May be Conjugated
PEGylation of Molecules
Non-covalent DNA binding agents of the invention may be joined to a PEG molecule (also referred to herein as pegylated non-covalent DNA binding agents of the invention) in order to enhance its stability and effectiveness.
Poly(ethylene glycol) (PEG) may be a linear or branched polyether terminated with hydroxyl groups and having the general structure:

HO—(CH2CH2O)n—CH2CH2—OH

A useful modification for PEG is monomethoxy PEG (mPEG) having the general structure:

CH3O—(CH2CH2O)n—CH2CH2—OH

The monofunctionality of mPEG makes it particularly suitable for conjugation with non PEG molecules because it can yield reactive PEGs that do not produce crosslinked products. mPEG can be further modified to have a functional group useful for conjugation with non PEG molecules.
To conjugate a PEG molecule to a non-PEG molecule such as a non-covalent DNA binding agent of the invention, it is necessary to activate the PEG by preparing a derivative of the PEG having a functional group at one or both termini. The functional group can be chosen based on the type of available reactive group on the molecule that will be conjugated to the PEG, In certain embodiments of this invention, it can be desirable to use the succinimidyl ester of the monopropionic acid derivative of PEG, as disclosed in Harris, J. M., et al., U.S. Pat. No. 5,672,662, which is incorporated herein fully by reference, or other succinimide activated PEG-carboxylic acids. In certain other embodiments, it can be desirable to use the p-nitrophenyl carbonate derivative of PEG, as disclosed in Kelly, S. J., et al. (2001) supra; PCT publication WO 00/07629 A2, supra, and in PCT publication WO 01/59078 A2 supra. Additional PEG derivatives include, but are not limited to, aldehyde derivatives of PEGs (Royer, G. P., U.S. Pat. No. 4,002,531; Harris, J. M., et al., U.S. Pat. No. 5,252,714), amine, bromophenyl carbonate, carbonylimidazole, chlorophenyl carbonate, fluorophenyl carbonate, hydrazide, iodoacetamide, maleimide, orthopyridyl disulfide, oxime, phenylglyoxal, thiazolidine-2-thione, thioester, thiol, triazine and vinylsulfone derivatives of PEGs.
In accordance with the practice of the invention, one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and up to 10) strands of one or more PEGs can be coupled to a non-covalent DNA binding agents of the invention. In one embodiment, one or two strands of PEG may be coupled to a non-covalent DNA binding agents of the invention.
In an embodiment of the invention, coupling of PEG to non-covalent DNA binding agents of the invention may be effected by, for example, reductive alkylation (also known as reductive amination) using standard methods (see e.g., Bentley, M. D., et al., U.S. Pat. No. 5,990,237; references 1-69).
In one embodiment, a PEG derivative suitable for conjugation with N-terminal amino acid groups of proteins or polypeptides (e.g. non-covalent DNA binding agents of the invention) is mPEG-propionaldehyde as shown below in a reductive alkylation reaction (see for example U.S. Pat. No. 5,252,714?). In this embodiment, sodium cyanoborohydride may be used as the reducing agent (Cabacungan, J. C., et al., (1982) Anal Biochem 124:272-278; U.S. Pat. No. 5,252,714). In accord with the practice of the invention, H2N—R can be non-covalent DNA binding agents of the invention.




Other PEG derivatives suitable for conjugation with N-terminal amino acid groups include, but are not limited to: PEG-acetaldehyde, PEG carboxylic acids (e.g., PEG propionic acid, PEG butanoic acid).
Reversible conjugation using PEG derivative molecules can be beneficial in some circumstances. Examples of PEG derivatives that can conjugate and release non-PEG molecules include, but are not limited to: PEG-succinimidyl succinate, PEG maleic anhydride, mPEG phenyl ether succinimidyl carbonates and mPEG benzamide succinimidyl carbonates.
Heterobifunctional PEGs are PEGs bearing dissimilar terminal groups. Heterobifunctional PEGs with appropriate functional groups can be used to link two entities where a hydrophilic, flexible, and biocompatible spacer is needed. Heterobifunctional PEGs can be used in a variety of ways including, but not limited to, linking molecules to surfaces (for immunoassays, biosensors or various probe applications, etc), targeting of drugs, liposomes, and viruses to specific tissues, liquid phase peptide synthesis and other applications.
In addition to the linear PEG molecules described above, branched and/or forked PEGs can be used to conjugate non-PEG molecules (e.g. non-covalent DNA binding agents of the invention). Branched PEG molecules have a single functional group at the end of two PEG chains. A branched PEG structure can be more effective than a linear PEG in protecting conjugated agents from proteolysis and in reducing antigenicity and immunogenicity of such conjugates. Forked PEGs have two reactive groups at one end of a single PEG chain. Forked PEG molecules can be used to bring two non PEG molecules in close proximity to each other by attaching the non PEG molecules to the single forked PEG molecule.
Examples of branched and/or forked PEG molecules are shown below.




Enhanced Activity of PEGylated Non-Covalent DNA Binding Agents of the Invention

Enhanced receptor binding activity and functional activity (e.g., increased or extended half-life) may be an advantage of the pegylated non-covalent DNA binding agents of the invention. Increased receptor binding activity and increased functional activity can be measured, or employed, in vitro, and increased potency, can be measured either in vitro or in vivo.
III. Anti-Inflammatory Agents
Anti-inflammatory agents useful in the combination therapy of the invention include, but are not limited to, dihydrofolic acid reductase inhibitors e.g., methotrexate; cyclophosphamide; cyclosporine; cyclosporin A; chloroquine; hydroxychloroquine; sulfasalazine (sulphasalazopyrine) gold salts D-penicillamine; leflunomide; azathioprine; anakinra; a Non-Steroidal Anti-Inflammatory Drug (NSAID); TNF blockers e.g., infliximab (REMICADE®) or etanercept; and a biological agent that targets an inflammatory cytokine. In accordance with the practice of the invention, therapeutically effective salts or prodrugs of these agents may also be used.
NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam and tramadol. In accordance with the practice of the invention, therapeutically effective salts or prodrugs of these agents may also be used.
IV. Anti-Cancer Agents
Anti-cancer agents useful in the combination therapy of the invention include, but are not limited to: histone deacetylase inhibitors (HDIs or HDACIs) (such as trichostatin A (7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide)); topoisomerase I inhibitors such as camptothecin (S)-4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione), topotecan (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochioride) and irinotecan ((S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4′bipiperidine]-1′-carboxylate); protein synthesis inhibitors such as cyclohexamide(4-[(2R)-2-[(1S,3S,5S)-3,5-Dimethyl-2-oxocyclohexyl]-2-hydroxyethyl]piperidine-2,6-dione); DNA alkylating agents such as mitomycin C ([6-Amino-8a-methoxy-5-methyl-4,7-dioxo-1,1a,2,4,7,8,8a,8b-octahydroazireno[2′,3′:3,4]pyrrolo[1,2-a]indol-8-yl]methyl carbamate); topoisomerase II inhibitors such as anthracycline antibiotics like doxorubicin ((8S,10S)-10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione) and etoposide (4′-demethyl-epipodophyllotoxin 9-[4,6-O—(R)-ethylidene-beta-D-glucopyranoside], 4′-(dihydrogen phosphate)); anti-metabolite agents (such as 6-thioguanine (6TG) (2-amino-6,7-dihydro-3H-purine-6-thione), and 5-fluorouracil (5-FU)(5-fluoro-1H-pyrimidine-2,4-dione); epidermal growth factor receptor (EGFR) inhibitors (such as gefitinib (N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine) and erlonitib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4-amine)); RNA synthesis inhibitor such as actinomycin D (2-amino-N,N′-bis[(6S,9R,10S,13R,18aS)-6,13-diisopropyl-2,5,9-trimethyl-1,4,7,11,14-pentaoxohexadecahydro-1H-pyrrolo[2,1-i][1,4,7,10,13]oxatetraazacyclohexadecin-10-yl]-4,6-dimethyl-3-oxo-3H-phenoxazine-1,9-dicarboxamide); anti-mitotic agents like tubulin inhibitors such as paclitaxel ((2α,4α,5β,7β, 10β,13α)-4,10-bis(acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-yl benzoate)(also known as Taxol) and vinca alkaloids like vincristine (methyl(1R,9R,10S,11R,12R,19R)-11-(acetyloxy)-12-ethyl-4-[(13S,15S,17S)-17-ethyl-17-hydroxy-13-(methoxycarbonyl)-1,11-diazatetracyclo[13.3.1.04,12.05,10]nonadeca-4(12),5,7,9-tetraen-13-yl]-8-formyl-10-hydroxy-5-methoxy-8,16-diazapentacyclo[10.6.1.01,9.02,7.016,19]nonadeca-2,4,6,13-tetraene-10-carboxylate) and vinblastine (dimethyl(2β,3β,4β,5α,12β,19α)-15-[(5S,9S)-5-ethyl-5-hydroxy-9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methanoazacycloundecino[5,4-b]indol-9-yl]-3-hydroxy-16-methoxy-1-methyl-6,7-didehydroaspidospermidine-3,4-dicarboxylate); DNA synthesis inhibitors like fludarabine ([(2R,3R,4S,5R)-5-(6-amino-2-fluoro-purin-9-yl)-3,4-dihydroxy-oxolan-2-yl]methoxyphosphonic acid) and hydroxyurea; Poly ADP ribose polymerase (PARP) inhibitors (such as olaparib (4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)-4-fluorophenyl]methyl(2H)phthalazin-1-one)); and DNA crosslinking agents such as such as cisplatin ((SP-4-2)-diamminedichloridoplatinum), carboplatin (cis-diammine(cyclobutane-1,1-dicarboxylate-O,O′)platinum(II)) and oxaliplatin ([(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O′)platinum(II)). In accordance with the practice of the invention, therapeutically effective salts or prodrugs of these anti-cancer agents may be used.
V. Genes
The invention provides for novel compositions and use of one or more non-covalent DNA binding agents, alone (as the only active agent(s)) or in combination with other anticancer or anti-inflammatory active agents, in the treatment of cancer or inflammatory disease in patients with, for example, mutations in genes including but not limited to:

    
    
        genes regulating DNA replication, recombination, repair and/or apoptosis such as PTEN, p53, BRCA1 and/or BRCA2, together with the associated BRCA1/rad51/MRE11/replication protein A (RPA) complex;
        genes regulating DNA mismatch repair such as mlh1, MSH2, MSH6, PMS1, PMS2;
        genes regulating translesion synthesis such as REV3 and its associated protein complexes at the replication fork;
        genes regulating cell proliferation such as KRAS and BRAF kinase pathways.
        Genes encoding kinases regulating DNA replication, recombination, repair and/or apoptosis such as ATM, ATR, Chk1 and/or Chk2 kinases;
        genes involved in base excision repair such as XRCC1;
        nucleotide excision repair genes such as ERCC1;
        homologous recombination genes such as RAD51, RAD52, RAD54, BRCA1, BRCA2, XRCC2 and XRCC3;
        genes regulating non-homologous recombination such as KU70, KU80, XRCC4 and DNA ligase4; and
        genes regulating transcription-coupled repair such as CSA, CSB and XPG.
    
    


The invention therefore provides for novel compositions and use of one or more non-covalent DNA binding agents alone, as the only active agent(s), or in combination with other anticancer or anti-inflammatory active agents, in the treatment of cancer or inflammatory disease in patients with, for example, a mutation in a gene or gene pathway including but not limited to PTEN, p53, BRCA1, BRCA2, MLH1, PMS1, PMS2, MSH2, MSH6, REV3, KRAS, BRAF, Chk1, Chk2, KU70, KU80, DNA ligase 4, CSA, CSB, XRCC1, XRCC2, XRCC3, XRCC4, RAD51, RAD52, RAD54, REV, ATM, ATR, XPF, Ercc1, XPA, XPB, XPD, XPF, XPG, MSH6/3, PCNA, BARD1, RAD50, NBS1, Mre11, BLM, PMS2, MLH1, MEDI, RFC, polγ/ε, RPA, DNA ligase I and the MRE1/RPA1/RAD51 complex.











TABLE 1







Symbol
Entrez Gene ID
NCBI Reference Sequence




















TP53
7157
NM_000546



MLH1
4292
NM_000249



MSH2
4436
NM_000251



BRCA1
672
NM_007294



REV3L
5980
NM_002912



PARP1
142
NM_001618



RAD51
5888
NM_002875



MRE11A
4361
NM_005591



ATM
472
NM_000051



ATR
545
NM_001184



PTEN
5728
NM_000314



ERCC1
2067
NM_001983



BRCA2
675
NM_000059



XRCC1
7515
NM_006297



KRAS
3845
NM_033360



BRAF
673
NM_004333



RAD50
10111
NM_005732



RAD51
5393
NM_134424










PTEN

Phosphatase and tensin homolog (PTEN) is a protein that is encoded by the PTEN gene. Mutations of this gene are a step in the development of many cancers. PTEN acts as a tumor suppressor gene through the action of its phosphatase protein product. This phosphatase is involved in the regulation of the cell cycle, preventing cells from growing and dividing too rapidly.
This gene was identified as a tumor suppressor that is mutated in a large number of cancers at high frequency. The protein encoded by this gene is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It contains a tensin like domain as well as a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating the Akt/PKB signaling pathway.
p53
p53 is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is important in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer. As such, p53 plays a role in conserving stability by preventing genome mutation.
BRCA1
BRCA1 (breast cancer 1) is a human tumor suppressor gene, which produces a protein, called breast cancer type 1 susceptibility protein. BRCA1 is expressed in the cells of breast and other tissue, where it helps repair damaged DNA, or destroy cells if DNA cannot be repaired. If BRCA1 itself is damaged, damaged DNA is not repaired properly and this increases risks for cancers.
The protein encoded by the BRCA1 gene combines with other tumor suppressors, DNA damage sensors, and signal transducers to form a large multi-subunit protein complex known as the BRCA1-associated genome surveillance complex (BASC). The BRCA1 protein associates with RNA polymerase II, and, through the C-terminal domain, also interacts with histone deacetylase complexes. This protein thus plays a role in transcription, DNA repair of double-stranded breaks, ubiquitination, transcriptional regulation as well as other functions.
BRCA2
BRCA2 (Breast Cancer 2 susceptibility protein) is a protein that in humans is encoded by the BRCA2 gene. BRCA2 belongs to the tumor suppressor gene family and the protein encoded by this gene is involved in the repair of chromosomal damage with an important role in the error-free repair of DNA double strand breaks.
DNA Mismatch Repair Genes
DNA mismatch repair is a system for recognizing and repairing erroneous insertion, deletion and mis-incorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage.
Mismatch repair is strand-specific. During DNA synthesis it is common that errors are introduced into the newly synthesized (daughter) strand.
Any mutational event that disrupts the superhelical structure of DNA carries with it the potential to compromise the genetic stability of a cell.
Examples of mismatched bases include a G/T or A/C. Mismatches are commonly due to tautomerization of bases during synthesis. The damage is repaired by recognition of the deformity caused by the mismatch, determination of the template and non-template strand, and excision of the wrongly incorporated base and replacement of the incorrect base with the correct nucleotide. The removal process involves more than just the mismatched nucleotide itself. A few or up to thousands of base pairs of the newly synthesized DNA strand can be removed.
Mismatch repair (MMR) genes are involved in recognition and repair of certain types of DNA damage or replication errors. These genes also function to help preserve the fidelity of the genome through successive cycles of cell division.
The protein products of MMR genes also repair branched DNA structures, prevent recombination of divergent sequences, direct non-MMR proteins in nucleotide excision and other forms of DNA repair, and are involved in regulation of meiotic crossover. Defects in MMR genes lead to Microsatellite Instability (MSI) and cancer.
MLH1
MutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli), also known as MLH1, is a human gene located on Chromosome 3. It is a gene commonly associated with hereditary nonpolyposis colorectal cancer.
This gene was identified as a locus frequently mutated in hereditary nonpolyposis colon cancer (HNPCC). It is a human homolog of the E. coli DNA mismatch repair gene mutL, consistent with the characteristic alterations in microsatellite sequences (RER+ phenotype) found in HNPCC. Alternatively spliced transcript variants encoding different isoforms have been described, but their full-length natures have not been determined.
PMS1
PMS1 protein homolog 1 is a protein that in humans is encoded by the PMS1 gene.
The protein encoded by this gene was identified by its homology to a yeast protein involved in DNA mismatch repair. This protein forms heterodimers with MLH1, a DNA mismatch repair protein, and some cases of hereditary nonpolyposis colorectal cancer have been found to have mutations in this gene.
PMS2
Mismatch repair endonuclease PMS2 is an enzyme that in humans is encoded by the PMS2 gene.
This gene is one of the PMS2 gene family members which are found in clusters on chromosome 7. The product of this gene is involved in DNA mismatch repair. The protein forms a heterodimer with MLH1 and this complex interacts with MSH2 bound to mismatched bases. Defects in this gene are associated with hereditary nonpolyposis colorectal cancer, with Turcot syndrome, and are a cause of supratentorial primitive neuroectodermal tumors. Alternatively spliced transcript variants have been observed.
MSH2
MSH2 is a gene commonly associated with Hereditary nonpolyposis colorectal cancer.
MSH2 was identified as a locus frequently mutated in hereditary nonpolyposis colon cancer (HNPCC). When cloned, it was discovered to be a human homolog of the E. coli mismatch repair gene mutS, consistent with the characteristic alterations in microsatellite sequences (RER+ phenotype) found in HNPCC. It is also associated with some endometrial cancers.
MSH3
DNA mismatch repair protein Msh3 is a protein that in humans is encoded by the MSH3 gene. MSH3 has been shown to interact with MSH2, PCNA and BRCA1.
MSH6
MSH6 is a gene commonly associated with hereditary nonpolyposis colorectal cancer.
MSH6 has been shown to interact with MSH2, PCNA and BRCA1.
VI. Cells and Cell Lines
Cell lines useful according to the invention include but are not limited to breast cancer cell lines (MMR- or PTEN-deficient or BRCA1 mutant), e.g., MDA-MB-231, MCF-7, MDA-MB-468; colon cancer cell lines (MMR-deficient; KRAS-mutant cells) e.g., HCT-116, SW-620, SW-480, SW48, SW-403, Colo205; lymphoblastoid cell lines (MSH2- or PTEN-deficient cells) e.g., CEM and Jurkat; ovarian and uterine cancer cell lines (DNA MMR-deficient cells) e.g., HeLa, SKOV-3; osteosarcoma cells (MMR-competent) e.g., U2OS; and lung cancer cells (MMR-competent or MMR-deficient) e.g., A549 and H1299.
Cell lines derived from patients with any of the cancers or inflammatory diseases recited herein are also useful according to the methods of the invention.
VII. Diseases
The invention provides for novel compositions and methods for treatment of a subject with a disease comprising administration of a pharmaceutically effective amount of one or more of a non-covalent DNA binding agent, for example, a non-covalent DNA minor groove binding agent, alone, as the only active agent(s) or in combination with one or more anti-cancer and/or anti-inflammatory active agents. For example, the invention provides for treating cancer with one or more non-covalent DNA-minor groove binding agents that result in DNA crosslinking or intercalation, alone, as the only active agent(s) or in combination with one or more anti-cancer active agents. The invention contemplates treating any one of one of cancer, tumor growth, cancer of the colon, breast, bone, brain and others (e.g., osteosarcoma, neuroblastoma, colon adenocarcinoma), chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), cardiac cancer (e.g., sarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma); lung cancer (e.g., bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma); various gastrointestinal cancer (e.g., cancers of esophagus, stomach, pancreas, small bowel, and large bowel); genitourinary tract cancer (e.g., kidney, bladder and urethra, prostate, testis; liver cancer (e.g., hepatoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bone cancer (e.g., osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma, multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma, benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors); cancers of the nervous system (e.g., of the skull, meninges, brain, and spinal cord); gynecological cancers (e.g., uterus, cervix, ovaries, vulva, vagina); hematologic cancer (e.g., cancers relating to blood, Hodgkin's disease, non-Hodgkin's lymphoma); skin cancer (e.g., malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis); and cancers of the adrenal glands (e.g., neuroblastoma).
In particular, the invention relates to novel compositions of one or more non-covalent DNA binding agents, alone, as the only active agent(s) or in combination with one or more anti-cancer active agents and their use to treat those cancers that are genetically-resistant and have a loss of at least one tumor suppressor gene function. Such cancers include tumors of the brain (such as gliomas and glioblastomas), blood (such as leukemias and lymphomas), bladder, breast, colorectal, endometrial, lung, melanomas, ovarian, prostate, renal and testicular cancers.
In one embodiment the invention provides for treating MMR-deficient colorectal cancer using a novel composition of one or more non-covalent DNA binding agents, alone, as the only active agent(s) or in combination with one or more anti-cancer active agents of the invention. One of the most studied genotypic subtypes of colorectal cancer is that characterized by a deficient mismatch repair (dMMR) pathway, usually found in combination with microsatellite instability (see Hewish et al., Nature Reviews 7: 197-208, 2010). MMR-deficient colorectal cancer can occur as a result of inherited or sporadic abnormalities in DNA repair pathways. The phenotypic characteristics of this cancer include proximal anatomical location, mucinous features and lymphocytic infiltration.
Preclinical and clinical studies have demonstrated that MMR-deficient colorectal cancer shows resistance to 5-fluorouracil. Heterogeneity exists within MMR-deficient colorectal cancer subtype, possibly due to secondary mutations from MMR-deficiency-associated mutator phenotype.
In another embodiment, the invention provides for treating ‘triple negative’ and ‘basal-like’ breast cancers with novel compositions of one or more non-covalent DNA binding agents, alone, as the only active agents, or in combination with one or more anti-cancer active agents of the invention. Triple-negative breast cancer is the subgroup of breast cancer that does not express clinically significant levels of the estrogen receptor (ER), progesterone receptor (PR) and HER2/neu (HER2) (Carey, L., Winer, E, Viale, G, Cameron, D. and Gianni, L. Triple-negative breast cancer: disease entity or title of convenience. Nature Reviews 7: 683-692, 2010).
BRCA1 protein expression levels are significantly lower in tumors of high histological grade that lack hormone receptors (triple negative and basal-like breast tumors). Further, basal-like breast cancers also have significant TP53 (P53) gene mutations and BRCA1 pathway dysfunction. BRCA1-pathway related cancers likely have DNA repair defects. These BRCA1 pathway dysfunction tumors show sensitivity to DNA crosslinking agents, for example platinum, in combination with antimetabolite drugs, such as gemcitabine, and poly ADP-ribose polymerase (PARP) inhibitors, such as olaparib and iniparib.
In another embodiment, the invention provides for treating human glioblastomas with novel compositions of one or more non-covalent DNA binding agents, alone, as the only active agent(s) or in combination with one or more anti-cancer active agents of the invention. One of the key markers for glioblastomas is the methylation status of MGMT. The MGMT methylation status predicts the sensitivity of human glioblastomas to alkylating agents, e.g., temozolomide.
The invention also contemplates treating any one of the inflammatory disease recited herein with novel compositions of one or more non-covalent DNA binding agents, alone, as the only active agent(s) or in combination with one or more anti-inflammatory active agents of the invention.
The invention also contemplates treating a subject having an infection (e.g. bacterial infection, viral infection, yeast infection, or parasitic infection) with therapeutically effective amount of one or more of a PBD such as NSC718813, NSC723734, NSC 723732 and NSC726260 so as to treat the subject with the infection.
The invention also contemplates treating a subject suffering from an infection (e.g. bacterial infection, viral infection, yeast infection, or parasitic infection) by administering to the subject a therapeutically effective amount of one or more of the following PBD's:




wherein R is H, OH, or OAc and n is 3 to 5;





wherein R is H, OH, and n is 1 to 4;





wherein R and R1 are independently H or —OH, and n is an integer from 3 to 5;





wherein n is 2 to 10; or





wherein R is H, OH, OAc, and R1 is H, and n is 3 to 5.

VIII. Dosages and Modes of Administration
In general, non-covalent DNA binding agents of the invention may be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either as one or more non-covalent DNA binding agents like the PBDs alone or in combination with one or more additional therapeutic agents, e.g., anti-cancer agents and/or anti-inflammatory agents. A therapeutically effective amount may vary widely depending on the disease, the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.001 mg to 1000 mg per subject. An indicated daily intravenous dosage in the larger mammal, e.g. humans, is in the range from about 0.0001 mg to about 100 mg per subject, conveniently administered, e.g. in divided doses up to 1-2 times a day or in retard form. Suitable unit dosage forms for intravenous administration comprise from about 0.001 mg to about 10 mg/ml active ingredient.
In certain embodiments, a therapeutic amount or intravenous dose of one or more of a non-covalent DNA binding agent of the present invention may range from about 0.001 mg to about 100 mg per subject, alternatively from about 0.01 mg to about 10 mg per subject. In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 0.001 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses. Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.
Upon improvement of a subject's condition, a maintenance dose of one or more of a non-covalent DNA binding agent, either alone or in combination with one or more additional therapeutic agents, e.g., a chemotherapeutic agent, may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be altered, for example reduced, as a function of the symptoms, to a level at which the improved condition is retained and when the symptoms have been alleviated to the desired level, treatment should cease. The subject may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
It will be understood, however, that the total daily usage of one or more non-covalent DNA binding agents alone or in combination with one or more anti-cancer and/or anti-inflammatory agents of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific inhibitory dose for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
In general, the anti-inflammatory agents of the invention may be administered in therapeutically effective amounts via any of the acceptable modes known in the art. Depending on the anti-inflammatory agent, an effective amount can be in a range of about 0.01 to about 5000 mg/day. This range can be modified to an amount of about 0.1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about 700 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about 1300 to 1400 mg/day, about 1400 to 1500 mg/day, about 1500 to 1600 mg/day, about 1600 to 1700 mg/day, about 1700 to 1800 mg/day, about 1800 to 1900 mg/day, about 1900 to 2000 mg/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to 3500 mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to 5000 mg/day. It would be clear to one skilled in the art that dosage will vary depending on the particular anti-inflammatory agent being used Specific examples of appropriate dosages, depending on the anti-inflammatory agent, are described below.
In another embodiment, an effective amount of an anti-inflammatory agent can be in a range of about 0.1 mg/week to 40 mg/week; 0.1 mg/week to 5 mg/week; 5 mg/week to 10 mg/week; 10 mg/week to 30 mg/week; 30 mg/week to 35 mg/week; 0.1 mg/week to 100 mg/week; or 30 mg/week to 50 mg/week. In another embodiment, an anti-inflammatory agent can be administered in an amount of about 50 mg/week or 25 mg twice weekly. It would be clear to one skilled in the art that dosage range will vary depending on the particular anti-inflammatory agent being used, for example see below.
Methotrexate is an antimetabolite molecule that interferes with DNA synthesis, repair and cellular replication. Methotrexate is an inhibitor of dihydrofolic acid reductase i.e. it is a folic acid antagonist. Methotrexate may be administered in an amount about 0.1 to 40 mg per week with a dosage ranging from about 5 to 30 mg per week. Methotrexate may be administered to a subject in various increments: about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, or about 35 to 40 mg/week. In one embodiment, an effective amount of methotrexate, may be about 10 to 30 mg/week.
Cyclophosphamide, an alkylating agent, may be administered in dosages ranging about 0.1 to 10 mg/kg body weight per day.
Cyclosporine (e.g. NEORAL®) also known as Cyclosporin A, may be administered in dosages ranging from about 1 to 10 mg/kg body weight per day. Dosages ranging about 2.5 to 4 mg per body weight per day may be used.
Chloroquine or hydroxychloroquine (e.g. PLAQUENIL®), may be administered in dosages ranging about 100 to 1000 mg daily. Preferred dosages range about 200-600 mg administered daily.
Sulfasalazine (e.g., AZULFIDINE EN-Tabs®) may be administered in amounts ranging about 50 to 5000 mg per day, with a dosage of about 2000 to 3000 mg per day for adults. Dosages for children may be about 5 to 100 mg/kg of body weight, up to 2 grams per day.
Injectable gold salts may be prescribed in dosages about 5 to 100 mg doses every two to four weeks. Orally administered gold salts may be prescribed in doses ranging about 1 to 10 mg per day.
D-penicillamine or penicillamine (CUPRIMINE®) may be administered in dosages about 50 to 2000 mg per day, with dosages about 125 mg per day up to 1500 mg per day.
Azathioprine may be administered in dosages of about 10 to 250 mg per day. For example, a dosage range of about 25 to 200 mg per day is acceptable.
Anakinra (e.g. KINERET®) is an interleukin-1 receptor antagonist. A possible dosage range for anakinra is about 10 to 250 mg per day. In one example, the dosage may be about 100 mg per day.
Infliximab (REMICADE®) is a chimeric monoclonal antibody that binds tumor necrosis factor alpha (TNFα) and inhibits the activity of TNFα. Infliximab may be administered in dosages about 1 to 20 mg/kg body weight every four to eight weeks. Dosages of about 3 to 10 mg/kg body weight may be administered every four to eight weeks depending on the subject.
Etanercept (e.g. ENBREL®) is a dimeric fusion protein that binds the tumor necrosis factor (TNF) and blocks its interactions with TNF receptors. In one example, the dosage range of etanercept may be about 10 to 100 mg per week for adults. In another example, the dosage may be about 50 mg per week. Dosages for juvenile subjects may range from about 0.1 to 50 mg/kg body weight per week with a maximum of about 50 mg per week. For adult patients, etanercept may be administered e.g., injected, in 25 mg doses twice weekly e.g., 72-96 hours apart in time.
Leflunomide (ARAVA®) may be administered at dosages from about 1 and 100 mg per day. In one embodiment, the dosage range is from about 10 to 20 mg per day.
It is contemplated that global administration of a therapeutic composition to a subject is not needed in order to achieve a highly localized effect. Localized administration of a therapeutic composition according to the invention is preferably by injection, catheter or by means of a drip device, drug pump or drug-saturated solid matrix from which the composition can diffuse implanted at the target site. When a tissue that is the target of treatment according to the invention is on a surface of an organism, topical administration of a pharmaceutical composition is possible. For example, antibiotics are commonly applied directly to surface wounds as an alternative to oral or intravenous administration, which methods necessitate a much higher absolute dosage in order to counter the effect of systemic dilution, resulting both in possible side-effects in otherwise unaffected tissues and in increased cost.
Systemic administration of a therapeutic composition according to the invention may be performed by methods of whole-body drug delivery well known in the art. These include, but are not limited to, intravenous drip or injection, subcutaneous, intramuscular, intraperitoneal, intracranial and spinal injection, ingestion via the oral route, inhalation, trans-epithelial diffusion (such as via a drug-impregnated, adhesive patch) or by the use of an implantable, time-release drug delivery device. Note that injection may be performed by conventional means.
Systemic administration is advantageous when a pharmaceutical composition must be delivered to a target tissue that is widely-dispersed, inaccessible to direct contact or, while accessible to topical or other localized application, is resident in an environment (such as the digestive tract) wherein the native activity of the nucleic acid or other agent might be compromised, e.g. by digestive enzymes or extremes of pH.
A novel therapeutic composition for use in the invention can be given in a single- or multiple doses. A multiple dose schedule is one in which a primary course of administration can include 1-10 or more separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the level of the therapeutic agent. Such intervals are dependent on the continued need of the recipient for the therapeutic agent, and/or the half-life of a therapeutic agent. The efficacy of administration may be assayed by monitoring the reduction in the levels of a symptom indicative or associated with the disease which it is designed to inhibit. The assays can be performed as described herein or according to methods known to one skilled in the art.
A therapeutically effective regimen may be sufficient to arrest or otherwise ameliorate symptoms of a disease. An effective dosage regimen requires providing the regulatory drug over a period of time to achieve noticeable therapeutic effects wherein symptoms are reduced to a clinically acceptable standard or ameliorated. The symptoms are specific for the disease in question. For example, when the disease is associated with tumor formation, the claimed invention is successful when tumor growth is arrested, or tumor mass is decreased by at least 50% and preferably 75%.
IX. Pharmaceutical Compositions
In another aspect, the invention provides for novel pharmaceutical compositions comprising one or more non-covalent DNA binding agents, alone or in combination with other anticancer or anti-inflammatory agents, or a pharmaceutically acceptable ester, salt, or prodrug thereof, together with a pharmaceutically acceptable carrier. This invention provides for a pharmaceutical composition comprising one or more non-covalent DNA binding agent, alone, as the only active agent(s) or in combination with one or more therapeutic active agents, e.g., a chemotherapeutic agent.
Non-covalent DNA binding agents of the invention can be administered as pharmaceutical compositions by any conventional route, in particular parenterally such as intravenously or by subcutaneous or intramuscular injections; enterally, e.g., orally, e.g., in the form of tablets or capsules, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form for localized delivery. Pharmaceutical compositions comprising a non-covalent DNA binding agent of the present invention in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coating methods. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a compound of the present invention with a carrier. A carrier can include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
One or more non-covalent DNA binding agents of the invention can be administered in therapeutically effective amounts, alone, as the only active agent(s) or in combination with one or more therapeutic active agents (pharmaceutical combinations), resulting in novel compositions. For example, synergistic effects can occur with other anti-proliferative, anti-cancer, immunomodulatory or anti-inflammatory substances. Where the compounds of the invention are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth.
The present invention encompasses pharmaceutically acceptable topical formulations of inventive compounds. The term “pharmaceutically acceptable topical formulation,” as used herein, means any formulation which is pharmaceutically acceptable for intradermal administration of a compound of the invention by application of the formulation to the epidermis. In certain embodiments of the invention, the topical formulation comprises a carrier system. Pharmaceutically effective carriers include, but are not limited to, solvents (e.g., alcohols, poly alcohols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffered solutions (e.g., hypotonic or buffered saline) or any other carrier known in the art for topically administering pharmaceuticals. A more complete listing of art-known carriers is provided by reference texts that are standard in the art, for example, Remington's Pharmaceutical Sciences, 16th Edition, 1980 and 17th Edition, 1985, both published by Mack Publishing Company, Easton, Pa., the disclosures of which are incorporated herein by reference in their entireties. In certain other embodiments, the topical formulations of the invention may comprise excipients. Any pharmaceutically acceptable excipient known in the art may be used to prepare the inventive pharmaceutically acceptable topical formulations. Examples of excipients that can be included in the topical formulations of the invention include, but are not limited to, preservatives, antioxidants, moisturizers, emollients, buffering agents, solubilizing agents, other penetration agents, skin protectants, surfactants, and propellants, and/or additional therapeutic agents used in combination with the inventive compound. Suitable preservatives include, but are not limited to, alcohols, quaternary amines, organic acids, parabens, and phenols. Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherols, and chelating agents like EDTA and citric acid. Suitable moisturizers include, but are not limited to, glycerine, sorbitol, polyethylene glycols, urea, and propylene glycol. Suitable buffering agents for use with the invention include, but are not limited to, citric, hydrochloric, and lactic acid buffers. Suitable solubilizing agents include, but are not limited to, quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates. Suitable skin protectants that can be used in the topical formulations of the invention include, but are not limited to, vitamin E oil, allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.
In certain embodiments, the pharmaceutically acceptable topical formulations of the invention comprise at least a compound of the invention and a penetration enhancing agent. The choice of topical formulation will depend on several factors, including the condition to be treated, the physicochemical characteristics of the inventive compound and other excipients present, their stability in the formulation, available manufacturing equipment, and costs constraints. As used herein the term “penetration enhancing agent” means an agent capable of transporting a pharmacologically active compound through the stratum corneum and into the epidermis or dermis, preferably, with little or no systemic absorption. A wide variety of compounds have been evaluated as to their effectiveness in enhancing the rate of penetration of drugs through the skin. See, for example, Percutaneous Penetration Enhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which surveys the use and testing of various skin penetration enhancers, and Buyuktimkin et ah, Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh T. K., Pfister W. R., Yum S. I. (Eds.), Interpharm Press Inc., Buffalo Grove, IU. (1997). In certain exemplary embodiments, penetration agents for use with the invention include, but are not limited to, triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe-vera gel), ethyl alcohol, isopropyl alcohol, octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethylsulfoxide. fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate) and N-methyl pyrrolidine.
In certain embodiments, the compositions may be in the form of ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. In certain exemplary embodiments, formulations of the compositions according to the invention are creams, which may further contain saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleyl alcohols, stearic acid being particularly preferred. Creams of the invention may also contain a non-ionic surfactant, for example, polyoxy-40-stearate. In certain embodiments, the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms are made by dissolving or dispensing the compound in the proper medium. As discussed above, penetration enhancing agents can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a compound of the present invention formulated together with one or more pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, or as an oral or nasal spray.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof, Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
According to the descriptions of novel compositions and methods of treatment of the present invention, disorders are treated or prevented in a subject, such as a human or other animal, by administering to the subject a therapeutically effective amount of one or more non-covalent DNA binding agent, alone, as the only active agent(s) or in combination with one or more other active agents, in such amounts and for such time as is necessary to achieve the desired result. The term “therapeutically effective amount” of a compound of the invention, as used herein, means a sufficient amount of the compound so as to decrease the symptoms of a disorder in a subject. As is well understood in the medical arts a therapeutically effective amount of a compound of this invention will be at a reasonable benefit/risk ratio applicable to any medical treatment.
The invention also provides for novel compositions of pharmaceutical combinations, e.g. a kit, comprising an agent which is a compound of the invention as disclosed herein, in free form, or in pharmaceutically acceptable salt form. The kit can comprise instructions for its administration to a subject suffering from or susceptible to a disease or disorder.
Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The non-covalent DNA binding agent compounds (e.g., including those delineated herein), or pharmaceutical salts thereof may be formulated into pharmaceutical compositions for administration to animals or humans. These pharmaceutical compositions, which comprise an amount of the non-covalent DNA binding agent compounds effective to treat or prevent a non-covalent DNA cross-link mediated condition and a pharmaceutically acceptable carrier, are another embodiment of the present invention.
This invention also encompasses novel pharmaceutical compositions containing, and methods of treating disorders through administering, pharmaceutically acceptable prodrugs of one or more non-covalent DNA binding agents of the invention alone, as the only active agent(s) or in combination with other available active agents. For example, non-covalent DNA binding agents of the invention having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of compounds of the invention. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also includes 4-hydroxyproline, hydroxyysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. Free hydroxy groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 1 15. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may be an alkyl ester, optionally substituted with groups including but not limited to ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, are also encompassed. Prodrugs of this type are described in J. Med. Chem. 1996, 39, 10. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All of these prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities.
Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term “stable”, as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).
The terms “isolated,” “purified,” or “biologically pure” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Particularly, in embodiments of the invention the compound is at least 85% pure, more preferably at least 90% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
X. Kits or Pharmaceutical Systems
The novel Compositions described in this application may be assembled into kits or pharmaceutical systems for use in disease treatment, e.g., cancer treatment or treatment of an inflammatory disease. Kits or pharmaceutical systems according to this aspect of the invention comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampules, bottles and the like. The kits or pharmaceutical systems of the invention may also comprise associated instructions for using one or more non-covalent DNA binding agents of the invention, alone, as the only active agent(s) or in combination with other active agents. The non-covalent DNA binding agents of the kits or pharmaceutical systems of the invention may have any one of the functional properties described for the non-covalent DNA binding agents of the methods of the invention.
In certain embodiments, the kits of the invention include a test for determining if a subject has a mutation in a particular gene of interest.
XI. Uses
The methods of the invention can be used to treat a subject with a disease, e.g., cancer and/or inflammatory disease.
XII. Animal Models
The invention provides for animal models for various diseases, including but not limited to cancer.
Additional animal models known in the art are also useful according to the invention, such as those models for inflammatory disorders such as rheumatoid arthritis, psorias, Crohn's disease and ulcerative colitis.
A. Rheumatoid Arthritis:
Animal models for Rheumatoid arthritis include but are not limited to collagen induced arthritis in mouse and rat, collagen antibody induced arthritis in mouse, spontaneous rheumatoid arthritis in K/B×N mice, arthritis induced by adoptive transfer of serum from K/B×N mice and spontaneous arthritis in TNFα transgenic mice.
Multiple Sclerosis:
Animal models for Multiple Sclerosis include but are not limited to experimental autoimmune encephalopathy in mouse and rat induced by injection of myelin oligodendrocyte glycoprotein and experimental autoimmune encephalopathy in mouse and rat induced by injection of proteolipid protein.
C. Inflammatory Bowel Disease (Crohn's Disease):
Animal models for Crohn's Disease include but are not limited to Dextran sodium sulfate induced colitis in mouse and rat and colitis induced by adoptive transfer of CD4+CD45RBhigh cells into SCID mice
D. Inflammatory Bowel Disease (Ulcerative Colitis):
An animal model for ulcerative colitis includes but is not limited to trinitrobenzene sulfonic acid induced colitis in mouse and rat.
E. Type I Diabetes: Spontaneous Type I Diabetes
An animal model for Type I Diabetes includes but is not limited to BB/Wor rat or NOD mice.
F. Graft Versus Host Disease
An animal model for graft versus host disease includes but is not limited to transfer of allogenic donor lymphocytes and stem cells into irradiated host mice and transfer of allogenic donor lymphocytes and stem cells into immune competent host mice.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Having now generally described the invention, the same will be more readily understood through reference to the following Examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
The following examples are put forth for illustrative purposes only and are not intended to limit the scope of what the inventors regard as their invention.
Example 1
Potency of Non-Covalent DNA Binding Agents in MMR-Proficient Tumor Cells-Pharmacological Profile
Three novel non-covalent DNA binding agents of the invention, NSC 718813, NSC 723734 and NSC 726260, are tested in five different EGFR-resistant, K-Ras mutant cancer cell lines. These cell lines represent colorectal (SW480, SW620 and HCT116) and breast cancer (MDA-MB-231 and MDA-MA-468). The growth inhibitory effects of novel non-covalent DNA binding agents of the invention in EGFR-resistant, mutant K-ras cancer cells are compared to those observed in tumor cells that either do not express EGFR (U2OS) and/or carry the wild-type KRAS gene, and/or have normal EGFR expression or wild-type K-ras (SW403). The tumor cell lineage and their respective mutations in EGF receptor and/or its signaling cascade genes are shown in Table 2.
In Vitro Cancer Screening Methods
The in vitro assays to evaluate the anticancer potential of non-covalent DNA binding agents were measured by using one or more of the assays described below.
Sulforhodamine B (SRB) Uptake Assay:
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37° C., 5% CO2, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration, and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/ml gentamicin. Additional four, 10-fold or 1⁄2 log serial dilutions are made to provide a total of five drug concentrations plus control. Aliquots of 100 μl of these different drug dilutions are added to the appropriate microtiter wells already containing 100 μl of medium, resulting in the required final drug concentrations.
Following drug addition, the plates are incubated for an additional 48 h at 37° C., 5% CO2, 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4° C. The supernatant is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentration levels. Percentage growth inhibition is calculated as:

[(Ti−Tz)/(C−Tz)]×100 for concentrations for which Ti>/=Tz 

[(Ti−Tz)/Tz]×100 for concentrations for which Ti<Tz. 

Three dose response parameters are calculated for each experimental agent.
Growth inhibition of 50% (GI50) is calculated from [(Ti−Tz)/(C−Tz)]×100=50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.
The drug concentration resulting in total growth inhibition (TGI) is calculated from Ti Tz.
The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment is calculated from [(Ti−Tz)/Tz]×100=−50.
Values are calculated for each of these three parameters if the level of activity is reached. However, if the effect is not reached or is exceeded, the value for that parameter is expressed as, greater or less than the maximum or minimum concentration tested.
Alamar Blue Cell Survival Assay in Human Tumor Cells:
Tumor cells are plated in 96 well plates at a density of 8,000 to 10,000 cells per well in 100 uL volume and grown overnight. On the second day, the cells are supplemented with medium containing an appropriate dilution of the compounds to be tested. The cells are treated with the test compounds for two more days and the growth medium was replaced with fresh medium containing 3% Alamar Blue, incubated for 2-3 hours and plates are read in a SpectraMax Gemini XS fluorescence plate reader (Molecular Devices).
Alamar Blue Cell Survival Assay in Yeast Cells:
The cells are diluted 100-fold in yeast complete medium. 100 μL of diluted cells are seeded in 96 well plates with or without a non-covalent DNA binding compound and incubated for 24 hours at 30° C. The following day, an equal volume of yeast complete medium containing 1% alamar blue is added and incubated at 30° C. for two hours. Fluorescence intensity is measured in a fluorescent reader to calculate the inhibition effect of non-covalent DNA binding agents in mutant and wild type yeast cells.
Half-Maximal Trypan Blue Exclusion Cytotoxic Concentration (CC50) Assay:
In this assay non-specific cytotoxicity of various test compounds is determined based upon trypan blue exclusion. For the trypan blue dye exclusion assay, the cells are seeded at 10*5 cells per well in a 24-well plate and incubated overnight. The medium is replaced with fresh medium containing serial dilutions of a test compound which is diluted in DMSO. DMSO alone is used as a control. The maximum amount of DMSO in each well does not exceed more than 10%. The cells are incubated with compound for 48 hours and the supernatant which may contain dead cells is collected. The attached cells are trypsinized and transferred to the supernatant. The number of cells which do not incorporate trypan blue dye are calculated as viable cell number by hemocytometer. From the dose-response curve, the 50% CC50 is determined.
siRNA Inhibition of MMR, p53, and REV Functions
siRNA specific for different genes is purchased from Dharmacon (Thermo. Fisher Scientific Dharmacon Products, Lafayette, Colo. 80026) and the protocol recommended by the supplier is utilized. Confluent cells are trypsinized and 5000 cells are seeded in a well in the presence or absence of siRNA in 100 μL medium. The cells are incubated with siRNA for two days. A non-covalent DNA binding agent of the invention is added in a 10 μL volume and incubated for another 48 hours. After treatment with the agent, the medium is replaced with 1% alamar blue containing medium to measure fluorescence after two hours. The difference in fluorescence intensity shows the growth inhibition.
Methods for Combination Experiments
Tumor cells are plated in 96 well plates at a density of 8,000 to 10,000 cells per well in 100 uL volume and grown overnight. On the second day, the cells are supplemented with medium containing an appropriate dilution of the compounds to be tested as follows: In each well 100 uL of medium is added to all the wells. 50 uL of 3× concentration of novel non-covalent DNA binding agents are added to the top row (row A). After mixing 50 uL is added to next row (row B) and 1/3 dilution is continued up to row F (six rows) leaving seventh and eighth rows. 50 uL 3× concentration of other compounds in the combination are added to seven wells (A to G) in the left column 1 and diluted (1/3 dilution) from left to right until column 6. This is repeated other half of the plate from 7 to 12. The cells are incubated with combination of compounds for two more days and the growth medium was replaced with fresh medium containing 3% Alamar Blue, incubated for 2-3 hours and plates are read in a SpectraMax Gemini XS fluorescence plate reader (Molecular Devices). Mean of two wells is taken for calculation of combination effect.
Results:
Novel non-covalent DNA binding agents have IC50 values ranging from 8 nM to 1075 nM in tumor cells that have wild type K-RAS gene. In tumor cells harboring mutations in genes in EGFR pathways, both K-RAS and K-RAS/BRAF with or without PTEN deficiency, the IC50 values for novel non-covalent DNA binding agents of the invention are similar or better than those observed in tumor cells with the wild type K-RAS, U2OS.
The colon cancer cell line HCT 116, which has double mutations in K-RAS and in the DNA mismatch repair gene MLH, is more susceptible to non-covalent DNA binding agents of the invention than the colon cancer cells which have a K-RAS mutation only. The tumor cells which are deficient in PTEN are more sensitive to novel non-covalent DNA binding agents of the invention then are other mutated tumor cell lines. Among the three compounds tested NSC 718813 and NSC 723734 have similar potency (<100 nM), while NSC 726260 is comparatively less potent, with IC50 values around 1 uM. These cellular potency estimates for novel non-covalent DNA binding agents of the invention, in tumor cells that have K-RAS mutations and/or PTEN or mismatch repair gene deficiencies, provides a novel approach to treating genetically-resistant cancers with such genetic mutations.
The results are presented in FIG. 1—U2OS, 2-Col205, 3-HeLa, 4-lymphoblastoid 4-CEM cells, 5-leukemia cells (CEM), 6-Jurkat Cells, 7-MDA-MB-468, 8-2E-H1299 cancer cells, 9A-SW403, 9B-SW403, 10A-SW620 and 10B-HCT116, and Table 2.
Novel Non-Covalent DNA Binding Agents of the Inventions are Effective in K-RAS Mutant Colon Cancer Cells:










TABLE 2









Deficiency
IC50 nM














Type of
Mutation (Gain of
(Loss of
NSC
NSC



Cell Line
Cancer
function)
function)
718813
723734
NSC 726260





U2OS
Osteosacroma
WT
Lack of
202 + 27.3
178 + 40.9
397 + 51.6





EGFR


SW403
Colon
WT (EGFR Over
—
210 + 35.4
 550 + 141.4
1025 + 106.1




Expression)


SW620
Colon
KRAS
—
236 ± 37.2
175 ± 25.0
1050 ± 50.0 


SW480
Colon
KRAS
—
 48 + 17.7
575 + 35.4
1075 + 35.0 


HCT116
Colon
KRAS
MLH1
17 + 2.5
160 + 42.4
 550 + 167.5


MDA231
Breast
KRAS & BRAF
—
54 + 2.3
394 + 17.0
501 + 29.0




(ERK+)


MDA-MB-468
Breast
ERK+ (EGFR
PTEN
 8 ± 1.2
22 ± 0.7
364 + 54.8




over expression)


CEM
Leukemia
—
PTEN
51 ± 0.6
49 ± 0.8
161 ± 0.4 


Jurkat
Leukemia
—
PTEN
17 ± 0.2
45 ± 4.0
114 ± 26.7





WT: Wild Type tumor cell line






Example 2
Non-Covalent DNA Binding Agents Cause Double Strand Breaks
As evidenced by the sensitivity of yeast RAD52 mutants to the cytotoxicity of novel DNA binding agents, these agents cause double stranded breaks.
Yeast cells that carry mutations in different genes involved in homologous recombination (rad 50, rad51, rad52, and rad57) and nucleotide excision/double strand repair (rad1) are grown to stationary culture overnight. Results are shown in Table 3.










TABLE 3









IC50 uM














Yeast
PBD-A
PBD-B
PBD-C
PBD-D

















mutation
718813
723734
723732
726260



rad1
11
15
R
15



rad50
90
17
R
20



rad51
 7
28
100
4.5



rad52
90
50
105
15



rad57
ND
ND
ND
0.3



Wild type yeast
R
R
R
45







R = Resistant



(No killing up to 250 uM)






Example 3
Half-Life of Non-Covalent DNA Binding Agents in Rats
Determination of Pharmacokinetics of Novel Non-Covalent DNA Binding Agents in Rats:
Intravenous and oral pharmacokinetic studies are conducted on the novel non-covalent DNA binding agents, NSC 718813, NSC 723734 and NSC 726260, in male Sprague-Dawley rats. The studies are conducted in a parallel design with two groups of four male rats each for intravenous and oral administration of the test agents. The protocols for the studies are approved by the appropriate institutional animal care and use committee.
Groups of rats designated to receive oral doses of the novel non-covalent DNA binding agents of the invention molecules receive an oral dose of 20 mg/kg in a formulation vehicle comprised of N,N-dimethylacetamide (DMA), polyethylene glycol 400 (PEG400), ethanol, Cremophor EL and water (10:10:10:5:65 v/v). The dose volume for the oral doses of the test compounds is 8 mL/kg. Groups of rats are randomized to receive intravenous doses of agents. These rats receive a single intravenous bolus dose of 3 mg/kg of the test compound in a vehicle comprised of DMA:PEG400:ethanol:Cremophor EL:0.9% sodium chloride (saline) (10:10:10:5:65 v/v). The dose volume for intravenous doses of test agents is 1 mL/kg.
Predose blood samples are obtained from all rats from both, oral and intravenous dosing groups. For the intravenously dosed rats, blood samples (100 uL each) are obtained at 0.083, 0.25. 0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 hours post-dose. For the oral dose groups, the sampling times are identical to the intravenous dose group, except that the 0.083 hour sample is not collected. Following the collection of the blood samples, an equal volume of water is added to the blood sample to hemolyze the blood sample and the samples are stored frozen at −70° C. until bioanalysis.
Plasma samples are analyzed for the concentration of the test non-covalent DNA binding agents of the invention using an HPLC method with mass spectrometric (MS/MS) detection, following a liquid:liquid extraction of the plasma samples using a dichloromethane:ethyl acetate (20:80) mixture. To a 100 μL aliquot of sample, 50 μL of an internal standard (NSC 723732) is added. After mixing the internal standard wen, 2.5 mL of the extracting solvent (dichloromethane:ethyl acetate 20:80 v/v) is added. The mixture is vortexed for one minute and the samples are centrifuged at 3000 rpm for 3 minutes. Approximately 2 mL of the supernatant is taken from the centrifuged tubes and the sample is dried under a nitrogen stream at 50° C. The residue is reconstituted with 100 μL of the mobile phase and 20 μL is injected into the HPLC system for analysis. The mobile phase is comprised of milli-Q water:acetonitrile:formic acid (20:80:0.05) adjusted to pH 7.5 with ammonia.
Liquid Chromatography Mass Spectrometric (LC/MS/MS) Conditions:
The analysis of the test agent concentration is conducted by an HPLC method using a Shimadzu Prominence HPLC system and the eluent is analyzed using an API 4000 LC-MS/MS system (Applied Biosystems). The samples are analyzed on a HyPurity Advance, 50×4.6 mm, 5 u, Thermoelectron column. An injection volume of 20 μL is used for the analytical sample and the flow rate of the mobile phase is 0.6 mL/minute. Mass spectrometric analysis is conducted on the eluent using the API 4000 LC-MS/MS system and the mass parameters are analyzed for MRM transitions using NSC 723732 as the internal standard, in a positive ionization mode at a temperature of 400 C.
Pharmacokinetics of Novel Non-Covalent DNA Binding Agents, NSC 718813, NSC 723734 and NSC 726260 Following Intravenous and Oral Administration in Male Sprague-Dawley Rats:
The pharmacokinetics of NSC 718813, NSC723734 and NSC726260 are evaluated in the rat following intravenous and oral administration to evaluate the metabolic stability and clearance profile of these novel agents. Furthermore, the formulation properties of these agents are evaluated to assess their aqueous solubility and ability to administer formulations of these non-covalent DNA binding agents in vehicles similar to those used for various chemotherapeutic agents. Non-covalent DNA binding compounds have somewhat limited aqueous solubility, and require the addition of non-aqueous solvents such as polyethylene glycol 400, Cremophor and dimethylacetamide to allow intravenous administration of these agents in rats.
Pharmacokinetics of NSC 718813
NSC 718813 achieves excellent exposure in the blood following intravenous administration of a dose of 3 mg/kg. Concentrations well above its in vitro GI50 and/or TGI are achieved in rat blood for at least 4 hours following intravenous administration (see Table 4 and FIG. 11 below).







TABLE 4







Pharmacokinetic parameters (mean ± SD) of NSC 718813/1 in male Sprague


Dawley rats following oral solution and intravenous bolus administration


















AUC0-t








Tmaxa
Cmax
(ng · h/
AUC0-inf
T1/2b
CLblood
Vdss


Route
(h)
(ng/mL)
mL)
(ng · h/mL)
(h)
(mL/min/kg)
(L/kg)
F (%)c





IV-bolus
0.08
5723 ± 1005
2376 ± 304 
2424 ± 309 
2.2 ± 0.4
20.0 ± 2.9
1.5 ± 0.4



(N = 4)
(0.08-0.13)


Oral
0.5 
112 ± 32 
303 ± 129
345 ± 142
1.8 ± 0.6
NAd
NA
2.0


(N = 5)
(0.25-2.0) 





amedian (range);


bharmonic mean;


cF = (AUC0-inf)oral × doseiv/(AUC0-inf)iv × doseoral, mean oral dose: 20.50 mg/kg; mean intravenous dose: 2.90 mg/kg;


dnot applicable






These novel non-covalent DNA binding agents of the invention are designed to address the metabolic instability and rapid clearance of the naturally occurring antitumor antibiotics like anthramycin and neothramycin. As shown in Table 4, the systemic clearance of NSC 718813 is estimated to be approximately 20 mL/min/kg, which is significantly lower than the hepatic blood flow in the rat—showing that NSC 718813 has a low to moderate clearance following intravenous administration. NSC-718813 has better metabolic stability than its naturally occurring antitumor antibiotic analogs. NSC 718813 at an oral dose of 20 mg/kg has low, but measurable blood levels for up to 8 hours post-dose (see FIG. 11) and has an estimated oral bioavailability of 2%. The poor oral bioavailability of NSC 718813 coupled with its low systemic clearance, suggests absorption-limited oral bioavailability, either due to poor absorption across the gut wall and/or luminal or gastrointestinal mucosal pre-systemic elimination.
The pharmacokinetic profile and estimated parameters following intravenous and oral administration for NSC723734 are shown in FIG. 12 and Table 5, below.







TABLE 5







Pharmacokinetic parameters (mean ± SD) of NSC 723734 in male Sprague


Dawley rats (N = 4) following oral solution and intravenous bolus administration
















Tmaxa
Cmax
AUC0-t
AUC0-inf
T1/2b
CLblood
Vdss



Route
(h)
(ng/mL)
(ng · h/mL)
(ng · h/mL)
(h)
(mL/min/kg)
(L/kg)
F (%)c





IV-bolus
0.083
4053 ± 472
4246 ± 311
4405 ± 330
6.3 ± 0.3
11.4 ± 0.5
3.2 ± 0.3
NAd



(0.083-0.083)


Oral
0.25 
90.5 ± 56 
196 ± 93
216 ± 84
2.3 ± 0.7
NA
NA
0.7



(0.25-0.25)





amedian (range);


bharmonic mean;


cF = (AUC0-inf)oral × doseiv/(AUC0-inf)iv × doseoral, mean oral dose: 20.34 mg/kg; mean intravenous dose: 3.00 mg/kg;


dnot applicable






Following intravenous administration, NSC723734 shows a low clearance (11 mL/min/kg) which is about 20% of normal liver blood flow in rat (55 mL/min/kg). The compound is well distributed with a mean volume of distribution (3 L/kg) that is about 4 times the total body water. The compound is eliminated with a mean (harmonic) elimination T1/2 of 6.3 h. The mean intravenous Cmax is 4053 ng/mL and the mean overall intravenous exposure (AUC0-inf) is 4405 ng·h/mL. After oral dosing, NSC723734 shows a median Tmax of 0.25 h, indicating that the compound undergoes rapid absorption. The mean oral Cmax is 91 ng/mL, and the mean overall exposure (AUC0-inf) is 216 ng·h/mL. The oral absolute bioavailability of NSC723734 in rats is estimated to be approximately 1%. Because the overall blood clearance of the compound in the rat is low, it is unlikely that the low bioavailability of the compound results from a significant first-pass effect. It is possible that low solubility or membrane permeability may determine the oral bioavailability.
Pharmacokinetics of NSC 726260
The pharmacokinetic profile and estimated parameters following intravenous and oral administration for NSC726260 are shown in FIG. 13 and Table 6, below.







TABLE 6







Pharmacokinetic parameters (mean ± SD) of NSC726260 in male Sprague-


Dawley rats (N = 4) following oral solution and intravenous bolus administration
















Tmaxa
Cmax
AUC0-t
AUC0-inf
T1/2b
CLblood
Vdss



Route
(h)
(ng/mL)
(ng · h/mL)
(ng · h/mL)
(h)
(mL/min/kg)
(L/kg)
F (%)c





IV-bolus
0.083
5587 ± 1195
5058 ± 874 
5112 ± 871
4.8 ± 0.5
10.4 ± 2.0
1.9 ± 0.7
NAd



(0.083-0.083)


Oral
4.0 
438 ± 146
2474 ± 844
2536 ± 896
4.6 ± 1.7
NA
NA
7.9



(4.0-4.0)





amedian (range);


bharmonic mean;


cF = (AUC0-inf)oral × doseiv/(AUC0-inf)iv × doseoral, mean oral dose: 19.55 mg/kg; mean intravenous dose: 3.12 mg/kg;


dnot applicable






Following intravenous administration, NSC726260 shows a low clearance (10.4 mL/min/kg) which is about 20% of normal liver blood flow in rat (55 mL/min/kg). The compound is well distributed with a mean volume of distribution (1.9 L/kg) that is about 3 times the total body water. The compound is eliminated with a mean (harmonic) elimination T1/2 of 4.8 h. The mean intravenous Cmax is 5587 ng/mL and the mean overall intravenous exposure (AUC0-inf) is 5112 ng·h/mL. After oral dosing, NSC 726260 shows a median Tmax of 4.0 h, indicating that the compound undergoes sustained absorption. The mean oral Cmax is 438 ng/mL, and the mean overall exposure (AUC0-inf) is 2536 ng·h/mL.
The oral absolute bioavailability of NSC726260 in rats is estimated to be approximately 8%. Because the overall blood clearance of the compound in the rat is low, it is unlikely that the low bioavailability of the compound results from a significant first-pass effect. It is possible that low solubility or membrane permeability may determine the oral bioavailability.
Example 4
siRNA Inhibition of MMR, p53, and REV Functions
siRNA specific for different genes is purchased from Dharmacon (Thermo Fisher Scientific Dharmacon Products, Lafayette, Colo. 80026) and the protocol recommended by the supplier is utilized. Confluent cells are trypsinized and 5000 cells are seeded in a well in the presence or absence of siRNA in 100 μL medium. The cells are incubated with siRNA for two days. A non-covalent DNA binding agent of the invention is added in a 10 μL volume and incubated for another 48 hours. After treatment with the agent, the medium is replaced with 1% alamar blue containing medium to measure fluorescence after two hours. The difference in fluorescence intensity shows the growth inhibition. The results are presented in FIGS. 14-18 (SEQ ID NO:3-12, respectively) and Table 7.









TABLE 7









IC50 (uM)










si RNA knock out
Fold improvement in IC50

















Cell line
Compound
Control
p53
rev
mlh1
msh2
p53
rev
mlh1
msh2




















U2OS
NSC 718813
0.30
0.03
0.06
0.1

10
5
3.0



Wild type
NSC 723734
0.07
0.06
0.001
0.015

1.2
>70
3.5



NSC 726260
0.4
0.35
0.003
0.003

1.1
135
135



Doxorubicin
0.7
>1 uM
>2 uM
>3 uM

0.7
0.35
0.23


H1299 (p53−)
NSC 718813
0.6
—
—
0.5
0.35


1.3
1.9



NSC 723734
0.9
—
—
0.45
0.35


2.0
2.6


HCT116 (mlh−)
NSC 718813
0.1
0.04
0.07
—
—
12.5
7.1



NSC 723734
0.3
0.18
0.18
—
—
2.2
2.2



NSC 726260
0.75
0.2
0.15
—
—
3.8
5.0



Camptothecin
0.25
0.2
0.15
—
—
1.3
1.7

## Claims

1. A composition for treating a cancer or an inflammatory disorder comprising a combination of agents in a pharmaceutically acceptable carrier, wherein said agents comprise:
(i) a non-covalent DNA-binding pyrrolobenzodiazepine dimer (PBD); and
(ii) topoisomerase II inhibitor, wherein the PBD is selected from the group consisting of NSC718813, NSC723734, NSC723732 and NSC726260, wherein the topoisomerase II inhibitor is selected from the group consisting of an anthracycline antibiotics, doxorubicin, etoposide and cisplatin, and wherein the combination provides a synergistic effect for treating a cancer or an inflammatory disorder.

2. A kit for treating a cancer or an inflammatory disorder comprising a combination of agents in a pharmaceutically acceptable carrier, wherein said agents comprise:
(i) a non-covalent DNA: binding pyrrolobenzodiazepine dimer (PBD); and
(ii) topoisomerase II inhibitor, wherein the PBD is selected from the group consisting of NSC718813, NSC723734, NSC723732 and NSC726260, wherein the topoisomerase II inhibitor is selected from the group consisting of an anthracycline antibiotics, doxorubicin, etoposide and cisplatin, and wherein the combination provides a synergistic effect for treating a cancer or an inflammatory disorder.

3. The kit of claim 2, wherein the pharmaceutically acceptable carrier is selected from the group consisting of alumina, aluminum stearate, lecithin, albumin, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, phosphate buffered saline solution, water, emulsions, salts or electrolytes, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sucrose, glucose, maltose, and lipids.

4. The kit of claim 2, additionally comprising an instruction for use of said agents.

5. The kit of claim 4, wherein the instruction comprises instructions for administration of said agents to a subject suffering from cancer.

6. The kit of claim 4, wherein said cancer is selected from the group consisting of: lung cancer, breast cancer, osteosarcoma, neuroblastoma, colon adenocarcinoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), sarcoma, myxoma, rhabdomyoma, fibroma, lipoma, teratoma; bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma, esophageal cancer, stomach cancer, pancreatic cancer, small bowel cancer, large bowel cancer; kidney cancer, bladder cancer, urethra cancer, prostate cancer, testis cancer; hepatoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma, multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma, benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, giant cell tumors, cancer of the skull, meninges cancer, brain cancer, spinal cord cancer, uterus cancer, cervical cancer, cancer of the ovaries, vulva cancer, vagina cancer, Hodgkin's disease, non-Hodgkin's lymphoma, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma and dermatofibroma.

7. The composition of claim 1, wherein the pharmaceutically acceptable carrier is selected from the group consisting of alumina, aluminum stearate, lecithin, albumin, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, phosphate buffered saline solution, water, emulsions, salts or electrolytes, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sucrose, glucose, maltose, and lipids.

8. The composition of claim 1, wherein said cancer is selected from the group consisting of: lung cancer, breast cancer, osteosarcoma, neuroblastoma, colon adenocarcinoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), sarcoma, myxoma, rhabdomyoma, fibroma, lipoma, teratoma; bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma, esophageal cancer, stomach cancer, pancreatic cancer, small bowel cancer, large bowel cancer; kidney cancer, bladder cancer, urethra cancer, prostate cancer, testis cancer; hepatoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma, multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma, benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, giant cell tumors, cancer of the skull, meninges cancer, brain cancer, spinal cord cancer, uterus cancer, cervical cancer, cancer of the ovaries, vulva cancer, vagina cancer, Hodgkin's disease, non-Hodgkin's lymphoma, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma and dermatofibroma.

9. The composition of claim 1, wherein the combination of said agents resulting in inhibition of cancer or inflammatory disorder despite a previous resistance or refractory response to an anti-cancer therapy.