Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
  • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
  • C07D295/15—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
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  • A61K31/4164—1,3-Diazoles
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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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Definitions

• kinases that affect cell proliferation and survival pathways drive tumorigenesis in numerous cancers.
• Specific targeting of these oncogenic kinases with inhibitors has led to dramatic responses in large fractions of patients with advanced disease.
• response to kinase inhibitors is often short-lived due to the rapid onset of resistance to these drugs.
• reactivation of the mitogen-activated protein kinase (MAPK) pathway is responsible for acquired resistance to a large number of clinically approved inhibitors, including those targeting mutant BRAF, mutant EGFR, EML4-ALK, or BCR-ABL kinases.
• the loss of anticancer efficacy caused by secondary mutations to MEK1 and MEK2 can be addressed by a small molecule approach that degrades MEK1 and MEK2 kinases to render them incapable of downstream signaling, and therefore would provide an advancement in cancer therapy.
• composition comprising:
• This disclosure also provides a method of treating a cancer comprising administering to a patient in need thereof, concurrently or sequentially, a therapeutically effective amount of the compound PAC-l, and an effective amount of a second active agent, wherein the second active agent is an inhibitor of a mutant kinase or an inhibitor of a fusion kinase;
• composition to prepare a medicament for the treatment of cancer, the composition comprising:
• composition is an enhancer of MEK kinase degradation.
• the disclosed methods provide for the use of the compositions described herein for use in medical therapy.
• the medical therapy can be treating cancer, for example, melanoma, leukemia, breast cancer, lung cancer, pancreatic cancer, prostate cancer, or colon cancer.
• the invention also provides for the use of a composition as described herein for the manufacture of a medicament to treat a disease in a mammal, for example, cancer in a human, cat, or dog.
• the medicament can include a pharmaceutically acceptable diluent, excipient, or carrier.
• FIG. 1 Enhancement of caspase-3 activity following co-treatment of cancer cells with PAC-l and diverse targeted kinase inhibitors. Negligible increases in caspase-3 activity or PARP-l cleavage was observed in (A) H1975 and (B) PC-9 GRNSCLC cells treated with DMSO, single-agent PAC-l (5 ⁇ M) or osimertinib. In cells treated with PAC-l + osimertinib, dramatic increases in caspase-3 activity was observed as early as 36 h post-treatment.
• FIG. 1 PAC-l combination therapies lead to caspase-3 activation and degradation of MEK1 and MEK2 kinases.
• A Procaspase-3 activation leads to dramatic reduction in MEK1 and MEK2 levels in A375 and SK-MEL-5 cells treated with PAC-l (5 ⁇ M) + vemurafenib (10 ⁇ M) for 48 hours. This reduction was not observed in cells treated with trametinib (30 nM) + vemurafenib.
• B H1975 and PC-9 GR cells treated with PAC-l (5 ⁇ M) + osimertinib (4 nM) for 48 hours led to procaspase-3 activation and corresponding degradation of MEK1 and MEK2 kinases.
• FIG. 3 Cells treated with PAC-l and vemurafenib or osimertinib have sustained inhibition of MAPK signaling.
• PAC-l combination therapies substantially delay or eliminate acquired resistance.
• A375 cells were treated with indicated concentrations of PAC-l, vemurafenib, trametinib, or the respective combinations for up to 30 days. Cells were fixed and stained with SRB dye before imaging.
• B Quantification of (A), data reported is the mean and standard error of three independent experiments.
• C PC-9 GR cells were treated with indicated concentrations of PAC-l, osimertinib, trametinib, or the respective combinations for up to 35 days. Cells were imaged as described in (B). See also Figure 10.
• D D
• FIG. 5 Proposed mechanism of action of PAC-l combination therapy with clinically approved kinase inhibitors examined herein. Shown kinase inhibitors target key oncogenic driver kinases, leading to transient inhibition of signaling through MEK. Of critical importance is the ability of PAC-l treatment to induce cleavage of MEK kinases. This MEK cleavage, in conjunction with upstream pathway inhibition, potently abolishes ERK phosphorylation and hinders pro-survival and proliferation signaling.
• Figure 6. Enhanced apoptotic cell death in cells treated with PAC-l and clinically approved targeted kinase inhibitors.
• FIG. 1 EGFR T790M cells and
• B H3122 cells were treated with indicated concentrations of PAC-l + osimertinib or PAC-l + ceritinib, respectively for 48 hours.
• C K-562 cells were treated with indicated concentrations of PAC-l + imatinib for 72 hours. Cells were then stained with Annexin V-FITC and PI dyes and analyzed via flow cytometry. Data shown is average of at least three independent experiments and error bars are s.e.m. The dashed horizontal lines on the bar graphs represent the level of apoptotic cell death expected from an additive effect of the compounds.
• B-C Degradation of MEK1/2 kinases in cells treated with lower concentrations of PAC-l + osimertinib or PAC-l + ceritinib for 48 hours.
• D Treatment of K-562 (BCR-ABL) cells with imatinib in combination with either PAC-l or trametinib for 48 hours led to procaspase-3 activation and MEK1/2 degradation.
• E After 48 hours, lower concentration of imatinib (80 nM) in combination with PAC-l also led to procaspase-3 and resultant degradation of MEK1/2 kinases.
• Figure 8 Protection from cell death in cells treated with PAC-l + targeted kinase inhibitors using caspase inhibitor Q-VD-OPh (25 ⁇ M) for 48 hours.
• C H3122 cells were treated PAC-l (5 ⁇ M), ceritinib (30 nM), Q-VD-OPh, and combinations thereof.
• E A375 cells treated with PAC-l (5 ⁇ M), vemurafenib (10 ⁇ M), Q-VD-OPh (25 ⁇ M), and combinations for 48 hours.
• PC-9 GR cells were treated PAC-l (5 ⁇ M), osimertinib (4 nM), Q-VD-OPh (25 ⁇ M), and combinations for 48 hours.
• H3122 cells were treated PAC-l (5 ⁇ M), ceritinib (30 nM), Q-VD-OPh (25 ⁇ M), and combinations for 48 hours.
• FIG. 9 Sustained inhibition of MEK1/2 and ERK1/2 phosphorylation.
• A MEK1/2 and ERK1/2 phosphorylation in EGFR T790M cells after 48 hour of treatment with PAC-l and lower concentration of osimertinib (2.5 nM).
• B-D PAC-l + ceritinib lead to sustained MEK1/2 phosphorylation in H3122 cells.
• B H3122 cells treated with PAC-l (5 ⁇ M) + ceritinib (30 nM) for 48 hours led to sustained inhibition of MEK1/2 and ERK1/2 phosphorylation.
• trametinib (30 nM) + ceritinib treatment did not inhibit MEK1/2 phosphorylation.
• C At a lower concentration of ceritinib (15 nM), the combination led to sustained inhibition of MEK1/2 and ERK1/2 phosphorylation.
• D D
• Figure 10. PAC-l + osimertinib delays resistance.
• Related Figure 4. (A) PC-9 GR cells and (B) H1975 cells following 28 days of treatment. Cells were fixed and stained with SRB dye before imaging.
• FIG. 11 Rapid onset of resistance to targeted kinase inhibitors limits their use in treating advanced cancers.
• This disclosure shows that combination of diverse kinase inhibitors with a procaspase-3 activating compound (PAC-l) leads to degradation of MEK1/2, dramatically delaying acquired resistance; wherein PAC-l enhances caspase-3 activity and apoptosis induced by diverse kinase inhibitors; Caspase-3 -mediated MEK degradation sustains inhibition of MEK phosphorylation; PAC-l combination therapies dramatically delay or eliminate acquired resistance.
• PAC-l procaspase-3 activating compound
• PAC-l is a selective procaspase-3 activating compound that synergizes with vemurafenib, a BRAF V600E inhibitor, in numerous melanoma cell lines harboring the V600E mutation in BRAF to delay onset of acquired resistance, suggesting the feasibility of this strategy.
• PAC-l in combination with four different clinically approved inhibitors targeting four different kinases that signal through the MAPK pathway. These combinations dramatically enhance caspase-3 activity and induce degradation of MEK1/2 kinases.
• references in the specification to "one embodiment”, “an embodiment”, etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.
• the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit.
• ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range.
• a recited range e.g., weight percentages or carbon groups
• any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths.
• each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
• all language such as “up to”, “at least”, “greater than”, “less than”, “more than”, “or more”, and the like include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above.
• all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals,
• substantially is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified.
• the term could refer to a numerical value that may not be 100% the full numerical value.
• the full numerical value may be less by aboutl%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.
• next active agent refers to the administration of one active agent used in the method followed by administration of another active agent.
• the next active agent can be administered substantially immediately after the first, or the next active agent can be administered after an effective time period after the first active agent; the effective time period is the amount of time given for realization of maximum benefit from the administration of the first active agent.
• contacting refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro , or in vivo.
• an “effective amount” refers to an amount effective to treat a disease, disorder, and/or condition, or to bring about a recited effect.
• an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art, especially in light of the detailed disclosure provided herein.
• the term "effective amount” is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host.
• an “effective amount” generally means an amount that provides the desired effect.
• an effective amount refers to an amount of the active agent described herein that are effective, either alone or in combination with a pharmaceutical carrier, upon single- or multiple-dose administration to a cell or a subject, e.g., a patient, at inhibiting the growth or proliferation, inducing the killing, or halting the growth of hyperproliferative cells.
• growth inhibition or killing can be reflected as a prolongation of the survival of the subject, e.g., a patient beyond that expected in the absence of such treatment, or any improvement in the prognosis of the subject relative to the absence of such treatment.
• treating include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition.
• the terms “treat”, “treatment”, and “treating” can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated.
• treatment can include medical, therapeutic, and/or prophylactic administration, as appropriate.
• subject or“patient” means an individual having symptoms of, or at risk for, a disease or other malignancy.
• a patient may be human or non-human and may include, for example, animal strains or species used as“model systems” for research purposes, such a mouse model as described herein.
• patient may include either adults or juveniles (e.g., children).
• patient may mean any living organism, preferably a mammal (e.g, human or non-human) that may benefit from the administration of compositions contemplated herein.
• mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
• non-mammals include, but are not limited to, birds, fish and the like.
• the mammal is a human.
• compositions of the disclosure are used interchangeably herein and refer to the placement of the compositions of the disclosure into a subject by a method or route which results in at least partial localization of the composition to a desired site.
• the compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.
• the compositions described herein may be administered with additional compositions to prolong stability and activity of the compositions, or in combination with other therapeutic drugs.
• inhibitor refers to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells.
• the inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.
• induce denote quantitative differences between two states, and can refer to at least statistically significant differences between the two states.
• an amount effective to inhibit the growth of hyperproliferative cells means that the rate of growth of the cells can be, in some embodiments, at least statistically significantly different from the untreated cells. Such terms can be applied herein to, for example, rates of proliferation.
• the phrase "inhibiting the growth or proliferation" of the hyperproliferative cell, e.g. neoplastic cell refers to the slowing, interrupting, arresting, or stopping its growth and metastasis, and does not necessarily indicate a total elimination of the neoplastic growth.
• cancer generally refers to any of a group of more than 100 diseases caused by the uncontrolled growth of abnormal cells. Cancer can take the form of solid tumors and lymphomas, and non-solid cancers such as leukemia. Unlike normal cells, which reproduce until maturation and then only as necessary to replace wounded cells, cancer cells can grow and divide endlessly, crowding out nearby cells and eventually spreading to other parts of the body.
• cancerous conditions examples include, but are not limited to, anal cancer, transitional cell bladder cancer, bone cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, head and neck cancer, Kaposi's sarcoma, leukemia, lung cancer such as bronchogenic lung cancer, small cell lung cancer, and non-small cell lung cancer, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, malignant lymphoma,
• osteogenic carcinomas e.g. cancer of the bone
• ophthalmic cancers e.g. retinoblastomas and other cancers of the eye
• ovarian cancer prostate cancer
• renal cancer skin cancers such as melanoma, soft tissue sarcomas, thyroid cancer, and Wilms' tumor.
• non-malignant hyperproliferative conditions e.g. precancerous conditions
• non-malignant hyperproliferative conditions include, but are not limited to, adenomas, chondromas, enchondromas, fibromas, myomas, myxomas, neurinomas, osteoblastomas, osteochondromas, osteomas, papillary tumors, and the like, including other cancers described herein.
• leukemia or “leukemic cancer” refer to all cancers or neoplasias of the hematopoetic and immune systems (blood and lymphatic system). These terms refer to a progressive, malignant disease of the blood-forming organs, marked by distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
• Myelomas refer to other types of tumors of the blood and bone marrow cells. Lymphomas refer to tumors of the lymph tissue. Examples of leukemia include acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myelogenous leukemia (CML).
• AML acute myelogenous leukemia
• ALL acute lymphoblastic leukemia
• CML chronic myelogenous leukemia
• the second active agent is an inhibitor of mutant c-kit.
• the composition is an enhancer of MEK kinase degradation.
• the composition is a mediator of caspase- 3 degradation of both MEK-l and MEK-2 kinases.
• the composition is an inhibitor of MEK-l and MEK-2 kinase phosphorylation, an inhibitor of ERK-l and ERK-2 kinase phosphorylation, or a combination thereof.
• the second active agent is an inhibitor of a mutant EGFR kinase, or tyrosine- protein kinase.
• the second active agent is osimertinib, gefitinib, erlotinib, afatinib, or a combination thereof.
• the second active agent is an inhibitor of a fusion kinase, wherein the fusion kinase is EML4-ALK or Bcr-Abl.
• the fusion kinase is EML4-ALK and the second active agent is ceritinib, crizotinib, alectinib, brigatinib, or a combination thereof.
• the fusion kinase is Bcr-Abl and the second active agent is imatinib, dasatinib, nilotinib, bosutinib, ponatinib, or a combination thereof.
• the carrier comprises water, a buffer, a sugar, a cellulose, a cyclodextrin, dimethyl sulfoxide, polyethylene glycol, tocopherol, a liposome, a micelle, or a combination thereof, or b) the excipient comprises, a binder, a lubricant, a sorbent, a vehicle, a disintegrant, a preservative, or a combination thereof.
• the concentration of PAC-l is about 0.1 ⁇ M to about 50 ⁇ M. In additional embodiments, the concentration of PAC-l is about 0.1 ⁇ M to about 5 ⁇ M, about 1 ⁇ M to about 10 ⁇ M, about 2 ⁇ M to about 15 ⁇ M, about 3 ⁇ M to about 20 ⁇ M, about 4 ⁇ M to about 25 ⁇ M, about 5 ⁇ M to about 30 ⁇ M, about 10 ⁇ M to about 40 ⁇ M, about 15 ⁇ M to about 50 ⁇ M, about 20 ⁇ M to about 75 ⁇ M, about 25 ⁇ M to about 100 ⁇ M, about 50 ⁇ M to about 100 ⁇ M, or about 0.1 nM to about 5 ⁇ M.
• the concentration of the second active agent is about 1 nM to about 100 ⁇ M. In some additional embodiments, the concentration of the second active agent is about 0.1 nM to about 100 ⁇ M, about 0.5 nM to about 0.5 ⁇ M, about 0.5 nM to about 1 ⁇ M, about 1 nM to about 10 ⁇ M, about 1 nM to about 20 ⁇ M, about 10 nM to about 50 ⁇ M, about 0.1 ⁇ M to about 10 ⁇ M, about 0.1 ⁇ M to about 20 ⁇ M, about 1 ⁇ M to about 30 ⁇ M, about 10 ⁇ M to about 50 ⁇ M, about 10 ⁇ M to about 75 ⁇ M, or about 15 ⁇ M to about 100 ⁇ M.
• This disclosure provides various embodiments of a method of inhibiting the growth or proliferation of cancer cells comprising contacting cancer cells with an effective amount of a composition of any one of the disclosed compositions, thereby inhibiting the growth or proliferation of the cancer cells.
• a method of inducing apoptosis in a cancer cell comprising contacting the cancer cell with an effective amount of a composition of any one of the disclosed compositions, wherein apoptosis is thereby induced in the cancer cell.
• a method of treating a cancer comprising administering to a patient in need thereof, concurrently or sequentially, a therapeutically effective amount of the compound PAC-l, and an effective amount of a second active agent, wherein the second active agent is an inhibitor of a mutant kinase or an inhibitor of a fusion kinase; wherein the cancer is thereby treated.
• the second active agent is an inhibitor of a mutant EGFR kinase, an inhibitor of mutant c-kit, an inhibitor of EML4-ALK fusion kinase, or an inhibitor of Bcr-Abl fusion kinase, wherein the mutant EGFR kinase optionally has the T790M mutation.
• a pro-apoptotic Bcl-2 protein (BIM) is upregulated.
• a cancer is treated by degrading or abolishing both MEK- 1 and MEK-2 kinases, thereby effectively inhibiting the MAPK signaling pathway and inducing apoptosis in a cancer cell, by inhibiting phosphorylation of MEK-l and MEK-2, ERK-l and ERK-2, or a combination thereof.
• the cancer is melanoma, leukemia, gastric cancer, kidney cancer, lung cancer, brain cancer, or metastatic forms thereof.
• the second active agent is osimertinib, gefitinib, erlotinib, afatinib, ceritinib, crizotinib, alectinib, brigatinib, dasatinib, nilotinib, bosutinib, ponatinib, trametinib, cobimetinib, binimetinib, or imatinib, wherein resistance to treatment of a cancer in a patient in need thereof is reduced, delayed, or eliminated.
• PAC-l synergizes with osimertinib, ceritinib, or imatinib in vitro or in vivo , wherein:
• the concentration of PAC-l is about 2 ⁇ M to about 5 ⁇ M
• the second active agent is osimertinib
• the concentration of osimertinib is about 1 nM to about 30 nM
• the concentration of PAC-l is about 2 ⁇ M to about 5 ⁇ M
• the second active agent is ceritinib
• the concentration of ceritinib is about 5 nM to about 30 nM
• the concentration of PAC-l is about 5 ⁇ M to about 7.5 ⁇ M
• the second active agent is imatinib
• the concentration of imatinib is about 60 nM to about 100 nM.
• concentrations of PAC-l and the second active agent(s) recited throughout this disclosure can also be recited and interpreted as ratios of PAC-l to the second active agent, for example, by converting the concentrations recited herein to their corresponding molar ratios of PAC-l to the second active agent(s).
• the compound PAC-l and the second active agent are concurrently administered to a cancer patient. In yet other embodiments, the compound PAC-l and the second active agent are sequentially administered to a cancer patient. In some more embodiments, the compound PAC-l is administered to a cancer patient before the second active agent. In even more embodiments, the compound PAC-l is administered to a cancer patient after the second active agent.
• the methods further comprise administering to the patient, concurrently or sequentially, a therapeutically effective amount of a MEK inhibitor, a V600E mutated BRAF kinase inhibitor, or a combination thereof.
• the MEK inhibitor is trametinib, cobimetinib, binimetinib, or a combination thereof.
• the mutated BRAF kinase inhibitor is vemurafenib, dabrafenib, encorafenib, or a combination thereof.
• composition to prepare a medicament for the treatment of cancer, the composition comprising:
• composition is an enhancer of MEK kinase degradation.
• the cancer is melanoma, leukemia, gastric cancer, kidney cancer, lung cancer, brain cancer, or metastatic forms thereof.
• Caspase-3 activity is significantly enhanced in cells treated with PAC-l and diverse kinase inhibitors
• PAC-l significantly increases the caspase-3 activity in osimertinib-treated non-small cell lung cancer (NSCLC) cell lines H1975
• caspase-3 Activation of an executioner caspase such as caspase-3 leads to the cleavage of hundreds of proteins in the cell. Intriguingly, the protein substrates for caspase-3 tend to be found in protein complexes or signaling pathways that govern cell fate and survival.
• H1975 and PC-9 GR cells were treated with PAC-l and osimertinib and probed for changes ERK1/2 and MEK1/2 phosphorylation.
• EGFR T790M cells treated with PAC-l and osimertinib led to sustained loss of ERK1/2 and MEK1/2 phosphorylation, as a result of MEK1 and MEK2 degradation.
• PAC-l and gefitinib no corresponding reduction in ERK1/2 or MEK1/2 phosphorylation is observed (Figure 3C), indicating that the effect with osimertinib is specific for the EGFR T790M target.
• H3122 cells EML4-ALK
• EML4-ALK H3122 cells
• PAC-l + ceritinib or trametinib + ceritinib were treated with PAC-l + ceritinib or trametinib + ceritinib and probed for changes in ERK1/2 and MEK1/2 phosphorylation.
• H3122 cells co-treated with PAC-l + ceritinib for 48 hours also led to sustained reduction in ERK1/2 and MEK1/2 phosphorylation (Figure 9B and Figure 9D), which can be attributed to the caspase-3 -mediated degradation of MEK1 and MEK2 kinases.
• PAC-l + vemurafenib is more efficacious than trametinib + vemurafenib in eliminating acquired resistance
• PAC-l + vemurafenib would be more efficacious than trametinib + vemurafenib in delaying resistance in A375 cells.
• the rationale behind the hypothesis is that PAC-l + vemurafenib significantly enhances apoptotic cell death leading to degradation of MEK kinases to further inhibit ERK1/2 phosphorylation.
• A375 cells were treated with PAC-l, vemurafenib, trametinib, and their respective combinations for up to 30 days.
• PAC-l or trametinib (5 nM) as single agents had minimal cytotoxic effect as compared to DMSO-treated cells following 8 days of treatment.
• treatment with osimertinib, PAC-l + osimertinib, or trametinib + osimertinib for 8 days was effective in inhibiting cell proliferation.
• resistant clones were visible in PC-9 GR cells after 28 days of treatment with single-agent osimertinib but not in cells treated with PAC-l and osimertinib ( Figures 4C and D).
• Resistant clones were visible in H3122 cells after 20 days of treatment with single-agent ceritinib but not in cells treated with either PAC-l and osimertinib or trametinib and ceritinib (Figure 4E). After 32 days of treatment, there was a dramatic increase in the number of resistant clones present in cells treated with single-agent ceritinib but few resistant clones were observable in cells treated with PAC-l + ceritinib ( Figure 4E). In cells treated with trametinib + ceritinib, a number of resistant clones were clearly visible, indicating the presence of ALKi + MEKi resistant H3122 cells ( Figures 4E and 4F).
• kinase inhibitors targeting BRAF V600E , EGFR T790M , EML4- ALK, and BCR-ABL leads to enhanced procaspase-3 activation and degradation of MEK1 and MEK2 kinases (Figure 5).
• the degradation of MEK kinases then leads to sustained inhibition of MEK1/2 and ERK1/2 signaling.
• the combined effect of increased apoptotic cell death and sustained inhibition of the MAPK pathway that is observed in the PAC-l combination therapies work in tandem to dramatically delay or eliminate resistance.
• the disclosed results show that co-treatment of a procaspase-3 activator, PAC-l, with diverse targeted kinase inhibitors at clinically relevant concentrations (see Table 2 for predicted effects of each kinase inhibitor on MEK1/2 phosphorylation and caspase activity) is broadly effective in enhancing caspase-3 activity and apoptotic cell death across diverse tumor histologies and driver mutations.
• the resultant caspase-3 activity leads to enzymatic degradation of both MEK1 and MEK2 kinases and sustained inhibition of both MEK1/2 and ERK1/2 phosphorylation. While sustained ERK1/2 inhibition can be achieved with MEK1/2 inhibitors, this disrupts the negative feedback on RAF kinases, leading to the paradoxical hyper-phosphorylation of MEK1/2.
• Trametinib was developed as a“feedback buster” to minimize MEK1/2 hyper-phosphorylation but the inhibitory effect is relatively transient as previously shown and consistent with the disclosed results ( Figure 3).
• this disclosure show that caspase-3 mediated degradation of MEK1 and MEK2 kinases is an excellent strategy to inactivate ERK1/2, without the corresponding rebound in MEK1/2 phosphorylation.
• the disclosed results are supported by the abolishment of ERK1/2 phosphorylation observed upon genomic knockdown of both MEK1 and MEK2. Due to the critical role of MEK1/2 kinases in regulating the MAPK pathway, its sustained inhibition can be advantageous in significantly delaying the onset of acquired resistance.
• Targeted kinase inhibitors have had a dramatic impact on cancer treatment, but resistance has seriously limited the durability of this effect.
• a potentially generalizable strategy has been identified to eliminate or substantially delay the resistance to targeted anticancer therapies, and have successfully demonstrated its efficacy in cancers driven by BRAF V600E , EGFR T790M , and EML4-ALK kinases.
• the compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient, or carrier.
• the compounds may be added to a carrier in the form of a salt or solvate.
• a pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a- ketoglutarate, and b-glycerophosphate.
• Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.
• salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound.
• a sufficiently basic compound such as an amine
• a suitable acid for example, a sufficiently basic compound such as an amine
• Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.
• the compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms.
• the forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.
• the compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier.
• a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
• compounds can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet.
• Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Such compositions and preparations typically contain at least 0.1% of active compound.
• compositions and preparations can vary and may conveniently be from about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10%, of the weight of a given unit dosage form.
• amount of active compound in such therapeutically useful compositions can be such that an effective dosage level can be obtained.
• the tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate.
• binders such as gum tragacanth, acacia, corn starch or gelatin
• excipients such as dicalcium phosphate
• a disintegrating agent such as com starch, potato starch, alginic acid and the like
• a lubricant such as magnesium stearate.
• a sweetening agent such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring, may be added.
• the unit dosage form When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
• a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
• the active compound may be incorporated into sustained-release preparations and devices.
• the active compound may be administered intravenously or intraperitoneally by infusion or injection.
• Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
• Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.
• compositions suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
• the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
• the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
• the proper fluidity can be
• microorganisms maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, buffers, or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and/or gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, optionally followed by filter sterilization.
• methods of preparation can include vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the solution.
• compounds may be applied in pure form, e.g., when they are liquids.
• a dermatologically acceptable carrier which may be a solid, a liquid, a gel, or the like.
• Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like.
• Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol/glycol blends, in which a compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
• Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
• the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.
• Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
• compositions for delivering active agents to the skin are known to the art; for example, see U.S. Patent Nos. 4,992,478 (Geria), 4,820,508
• compositions can be used in combinations with the compounds described herein where an ingredient of such compositions can optionally be replaced by a compound described herein, or a compound described herein can be added to the composition.
• Useful dosages of the compositions described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949 (Borch et al.).
• the amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.
• a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
• the compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
• the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.
• the compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg/m 2 , conveniently 10 to 750 mg/m 2 , most conveniently, 50 to 500 mg/m 2 of active ingredient per unit dosage form.
• the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
• the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
• the invention provides therapeutic methods as described herein for treating cancer in a mammal, which involve administering to a mammal having cancer an effective amount of a compound or composition described herein.
• a mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like.
• Cancer refers to any various type of malignant neoplasm, for example, lung cancer, breast cancer, melanoma and leukemia, and in general is characterized by an undesirable cellular proliferation, e.g., unregulated growth, lack of differentiation, local tissue invasion, and metastasis.
• the ability of a compound of the invention to treat cancer may be determined by using assays well known to the art. For example, the design of treatment protocols, toxicity evaluation, data analysis, quantification of tumor cell kill, and the biological significance of the use of transplantable tumor screens are known.
• A375, K-562, and SK-MEL-5 were obtained from ATCC.
• PC-9 GR, H1975, and H3122 were provided by Prof. Eric Haura (Moffitt Cancer Center).
• PC-9 GR, H1975, and H3122 were cultured in RPMI 1640 supplemented with 10% FBS (Gemini).
• A375 and SK- MEL-5 were cultured in DMEM + 10% FBS.
• K-562 was cultured in IMDM + 10% FBS. All cells were cultured at 37 °C with 5% CO2.
• Sex of human cell lines A375 (Female, 54 years old), K-562 (Female, 53 years old), SK-MEL-5 (Female, 24 years old), H3122 (Female, age unknown), H1975 (Female, age unknown), PC-9 GR (Female, age unknown).
• X/Y at the University of Arizona Genetics Core (UAGC). >1 million cells were harvested and lysed using the cell lysis buffer (50 mM Tris, 50 mM EDTA, 25 mM sucrose, 100 mM NaCl, 1% SDS, pH 8). DNA extraction and short tandem repeats (STRs) profiling for each cell line were carried out at the UAGC. The resulting autosomal STR profiles were compared to reference databases such as ATCC, DSMZ, and JCRB.
• STRs short tandem repeats
• SRB sulforhodamine B
• SRB sulforhodamine B
• EGFR T790M cell lines 4,000 cells were seeded in each well of 96-well plates and allowed to adhere overnight. The next day, indicated concentrations of PAC-l or osimertinib, were added and treated for 0, 2, 4, 24, 30, 35, 44 and 48 hour. K-562 cells were seeded at 3,000 cells per well and treated with indicated concentrations of PAC-l or imatinib for 0, 2, 4, 24, 48, 68, and 72 hour. H3122 cells were seeded at 4,000 cells per well and allowed to adhere overnight. The cells were then treated with indicated concentrations of PAC-l or ceritinib for 0, 2, 4, 24, 44, and 48 hour. 10 ⁇ M raptinal was used as the positive control throughout the experiment.
• Cells were lysed using RIPA buffer containing phosphatase (BioVision) and protease inhibitor cocktail (Calbiochem). Protein concentration was determined using the BCA assay (Pierce). Cell lysates containing 8-20 pg of protein were loaded into each lane of 4-20% gradient gels (BioRad) and ran for SDS-PAGE. Proteins were transferred onto PDVF membrane (Millipore) for Western blot analysis. Blots were blocked with BSA for one hour followed by incubation with primary antibody overnight (manufacturer’s recommended dilutions). Secondary antibody was incubated for one hour. Blots were then imaged with a ChemiDoc Touch after incubation with SuperSignal West Pico Solution following manufacturer’s protocols.
• EGFR T790M cell lines 40,000 cells were seeded in l2-well plates and allowed to adhere overnight. The next day, indicated concentrations of PAC-l or osimertinib were added and allowed to incubate at 37°C for 48 hours. K-562 cells were seeded at 30,000 and incubated with PAC-l or imatinib for 72 hours. In l2-well plates, 40,000 H3122 cells were seeded and allowed to adhere overnight. The next day, they were incubated with PAC-l or ceritinib for 48 hours.
• A375 (BRAF V600E ) cells were seeded with 70,000 cells/well in l2-well plates and allowed to adhere overnight. The next day, indicated concentrations of PAC-l, vemurafenib, trametinib, and/or Q-VD-OPh were added and cells were incubated at 37°C for 48 hours. For the EGFR T790M cell line, PC-9 GR, 40,000 cells were seeded in l2-well plates and allowed to adhere overnight. The next day, indicated concentrations of PAC-l, osimertinib, and Q-VD- OPh were added and allowed to incubate at 37°C for 48 hours.
• H3122 cells were seeded and allowed to adhere overnight. The next day, they were incubated with PAC-l, ceritinib, and/or Q-VD-OPh for 48 hours. K-562 cells were seeded with 35,000 cells/well in l2-well plates. The next day, indicated concentrations of PAC-l, imatinib, and/or Q-VD-OPh were added and cells were incubated at 37°C for 48 hours.
• This assay was performed as described previously. Briefly, 100-250 cells were seeded and allowed to adhere overnight. The next day, cells were treated with indicated
• PC-9 GR or H1975 cells were seeded at 2,000 cells per well and allowed to adhere overnight. The next day, cells were treated with indicated concentrations of PAC-l or osimertinib for 8 or 28 days. Media was refreshed every 3-4 days with new compounds.
• PC-9 GR cells were seeded at 10,000 cells per well in 6 well plates and allowed to adhere overnight. The next day, cells were treated with indicated concentrations of PAC-l, osimertinib, or 5 nM trametinib for 8, 28, or 35 days. Media was refreshed every 3-4 days with new compounds. At the end of the incubation period, cells were fixed 10%
• H3122 cells were seeded at 10,000 cells per well in 6 well plates and allowed to adhere overnight. The next day, cells were treated with indicated concentrations of PAC-l, osimertinib, or 5 nM trametinib for 8, 20, or 32 days. Media was refreshed every 3-4 days with new compounds. At the end of the incubation period, cells were fixed 10%
• compositions illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of the combination compounds described herein, (e.g., PAC-l and a second active agent), or pharmaceutically acceptable salts or solvates thereof (hereinafter referred to as 'Composition X', which can be one active agent or a combination of two active agents):
• composition illustrates representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of the combination compounds described herein (e.g., PAC-l and the second active agent), or pharmaceutically acceptable salts or solvates thereof:
• compositions may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Compound X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest. While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

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