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WO2021202961A1 - Procédés de traitement d'un mélanome métastatique résistant à l'immunothérapie - Google Patents

Procédés de traitement d'un mélanome métastatique résistant à l'immunothérapie Download PDF

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WO2021202961A1
WO2021202961A1 PCT/US2021/025514 US2021025514W WO2021202961A1 WO 2021202961 A1 WO2021202961 A1 WO 2021202961A1 US 2021025514 W US2021025514 W US 2021025514W WO 2021202961 A1 WO2021202961 A1 WO 2021202961A1
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inhibitor
compound
braf
formula
human subject
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Ann Richmond
Rebecca SHATTUCK-BRANDT
Anna VILGELM
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United States Represented By Department Of Veterans Affairs AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/451—Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • MDM2 Mouse double minute 2 homolog
  • p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of numerous genes involved in cell cycle arrest, apoptosis, senescence, and DNA repair. Unlike normal cells, which have infrequent cause for p53 activation, tumor cells are under constant cellular stress from various insults including hypoxia and pro-apoptotic oncogene activation. Thus, there is a strong selective advantage for inactivation of the p53 pathway in tumors, and it has been proposed that eliminating p53 function may be a prerequisite for tumor survival. In support of this notion, three groups of investigators have used mouse models to demonstrate that absence of p53 function is a continuous requirement for the maintenance of established tumors. When the investigators restored p53 function to tumors with inactivated p53, the tumors regressed.
  • p53 is inactivated by mutation and/or loss in 50% of solid tumors and 10% of liquid tumors.
  • MDM2 an oncoprotein, inhibits p53 function, and it is activated by gene amplification at incidence rates that are reported to be as high as 10%. MDM2, in turn, is inhibited by another tumor suppressor, pl4ARF. It has been suggested that alterations downstream of p53 may be responsible for at least partially inactivating the p53 pathway in p53 WT tumors (p53 wild type). In support of this concept, some p53 WT tumors appear to exhibit reduced apoptotic capacity, although their capacity to undergo cell cycle arrest remains intact.
  • One cancer treatment strategy involves the use of small molecules that bind MDM2 and neutralize its interaction with p53.
  • MDM2 inhibits p53 activity by three mechanisms: 1) acting as an E3 ubiquitin ligase to promote p53 degradation; 2) binding to and blocking the p53 transcriptional activation domain; and 3) exporting p53 from the nucleus to the cytoplasm. All three of these mechanisms would be blocked by neutralizing the MDM2-p53 interaction.
  • this therapeutic strategy could be applied to tumors that are p53 WT , and studies with small molecule MDM2 inhibitors have yielded promising reductions in tumor growth both in vitro and in vivo. Further, in patients with p53-inactivated tumors, stabilization of wild type p53 in normal tissues by MDM2 inhibition might allow selective protection of normal tissues from mitotic poisons.
  • MDM2 means a human MDM2 protein and p53 means a human p53 protein. It is noted that human MDM2 can also be referred to as HDM2 or hMDM2. Several MDM2 inhibitors are in human clinical trials for the treatment of various cancers.
  • the present invention provides a method of treating metastatic melanoma.
  • the present invention relates to a method of treating immunotherapy resistant metastatic melanoma comprising the step of administering to a human subject in need thereof, a therapeutically effective amount of a MDM2 inhibitor or a MDM2 inhibitor in combination with a BRAF inhibitor and a MEK inhibitor.
  • the present invention relates to a method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor.
  • the MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof.
  • the method further comprises detecting the BRAF genotype in the human subject.
  • the human subject exhibits a wild-type BRAF ⁇ 600 (BRAF ⁇ ) genotype.
  • the method further comprises detecting the NRAS genotype in the human subject.
  • the human subject exhibits a wild-type NRAS ( NRAS WT ) genotype.
  • the method further comprises detecting the NF1 genotype in the human subject.
  • the human subject exhibits a wild-type NF1 genotype (A/F1 WT ).
  • the human subject exhibits BRAF ⁇ 7 , A/RAS ⁇ and NF1 WT .
  • the human subject exhibits a mutant NRAS. In an embodiment, the human subject exhibits a mutant NF1.
  • the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).
  • TILs tumor-infiltrating lymphocytes
  • TCR T-cell receptor
  • PBL peripheral blood lymphocytes
  • CAR chimeric antigen receptor
  • the immunotherapy is an immune checkpoint protein inhibitor therapy.
  • the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.
  • the immunotherapy is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof.
  • the present invention relates to a method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, a BRAF inhibitor and a MEK inhibitor.
  • the MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof.
  • the BRAF inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and combinations thereof.
  • the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, selumetinib, pimasertib, binimetinib, and combinations thereof.
  • the method further comprises detecting the BRAF genotype in the human subject.
  • the human subject exhibits BRAF V ⁇ 0 mutation.
  • the human subject exhibits NRAS QG1 mutation.
  • the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).
  • TILs tumor-infiltrating lymphocytes
  • TCR T-cell receptor
  • PBL peripheral blood lymphocytes
  • CAR chimeric antigen receptor
  • the immunotherapy is an immune checkpoint protein inhibitor therapy.
  • the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.
  • the immunotherapy is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-PD- L2 antibody.
  • the anti-PD- L2 antibody is rHlgM12B7A.
  • the compound of Formula (I) is in a free form.
  • the MDM2 inhibitor is a pharmaceutically acceptable salt of a compound of Formula (I).
  • the compound of Formula (I) is in an amorphous form.
  • the compound of Formula (I) is administered once daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.
  • the compound of Formula (I) is administered twice daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.
  • the human is treated with the MDM2 inhibitor for a period selected from the group consisting of about 3 days, about 5 days, about 7 days, 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, and about 56 days.
  • the compound of Formula (I) is orally administered.
  • the MDM2 inhibitor is administered before administration of the BRAF inhibitor and MEK inhibitor.
  • the MDM2 inhibitor is administered after administration of the BRAF inhibitor and MEK inhibitor.
  • the MDM2 inhibitor is administered concurrently with administration of the BRAF inhibitor and MEK inhibitor.
  • the therapeutically effective amount of the MDM2 inhibitor is 120 mg.
  • FIG. 1A illustrates patient demographics and molecular characterization of PDX tumors.
  • a panel of 15 PDX melanoma tumors is arranged by distinct genetic phenotypes based on the mutation status.
  • the patient demographics are shown including Clark's Level tumor stage and prior treatments. Prior treatments are list in the order they were received.
  • the genetic results are based upon NextGen sequencing of the primary tumor sample or PDX P2 passage (PDX1577, 1668, 2316 and 2552, indicated by a *) using a Comprehensive Cancer Panel, and the results for 10 melanoma driver mutations are shown.
  • STOP LOST indicates a nonsense mutation within a stop codon
  • Splice Site indicates a mutation (deletion) involving a splice site.
  • FIG. IB illustrates the TP53 mutations by NGS.
  • the specific TP53 mutations detected by NextGen sequencing are shown for each PDX.
  • the identification of the mutations, the small nucleotide polymorphism (SNP) Effect and Impact, and the designation of germline or somatic mutation was determined by using QIAGEN NGS Data Web Analysis Web Portal.
  • SnpEff is an open-source tool that annotates variants and predicts their effects on genes by using an interval forest approach.
  • the variant frequency is also listed for each non-synonymous mutation (NSM).
  • the PDX numbers listed below the horizontal line are those tumors that express the P53P72R polymorphism.
  • the gray shading highlights those PDX tumors expressing point NSMs within the TP53 gene.
  • NGS was performed on the patient tumor (P0) except for PDX1577, 1668, 2316, and 2552. For these four PDX, the second passage (P2) was sequenced.
  • FIG. 2A illustrates those PDX lines that responded to the standard therapy dabrafenib and trametinib (D+T) but not to the compound of Formula (I) (Group I), with PDX1839 as an example.
  • FIG. 2B illustrates those PDX lines that responded to the combination therapy (the compound of Formula (I) + dabrafenib + trametinib) synergistically (Group II), but not to the compound of Formula (I) alone or trametinib alone or standard therapy dabrafenib and trametinib (D+T), with PDX1946 as an example.
  • the combination therapy the compound of Formula (I) + dabrafenib + trametinib
  • D+T standard therapy dabrafenib and trametinib
  • FIG. 2C illustrates those PDX lines that responded the compound of Formula (I) alone, but not to standard therapy dabrafenib and trametinib (D+T) (Group III), with PDX2316 as an example.
  • FIG. 2D illustrates those PDX lines that responded the compound of Formula (I) alone, but not to standard therapy dabrafenib and trametinib (D+T) (Group III), with PDX1595 and 1668 as examples, and also illustrates the compound of Formula (I) treatment increases nuclear localization of P53.
  • the standard therapy for PDX1595 was trametinib and for PDX1668 the standard therapy was dabrafenib + trametinib.
  • each panel in A-C includes data showing the effect of drug treatments on Tumor Growth, Final Tumor Weight, %Ki67 Staining and the Tumor Growth Statistical Analysis.
  • Tumor volume was analyzed on the natural log scale to better meet normality assumptions and the predicted mean and standard error of tumor volume over time for each treatment group is shown.
  • Dot plot of tumor weight (g) by treatment and % positive Ki67 by treatment are shown.
  • a t-ratio table for pairwise comparison in tumor growth rate between treatments based on the mixed-effect model with post hoc tests is shown.
  • FIG. 3A illustrates a summary of statistical summary of t. ratios obtained from the statistical analysis of treatment difference comparisons of the tumor growth rate based on the tumor volume for each PDX treatment comparison.
  • the BRAF and TP53 mutational status of each PDX is also listed.
  • the group assignment, shown in the final column, is based on the response to the compound of Formula (I) and T+/-D treatment.
  • FIG. 3B illustrates the synergy analysis of the effect of the compound of Formula (I) and dabrafenib + trametinib on the PDX lines.
  • the mean estimated tumor growth rate of 4 independent PDX Group II lines is graphed with a 95% confidence interval by treatment.
  • Mice implanted with PDX1351, 1577, 1946, and 2552 were treated in vivo with Vehicle, the compound of Formula (I), D+T, or the compound of Formula (l)+D+T as described in FIGs. 2A-2D.
  • FIG. 4A illustrates synergy associated with changes in cell morphology is consistent with karyorrhexis and large vacuole formation.
  • FI&E is shown for Vehicle and the compound of Formula (I) + D+T treated mice implanted with PDX1351, PDX1946, PDX2552 and PDX1577 (Group III).
  • the t-ratio for the statistical difference between the vehicle or the compound of Formula (I) treatment group compared to the compound of Formula (l)+D+T treatment group is shown.
  • 20X images are shown and the scale marker is lOOpm.
  • FIG. 4B illustrates alterations in protein expression associated with synergism.
  • a volcano plot of RPPA data obtained from vehicle-treated and the compound of Formula (I) + D+T treated Group III PDX tumors samples for PDX1179, PDX1351, and PDX2252 is shown. Three tumors were analyzed for each PDX treatment.
  • FIG. 4C illustrates alterations in protein expression associated with synergism.
  • a heat map of RPPA data obtained from vehicle-treated and the compound of Formula (I) + D+T treated Group III PDX tumors samples for PDX1179, PDX1351, and PDX2252 is shown. Three tumors were analyzed for each PDX treatment.
  • FIG. 5A illustrates the results of oncoprint cluster analysis.
  • DNA sequence analysis was performed using NextGen sequencing. Paired targeted analysis for all 15 PDX tumors was performed using Oncoprint on the cBioPortal (http://www.cbioportal.org/) hosted by Sloan Kettering Institute. These results show the analysis of the 10 genes listed in Table 1.
  • the inset table lists the brackets and terms used to describe the copy number variations.
  • FIG. 5B is a volcano plots of RPPA data obtained from vehicle-treated and the compound of Formula (I) treated Group I and II.
  • FIG. 5C is a heat map of RPPA data obtained from vehicle-treated and the compound of Formula (I) treated Group I and II.
  • FIG. 5D is a volcano plots of RPPA data obtained from vehicle-treated and the compound of Formula (I) treated Group III.
  • FIG. 5E is a heat map of RPPA data obtained from vehicle-treated and the compound of Formula (I) treated Group III.
  • three tumors were analyzed for each PDX treatment.
  • Group I and II PDX tumors were PDX1179, 1351 and 2552 and Group III PDX tumors were PDX1129,
  • FIG. 6A illustrates the tumor growth of PDX1577. Tumor volume was analyzed on the natural log scale to better meet normality assumptions and the predicted mean and standard error of tumor volume over time for each treatment group is shown.
  • FIG. 6B illustrates the final tumor weight of PDX1577 under different treatment. Dot plot of tumor weight (g) by treatment (gray line: mean with standard error).
  • FIG. 6C is a T-ratio table for PDX tumors treated with Navitoclax. A t-ratio table for pairwise comparison in tumor growth rate between treatments based on the mixed-effect model with post hoc tests is shown.
  • FIG. 7A illustrates NGS summary.
  • the mean read depth and number of high confidence variants are listed for each PDX.
  • FIG. 7B illustrates the mutations and copy number variations (cnv) for ten driver genes and MDM2. All NSMs and CNV noted by NGS for the 10 driver genes and MDM2 are listed. CNV is not available for PDX0807, 1129, 2195 and 9164. The following brackets were used for labeling CNV. HOMDEL 0-0.5 copies; HETLOSS 0.51-1.5 copies; GAIN: 2.5-3.5 copies; AMP >3.5 copies (see FIG. 5A). There were no NSMs for MDM2 in any PDX tumor. The primary tumor sample (P0) was analyzed except for PDX1577, 1668, 2316 and 2552. For these 4 PDX tumors, the second passage (P2) was analyzed and these PDX tumors are indicated by an *.
  • FIG. 8A illustrates short tandem repeat (STR) analysis results.
  • P2 second passage
  • P0 the originating patient tumor
  • P0 the originating patient tumor
  • P0 the originating patient tumor
  • P0 the originating patient tumor
  • P0 blood when available.
  • Two distinct isolates from each tumor were analyzed. The clonal variation between either the blood, patient sample or PDX tumor are noted and these variations all represented the loss of an allele. No gain of an allele was detected.
  • FIG. 8B illustrates the validation of PDX tumors via immunohistochemistry.
  • FIG. 9A illustrates the effect of drug treatments on estimated tumor growth for PDX1839 tumor.
  • FIG. 9B illustrates the effect of drug treatments on estimated tumor growth for PDX9164 tumor.
  • FIG. 9C illustrates the effect of drug treatments on estimated tumor growth for PDX2195 tumor.
  • FIG. 9D illustrates the effect of drug treatments on estimated tumor growth for PDX2252 tumor.
  • FIG. 9E illustrates the effect of drug treatments on estimated tumor growth for PDX1351 tumor.
  • FIG. 9F illustrates the effect of drug treatments on estimated tumor growth for PDX1179 tumor.
  • FIG. 9G illustrates the effect of drug treatments on estimated tumor growth for PDX1946 tumor.
  • FIG. 9H illustrates the effect of drug treatments on estimated tumor growth for PDX2552 tumor.
  • FIG. 91 illustrates the effect of drug treatments on estimated tumor growth for PDX1577 tumor.
  • FIG. 9J illustrates the effect of drug treatments on estimated tumor growth for PDX2316 tumor.
  • FIG. 9K illustrates the effect of drug treatments on estimated tumor growth for PDX1129 tumor.
  • FIG. 9L illustrates the effect of drug treatments on estimated tumor growth for PDX1767 tumor.
  • FIG. 9M illustrates the effect of drug treatments including Navitoclax on estimated tumor growth for PDX1129 tumor.
  • FIG. 9N illustrates the effect of drug treatments including Navitoclax on estimated tumor growth for PDX1351 tumor.
  • FIG. 90 illustrates the effect of drug treatments including Navitoclax on estimated tumor growth for PDX1577 tumor.
  • FIG. 9P illustrates the effect of drug treatments including Navitoclax on estimated tumor growth for PDX1668 tumor.
  • Tumor volume was analyzed on the natural log scale to better meet normality assumptions.
  • the predicted mean and standard error of tumor volume over time for each treatment group is shown.
  • a statistics table for pairwise comparison in tumor growth rate between treatments based on the mixed-effect model with post hoc tests is shown below each growth curve.
  • the final tumor weight for all PDX tumors is shown. Dot plot of tumor weight (g) by treatment (gray line: mean with standard error) is shown.
  • FIG. 10 illustrates alterations in protein expression associated with D+T response in Group I PDX tumors.
  • RPPA data obtained from Group I PDX tumors (PDX1839, 2195 and 2252) treated with D+T or vehicle control were analyzed and the volcano plot is shown. Three tumors for each PDX treatment were analyzed.
  • FIG. 11 illustrates that protein expression patterns did not predict the compound of Formula (I) responsiveness in Group III PDX tumors compared to Group I and II PDX tumors.
  • RPPA data obtained from vehicle-treated Group I PDX tumors (PDX1839, 2195 and 2252) and Group II PDX tumors (PDX 1179, 1351, and 2552) were compared to vehicle-treated Group III PDX tumors (PDX1129, 1595, 1668 and 2316). These data were analyzed and the volcano plot is shown. Three tumors for each PDX treatment were analyzed. No significant differences in protein expression were noted.
  • FIG. 12 illustrates the association between mutation status and t ratio.
  • the correlation between the BRAF V600 mutation status (“Yes", for mutation and "No", for wild-type BRAF or mutations outside of the V600 codon) are plotted relative to the t-ratio for the difference in tumor growth rate between the compound of Formula (I) treated and vehicle treated mice.
  • administered in combination with encompass administration of two or more active pharmaceutical ingredients to a subject so that both agents and/or their metabolites are present in the subject at the same time.
  • Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more agents are present.
  • combination or “pharmaceutical combination” is defined herein to refer to either a fixed combination in one dosage unit form, a non-fixed combination or a kit of parts for the combined administration where the therapeutic agents may be administered together, independently at the same time or separately within time intervals, which preferably allows that the combination partners show a cooperative, e.g. synergistic effect.
  • the single compounds of the pharmaceutical combination of the present disclosure could be administered simultaneously or sequentially.
  • the pharmaceutical combination of the present disclosure may be in the form of a fixed combination or in the form of a non-fixed combination.
  • an effective amount refers to that amount of an active pharmaceutical ingredient or combination of active pharmaceutical ingredients as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment.
  • a therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, and other factors which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will induce a particular response in target cells, (e.g., the reduction of platelet adhesion and/or cell migration).
  • the specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.
  • fixed combination means that the therapeutic agents, e.g., the single compounds of the combination, are in the form of a single entity or dosage form.
  • IC50 refers to the half maximal inhibitory concentration, i.e. inhibition of 50% of the desired activity.
  • EC50 refers to the drug concentration at which one-half the maximum response is achieved.
  • compounds described herein include of the isomers, stereoisomers, and enantiomers thereof.
  • non-fixed combination means that the therapeutic agents, e.g., the single compounds of the combination, are administered to a patient as separate entities or dosage forms either simultaneously or sequentially with no specific time limits, wherein preferably such administration provides therapeutically effective levels of the two therapeutic agents in the body of the subject, e.g., a mammal or human in need thereof.
  • “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, and absorption delaying agents. The use of such media and agents for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional media or agent is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Supplementary active ingredients can also be incorporated into the described compositions. Unless otherwise specified, or clearly indicated by the text, reference to therapeutic agents useful in the pharmaceutical combination of the present disclosure includes both the free base of the compounds, and all pharmaceutically acceptable salts of the compounds.
  • salts refers to salts derived from a variety of organic and inorganic counter ions known in the art.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.
  • QD means quaque die, once a day, or once daily.
  • BID bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID means bis in die, twice a day, or twice daily.
  • TID
  • Solvate refers to a compound in physical association with one or more molecules of a pharmaceutically acceptable solvent.
  • a prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • Compounds of the invention also include crystalline and amorphous forms, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as combinations thereof.
  • Crystalstalline form and polymorph are intended to include all crystalline and amorphous forms of the compound, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms, as well as combinations thereof, unless a particular crystalline or amorphous form is referred to.
  • the present invention relates to pharmaceutical combinations or pharmaceutical compositions that are particularly useful as a medicine. Specifically, the combinations or compositions of the present disclosure can be applied in the treatment of immunotherapy resistant metastatic melanoma.
  • the mutation at codon Q61 resulting in the Q61R/K/L substitutions, disrupts the GTPase activity of RAS, resulting in a constitutively active conformation; whereas, mutations at codon G12 or G13, affect the Walker A-motif of the protein, thus decreasing its sensitivity to GTPase-accelerating proteins.
  • the primary alteration and the most common of the BRAF mutations is V600E/K/M. This activating mutation accounts for nearly 90% of all the BRAF mutations in melanomas. Inhibitors of BRAF ⁇ /K/M were developed including dabrafenib, vemurafenib, and encorafenib, and shown successfully in Phase III clinical trials.
  • MEK kinases (MEK1 and MEK2), which function immediately downstream of BRAF, also have been studied as potential targets for inhibition, especially in combination with BRAF inhibition.
  • Three BRAF-MEK inhibitor combinations (dabrafenib- trametinib, vemurafenib-cobimetinib, and encorafenib and binimetinib) were successful in Phase III clinical trials. Treatment of patients with wild-type BRAF has proved much more difficult, especially for those with NRAS mutations. There are currently no targeted therapies that directly target mutant NRAS; however, MEK inhibitors (trametinib and pimasertib) have been shown to have some effect in wild type BRAF and mutant NRAS melanoma.
  • the P53 tumor suppressor protein is one such option.
  • over 80% of human melanomas express TP53 wt , but P53 degradation can be enhanced through overexpression of the murine double minute (MDM) proteins, MDM2 or MDMX.
  • MDM murine double minute
  • the compound of Formula (I) also has been reported to be an effective MDM2 inhibitor in glioblastoma cell lines, patient-derived stem cells (27), and a wide variety of TP53 wt but not in homozygous TP53 mutant tumor cell lines
  • the anti-tumor efficacy of combining the compound of Formula (I) with BRAF and MEK inhibitors using melanoma patient-derived xenograft models (PDX) was studied.
  • (I) is an effective potential agent for the treatment of melanoma tumors that are either S/?AF wt or Pan ⁇ T (BRAF wt , NRAS wt , NFl wt ). Furthermore, the compound of Formula (I) is an effective agent in combination with dabrafenib and trametinib for the treatment of BRAF ⁇ OOmut tumors.
  • the present invention relates to a method of treating immunotherapy resistant metastatic melanoma comprising the step of administering to a human subject in need thereof, therapeutically effective amounts of a MDM2 inhibitor or a MDM2 inhibitor in combination with a BRAF inhibitor and a MEK inhibitor.
  • the human subject is previously treated with immunotherapy described herein for treating metastatic melanoma.
  • the previously treated metastatic melanoma has progressed after treated with immunotherapy described herein.
  • the human subject is previously treated with immunotherapy described herein for treating the metastatic melanoma, but the metastatic melanoma relapses or progresses or metastasizes.
  • the immunotherapy resistant metastatic melanoma is melanoma including different types of melanoma such as superficial spreading melanoma, nodular melanoma, acral-lentiginous melanoma, lentigo maligna melanoma, amelanotic and desmoplastic melanomas, or metastatic melanoma.
  • the present invention relates to a method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor.
  • the MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof.
  • the method further comprises detecting the BRAF genotype in the human subject.
  • the human subject exhibits a wild-type BRAF ⁇ 600 (BRAF ⁇ ) genotype.
  • the method further comprises detecting the NRAS genotype in the human subject.
  • the human subject exhibits a wild-type NRAS (NRAS ⁇ ) genotype.
  • the method further comprises detecting the NF1 genotype in the human subject.
  • the human subject exhibits a wild-type NF1 genotype (NFl ⁇ ).
  • the human subject exhibits BRAF ⁇ , A/RAS ⁇ and NFl ⁇ .
  • the human subject exhibits a mutant NRAS.
  • the human subject exhibits a mutant NF1.
  • the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).
  • TILs tumor-infiltrating lymphocytes
  • TCR T-cell receptor
  • PBL peripheral blood lymphocytes
  • CAR chimeric antigen receptor
  • the immunotherapy is an immune checkpoint protein inhibitor therapy.
  • the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-PD- L2 antibody.
  • the anti-PD- L2 antibody is rHlgM12B7A.
  • the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.
  • the immunotherapy is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof.
  • the present invention relates to a method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, a BRAF inhibitor and a MEK inhibitor.
  • the MDM2 inhibitor is a compound of Formula (I):
  • the BRAF inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and combinations thereof.
  • the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, selumetinib, pimasertib, binimetinib, and combinations thereof.
  • the method further comprises detecting the BRAF genotype in the human subject.
  • the human subject exhibits BRAF V ⁇ 0 mutation.
  • the human subject exhibits NRAS QS1 mutation.
  • the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).
  • TILs tumor-infiltrating lymphocytes
  • TCR T-cell receptor
  • PBL peripheral blood lymphocytes
  • CAR chimeric antigen receptor
  • the immunotherapy is an immune checkpoint protein inhibitor therapy.
  • the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.
  • the immunotherapy is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-PD- L2 antibody.
  • the anti-PD- L2 antibody is rHlgM12B7A.
  • the compound of Formula (I) is in a crystalline form.
  • the compound of Formula (I) is in a free form.
  • the MDM2 inhibitor is a pharmaceutically acceptable salt of a compound of Formula (I).
  • the compound of Formula (I) is in an amorphous form.
  • the compound of Formula (I) is administered once daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.
  • the compound of Formula (I) is administered twice daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.
  • the human is treated with the MDM2 inhibitor for a period selected from the group consisting of about 7 days, 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, and about 56 days.
  • the compound of Formula (I) is orally administered.
  • the MDM2 inhibitor is administered before administration of the BRAF inhibitor and MEK inhibitor.
  • the MDM2 inhibitor is administered after administration of the BRAF inhibitor and MEK inhibitor. [0143] In an embodiment, the MDM2 inhibitor is administered concurrently with administration of the B/? F inhibitor and MEK inhibitor.
  • the therapeutically effective amount of the MDM2 inhibitor is 120 mg or more.
  • the combination may be administered by any route known in the art.
  • the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered by oral, intravenous, intramuscular, intraperitoneal, intravitreal, subcutaneous or transdermal means.
  • the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is administered orally.
  • the MDM2 inhibitor is in the form of a pharmaceutically acceptable salt.
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type BRAF W0 ° (BRAF ⁇ ) genotype.
  • a MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type BRAF W0 ° (BRAF ⁇ ) genotype.
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):
  • NRAS ⁇ wild-type NRAS
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type NF1 genotype (NF1 WT ).
  • a MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type NF1 genotype (NF1 WT ).
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein the human subject exhibits NRAS 061 mutation.
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type BRAF W ⁇ (BRAF ⁇ ) genotype.
  • BRAF ⁇ wild-type BRAF W ⁇
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):
  • NRAS ⁇ wild-type NRAS
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type NF1 genotype (NF1 WT ).
  • a MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type NF1 genotype (NF1 WT ).
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein the human subject exhibits NRAS 061 mutation.
  • the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):
  • the compound of Formula (I) has the structure and name shown below.
  • the compound of Formula (I) is in an amorphous form.
  • the MDM2 inhibitor is the compound of Formula (I) in a crystalline form.
  • the MDM2 inhibitor is the compound of Formula (I) in a crystalline anhydrous form.
  • the MDM2 inhibitor is the compound of Formula (I) in a crystalline anhydrous form characterized by a powder X-ray diffraction pattern comprising peaks at diffraction angle 2 theta degrees at approximately 11.6, 12.4, 18.6, 19.0, 21.6 and 23.6.
  • the MDM2 inhibitor is the compound of Formula (I) in a crystalline anhydrous form having the X-ray diffraction pattern substantially shown in FIG. 1. The method of making such crystalline form was disclosed in the International Application W02014200937, the disclosure of which is incorporated herein by reference in its entirety.
  • the compound of Formula (I) is also referred as AMG or AMG-232 or AMG 232 in the drawings.
  • the MDM2 inhibitors of the present invention can be used in combination with MAP kinase pathway inhibitors.
  • Examples of proteins in the MAP kinase pathway that can be inhibited and the inhibitors of such proteins used in combination with an MDM2 inhibitors are BRAF inhibitors, Pan-RAF inhibitors, and MEK inhibitors.
  • a pan-RAF inhibitor shows inhibitory activity on more than one RAF isoform.
  • a BRAF inhibitor exhibits more inhibitor activity (or selectivity) towards SRAFthan the other RAF proteins.
  • the MDM2 inhibitors of the present invention can be used in combination with BRAF inhibitors, such as those found in published PCT application WO2008/153,947.
  • a particular compound is AMG 2112819 (also known as 2112819) (Example 56).
  • Another particular BRAF inhibitor that can be used in the combinations of the present invention is dabrafenib.
  • Another BRAF inhibitor that can be used in the combinations of the present invention is vemurafenib.
  • the BRAF inhibitor is encorafenib.
  • Encorafenib has the chemical structure and name shown as:
  • the BRAF inhibitor is vemurafenib.
  • Vemurafenib has the chemical structure and name shown as:
  • the BRAF inhibitor is dabrafenib.
  • Dabrafenib has the chemical structure and name shown as:
  • the BRAF inhibitor is sorafenib.
  • Sorafenib has the chemical structure and name shown as:
  • the MDM2 inhibitors of the present invention can be used in combination with MEK inhibitors, such as those found in published PCT application W02002/006213.
  • a particular compound is N-(((2R)-2,3- dihydroxypropyl)oxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide, also known as AMG 1009089 or 1009089, (Example 39).
  • the MDM2 inhibitors of the present invention can be used in combination with MEK inhibitors.
  • Particular MEK inhibitors that can be used in the combinations of the present invention include PD0325901, trametinib, pimasertib, MEK162, TAK-733, GDC-0973 and AZD8330.
  • a particular MEK inhibitor that can be used along with MDM2 inhibitor in the combinations of the present invention is trametinib (also called AMG 2712849 or 2712849).
  • MEK inhibitor is N-(((2R)-2,3- dihydroxypropyl)oxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide, also known as AMG 1009089, 1009089 or PD0325901.
  • the MEK inhibitor is trametinib.
  • Trametinib has the chemical structure and name shown as:
  • the MEK inhibitor is cobimetinib.
  • Cobimetinib has the chemical structure and name shown as:
  • the MEK inhibitor is selumetinib.
  • Selumetinib has the chemical structure and name shown as:
  • the MEK inhibitor is pimasertib.
  • Pimasertib has the chemical structure and name shown as:
  • the MEK inhibitor is binimetinib.
  • Binimetinib has the chemical structure and name shown as:
  • the immunotherapy described herein refers to an immune checkpoint immunotherapy wherein an immune checkpoint protein inhibitor is administered to a subject in need thereof.
  • the immune checkpoint protein inhibitor is an agent that modulates a target selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, LAG 3, B7-H3, B7-H4, KIR, 0X40, IDO-1, IDO-2, CEACAM1, INFR5F4, BTLA, OX40L, and TIM3 or combinations thereof.
  • the immunotherapy is a T-cell engager.
  • the immune checkpoint protein inhibitor is a PD-1 inhibitor selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab.
  • the immune checkpoint protein inhibitor is a CTLA-4 inhibitor selected from the group consisting of ipilimumab and tremelimumab.
  • the immune checkpoint protein inhibitor comprises a PD-1 immune checkpoint protein inhibitor and a CTLA-4 immune checkpoint protein inhibitor.
  • the immune checkpoint protein inhibitor is a PD-L1 inhibitor selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.
  • the immune checkpoint protein inhibitor is an anti-PD-L2 antibody.
  • the anti-PD- L2 antibody is rHlgM12B7A.
  • the PD-1 inhibitor may be any PD-1 inhibitor or PD-1 blocker known in the art. In particular, it is one of the PD-1 inhibitors or blockers described in more detail in the following paragraphs.
  • the terms "inhibitor” and “blocker” are used interchangeably herein in reference to PD-1 inhibitors.
  • references herein to a PD-1 inhibitor that is an antibody may refer to a compound or antigen binding fragments, variants, conjugates, or biosimilars thereof.
  • references herein to a PD-1 inhibitor may also refer to a compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug thereof.
  • compositions and methods described include a PD-1 inhibitor that binds human PD-1 with a KD of about 100 pM or lower, binds human PD-1 with a KD of about 90 pM or lower, binds human PD-1 with a KD of about 80 pM or lower, binds human PD-1 with a KD of about 70 pM or lower, binds human PD-1 with a KD of about 60 pM or lower, binds human PD-1 with a KD of about 50 pM or lower, binds human PD-1 with a KD of about 40 pM or lower, or binds human PD-1 with a KD of about 30 pM or lower.
  • compositions and methods described include a PD-1 inhibitor that binds to human PD-1 with a k aS soc of about 7.5 c 10 5 l/M-s or faster, binds to human PD-1 with a k aS soc of about 7.5 x 10 5 l/M-s or faster, binds to human PD-1 with a k assoc of about 8 c 10 5 l/M-s or faster, binds to human PD-1 with a k ass0 c of about 8.5 c 10 5 l/M-s or faster, binds to human PD-1 with a k ass0 c of about 9 c 10 5 l/M-s or faster, binds to human PD-1 with a k ass0 c of about 9.5 c 10 5 l/M-s or faster, or binds to human PD-1 with a k ass0 c of about 1 c 10
  • compositions and methods described include a PD-1 inhibitor that binds to human PD-1 with a k dissoc of about 2 c 10 5 I/s or slower, binds to human PD-1 with a k dissoc of about 2.1 x 10 5 I/s or slower, binds to human PD-1 with a k dissoc of about 2.2 c 10 5 I/s or slower, binds to human PD-1 with a k dissoc of about 2.3 x 10 5 I/s or slower, binds to human PD-1 with a k dissoc of about 2.4 x 10 5 I/s or slower, binds to human PD-1 with a k dissoc of about 2.5 x 10 5 I/s or slower, binds to human PD-1 with a k dissoc of about 2.6 x 10 5 I/s or slower or binds to human PD-1 with a k dissoc with a k dissoc
  • compositions and methods described include a PD-1 inhibitor that blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 10 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 9 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 8 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 7 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 6 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower, blocks or inhibits binding of human PD-
  • an anti-PD-1 antibody comprises nivolumab (Bristol-Myers Squibb) or antigen-binding fragments, conjugates, or variants thereof.
  • Nivolumab is referred to as 5C4 in International Patent Publication No. WO 2006/121168.
  • Nivolumab is assigned CAS registry number 946414-94-4 and is also known to those of ordinary skill in the art as BMS-936558, MDX-1106 or ONO- 4538.
  • Nivolumab is a fully human lgG4 antibody blocking the PD-1 receptor.
  • the anti-PD-1 antibody is an antibody disclosed and/or prepared according to U.S. Patent No. 8,008,449 or U.S. Patent Application Publication No. 2009/0217401 or 2013/0133091, the disclosures of which are specifically incorporated by reference herein.
  • the monoclonal antibody includes 5C4 (referred to herein as nivolumab), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in U.S. Patent No. 8,008,449, the disclosures of which are hereby incorporated by reference.
  • the PD-1 antibodies 17D8, 2D3, 4H1, 5C4, and 4A11 are all directed against human PD-1, bind specifically to PD-1 and do not bind to other members of the CD28 family.
  • the sequences and CDR regions for these antibodies are provided in U.S. Patent No. 8,008,449, in particular FIG. 1 through FIG. 12; all of which are incorporated by reference herein in their entireties.
  • the anti-PD-1 antibody comprises pembrolizumab, which is commercially available from Merck, or antigen-binding fragments, conjugates, or variants thereof.
  • Pembrolizumab is referred to as h409AI I in International Patent Publication No. WO 2008/156712, U.S. Patent No. 8,354,509 and U.S. Patent Application Publication No. 2010/0266617, 2013/0108651 and 2013/0109843.
  • Pembrolizumab has an immunoglobulin G4, anti-(human protein PDCD1 (programmed cell death 1)) (human-Mus musculus monoclonal heavy chain), disulfide with human-Mus musculus monoclonal light chain, dimer structure.
  • PDCD1 programmeed cell death 1
  • pembrolizumab may also be described as immunoglobulin G4, anti-(human programmed cell death 1); humanized mouse monoclonal [228-L- proline(FllO-S>P)]y4 heavy chain (134-218')-disulfide with humanized mouse monoclonal k light chain dimer (226-226":229-229”)-bisdisulfide.
  • Pembrolizumab is assigned CAS registry number 1374853-91-4 and is also known as lambrolizumab, MK-3475, and SCFI-900475.
  • the anti-PD-1 antibody is an antibody disclosed in U.S. Patent No. 8,354,509 or U.S. Patent Application Publication No. 2010/0266617, 2013/0108651, 2013/0109843, the disclosures of which are specifically incorporated by reference herein.
  • the anti-PD-1 antibody is pidilizumab, which is also known as CT-011 (CureTech Ltd.), and which is disclosed in U.S. Patent No. 8,686,119 B2, the disclosures of which are specifically incorporated by reference herein.
  • anti-PD-1 antibodies and other PD-1 inhibitors include those described in U.S. Patent No. 8,287,856, 8,580,247, and 8,168,757 and U.S. Patent Application Publication No. 2009/0028857, 2010/0285013, 2013/0022600 and 2011/0008369, the teachings of which are hereby incorporated by reference.
  • antibodies that compete with any of these antibodies for binding to PD-1 are also included.
  • the anti-PD-1 antibody is an antibody disclosed in U.S. Patent No. 8,735,553, the disclosures of which are incorporated herein by reference.
  • the PD-1 inhibitor may also be a small molecule or peptide, or a peptide derivative, such as those described in U.S. Patent No . 8,907,053; 9,096,642; and 9,044,442 and U.S. Patent Application Publication No. 2015/0087581; 1,2,4 oxadiazole compounds and derivatives such as those described in U.S. Patent Application Publication No. 2015/0073024; cyclic peptidomimetic compounds and derivatives such as those described in U.S. Patent Application Publication No. 2015/0073042; cyclic compounds and derivatives such as those described in U.S. Patent Application Publication No.
  • the PD-1 inhibitor is selected from group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, PDR001, AUNP-12 and combinations thereof.
  • the PD-1 inhibitor is nivolumab.
  • the PD-1 inhibitor is pembrolizumab.
  • the PD-1 inhibitor is Pidilizumab.
  • the PD-1 inhibitor is AMP-224.
  • the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor or blocker known in the art. In particular, it is one of the PD-L1 or PD-L2 inhibitors or blockers described in more detail in the following paragraphs.
  • the terms "inhibitor” and “blocker” are used interchangeably herein in reference to PD-L1 and PD-L2 inhibitors.
  • references herein to a PD-L1 or PD-L2 inhibitor that is an antibody may refer to a compound or antigen-binding fragments, variants, conjugates, or biosimilars thereof.
  • references herein to a PD-L1 or PD-L2 inhibitor may refer to a compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug thereof.
  • the anti-PD-Ll antibody is durvalumab, which is also known as MEDI4736 (Medimmune) or antigen-binding fragments, conjugates, or variants thereof.
  • the anti-PD-Ll antibody is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013/0034559, the disclosures of which are specifically incorporated by reference herein.
  • the clinical efficacy of durvalumab (MEDI4736) has been described in: Page, Ann. Rev. Med., 2014, 65, 185-202; Brahmer, J. Clin. Oncol. 2014, 32, 5s (supplement, abstract 8021); and McDermott, Cancer Treatment Rev., 2014, 40, 1056-64.
  • the anti-PD-Ll antibody is atezolizumab, also known as MPDL3280A or RG7446 (Genentech) or antigen-binding fragments, conjugates, or variants thereof.
  • the anti-PD-Ll antibody is an antibody disclosed in U.S. Patent No. 8,217,149, the disclosure of which is specifically incorporated by reference herein.
  • the anti-PD-Ll antibody is an antibody disclosed in U.S. Patent Application Publication No. 2010/0203056, 2013/0045200, 2013/0045201, 2013/0045202 or 2014/0065135, the disclosures of which are specifically incorporated by reference herein.
  • the anti-PD-Ll antibody is avelumab, also known as MSB0010718C (Merck KGaA/EMD Serono) or antigen-binding fragments, conjugates, or variants thereof.
  • the anti-PD-Ll antibody is an antibody disclosed in U.S. Patent Application Publication No.
  • the anti-PD-Ll antibody is MDX-1105, also known as BMS-935559, which is disclosed in U.S. Patent No. 7,943,743, the disclosures of which are specifically incorporated by reference herein.
  • the anti-PD-Ll antibody is selected from the anti-PD-Ll antibodies disclosed in U.S. Patent No. 7,943,743 which is specifically incorporated by reference herein.
  • the anti-PD-Ll antibody is a commercially-available monoclonal antibody, such as INVIVOMAB anti-m-PD-Ll clone 10F.9G2 (BioXCell).
  • INVIVOMAB anti-m-PD-Ll clone 10F.9G2
  • a number of commercially-available anti-PD- Ll antibodies are known to one of ordinary skill in the art.
  • the anti-PD-L2 antibody is a commercially-available monoclonal antibody, such as BIOLEGEND 24F.10C12 Mouse lgG2a, k isotype (Biolegend), anti-PD-L2 antibody (Sigma-Aldrich), or other commercially-available anti-PD-L2 antibodies known to one of ordinary skill in the art.
  • the PD-L1 inhibitor is an anti-PD-Ll antibody.
  • the PD-L1 inhibitor is selected from the group consisting of Atezolizumab, Avelumab, Durvalumab, BMS-936559 and combinations thereof.
  • the anti-PD-Ll inhibitor is durvalumab (MEDI4736).
  • the anti-PD-Ll inhibitor is BMS-936559 (also known as MDX-1105-01).
  • the anti-PD-Ll inhibitor is Atezolizumab.
  • the anti-PD-Ll inhibitor is Avelumab.
  • the immunotherapy is a PD-L2 inhibitor.
  • the PD-L2 inhibitor is an anti-PD-L2 antibody.
  • the anti-PD- L2 antibody is rHlgM12B7A.
  • the at least one immune checkpoint protein inhibitor is an inhibitor of CTLA-4. In some embodiments, the at least one immune checkpoint protein inhibitor is an antibody against CTLA-4. In some embodiments, the at least one immune checkpoint protein inhibitor is a monoclonal antibody against CTLA-4. In other or additional embodiments, the at least one immune checkpoint protein inhibitor is a human or humanized antibody against CTLA-4. In one embodiment, the anti-CTLA-4 antibody blocks the binding of CTLA-4 to CD80 (B7-1) and/or CD86 (B7-2) expressed on antigen presenting cells.
  • Exemplary antibodies against CTLA-4 include: Bristol Meyers Squibb's anti- CTLA-4 antibody ipilimumab (also known as YervoyTM, MDX-010, BMS-734016 and MDX-101); anti-CTLA4 Antibody, clone 9H10 from Millipore; Pfizer's tremelimumab (CP-675,206, ticilimumab); and anti-CTLA4 antibody clone BNI3 from Abeam.
  • Anti-CTLA4 antibody clone BNI3 from Abeam.
  • the anti-CTLA-4 antibody is an anti-CTLA-4 antibody disclosed in any of the following patent publications (which is incorporated by reference in its entirety): WO 2001014424; WO 2004035607; US2005/0201994; EP 1212422; WO 2003086459; WO 2012120125; WO 2000037504; WO 2009100140; WO 200609649; WO 2005092380; WO 2007123737; WO 2006029219; W020100979597; W0200612168; and WO1997020574. Additional CTLA-4 antibodies are described in U.S. Patent No. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; in PCT Publication No.
  • the anti-CTLA-4 antibody is an, for example, those disclosed in: WO 98/42752; U.S. Patent No. 6,682,736 and 6,207,156; Hurwitz, "CTLA-4 blockade synergizes with tumor- derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma," Proc. Natl. Acad. Sci.
  • the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO 1996040915.
  • the CTLA-4 inhibitor may be B7-like peptides or nucleic acid molecules disclosed in U.S. Patent No. 6,630,575.
  • the immunotherapy is a T- cell engager.
  • the T cell engager is selected from an antigen binding domain or ligand that binds to (e.g., and in some embodiments activates) one or more of CD3, TCRa, TCRp, TCRy, TCRC, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226.
  • the T cell engager is selected from an antigen binding domain or ligand that binds to and does not activate one or more of CD3, TCRa, TCRp, TCRy, TCRC, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In some embodiments, the T cell engager binds to CD3.
  • the T cell engager is selected from the group consisting of: Catumaxomab mAb (anti CD3X anti-EpCAM), FBTA05 / Lymphomun (CD3X anti-anti-CD20), duly cable Eritrea mAb (Ertumaxomab) (anti-CD3 X anti-HER2 / neu), Ektomun (anti-CD3 X anti-GD2), Bona spit mAb (blinatumomab) and B. thuringiensis FIG mAb (solitomab).
  • compositions for Oral Administration are provided.
  • the invention provides a pharmaceutical composition for oral administration a combination comprising a MDM2 inhibitor and a pharmaceutical excipient suitable for oral administration.
  • the MDM2 inhibitor is a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
  • the invention provides a pharmaceutical composition for oral administration a combination comprising a MDM2 inhibitor, a BRAF inhibitor, and a MEK inhibitor, and a pharmaceutical excipient suitable for oral administration.
  • the MDM2 inhibitor is a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
  • the BRAF inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and combinations thereof.
  • the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, selumetinib, pimasertib, binimetinib, and combinations thereof.
  • the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption.
  • Pharmaceutical compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion.
  • Such dosage forms can be prepared by any of the methods, but all methods include the step of bringing the active ingredient(s) into association with the carrier, which constitutes one or more necessary ingredients.
  • compositions are prepared by uniformly and intimately admixing the active ingredient(s) with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent.
  • Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the invention further encompasses anhydrous pharmaceutical compositions and dosage forms since water can facilitate the degradation of some compounds.
  • water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time.
  • Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
  • Pharmaceutical compositions and dosage forms of the invention which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
  • An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained.
  • anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits.
  • suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.
  • the pharmaceutical compositions may further comprise a carrier.
  • the carrier can take a wide variety of forms depending on the form of preparation desired for administration.
  • any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose.
  • suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.
  • a MDM2 inhibitor, a BRAF inhibitor, or a MEK inhibitor administered will be independently dependent on the human being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compounds and the discretion of the prescribing physician.
  • an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, such as about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, such as about 0.05 to about 2.5 g/day.
  • dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect - e.g., by dividing such larger doses into several small doses for administration throughout the day.
  • the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered in multiple doses for treating immunotherapy resistant metastatic melanoma. In an embodiment, dosing may be once, twice, three times, four times, five times, six times, or more than six times per day. In some embodiments, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, once every other day, once weekly, twice weekly, three times weekly, four times weekly, biweekly, or monthly.
  • the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor may independently continue as long as necessary.
  • the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more days.
  • the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered for about 3 days, 5 days, 7 days, 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days.
  • the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered for more than about 6, 10, 14, 28 days, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months or one year.
  • an effective dosage of the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 10 mg to about 200 mg, about 20 mg to about 150 mg, about 30 mg to about 120 mg, about 10 mg to about 90 mg, about 20 mg to about 80 mg, about 30 mg to about 70 mg, about 40 mg to about 60 mg, about 45 mg to about 55 mg, about 48 mg to about 52 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 202 mg.
  • a MDM2 inhibitor or a pharmaceutically acceptable salt thereof is administered at a dosage of 10 to 500 mg BID, including a dosage of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg BID.
  • a MDM2 inhibitor or a pharmaceutically acceptable salt thereof is administered at a dosage of 10 to 500 mg QD, including a dosage of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg QD.
  • An effective amount of the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including buccal, sublingual, and transdermal routes, by intra-arterial injection, intravenously, parenterally, intramuscularly, intravitreal, subcutaneously or orally.
  • the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject intermittently, known as intermittent administration.
  • intermittent administration it is meant a period of administration of a therapeutically effective dose, followed by a time period of discontinuance, which is then followed by another administration period and so on.
  • the dosing frequency can be independently selected from three times daily, twice daily, daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly or monthly.
  • period of discontinuance or “discontinuance period” or “rest period” it is meant to the length of time when discontinuing of the administration of the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor.
  • the time period of discontinuance may be longer or shorter than the administration period or the same as the administration period.
  • the discontinuance period may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, one month, two months, three months, four months or more days.
  • the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a human subject in need thereof for treating immunotherapy resistant metastatic melanoma for a first administration period, then followed by a discontinuance period, then followed by a second administration period, and so on.
  • the immunotherapy resistant metastatic melanoma is metastatic melanoma.
  • the first administration period, the second administration period, and the discontinuance period are independently selected from the group consisting of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, one month, two months, three months, four months and more days, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject three times daily, twice daily, daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly or monthly.
  • the first administration period is at same length as the second administration period. In an embodiment, the first administration period is shorter than the second administration period. In an embodiment, the first administration period is longer than the second administration period.
  • the first administration period and the second administration period are about three weeks, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject daily; and the discontinuance is about two weeks.
  • the first administration period and the second administration period are about three weeks, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject weekly; and the discontinuance is about two weeks.
  • the first administration period and the second administration period are about four weeks, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject daily; and the discontinuance is about two weeks.
  • the first administration period and the second administration period are about four weeks, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject weekly; and the discontinuance is about two weeks.
  • the MDM2 inhibitor is the compound of Formula (I).
  • the BRAF inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and combinations thereof.
  • the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, selumetinib, pimasertib, binimetinib, and combinations thereof.
  • the MDM2 inhibitor is administered to a human intermittently; while the BRAF inhibitor and the MEK inhibitor are administered to a human non-intermittently. In an embodiment, while the BRAF inhibitor and the MEK inhibitor are administered to a human intermittently; while the MDM2 inhibitor is administered to a human non-intermittently. In an embodiment, the MDM2 inhibitor, the BRAF inhibitor, and the MEK inhibitor are administered to a human intermittently. In an embodiment, the MDM2 inhibitor, the BRAF inhibitor, and the MEK inhibitor are administered to a human non-intermittently.
  • D dabrafenib (Tafinlar), V600B-Raf mutant Inhibitor; G, GM-CSF (Oncovax); I, interferon; Ipi, ipilimumab (Yervoy), monoclonal Ab CTLA-4; N, nivolumab (Opdivo), monoclonal Ab PD-1; Pern, pembrolizumab (Keytruda), lgG4 isotype antibody PD-1; S, sunitinib (Sutent), multi-targeted receptor tyrosine kinase (RTK) inhibitor; S+T, sorafenib (Nexavar) + tivantinib, tyrosine-protein kinase inhibitor, MET inhibitor; T, trametinib (Mekinist), MEK1/2 inhibitor; V, vemurafenib (Zelboraf), B-Raf inhibitor.
  • G GM-CSF
  • Example 1 Immunotherapy Resistant Metastatic Melanomas Respond to HDM2 Inhibition as a Single Agent or in Combination with BRAF/ MEK inhibition
  • mice were all 3-6 month-old females weighing 20-26 grams the compound of Formula (I) and navitoclax were prepared in 5% DMSO and 95% corn oil and administered five days a week by oral gavage in a total volume of 100- 200uL based upon the weight of the mouse.
  • Corn oil with 5% DMSO was used as the vehicle control.
  • Dabrafenib and trametinib were prepared in 0.5% hydroxypropyl methylcellulose.
  • At least 5 mice per treatment group were used, with each mouse bearing 2 tumors.
  • Mouse body weight was measured daily for gavage dosing and recorded twice a week and tumor measurements were taken twice a week with micro-calipers. Tumor volume was estimated as 0.5 x length x width x depth.
  • PDX models 1577, 1668, 1767, 1595, 2316 and 2252 were provided to the Patient- Derived Models Repository at NCI-Frederick. All models were established according to the recommended Minimal Information Standard (Meehan, PDX-MI: Minimal information for patient- derived tumor xenograft models. Cancer Res. 2017, 77:e62).
  • DNA extraction from tumor tissue was achieved using standard phenol: chloroform extraction methods and ethanol precipitation or using the DNeasy Blood and Tissue Kit. For a subset of tumors with high melanin content, the DNA was further purified using the Qjagen DNeasy PowerClean Pro Cleanup K Kit. DNA from matching blood samples, when available, was extracted using the QIAamp DNA Mini and Blood Mini Kit. DNA from matched patient blood, when available, P0 (patient's original tumor), and P2A and P2B xenograft tumors were subject to STR Profiling, at the Intended Center for Biotechnology Research at the University of Florida, leveraging the PowerPlex 16 FIS System (Promega). PDX specimens were considered a match to the patient specimen when one allele from each 16 STR locus was present in the P0 specimen. The amelogenin alleles for all of the specimens match the patient sex that was reported.
  • Immunohistochemical (IHC) analyses were performed by Vanderbilt's Translational Pathology Shared Resource (TPSR). Slides were placed on the Leica Bond Max IHC Stainer. All steps besides dehydration, clearing and cover-slipping were performed on the Bond Max. Slides were deparaffinized and heat-induced antigen retrieval was performed on the Bond Max using their Epitope Retrieval 2 solution for 30 minutes. The sections were incubated with Ready-to-Use antibody as indicated below. The Bond Refine Polymer detection system was used for visualization. Slides were then dehydrated, cleared and coverslipped. I HC slides were scanned at the Digital Pathology Shared resource.
  • TPSR Vanderbilt's Translational Pathology Shared Resource
  • the automated quantification of the percentages of the KI67-positive cells was performed by Leica Biosystems' Digital Image Flub, using the software available with the Leica SCN400 Slide Scanner.
  • the antibodies used were as follows: anti-p53 (Leica Biosystems) for 30 minutes; anti-MelanA (Leica) for 15 minutes; anti-Ki67 (StatLab) for 30 minutes; anti-SOX-10 (Cell Marque) for one hour.
  • Flash-frozen tumor tissue was processed in RIPA buffer using a Precellys Homogenizer. Lysates were submitted to MD Anderson Cancer Center RPPA core for analysis with a 382 antibody panel. For each condition, three independent tumors were analyzed from three PDX tumors, resulting in 9 samples analyzed for each treatment. The only exception to this was for the compound of Formula (I) + dabrafenib +Trametinb treatment of PDX1351 where only two independent tumors were analyzed. RPPA data was analyzed after a log 2 transformation. All comparisons were performed based on a mixed- effect model to take into account the correlation structure with the measured data from the sample PDX. Using model-based (least-square) means, the average adjusted difference (log 2 FC: fold change in a log2 scale) between treatments (or groups) was estimated and compared using the Wald test.
  • PDX Melanoma patient-derived xenografts
  • SNaPshot is designed to screen for 43 somatic mutations in 6 genes (BRAF, NRAS, mast/stem cell growth factor receptor KIT, guanine nucleotide-binding protein q polypeptide (GNAQ), Guanine nucleotide-binding protein subunit alpha-11 (GNA11), and Catenin beta-1 (CTNNB1)( Lovly, Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials. PLoS One. 2012, 7).
  • SNaPshot analysis indicated that three tumors (1129, 1668 and 1767) were BRAFwt, NRASwt, and NFlwt.
  • NGS Next-Generation Sequencing
  • NGS did provide additional genetic information relevant to this study.
  • the UV carcinogenic etiology of melanoma results in melanoma having the highest prevalence of somatic mutation across cancer types (Alexandrov, Signatures of mutational processes in human cancer. Nature. 2013, 500:415- 21).
  • NGS of the patient and PDX tumors revealed a wide variation in the number of mutations.
  • PDX1767 had the fewest non-synonymous mutations (NSMs), with 295 high confidence variants at a mean read depth in the target region of 83, while PDX1668 had the most NSMs with 2,352 high confidence variants at a mean read depth of 199 (FIGs. 7A-B).
  • NSMs non-synonymous mutations
  • PDX1668 had the most NSMs with 2,352 high confidence variants at a mean read depth of 199 (FIGs. 7A-B).
  • Neither of these tumors had the canonical mutations in B
  • NGS identified additional mutations in driver genes. For PDX1668, NGS identified a loss of the stop codon in NF1, and two mutations in BRAF, S147N, and N140T, with unknown significance. Further analysis of PDX1129 by NGS identified two mutations in BRAF, D549N and K483T. While BRAF mutations that are distinct from the V600 mutations are of unknown significance, the D549N mutation has also been identified in the NZM41 cell line (Stones, Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors. 2013, 4:1-6).
  • PDX2316 also was retrospectively analyzed by NGS and was found to lack mutations in BRAF and NRAS, although mutations in NF1, CDKN2A, MTOR, and KIT were detected (FIG. 1).
  • the tumors used in this study closely mimic the expected distribution of mutations within the driver mutation genes BRAF, NRAS, and NF1, with 53% exhibiting mutated BRAF V600 and 13% exhibiting mutated NRAS QS1 mutations.
  • PDX2316 was BRAF wt
  • NRAS wt and PDX1767 was BRAF wt .
  • the cBioPortal for Cancer Genomics was used to identify the 10 most frequently mutated genes in melanoma.
  • the data set included 1414 patient/case sets for melanoma, including acral, desmoplastic and lentigo maligna but not uveal melanoma.
  • the distribution of occurrence of mutations in these top 10 genes is shown in FIG. 1A and FIG.
  • a hot spot in the cBioPortal is defined as, "this mutated amino acid was identified as a recurrent hotspot (statistically significant) and a 3D clustered hotspot in a population-scale cohort of tumor samples of various cancer types using methodology based in part on Chang, Nat Biotechnol, 2016 and Gao, Genome Medicine, 2017 (Fredriksson, Systematic analysis of noncoding somatic mutations and gene expression alterations across 14 tumor types. Nat Genet. 2014, 46; Chang, Identifying recurrent mutations in cancer reveals widespread lineage diversity and mutational specificity. Nat Biotechnol. Nature Publishing Group; 2016, 34:155-63).
  • CNV copy number variations
  • PDX0807 had two mutations, an insertion at codon 138 (A-ADG) and an NSM at codon T140 (T-S).
  • the mutations in PDX0807 occurred at a low frequency of 26-27%.
  • PDXs 1668, 1839, and 1946 had mutations within splice sites. While the exact nature and frequency of these mutations in not reported in the NGS data, these three PDX tumors also had a loss of heterozygosity in the TP53 locus of 1.46, 1.38 and 1.08 respectively. All mutations and alterations in copy number within the 10 focus genes and MDM2, regardless of snpEff scores, for each PDX are listed in FIG. 7B. Interestingly, only copy number alterations were noted for MDM2.
  • PDX tumors were characterized by Short Tandem Repeat (STR) analysis to confirm that the resulting PDX tumors were derived from the patient sample (FIG. 8A).
  • DNA samples were extracted from the primary tumor specimen, the patient's blood when available, and two independent second passage isolates from the established PDX for PDX samples 9164, 0807, 1577, 1595, 1668, 1767, 2316, and 2552.
  • a total of 16 STR loci (D351358, TH01, D18551, Penta E, D55828, D135317, D75820, D165539, CSF1P0, Dmelogenin, vWA, D851179, TPOX, FGA) were co-amplified in each sample.
  • STR profiles demonstrate that each analyzed PDX was derived from the primary specimen.
  • mouse PDX tumors do undergo genetic evolution, and in accordance with this, there were changes noted between the STR profiles of the human tumor sample and the PDX, with occasional loss of an allele, consistent with clonal selection.
  • PDXs 2552, 1767, 1668 and 1595 Representative results are shown for PDXs 2552, 1767, 1668 and 1595 (FIG. 8B).
  • the markers used for melanocytic differentiation were Melan-A/Mart 1 and SOX10.
  • patient tumors (P0) and the second PDX passage (P2) were stained by H&E and for Ki-67, a proliferation marker used to determine the mitotic rate. All melanoma tumors and resulting PDXs were positive for SOX10 (Willis, SOX10: A useful marker for identifying metastatic melanoma in sentinel lymph nodes.
  • a tumor did not express detectable melanin, the PDX did not either, as shown for PDX2552 and PDX1668.
  • PDX1595 had a low but detectable level of melanin as did the patient tumor. All tumors and derived PDXs stained positive for Ki-67.
  • 50 mg/kg the compound of Formula (I) comparable to a human dose of 250mg/kg, was administered by oral gavage to the mice once the PDX tumor reached a volume of 50-100mm 3 .
  • mice were treated 5- days/week and treatment was continued until one or more tumors reached the endpoint size limit defined by the IACUC protocol (1.5 cm diameter).
  • the compound of Formula (I) response to an FDA approved therapy was compared to, either dabrafenib (30 mg/kg) and trametinib (1 mg/kg) (D+T) or trametinib (T) alone (for NRAS mutant tumors) administered by oral gavage 5-days/week.
  • dabrafenib and trametinib (D+T) were administered in vivo either with or without 50 mg/kg the compound of Formula (I).
  • the response to all drugs was compared to the appropriate vehicle control group.
  • Toxicity was not detected with any treatment group based on as AST and ALT levels, weight loss or morbidity; however, at higher doses of the compound of Formula (I) (100 mg/kg) significant morbidity was noted (data not shown).
  • Representative data from these studies for PDX1839, PDX1946, PDX2316, PDX1668, and PDX1595 are shown in FIG. 2. (Data from all the PDX tumors are shown in FIGs. 9A-P). For each drug study, the tumor growth rate was statistically determined, the final tumor weight was measured, and FFPE sections were stained and quantified to measure proliferation based on Ki67 expression.
  • t-ratio is a ratio of the difference in tumor slope between control and a treatment group relative to its standard error, calculated by estimated marginal means based on the mixed effect model.
  • response to a drug was based on a positive t-ratio greater than 2.6.
  • Any treatment which resulted in a decreased tumor growth compared to vehicle control is indicated by a positive t-ratio.
  • any treatment which resulted in increased tumor growth compared to vehicle control is indicated by a negative t-ratio.
  • FIG. 2A represents those PDX tumors that responded to the standard therapy (D+T) but not to the compound of Formula (I).
  • This group of PDX tumors is referred to as Group I.
  • FIG. 2B represents those PDX lines that did not respond to either the compound of Formula (I) or the standard therapy alone but responded synergistically to the combination of the compound of Formula (I) and the standard therapy (Group II).
  • Group II was comprised of 5 PDX lines.
  • Four of the five lines exhibited BRAF V600 mutation and one line, PDX 1179, had a nras QS1 mutation.
  • mice were treated only with trametinib (T).
  • neither the compound of Formula (I) or the standard therapy (D+T or T) resulted in a significant decrease in either growth rate (adj.
  • the second group of tumors shown in FIG. 2B includes those PDX tumors that did not respond to either the compound of Formula (I) or the standard therapy alone but responded synergistically to the combination of the compound of Formula (I) and the standard therapy (Group II).
  • Group II was comprised of 6 PDX tumors and PDX1946 is representative of the response to therapy for Group II tumors.
  • Five of the six lines exhibited BRAF V600 mutations.
  • the sixth line, PDX 1179 had an NRAS QS1 mutation and a decreased BRAF copy number to 1.36 (see FIG. 7B) and was treated with trametinib but not dabrafenib.
  • FIG. 7B the sixth line
  • the third group of tumors did respond to the compound of Formula (I) alone with growth inhibition (Group III) (FIG. 2C and D).
  • the final three lines, PDXs 0807, 1129 and 1668 had BRAF mutations that are not in the V600 position (FIG. 1 and FIG. 3).
  • mice carrying these three PDX tumors were treated with the compound of Formula (I) and D+T alone and PDX0807 and 1129 were treated with the combination.
  • PDX2316, PDX1668, and PDX1595 are shown as examples of the three different genetic subgroups within the group that responded to the compound of Formula (I) [FIG. 2C (PDX2316) and D (PDX1595 and PDX1668)].
  • the compound of Formula (I) treatment resulted in a significant decrease in tumor growth rate (adj. p ⁇ 0.001 for PDX2316 and 1595 and adj.
  • the P53 protein level was monitored by IHC in two Group III PDX tumors that had been treated in vivo with the compound of Formula (I) (FIG. 2D).
  • the P53 protein level was very low in tumors from the vehicle-treated mice but the compound of Formula (I) treatment resulted in a substantial increase in P53 protein and nuclear localization.
  • neither dabrafenib or trametinib altered P53 levels or localization.
  • Group I PDXs did not respond to the compound of Formula (I), responded to D+T, and treatment with the combination of D+T and the compound of Formula (I) resulted in a small augmentation of tumor growth inhibition.
  • Group II PDXs did not respond to either the compound of Formula (I) alone or D+T alone but responded to the combination of the compound of Formula (I) and D+T (AMG+D+T).
  • Group III PDXs responded to the compound of Formula (I) monotherapy, as indicated by the gray shading. A larger t-ratio indicates that there was a larger difference between the treatment group and the comparison group.
  • tumor growth rate with 95% confidence intervals for each treatment was estimated (FIG. 3B).
  • the Group II PDX tumors did not respond to the compound of Formula (I) or D+T alone, but when administered together there was synergistic inhibition of tumor growth.
  • the power of this analysis is based on analyzing treatment effectiveness for multiple independent PDX tumors (FIG. 3).
  • analysis of H&E staining of those PDX tumors which had the highest t-ratios when comparing the effect of the compound of Formula (I) treatment to the effect of the compound of Formula (I) + D+T revealed morphologic differences after the compound of Formula (I) and D+T therapy (FIG. 4).
  • H&E staining of tumors isolated from vehicle-treated and D+T treated cells revealed that the tumor cells were tightly packed with minimal stroma, uniform in size and appearance, and had large nuclei with a high mitotic rate and a high Ki67 expression.
  • PDX tumors exhibiting a synergistic growth-inhibitory response to the compound of Formula (I) and D+T were characterized by mostly karyorrhectic nuclei with vacuolar changes around the karyorrhectic debris.
  • RPPA analysis of protein and phosphoprotein expression was conducted (FIG. 4B).
  • protein markers for different modalities of cell death including bcl- 2-like protein 4 (BAX), B-cell lymphoma 2 (BCL2), p53 upregulated modulator of apoptosis (PUMA), Poly (ADP-ribose) polymerase (PARP), Beclin, janus kinase (JAK), induced myeloid leukemia cell differentiation protein (MCL1), and the Caspase, signal transducer and activator of transcription (ST AT), and heat shock proteins (HSP) families of proteins, while present in the antibody panel, were notably absent from the list of positive hits when comparing vehicle-treated PDX tumors to the compound of Formula (I) +D +T treated tumors.
  • BAX bcl- 2-like protein 4
  • BCL2 B-cell lymphoma 2
  • PUMA p53 upregulated modulator of apoptosis
  • PARP Poly (ADP
  • hypoxia-inducible factor 1 subunit alpha HIF-lot
  • MCT4 monocarboxylate transporter 4
  • pNDRGl phosphorylated N-myc downstream-regulated gene-1
  • pMAPK mitogen-activated protein kinase
  • Group I PDX tumors 1839, 2195, and 2252 responded to D+T as a single agent, and exhibited decreased phosphorylation of MAPK, but did not exhibit altered expression of H IF- 1-a, MCT-4, LDFIA nor was there a decrease in phosphorylation of NDRG in response to D+T without the compound of Formula (I) (FIG. 10).
  • the Oncoprint Cluster Analysis shown in FIG. 5 represents the analysis with the 10 most relevant genetic alterations, as discussed in FIG. 1. No further insights were gained by including all 300 genes analyzed by NGS (data not shown). This analysis indicates that the mutational status of BRAF is highly correlated with responsiveness to the compound of Formula (I). Of the 9 PDX tumors that were resistant to the compound of Formula (I) (Groups I and II), all but one had a V600 mutation in BRAF (FIG. 1) ⁇
  • the other notable protein induced by the compound of Formula (I) was the receptor tyrosine kinase ephrin type-A receptor 2, (EPHA2) the erythropoietin-producing hepatocellular receptor A2, which has been reported to play a critical role in oncogenic signaling in many types of solid tumors (Zhou, Emerging and diverse functions of the EphA2 noncanonical pathway in cancer progression. Biol Pharm Bull. 2017, 40:1616-24).
  • Cyclin B1 Cyclin B1
  • PLK1 which belongs to the CDC5/Polo subfamily
  • forkhead box protein Ml FoxMl
  • Binding of FoxMl to G2/M gene promoters is dependent upon B-Myb. Biochim Biophys Acta - Gene Regul Mech. 2012, 1819:855-62). All three proteins are involved in the regulation of M-phase of the cell cycle (Liao, Regulation of the master regulator FOXM1 in cancer. Cell Commun Signal.
  • PDX0807 and PDX1668 both of which responded to the compound of Formula (I) as a single agent had t-ratios of 2.598 and 2.829, respectively, while the TP53wt PDX tumors in Group III, PDX 1595, 2316, 1129 and 1767, had t-ratios greater than 3.5 (FIG. 3).
  • BRAF V600 mutant melanoma tumors often develop resistance to MAPK inhibitors like dabrafenib and trametinib. It was postulated that D+T resistant tumors might respond to a Bcl-2 inhibitor to block tumor growth and proliferation through an alternate pathway. Indeed, it has been demonstrated that combining the MDM2 inhibitor, nutlin RG7388, with the BCL-2 inhibitor, ABT-199, provides better therapeutic efficacy than either drug alone in acute myeloid leukemia (AML) (Pan, Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy. Cancer Cell, Elsevier Inc.; 2017, 32:748-760.
  • AML acute myeloid leukemia
  • navitoclax was not effective as a single agent in any PDX tumor
  • navitoclax in combination with the compound of Formula (I) in BRAF V600E (PDX1351 and 1577) tumors was more effective than either drug alone and resulted in tumor regression in three PDX models, as shown by example for PDX1577 (FIG. 6).
  • the growth of only the mutant BRAF V600E tumors was inhibited by the combination of navitoclax and the compound of Formula (I).
  • the dose-escalation design for the compound of Formula (I) in the Phase I/ll trial included treatment for seven days of each 3-week cycle (7/21) at 120, 240, or 480 mg. Based on this, the maximally tolerated dose was 180 mg in combination with tremetanib or tremetanib + dabrafenib.
  • Group I consisted of three BRAF V600E mutant PDX tumors, which responded to dabrafenib and trametinib but not to the compound of Formula (I) as a single agent. In combination, these agents further inhibited tumor growth.
  • Group II consisted of BRAF V600E/M and one NRAS Q61H mutant tumors that did not respond to either the compound of Formula (I) or dabrafenib and trametinib alone, but when used in combination, the inhibition of tumor growth was demonstrated to be statistically significant and synergistic.
  • the BRAF V600wt PDX tumors responded to the compound of Formula (I) alone with significant inhibition of tumor growth.
  • the mutational status of BRAF was an effective predictor of the compound of Formula (I) monotherapy response.
  • the PDX panel only those tumors that were BRAF V600wt were responsive to the compound of Formula (I) alone, while, except for PDX1179, tumors with BRAF V600 mutations were resistant to the compound of Formula (I), but responded synergistically to the compound of Formula (I) combined with dabrafenib and trametinib.
  • Pan did not explore the mechanistic role of P53 in regulating the MAPK pathway, others have identified P53-regulated transcription of four phosphatases, wild-type p53-induced phosphatase 1 (Wipl), mitogen-activated protein kinase phosphatase 1 (MKP1), phosphatase of activated cells 1 also known as dual specificity phosphatase 2 (PAC1/DUSP2), and DUSP5, that negatively regulate MAPK signaling (Gen, The functional interactions between the p53 and MAPK signaling pathways. Cancer Biol Ther. 2004, 3:156-61).
  • Wipl wild-type p53-induced phosphatase 1
  • MKP1 mitogen-activated protein kinase phosphatase 1
  • PAC1/DUSP2 dual specificity phosphatase 2
  • DUSP5 dual specificity phosphatase 2
  • the RPPA panel did not include p-Myc or N-Myc
  • the N-Myc downstream-regulated gene NDRG-1 was decreased by the compound of Formula (I) and D+T treatment. Additionally, Myc has been shown to stabilize HIF-Ia (Doe, Myc posttranscriptionally induces HIF1 protein and target gene expression in normal and cancer cells. Cancer Res. 2012, 72:949-57) , which explains the decrease in HIF-Ia in the compound of Formula (I) and D+T treated tumors in the studies described here.
  • N-myc downstream regulated gene (NDRG) family Diverse functions, multiple applications. FASEB J. 2010, 24:4153-66; Gordan, HIF and c-Myc: Sibling Rivals for Control of Cancer Cell Metabolism and Proliferation. Cancer Cell. 2007, 12:108-13) and are key regulators of the glycolytic switch in tumors (Yu, The glycolytic switch in tumors: How many players are involved? J Cancer. 2017, 8:3430-40).
  • Bcl-2 inhibitors like dabrafenib and trametinib, can overcome the resistance to the compound of Formula (I).
  • Preclinical studies have shown that mice implanted with OCL-AML3 leukemic cells that are resistant to both the MDM2 inhibitor, RG7388, and the Bcl-2 inhibitor, ABT-199, exhibited enhanced overall survival with the two agents combined.
  • MDM2 inhibitor RG7388
  • Bcl-2 inhibitor ABT-199
  • FoxMl expression is increased in a variety of solid tumors, including melanoma, and inhibition of FoxMl leads to a decrease in cell proliferation and migration, metastasis and angiogenesis. Furthermore, analysis of the TGCA database illustrated that high levels of FoxMl are related to poor prognosis in most solid tumors (Liao, Regulation of the master regulator FOXM1 in cancer. Cell Commun Signal. 2018, 16:1-15). P53, both directly and indirectly through p21, has been reported to decrease FoxMl expression (Kurinna, P53 Regulates a Mitotic Transcription Program and Determines Ploidy in Normal Mouse Liver. Hepatology.
  • FoxMl is a target of p53-mediated repression. Oncogene. Nature Publishing Group; 2009, 5:4295-305). Subsequently, FoxMl induces the expression of the Cdkl activators, cyclin B and Cdc25 (Laoukili, FoxMl is required for execution of the mitotic programme and chromosome stability. Nat Cell Biol. 2005, 7:126-36), so a decrease in FoxMl expression would result in a decrease in cyclin B, as seen in the RPPA data. Cdc25 was also statistically decreased in the RPPA analysis, but the fold decrease was slight and log2(FC) was not greater than the cutoff -0.4.
  • cyclin B has been proposed to be a critical target of FoxMl at the G2/M transition (Murakami, Regulation of yeast forkhead transcription factors and FoxMl by cyclin-dependent and polo-like kinases. Cell Cycle. 2010, 9:3233-42).
  • Polo-like kinases (PLK) also help to regulate the cell cycle, mainly at the G2/M checkpoint and FoxMl and PLKs exist in a positive feedback loop where transcriptionally activated FoxMl controls the expression of PLKs while PLK1 binds and phosphorylates FoxMl and activates FoxMl as a transcription factor (Z, Malureanu, Plkl-dependent phosphorylation of FoxMl regulates a transcriptional programme required for mitotic progression. Nat Cell Biol. 2008, 10:1076-82). Therefore, the compound of Formula (I), through the P53 dependent inhibition of FoxMl expression, has a direct effect in regulating the G2/M checkpoint.
  • the tumors used in this study were very heterogeneous and there was no PDX tumor which was purely BRAF mutant and there was only one line which was a complete TP53 mutant (PDX9164).
  • the PDX tumors with a BRAF V600 mutation the PDX with the highest mutation frequency was PDX1577 with a BRAF V600 mutation frequency of 0.951 and the lowest was PDX1351 with a BRAF V600 mutation frequency of 0.316. There was an average BRAF V600 mutation frequency of 0.555. Similar heterogeneity was seen with TP53.
  • 4 PDX tumors with mutations in the TP53 gene were included.
  • PDX9164 had a TP53 mutation frequency of 1 at codon 192 (Q192*) and this mutation has a FATHMM pathogenic score of 0.93 suggesting it is highly pathogenic.
  • the other mutations occurred at lower frequencies, 0.265 for PDX0807 (A138ADG and T140S); 0.607 for PDX 2252 (R196*); and 0.484 and 0.235 for PDX1839 (R306* and Y236H). This heterogeneity complicates the interpretation of the effectiveness of the compound of Formula (I) in mutant versus wild type TP53 tumors.
  • the causative factors that contribute to MAPKi-resistance can be broadly classified into three categories: mutational events and non-mutational events, which are tumor inherent and lead to either MAPK pathway reactivation or activation of a parallel signaling pathway, and changes in the surrounding microenvironment. Based on the pleiotropic nature of drug resistance it is interesting, although not surprising, the two PDX tumors previously treated with D+T remained resistant. It is also interesting that none of the BRAF V600E mutant tumors that responded to D+T were derived from patients who had prior exposure to D+T.

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Abstract

L'invention concerne des procédés thérapeutiques et des compositions pharmaceutiques pour le traitement d'un mélanome métastatique résistant à l'immunothérapie.
PCT/US2021/025514 2020-04-02 2021-04-02 Procédés de traitement d'un mélanome métastatique résistant à l'immunothérapie Ceased WO2021202961A1 (fr)

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