Biomarkers for cancer therapy using MDM2 antagonists

Abstract

The present invention provides DNA damage response (DDR) pathway genes and their gene products as biomarkers for predicting effective cancer treatment with MDM2 antagonists. Identifying one or more DDR pathway biomarkers in cancer patients allows for determining whether the patient's cancer is likely to be successfully treated with MDM2 antagonists. Thus, the present invention generally relates to a companion diagnostic for MDM2 antagonist therapy. In particular, the DDR pathway comprises one or more genes from the homologous recombination repair (HRR) pathway; the non-homologous end joining (NHEJ) pathway; the mismatch repair (MMR) pathway; the Fanconi anemia (FA) pathway; and / or the base excision repair (BER) pathway.

Description

[Technical Field]

[0001] The present invention relates to biomarkers for cancer therapy.In particular, the present invention provides biological markers that identify cancer cells as potentially sensitive to MDM2 antagonists.These biomarkers can be incorporated into methods, systems and kits for predicting response to treatment, and personalized cancer treatment. [Background technology]

[0002] Precision medicine, or personalized medicine, is a new approach to disease treatment and prevention that takes into account individual differences in each patient's genes, environment, and lifestyle. It is often described as delivering the right drug in the right amount at the right time.

[0003] A particular focus of precision medicine is the need to predict whether a given patient will respond to a particular drug. Tests that can predict whether a particular drug will effectively treat an individual patient are often called companion diagnostics. Effective companion diagnostics are highly desirable because they can improve patient outcomes while simultaneously saving the significant economic costs of providing ineffective treatments. An effective companion diagnostic for a new therapeutic agent can also increase the likelihood that the therapy will be tested in the appropriate population and ultimately approved.

[0004] Precision medicine and companion diagnostics often rely on biomarkers that can reliably predict whether a patient is likely to respond to a particular treatment. Identifying reliable biomarkers for any therapy and disease is a significant challenge.

[0005] WO-A-2016 / 056673 describes a composite gene signature that purports to provide a predictive molecular tool for clinical application. The disclosure also relates to methods for predicting the sensitivity of a cancer or tumor to anti-cancer drugs, particularly inhibitors of MDM2 activity and antagonists of the interaction between MDM2 and p53 proteins, that may have an impact on the treatment of the cancer or tumor.

[0006] US-A-2015 / 0211073 also describes gene panels, generally comprising at least four genes, as biomarkers for predicting the response of cancer to MDM2 antagonists.

[0007] Iorio et al. (Cell. 2016 Jul 28;166(3):740-75) report in "A Landscape of Pharmacogenomic Interactions in Cancer" that cancer-driven alterations (integrating somatic mutations, copy number alterations, DNA methylation, and gene expression) identified in 11,289 tumors from 29 tissues were mapped to 1,001 molecularly annotated human cancer cell lines and correlated with sensitivity to 265 drugs. While such studies provide a resource for linking genotype and cellular phenotype and identifying treatment options for selected cancer subpopulations, developing clinically relevant molecularly targeted cancer therapies remains a challenging task.

[0008] There is still a need to identify robust biomarkers for use in precision medicine. Summary of the Invention

[0009] The present invention is based on the identification of biomarkers that can be used to predict effective cancer treatment with MDM2 antagonists. Identifying one or more of these biomarkers in a cancer patient allows for the determination of whether the patient's cancer is likely to be treated or likely to be successfully treated with an MDM2 antagonist. Thus, in certain aspects, the present invention generally relates to companion diagnostics for MDM2 antagonist therapy.

[0010] The biomarkers identified in the present invention are DNA damage response (DDR) pathway genes and their gene products. These proteins and the genes encoding them are all known in the art. As used herein, these biomarkers are referred to as "biomarkers of the present invention" and / or "DDR biomarkers." Reduced DDR function in cancer cells indicates sensitivity to MDM2 antagonism. Thus, depletion, loss, or reduced function of one or more DDR genes or gene products indicates sensitivity to MDM2 antagonism.

[0011] In particular, in one aspect, the present invention provides an MDM2 antagonist for use in a method for treating cancer, wherein the cancer is depleted of one or more DDR genes, gene products or activity.Depletion can be the depletion of the gene itself, the depletion of the gene product (i.e., reduced expression), or the mutation that causes reduced activity (i.e., loss-of-function mutation).This depletion can be due to abnormality that causes reduced activity, such as reduced copy number or epigenetic silencing.

[0012] There is a broad group of abnormalities that can cause loss / reduction of activity, such as loss of activity due to copy number reduction or loss-of-function mutations. Loss-of-function mutations can be considered as depletion of the wild-type gene product. Thus, when biomarker depletion is described, it includes mutations in the biomarker such that the wild-type is reduced or undetectable.

[0013] In one aspect, the present invention provides an MDM2 antagonist for use in a method of treating cancer, wherein the cancer is depleted in one or more genes, gene products, or activities in the homologous recombination (HR) pathway (also called the homology-directed repair (HRR) pathway). These cancer cells have reduced HR function and can therefore be identified as HR-deficient. In one embodiment, the cancer is BRCA1-depleted. Human BRCA1 is Entrez gene ID 672. In one embodiment, the cancer is BRCA2-depleted. Human BRCA2 is Entrez gene ID 675. In one embodiment, the cancer is ATM-depleted. ATM is the Ataxia Telangiectasia Mutated protein. Human wild-type ATM is Entrez gene ID 472. One embodiment of the present invention relates to the depletion of any two of BRCA1, BRCA2, and ATM. One embodiment of the present invention relates to the depletion of all three of BRCA1, BRCA2, and ATM. One embodiment of the present invention relates to loss-of-function mutations in BRCA1, BRCA2, or ATM compared to WT. One embodiment of the present invention relates to loss-of-function mutations in any two of BRCA1, BRCA2, and ATM compared to WT. One embodiment of the present invention relates to loss-of-function mutations in all three of BRCA1, BRCA2, and ATM compared to WT. One embodiment of the present invention relates to copy number reduction or epigenetic silencing in one, two, or three of BRCA1, BRCA2, or ATM compared to WT. One embodiment of the present invention relates to loss-of-function mutations in BRCA1 and / or BRCA2. One embodiment of the present invention relates to copy number reduction or epigenetic silencing in BRCA1 and / or BRCA2. In one embodiment, homologous recombination repair deficiency mimics BRCA1 or BRCA2 loss.

[0014] In one embodiment, the HR pathway gene is selected from one or more of the following genes: LIG1, MRE11A, NBN, PARG, PARP1, PARPBP, RAD50, TP53BP1, XRCC2, XRCC3, EXO1, PCNA, POLD1, POLD2, POLD3, POLD4, RFC1, RFC2, RFC3, RFC4, RFC5, RPA1, RPA2, RPA3, RPA4, BARD1, BLM, BRCA1, BRCA2, BRIP1, DMC1, DNA2, EID3, EME1, EME2, ERCC1, H2AFX, HELQ, HFM1, INO80, KAT5, MUS81, NFATC2 IP, NSMCE1, NSMCE2, NSMCE3, NSMCE4A, PALB2, PARP2, PAXIP1, POLH, POLQ, PPP4C, PPP4R1, PPP4R2, PPP4R4, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54B, RAD54L, R BBP8, RDM1, RECQL, RECQL4, RECQL5, RMI1, RMI2, RTEL1, SHFM1, SLX1A, SLX1B, SLX4, SMARCAD1, SMC5, SMC6, SPO11, SWSAP1, TOP3A, TOP3B, UIMC1, WRN, and / or ZSWIM7. All of these HR pathway genes are known in the art and are described, for example, in Knijnenburg et al Cell Rep. 2018 Apr 3;23(1):239-254.e6.

[0015] In some embodiments, the HR-deficient cancer cells are ATM-deficient. rather than , i.e., wild-type ATM is expressed at normal (or elevated) levels in these cancer cells. In these cells, HR deficiency is provided by functional loss of one or more different HR genes, e.g., BRCA1 and / or BRCA2. These cancer cells, in some embodiments, comprise HR depletion consisting of a gene, gene product, or activity in the HR pathway other than ATM.

[0016] In some embodiments, the HR-deficient cancer cells are ATR-deficient. rather than, i.e., in these cancer cells, wild-type ATR is expressed at normal (or elevated) levels. In these cells, HR deficiency is provided by functional loss of one or more different HR genes, e.g., BRCA1 and / or BRCA2. These cancer cells, in some embodiments, comprise HR depletion consisting of a gene, gene product, or activity in the HR pathway other than ATR. ATR is an "Ataxia Telangiectasia and Rad3-related" protein. Exemplary sequences for human ATR are available in the UniProtKB database under accession number Q13535 (ATR-HUMAN) and in the GenBank database under NCBI accession number AAK26749.1, and have also been published in literature, such as Bentley et al., EMBO J., 15: 6641-6651 (1996) and Cimprich et al., Proc. Natl. Acad. Sci. USA 107: 18575-18480 (1996).

[0017] In some embodiments, these cancer cells comprise an HR depletion consisting of a gene, gene product, or activity in the HR pathway other than ATR.

[0018] In some embodiments, HR-deficient cancer cells are neither ATM-deficient nor ATR-deficient, i.e., wild-type ATR and wild-type ATM are expressed at normal (or elevated) levels in these cancer cells, in which HR deficiency is provided by functional loss of one or more different HR genes, e.g., BRCA1 and / or BRCA2.

[0019] In some embodiments, the cancer cells comprise an HR depletion consisting of genes, gene products or activity in the non-ATM, non-ATR HR pathway.

[0020] In some embodiments, the cancer is determined to be depleted in one or more genes or gene products in one or more DNA damage repair (DDR) pathways, or the cancer has at least one loss-of-function mutation in at least one DDR pathway gene, and the one or more DDR genes or gene products are not ATM and / or ATR. This means that the genes or gene products may comprise ATM and / or ATR and one or more other DDR pathway biomarkers, or if it is a single biomarker, it is not ATM or ATR, and if it is a biomarker pair, they are not ATM and ATR, but may comprise ATM or ATR and another DDR pathway biomarker. In some embodiments, the cancer may be depleted in one gene or gene product in one DNA damage repair (DDR) pathway, or have at least one loss-of-function mutation in one DDR pathway gene, and the DDR pathway gene or gene product is not ATM or ATR.

[0021] Thus, in some embodiments, when there is a single biomarker according to the invention, it is neither ATM nor ATR. In some embodiments, when there is a single biomarker according to the invention, it is not ATM. In some embodiments, when there is a single biomarker according to the invention, it is not ATR.

[0022] In some embodiments, the cancer may be depleted of two genes or gene products in one or more DNA damage repair (DDR) pathways, or the cancer has at least one loss-of-function mutation in at least two DDR pathway genes, and the DDR pathway gene or gene product is not ATM or ATR.

[0023] In some embodiments, the cancer may be depleted in two genes or gene products in one or more DNA damage repair (DDR) pathways, or the cancer has at least one loss-of-function mutation in at least two DDR pathway genes, the DDR pathway genes or gene products including one or more of ATM and / or ATR, and one or more DDR pathway genes or gene products that are neither ATM nor ATR. In another aspect, the cancer is depleted in one or more genes, gene products, or activity in the Fanconi Anemia (FA) pathway. This pathway includes at least the genes FANCA (Entrez gene ID 2175), FANCB (gene ID 2187), FANCC (gene ID 2176), FANCD1 (also known as BRCA2, gene ID 675), FANCD2 (gene ID 2177), FANCE (gene ID 2178), FANCF (gene ID 2188), FANCG (gene ID 2189), FANCI (gene ID 55215), FANCJ (gene ID 83990), FANCL (gene ID 55120), FANCM (gene ID 57697), FANCN (gene ID 79728), FANCO (gene ID 889), FANCP (gene ID 84464), FANCQ (gene ID 2072), and FANCR (gene ID 84464). 5888), FANCS (also known as BRCA1, gene ID 672), FANCT (gene ID 29089), FANCU (gene ID 7516), FANCV (gene ID 10459), and FANCW (gene ID 55159). One embodiment of the invention relates to loss-of-function mutations in one or more FA pathway genes compared to WT.

[0024] In another aspect, the present invention provides an MDM2 antagonist for use in a method for treating cancer, wherein the cancer is depleted in one or more genes, gene products, or activities in the non-homologous end joining (NHEJ) pathway. Thus, these cancer cells can be characterized as HEJ-deficient. In one embodiment, the cancer is depleted in ATRX. ATRX is Alpha thalassemia / mental retardation syndrome X-linked. Human wild-type ATRX is Entrez gene ID 546. One embodiment of the present invention relates to loss-of-function mutations of ATRX compared to WT.

[0025] In a further aspect, the present invention provides an MDM2 antagonist for use in a method of treating cancer, wherein the cancer is depleted in one or more genes, gene products, or activities in the mismatch repair (MMR) pathway. Accordingly, these cancer cells can be characterized as MMR-deficient. In one embodiment, the cancer is depleted in one or more of MSH2 (Entrez gene ID 4436), MSH3 (gene ID 4437), MSH6 (gene ID 2956), MLH1 (gene ID 4292), PMS2 (gene ID 5395), and / or MLH3 (gene ID 27030). In a further embodiment, the cancer is depleted in POLD1 (Entrez gene ID 5424) or POLE (gene ID 5426), e.g., has one or more POLD1 and / or POLE mutations. One embodiment of the present invention relates to loss-of-function mutations in one or more MMR pathway genes compared to WT.

[0026] In some embodiments, the cancer comprises mutational signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutation signature SBS20. These single base substitution (SBS) signatures are known from the Catalog of Somatic Mutations in Cancer (COSMIC), GRCh37 v91. The COSMIC SBS signatures are accessible at cancer.sanger.ac.uk / cosmic and were generated as described by Alexandrov et al. Nature volume 578, pages 94-101 (2020).

[0027] In a further aspect, the present invention provides an MDM2 antagonist for use in a method for treating cancer, wherein the cancer is deficient in one or more genes, gene products, or activities in the base excision repair (BER) pathway.Therefore, these cancer cells can be characterized as BER-deficient.One embodiment of the present invention relates to loss-of-function mutations in one or more BER pathway genes compared to WT.

[0028] In one embodiment of all aspects, the loss or depletion of DDR genes or functional gene products in cancer is manifested by the microsatellite instability (MSI) and / or tumor mutation burden of the cancer, commonly "MSI-High" and / or "High tumor mutation burden."

[0029] For some biomarkers, protein is typically measured. This can be accomplished, for example, using immunohistochemistry (IHC). In some embodiments, mutation analysis (e.g., DNA sequencing) can be used to detect biomarker status.

[0030] In some embodiments, the present invention provides an MDM2 antagonist for use in a method of treating cancer, wherein the cancer has depletion of one or more DDR biomarkers and, optionally, elevated expression of one or more interferon signature (IFN signature) genes. The IFN signature genes comprise CXCL10, CXCL11, RSAD2, MX1, BATF2, IFI44L, IFITM1, ISG15, CMPK2, IFI27, CD74, IFIH1, CCRL2, IFI44, HERC6, ISG20, IFIT3, HLA-C, OAS1, IFI35, IRF9, EPSTI1, USP18, BST2, CSF1, C1S, DHX58, TRIM14, OASL, IRF7, LGALS3BP, DDX60, LAP3, LAMP3, PARP12, PARP9, SP110, PLSCR1, WARS, STAT1, IRF3, IRF5, MSC, JUN, SPI1, IRF1, COMMD3-BMI1, STAT2, RUNX3, SREBF1, and FLI1. These biomarkers are collectively referred to herein as the “interferon signature.” Generally, IFN signature biomarkers are detected as mRNA.

[0031] Measurement techniques for one or more nucleic acid biomarkers, for example, IFN signature biomarkers, can include quantitative techniques such as RT-PCR or Nanostring analysis, as known in the art. DNA can also be measured. In some embodiments, copy number variation (CNV) analysis and / or mutation analysis (e.g., DNA sequencing) can be used to detect the status of biomarker genes.

[0032] In some embodiments, the present invention provides an MDM2 antagonist for use in a method of treating cancer, wherein the cancer is depleted in one or more DDR biomarkers, and optionally also has reduced expression of one, two, or three of CDKN2A, BAP1, and SKP2. Optionally, the cancer is depleted in one or more DDR biomarkers, and optionally has reduced expression of one, two, or three of CDKN2A, BAP1, and SKP2. Such cancers may have increased expression of one or more interferon signature (IFN signature) genes described herein. In one embodiment, an MDM2 antagonist is provided for use in a method of treating cancer, wherein the cancer is depleted in one or more DDR biomarkers, and optionally is also depleted in CDKN2A, BAP1, and / or has increased expression of one, two, three, four, five, or more interferon signature genes.

[0033] SKP2 depletion may refer to loss or complete loss of the SKP2 gene, mutation and loss of function of the SKP2 gene, or SKP2 depletion may refer to low gene expression and low protein expression and function resulting from loss or mutation of that gene or others. In one embodiment, an MDM2 antagonist is provided for use in a method of treating cancer, wherein the cancer is depleted in one or more DDR biomarkers, and optionally exhibits decreased, reduced, low, or absent SKP2 expression.

[0034] The CDKN2A gene encodes the p16(INK4A) and p14(ARF) proteins, and reference to the gene CDKN2A includes the proteins encoded by CDKN2A. CDKN2A loss can be assessed by low protein expression product levels, i.e., expression levels lower than control expression levels of p16(INK4A) and / or p14(ARF), i.e., the consequence of CDKN2A gene loss is loss of p16 and / or p14.

[0035] For CDKN2A, protein is generally measured. This can be achieved, for example, by immunohistochemistry (IHC). In some embodiments, mutation analysis (e.g., DNA sequencing) can be used to detect CDKN2A status.

[0036] For BAP1, protein can generally be measured. This can be achieved, for example, using immunohistochemistry (IHC). In some embodiments, cellular location can also be measured. In some embodiments, mutation analysis (e.g., DNA sequencing) can be used to detect BAP1 status. Biomarkers identified herein as having high expression may be referred to as interferon signature, or IFN signature, biomarkers. They are also referred to as type 1 interferon pathway genes. Generally, these biomarkers are detected as mRNA. Therefore, measurement techniques for one or more IFN signature biomarkers include quantitative techniques such as RT-PCR or Nanostring analysis, as known in the art. DNA can also be measured. In some embodiments, copy number variation (CNV) analysis and / or mutation analysis (e.g., DNA sequencing) can be used to detect the status of biomarker genes.

[0037] The biomarkers of the present invention can be measured directly or indirectly. Indirect measurement generally involves detecting a molecule functionally upstream or downstream of the biomarker, the level of which correlates with the level of the biomarker. For example, a substrate on which the biomarker acts can be used as an indirect measurement of the biomarker. In one embodiment, BAP1 levels can be measured by detecting the level of histone H2A ubiquitination, and increased H2A ubiquitination generally reflects decreased BAP1. In another embodiment, BAP1 depletion can be assessed by determining increased expression or activity of EZH2. In one embodiment, SKP2 can be indirectly detected through the detection of one or more SKP2 substrates. A typical SKP2 substrate is p27. In one embodiment of the invention, SKP2 levels are assessed by measuring the levels of one or more of p27, p21, p57, E2F-1, MEF, P130, Tob1, cyclin D, cyclin E, Smad4, Myc, Mcb, RASSF1A, Foxo1, Orc1p, Cdt1, Rag2, Brca2, CDK9, MPK1, and / or UBP43.

[0038] Another indirect measure of DDR biomarkers is a downstream readout of DDR defects. One such readout is tumor mutation burden (TMB). Another downstream readout is microsatellite instability (MSI). Clinical assays for stratifying patients based on these features are already used in clinics. For example, MSI-high (or MSI-H) is a well-known clinical definition of cancer that can be determined using techniques including next-generation sequencing, fluorescent multiplex PCR and capillary electrophoresis, immunohistochemistry, or single-molecule molecular inversion probes. Furthermore, TMB can be measured using NGS methods. TMB can be used as a marker of genomic instability and MSI. MSI testing methods (MSK-Impact and F1CDx) include both microsatellite instability (MSI) and tumor mutation burden (TMB).

[0039] The data in the following examples demonstrate that depletion, e.g., loss (also known as total or complete loss) of one or more DDR biomarker activities predicts cancer cell sensitivity to MDM2 antagonists. Thus, low levels of one or more DDR biomarkers can be used to identify cancers suitable for treatment with MDM2 antagonists. In certain embodiments, the DDR biomarkers can be from one or more of the following pathways: HR, NHEJ, FA, MMR, and / or BER. The multiple DDR biomarkers measured can all be from the same pathway or from different pathways. In one embodiment, the one or more DDR biomarkers measured comprise ATM, BRCA1, and / or BRCA2, typically loss-of-function ATM, BRCA1, and / or BRCA2 mutations. In another embodiment, the one or more DDR biomarkers measured comprise ATRX, typically loss-of-function ATRX mutations. In another embodiment, one or more DDR biomarkers are in the FA pathway. In further embodiments, MMR biomarker depletion is indicated by MSI (e.g., MSI-High) and / or increased tumor mutation burden compared to non-sensitive cells. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In a further embodiment, the one or more DDR biomarkers measured comprise POLD1 and / or POLE. In some embodiments, the one or more DDR biomarkers measured comprise the mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency and / or the POLD1 mutation signature SBS20.

[0040] In some embodiments, a decrease in the level of one or more biomarkers of the invention is determined relative to cancer cells. This cancer:non-cancerous comparison can be particularly useful. The non-cancerous cells are generally cells of the same type as the cancer cells. The non-cancerous cells may be from the same patient, a different patient, or may represent known values ​​for non-cancerous cells of that type. Thus, biomarker levels, e.g., expression or activity, can be compared to control levels determined in healthy individuals or normal, non-proliferating tissue.

[0041] In some other embodiments, the level, e.g., decreased expression, of one or more biomarkers of the present invention is determined in comparison with or in a cancer cell sample from an MDM2 inhibitor non-responder. The non-responder cancer cells are generally cells of the same cancer type as the cancer cells being tested. The non-responder cancer cells are generally from one or more patients different from the sample being tested, or may be known values ​​for non-responder cancer cells of that cancer type.

[0042] In some embodiments, a patient can be identified as a candidate for treatment with an MDM2 antagonist if the expression or activity levels of one or more DDR biomarkers are below the upper limit of normal (ULN). In one embodiment, the one or more DDR biomarkers are from one or more of the following pathways: HR, NHEJ, FA, MMR, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways.

[0043] In another embodiment, the one or more DDR biomarkers measured comprise ATM. In some embodiments, the presence of ATM with a loss-of-function mutation compared to wild-type predicts sensitivity to MDM2 antagonism. In another embodiment, the one or more DDR biomarkers measured comprise BRCA1. In some embodiments, the presence of BRCA1 with a loss-of-function mutation compared to wild-type predicts sensitivity to MDM2 antagonism. In another embodiment, the one or more DDR biomarkers measured comprise BRCA2. In some embodiments, the presence of BRCA2 with a loss-of-function mutation compared to wild-type predicts sensitivity to MDM2 antagonism.

[0044] In another embodiment, the one or more DDR biomarkers measured comprise ATRX. In some embodiments, the presence of ATRX with a loss-of-function mutation compared to wild-type predicts sensitivity to MDM2 antagonism. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In a further embodiment, the one or more DDR biomarkers measured comprise POLD1 and / or POLE. In some embodiments, the one or more DDR biomarkers measured comprise the mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or the POLD1 mutation signature SBS20.

[0045] In another embodiment, one or more of the DDR biomarkers are in the FA pathway.

[0046] In further embodiments, depletion of DDR biomarkers, e.g., MMR biomarkers, is identified by MSI (e.g., MSI-High) and / or increased tumor mutation burden (e.g., high TMB, or increased compared to non-susceptible cancer cells).

[0047] Optionally, the method may comprise administering to the patient a therapeutically effective amount of an MDM2 antagonist.

[0048] In all aspects and embodiments described herein, the cancer is generally a p53 wild-type cancer.

[0049] In one embodiment, the invention provides an MDM2 antagonist for use in the treatment of cancer, particularly p53 wild-type cancer, wherein the cancer is characterized by one or more of the biomarkers of the invention in a biological sample obtained from a patient.

[0050] According to another embodiment of the present invention, there is provided a method of treating cancer in a patient, said method comprising selecting a patient based on an expression profile of two or more biomarkers of the present invention (in certain embodiments, the patient is Decreased expression or activity of one or more DDR biomarkers in a biological sample obtained from the patient and optionally then administering to said patient a therapeutically effective amount of an MDM2 antagonist. In one embodiment, the one or more DDR biomarkers are from one or more of the following pathways: HR, NHEJ, FA, MMR, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. In a further embodiment, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. In another embodiment, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In a further embodiment, the one or more DDR biomarkers measured comprise one or more POLD1 and / or POLE mutations. In some embodiments, the one or more DDR biomarkers measured comprise mutational signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency and / or POLD1 mutational signature SBS20. In another embodiment, one or more of the DDR biomarkers are in the FA pathway. In a further embodiment, biomarker depletion is identified by elevated MSI and / or increased tumor mutational burden.

[0051] According to a further embodiment of the present invention, there is provided an MDM2 antagonist for use in treating cancer in a patient, wherein the patient has been selected for having reduced or low levels of one or more DDR biomarkers in a biological sample obtained from the patient. In one embodiment, the one or more DDR biomarkers are from one or more of the following pathways: HR, NHEJ, FA, MMR, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. In a further embodiment, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. In another embodiment, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In further embodiments, the one or more DDR biomarkers measured comprise POLD1 and / or POLE. In some embodiments, the one or more DDR biomarkers measured comprise measuring mutational signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutational signature SBS20. In another embodiment, one or more of the DDR biomarkers are in the FA pathway. In further embodiments, biomarker depletion is identified by elevated MSI (e.g., MSI-High) and / or high tumor mutation burden (TMB).

[0052] In certain embodiments, patient tissue samples are tested before treatment to determine the biomarker expression profile of cancer.The sample can generally comprise one or more cancer cells, cancer DNA, or circulating tumor DNA.The sample can be a blood sample.The sample can be a tumor sample, for example, a tumor biopsy.The test can comprise an assay for detecting protein, mRNA, DNA, and / or ctDNA.

[0053] In another aspect, the invention provides the use of the expression level of one or more biomarkers of the invention in a cancer cell sample from a human patient as a biomarker for assessing whether the cancer will be susceptible to treatment with an MDM2 antagonist.

[0054] In a further aspect, the invention provides a method for prognosing or assessing the responsiveness of a human cancer patient to treatment with an MDM2 antagonist, comprising assessing the expression level of one or more biomarkers of the invention in a sample from the cancer patient, and determining whether the expression level examined indicates that the cancer should be treated with an MDM2 antagonist.

[0055] In some embodiments, one or more biomarkers of the present invention indicate that a cancer is likely to effectively undergo apoptosis, and thus, in some embodiments, the present invention can identify patients for whom treatment may be particularly effective.

[0056] In some embodiments, the evaluating step comprises an in vitro assay to determine the expression level of one or more biomarkers.

[0057] In some embodiments, the evaluating step comprises comparing the expression level to expression levels known to be associated with responsiveness or non-responsiveness to treatment with an MDM2 antagonist, hi some embodiments, the evaluating step comprises comparing the examined expression level to a threshold value representing an expression level associated with susceptibility to treatment with an MDM2 antagonist in the same manner as the observed expression level to assess whether the cancer is indicative of being treatable with an MDM2 antagonist.

[0058] In some embodiments, patients are classified into groups based on their biomarker profiles, which may include classifying patients as likely to respond well (or strongly) or not to respond to treatment with an MDM2 antagonist.

[0059] In a further aspect, the invention provides a method for determining whether a human cancer patient is suitable for treatment with an MDM2 antagonist, comprising: detecting the expression or activity of one or more biomarkers of the invention in a sample of cancer cells from the patient; and assessing whether a patient's cancer may be treatable with an MDM2 antagonist based on the expression or activity level of a biomarker in the sample; Optionally, the method of this aspect comprises the further step of treating cancer in a patient with an MDM2 antagonist.

[0060] In a further embodiment, the invention provides an MDM2 antagonist for use in combination with an anti-cancer compound in treating cancer in a patient, wherein said cancer in said patient is characterized as a p53 wild-type cancer selected as having one or more biomarkers of the invention.

[0061] In a further embodiment, the invention provides a method of treating cancer in a patient, wherein said cancer in said patient is optionally a p53 wild-type cancer, and the patient has been selected as having one or more biomarkers of the invention at levels indicative of efficacious MDM2 antagonist treatment, comprising administering to the selected patient a therapeutically effective amount of an MDM2 antagonist and optionally another anti-cancer agent.

[0062] In a further embodiment, the invention provides a method for identifying a patient suffering from cancer suitable for treatment with an MDM2 antagonist, said method comprising detecting, and optionally quantifying, the expression of one or more biomarkers of the invention.

[0063] In a further embodiment, the present invention provides a method for selecting a patient (e.g., a patient suffering from cancer), said method comprising the step of selecting a patient by detecting, and optionally quantifying, the expression of one or more biomarkers of the present invention.

[0064] In a further embodiment, the invention provides a method for determining the likelihood that a cancer patient will respond to therapy with an MDM2 antagonist, the method comprising: obtaining a measure of a decrease in expression of one or more of the DDR biomarkers in a sample of cancer cells from the patient compared to corresponding non-cancerous cells; and and determining whether the patient is likely to respond to therapy with an MDM2 antagonist based on the measurement. The one or more DDR biomarkers optionally are from one or more of the following pathways: HR, NHEJ, MMR, FA, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. Optionally, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In a further embodiment, the one or more DDR biomarkers measured comprise one or more POLD1 and / or POLE mutations. In some embodiments, the one or more DDR biomarkers measured comprise mutational signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutational signature SBS20. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, one or more of the DDR biomarkers are in the MMR pathway, and depletion of said one or more biomarkers is identified by MSI and / or high tumor mutational burden.

[0065] In a further embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: Determining one or more biomarkers of the invention; administering a therapeutically effective amount of an MDM2 antagonist to a patient having one or more biomarkers of the invention. The present invention provides a drug administration process comprising:

[0066] In yet a further aspect, the invention provides a method for detecting expression of one or more biomarkers of the invention in a human patient suffering from cancer. The method generally comprises: (a) obtaining a cancer cell sample from a human patient; and (b) detecting whether the biomarker is expressed in the sampled cancer cells by contacting the sample with one or more reagents for detecting expression of the biomarker. The compound comprises:

[0067] In yet a further aspect, the invention provides a kit or device for detecting the expression level of at least one biomarker for sensitivity to MDM2 antagonism in a sample from a human patient, said kit or device comprising one or more detection reagents for detecting one or more biomarkers of the invention.

[0068] In a further aspect, the present invention resides in a system for assessing whether a human cancer patient is susceptible to treatment with an MDM2 antagonist, the system comprising: detection means capable of and adapted to detect one or more biomarkers of the invention in a sample from a human patient; a processor capable of, and adapted to, determine an indication of the likelihood that the patient is treatable with an MDM2 antagonist from the determined one or more biomarkers. The compound comprises:

[0069] The system optionally includes a data connection to an interface, particularly a graphical user interface, capable of presenting information, preferably information such as the subject's age, and optionally other patient information such as gender and / or medical history information, the interface being part of the system or being a remote interface. Optionally, one or more of the items, particularly the processor, may function "in the cloud," i.e., by means of an internet-based application rather than on a fixed machine.

[0070] The present invention also provides methods, combinations, and kits for identifying and screening patients.

[0071] In a further embodiment, the present invention provides a method of screening or identifying a patient for treatment with an MDM2 antagonist, comprising: determining whether the patient has decreased expression of one or more DDR biomarkers in a biological sample obtained from the patient; In one embodiment, the one or more DDR biomarkers are derived from one or more of the following pathways: HR, NHEJ, MMR, FA, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. In a further embodiment, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. In another embodiment, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In a further embodiment, the one or more DDR biomarkers measured comprise POLD1 and / or POLE. In some embodiments, the one or more DDR biomarkers measured comprise mutational signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency and / or POLD1 mutational signature SBS20. In another embodiment, one or more of the DDR biomarkers are in the FA pathway. In a further embodiment, depletion of MMR biomarkers is identified by MSI and / or high tumor mutational burden.

[0072] In a further embodiment, the present invention provides a method for identifying a patient responder, comprising testing the patient for decreased expression of one or more DDR biomarkers in a biological sample obtained from the patient. In one embodiment, the one or more DDR biomarkers are from one or more of the following pathways: HR, NHEJ, MMR, FA, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. In a further embodiment, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. In another embodiment, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In further embodiments, the one or more DDR biomarkers measured comprise one or more POLD1 and / or POLE mutations. In some embodiments, the one or more DDR biomarkers measured comprise mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutation signature SBS20. In another embodiment, the one or more DDR biomarkers are in the FA pathway. In further embodiments, depletion of MMR biomarkers is identified by MSI and / or high tumor mutation burden.

[0073] In a further embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (a) To identify patients in need of treatment for cancer, possibly p53 wild-type cancers such as mesothelioma; (b) determining that the patient has decreased expression of one or more DDR biomarkers in a biological sample obtained from said patient; and treating the patient with a therapeutically effective amount of an MDM2 antagonist. The present invention provides a method of treatment comprising:

[0074] In a further embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (a) identifying patients in need of treatment for cancer, possibly breast, ovarian, prostate or pancreatic cancer; (b) determining that the patient has decreased expression of one or more DDR biomarkers in a biological sample obtained from said patient; and treating the patient with a therapeutically effective amount of an MDM2 antagonist. The present invention provides a method of treatment comprising:

[0075] Optionally, the one or more DDR biomarkers are from one or more of the following pathways: HR, NHEJ, MMR, FA, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. Optionally, the one or more DDR biomarkers measured comprise ATM. Optionally, the one or more DDR biomarkers measured comprise BRCA1. Optionally, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In a further embodiment, the one or more DDR biomarkers measured comprise one or more POLD1 and / or POLE mutations. In some embodiments, the one or more DDR biomarkers measured comprise mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutation signature SBS20. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, when one or more of the DDR biomarkers are in the MMR pathway, depletion of said one or more biomarkers is distinguished by MSI and / or high tumor mutation burden.

[0076] In a further embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (a) Identifying patients who require treatment for cancer, and possibly mesothelioma; (b) determining in said patient one or more biomarkers of the invention, optionally one or more DDR biomarkers from one or more of the HR, NHEJ, FA, MMR and / or BER pathways; a. optionally, the one or more DDR biomarkers measured comprise one, two, three, or four of BRCA1, BRCA2, ATM, and ATRX; and / or b. optionally, one or more of the DDR biomarkers are in the FA pathway; and / or c. optionally, one or more of the DDR biomarkers are in the MMR pathway, e.g., one or more of MSH2, MSH3, MSH6, MLH1, PMS2, MLH3, POLE and / or POLD1, and / or mutation signatures SBS6, SBS26 and / or SBS20, and optionally, depletion of the one or more MMR pathway DDR biomarkers is distinguished by MSI and / or high tumor mutational burden; (c) selecting an MDM2 antagonist as treatment for a patient based on the recognition that the MDM2 antagonist is effective in a patient having one or more biomarkers of the invention; (d) treating the patient with a therapeutically effective amount of an MDM2 antagonist. The present invention provides a method of treatment comprising:

[0077] In a further embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (a) identifying patients in need of treatment for cancer, possibly breast, ovarian, prostate, or pancreatic cancer; (b) determining in the patient one or more biomarkers of the invention, optionally one or more DDR biomarkers from one or more of the HR, NHEJ, FA, MMR and / or BER pathways; a. optionally, the one or more DDR biomarkers measured comprise or consist of one, two, three or four of BRCA1, BRCA2, ATM and ATRX; and / or b. optionally, one or more of the DDR biomarkers are in the FA pathway; and / or c. optionally, one or more of the DDR biomarkers are in the MMR pathway, e.g., one or more of MSH2, MSH3, MSH6, MLH1, PMS2, MLH3, POLE and / or POLD1, and / or mutation signatures SBS6, SBS26 and / or SBS20, and optionally, depletion of the one or more MMR pathway DDR biomarkers is distinguished by MSI and / or high tumor mutational burden; (c) selecting an MDM2 antagonist as treatment for a patient based on the recognition that the MDM2 antagonist is effective in a patient having one or more biomarkers of the invention; (d) treating the patient with a therapeutically effective amount of an MDM2 antagonist. The present invention provides a method of treatment comprising:

[0078] In a further embodiment, the present invention provides a method for selecting a treatment for a cancer patient, comprising: (a) assaying one or more biological samples, thereby determining one or more biomarkers of the invention in a patient; (b) selecting a therapeutically effective amount of an MDM2 antagonist for treatment of the patient based on that determination. The method comprises:

[0079] In one embodiment, the DDR biomarkers are from the HR pathway and include LIG1, MRE11A, NBN, PARG, PARP1, PARPBP, RAD50, TP53BP1, XRCC2, XRCC3, EXO1, PCNA, POLD1, POLD2, POLD3, POLD4, RFC1, RFC2, RFC3, RFC4, RFC5, RPA1, RPA2, RPA3, RPA4, BARD1, BLM, BRCA1, BRCA2, BRIP1, DMC1, DNA2, EID3, EME1, EME2, ERCC1, H2AFX, HELQ, HFM1, INO80, KAT5, MUS81, NFATC2IP, NSMCE1 , NSMCE2, NSMCE3, NSMCE4A, PALB2, PARP2, PAXIP1, POLH, POLQ, PPP4C, PPP4R1, PPP4R2, PPP4R4, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54B, RAD54L, RBBP8, RDM1, RECQL, RECQL4, RECQL5, RMI1, RMI2, RTEL1, SHFM1, SLX1A, SLX1B, SLX4, SMARCAD1, SMC5, SMC6, SPO11, SWSAP1, TOP3A, TOP3B, UIMC1, WRN, and / or ZSWIM7.

[0080] In a further embodiment, the invention provides a process for selecting a patient (e.g., a patient suffering from cancer) for treatment with an MDM2 antagonist, characterized in that the patient is selected for reduced or low expression of one or more DDR biomarkers in a biological sample obtained from the patient. Optionally, the one or more DDR biomarkers are from one or more of the following pathways: HR, NHEJ, FA, MMR, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. Optionally, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In further embodiments, the one or more DDR biomarkers measured comprise POLD1 and / or POLE. In some embodiments, the one or more DDR biomarkers measured comprise mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutation signature SBS20. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, when one or more of the DDR biomarkers are in the MMR pathway, depletion of said one or more biomarkers is identified by MSI and / or high tumor mutation burden.

[0081] In a further embodiment, the present invention provides an MDM2 antagonist for use in treating cancer in a patient, wherein the patient is characterized by decreased expression or activity of one or more DDR biomarkers in a biological sample obtained from the patient. Optionally, the one or more DDR biomarkers are from one or more of the following pathways: HR, NHEJ, FA, MMR, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. Optionally, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In further embodiments, the one or more DDR biomarkers measured comprise one or more POLD1 and / or POLE mutations. In some embodiments, the one or more DDR biomarkers measured comprise mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutation signature SBS20. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, when one or more of the DDR biomarkers are in the MMR pathway, depletion of said one or more biomarkers is identified by MSI and / or high tumor mutation burden.

[0082] In a further embodiment, the present invention provides a kit for treating cancer in a patient, said kit comprising a biosensor for the detection and / or quantification of one or more biomarkers of the invention and / or reagents for the detection of one or more biomarkers of the invention, optionally together with instructions for use of the kit according to the methods as defined herein.

[0083] In a further embodiment, the invention provides a method of determining responsiveness of an individual with cancer to treatment with an MDM2 antagonist, comprising detecting decreased expression or activity of one or more DDR biomarkers in a biological sample obtained from the patient, optionally the one or more DDR biomarkers being from one or more of the following pathways: HR, NHEJ, FA, MMR, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. Optionally, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In further embodiments, the one or more DDR biomarkers measured comprise one or more POLD1 and / or POLE mutations. In some embodiments, the one or more DDR biomarkers measured comprise mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutation signature SBS20. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, when one or more of the DDR biomarkers are in the MMR pathway, depletion of said one or more biomarkers is identified by MSI and / or high tumor mutation burden.

[0084] In a further embodiment, the invention provides a method for determining the responsiveness of an individual having cancer to treatment with an MDM2 antagonist, comprising: identifying a patient having decreased expression or activity of one or more DDR biomarkers in a biological sample obtained from said patient; and administering to said patient a therapeutically effective amount of an MDM2 antagonist. The method comprises:

[0085] The one or more DDR biomarkers optionally are from one or more of the following pathways: HR, NHEJ, MMR, FA, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. Optionally, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In a further embodiment, the one or more DDR biomarkers measured comprise POLD1 and / or POLE. In some embodiments, the one or more DDR biomarkers measured comprise mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutation signature SBS20. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, when one or more of the DDR biomarkers are in the MMR pathway, depletion of said one or more biomarkers is distinguished by MSI and / or high tumor mutation burden.

[0086] In a further embodiment, the present invention provides a method of treating cancer in a patient, the method comprising selecting a patient having decreased expression or activity of one or more DDR biomarkers in a biological sample obtained from the patient. Optionally, the one or more DDR biomarkers are from one or more of the following pathways: HR, NHEJ, MMR, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. Optionally, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In further embodiments, the one or more DDR biomarkers measured comprise one or more POLD1 and / or POLE mutations. In some embodiments, the one or more DDR biomarkers measured comprise mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutation signature SBS20. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, one or more of the DDR biomarkers are in the MMR pathway, and depletion of said one or more biomarkers is identified by MSI and / or high tumor mutation burden.

[0087] In a further embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) directing the determination of expression or activity of one or more DDR biomarkers, optionally the one or more DDR biomarkers from one or more of the following pathways: HR, NHEJ, FA, MMR, and / or BER; and (ii) administering a therapeutically effective amount of an MDM2 antagonist to a patient having a decreased level of one or more DDR biomarkers. The present invention provides a drug administration process comprising:

[0088] In a further embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) MDM2 antagonists; (ii) a patient package insert detailing instructions for use of an MDM2 antagonist in treating patients identified using the biomarker profile described herein; The present invention provides a packaged pharmaceutical formulation comprising:

[0089] In a further embodiment, the present invention provides a method of treating cancer in a patient, said method comprising: (i) contacting a sample from a patient with a primer, antibody, substrate, or probe to determine the expression or activity level of one or more DDR biomarkers; a. optionally, the one or more DDR biomarkers are from one or more of the following pathways: HR, FA, NHEJ, MMR, and / or BER; b. optionally, the one or more DDR biomarkers measured comprise ATM, BRCA1 BRCA2, and / or ATRX; c. optionally, the one or more DDR biomarkers measured comprise MSH2, MSH3, MSH6, MLH1, MLH3, PMS2, POLE and / or POLD1 mutations, and optionally, the cancer comprises mutation signatures SBS6, SBS26 and / or SBS20; d. optionally, one or more of the DDR biomarkers are in the FA pathway; and / or e. optionally, depletion of said one or more biomarkers can be identified by MSI and / or high tumor mutational burden; (ii) selecting a patient having a decreased level of one or more DDR biomarkers in a biological sample obtained from said patient; (iii) then administering to said patient selected in step (ii) a therapeutically effective amount of an MDM2 antagonist. The compound comprises:

[0090] In a further embodiment, the invention provides a method for identifying a patient for treatment with an MDM2 antagonist, said method comprising: (a) contacting a sample from a patient with a plurality of oligonucleotide primers, said plurality of primers comprising at least one pair of oligonucleotide primers for any one or more DDR biomarkers; (b) performing PCR on the sample to amplify gene expression products / transcripts in the sample; (c) determining the level of an expression product of at least one of said genes; and (d) identifying the patient as a candidate for treatment with an MDM2 antagonist if the expression level of the at least one gene is below the upper limit of normal (ULN). The compound comprises:

[0091] A patient may optionally be identified as a candidate for treatment with an MDM2 antagonist if the expression levels of one or more DDR biomarkers are lower (e.g., below) the upper limit of normal (ULN). Optionally, the one or more DDR biomarkers are from one or more of the following pathways: HR, NHEJ, FA, MMR, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. Optionally, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In further embodiments, the one or more DDR biomarkers measured comprise one or more POLD1 and / or POLE mutations. In some embodiments, the one or more DDR biomarkers measured comprise mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutation signature SBS20. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, when one or more of the DDR biomarkers are in the MMR pathway, depletion of said one or more biomarkers is identified by MSI and / or high tumor mutation burden.

[0092] In a further embodiment, the invention provides a method for identifying a patient for treatment with an MDM2 antagonist, said method comprising: (a) contacting a sample from a subject with an antibody to one or more biomarkers of the invention; (b) performing an assay on said sample; (c) determining the level of one or more biomarkers of the invention; and (d) identifying said patient as a candidate for treatment with an MDM2 antagonist if the level of one or more biomarkers of the invention is elevated or decreased below the upper limit of normal (ULN). The compound comprises:

[0093] The assay in part (b) can be or comprise an immunohistochemistry assay. In some embodiments, the assay can be or comprise an ELISA. When a sample from a patient is contacted with antibodies to one or more DDR biomarkers, an immunohistochemistry assay is typically performed on the sample, and if the level of one or more DDR biomarkers is below the upper limit of normal (ULN) (or absent), the patient is identified as a candidate for treatment with an MDM2 antagonist.

[0094] Once a patient is identified for treatment, the methods described herein further comprise treating the patient's cancer with an MDM2 antagonist.

[0095] In a further embodiment, the invention provides a method for selecting a cancer patient for receiving MDM2 antagonist therapy for cancer, comprising: (a) determining the level of one or more DDR biomarkers in a biological sample from the patient; The one or more DDR biomarkers are optionally from one or more of the HR, FA, NHEJ, MMR and / or BER pathways; and / or The one or more DDR biomarkers measured optionally comprise ATM, BRCA1, BRCA2 and / or ATRX; and / or The one or more DDR biomarkers measured optionally comprise MSH2, MSH3, MSH6, MLH1, PMS2, MLH3, POLE and / or POLD1, and optionally the cancer comprises the mutation signatures SBS6, SBS26 and / or SBS20; and / or One or more of the DDR biomarkers are optionally in the FA pathway; and / or If one or more of the DDR biomarkers are in the MMR pathway, depletion of said one or more biomarkers is optionally distinguished by MSI and / or high tumor mutational burden; and (b) selecting patients having a level of one or more DDR biomarkers in a biological sample from the patient that is less than a predetermined value in the biological sample from patients having a level that is equal to or greater than a predetermined value; The compound comprises:

[0096] In a further embodiment, the present invention provides a method for predicting the efficacy of an MDM2 antagonist against cancer in a patient, or for predicting a cancer patient's response to an MDM2 antagonist against cancer, comprising determining the level of one or more DDR biomarkers in a biological sample from the patient, wherein efficacy in the patient is indicated if the biological sample level of the one or more DDR biomarkers is equal to or generally low than a predetermined value. Optionally, the one or more DDR biomarkers are derived from one or more of the following pathways: HR, NHEJ, MMR, and / or BER. The one or more DDR biomarkers measured may all be from the same pathway or may be from different pathways. Optionally, the one or more DDR biomarkers measured comprise ATM. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA1. In a further embodiment, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATRX. In one embodiment, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2, and / or MLH3. In a further embodiment, the one or more DDR biomarkers measured comprise POLD1 and / or POLE. In some embodiments, the one or more DDR biomarkers measured comprise mutation signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency and / or POLD1 mutation signature SBS20. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, when one or more of the DDR biomarkers are in the MMR pathway, depletion of said one or more biomarkers is identified by MSI and / or high tumor mutation burden.

[0097] In one embodiment, the patient has one or more of the following markers: LIG1, MRE11A, NBN, PARG, PARP1, PARPBP, RAD50, TP53BP1, XRCC2, XRCC3, EXO1, PCNA, POLD1, POLD2, POLD3, POLD4, RFC1, RFC2, RFC3, RFC4, RFC5, RPA1, RPA2, RPA3, RPA4, BARD1, BLM, BRCA1, BRCA2, BRIP1, DMC1, DNA2, EID3, EME1, EME2, ERCC1, H2AFX, HELQ, HFM1, INO80, KAT5, MUS81, NFATC2IP, NSMCE1, NSMCE2, NSMCE3, NSMCE4A, PALB2, P Selection for MDM2 antagonist treatment is based on measurement of another HR pathway biomarker gene selected from ARP2, PAXIP1, POLH, POLQ, PPP4C, PPP4R1, PPP4R2, PPP4R4, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54B, RAD54L, RBBP8, RDM1, RECQL, RECQL4, RECQL5, RMI1, RMI2, RTEL1, SHFM1, SLX1A, SLX1B, SLX4, SMARCAD1, SMC5, SMC6, SPO11, SWSAP1, TOP3A, TOP3B, UIMC1, WRN, and / or ZSWIM7.

[0098] In a further embodiment, the invention provides a method for selecting a patient having cancer in need of treatment with an MDM2 antagonist, the method comprising testing a tumor sample obtained from the patient for low levels of one or more DDR biomarkers.

[0099] In a further embodiment, the present invention provides a method of treating cancer, comprising: (i) testing a tumor sample obtained from a patient with or suspected of having cancer for the loss of one or more DDR biomarkers; and (ii) administering an MDM2 antagonist to the patient from whom the sample was obtained. Optionally, the one or more DDR biomarkers in (i) are from one or more of the HR, NHEJ, MMR, and / or BER pathways. Optionally, the one or more DDR biomarkers measured comprise ATM, BRCA1, BRCA2, and / or ATRX. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, when one or more of the DDR biomarkers are from the MMR pathway, the depletion of the one or more biomarkers is identified by MSI and / or high tumor mutational burden. Optionally, the one or more DDR biomarkers measured comprise one or more of MSH2, MSH3, MSH6, MLH1, PMS2 MLH3, POLD1 and / or POLE. Optionally, the one or more DDR biomarkers measured comprise mutational signatures SBS6 and / or SBS26 associated with DNA mismatch repair deficiency, and / or POLD1 mutational signature SBS20.

[0100] In a further embodiment, the invention provides a method for identifying patients having cancer who are most likely to benefit from treatment with an MDM2 antagonist, the method comprising measuring the level of one or more of the biomarkers of the invention in a tumor sample obtained from the patient, and identifying whether the patient is likely to benefit from treatment with an MDM2 antagonist depending on the levels present.

[0101] Some embodiments of the invention comprise detecting the presence of mutations in one or more DDR biomarkers that are indicative of loss of one or more DDR biomarkers, which can be compared to control levels determined in normal, non-proliferative tissue or in the absence of mutations.

[0102] The present invention provides various methods for determining whether a cancer patient will be amenable to treatment with an MDM2 antagonist; a method for predicting the sensitivity of tumor cell growth to inhibition by an MDM2 antagonist; a method for predicting the responsiveness of a subject's cancer to a cancer therapy comprising an MDM2 antagonist; a method for developing a treatment plan for a subject with cancer; and an in vitro method for identifying patients who will be responsive or sensitive to a treatment regimen with an MDM2 antagonist. These methods generally comprise comparing the level of one or more biomarkers of the present invention in a sample, typically a tumor sample, with a reference level, and predicting the responsiveness of the cancer to treatment with a cancer therapy comprising an MDM2 antagonist. In one embodiment, these methods comprise analyzing one or more, for example, two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more, or eleven or more, of the biomarkers described herein. In one embodiment, the one or more biomarkers include ATM. In one embodiment, the one or more biomarkers do not include ATM. In one embodiment, one or more biomarkers do not include ATR. In one embodiment, one or more biomarkers include BRCA1 or BRAC2. In one embodiment, two or more biomarkers include BRCA1 and BRAC2. In one embodiment, two or more biomarkers include BAP1 and CDKN2A. In one embodiment, two or more biomarkers include ATM. In one embodiment, two or more biomarkers include ATR.

[0103] In a further embodiment, the present invention provides an in vitro method for predicting the likelihood that a patient having a tumor that is a candidate for treatment with an MDM2 antagonist will respond to treatment with the compound, comprising the steps of: (a) determining the level of one or more DDR biomarkers in one or more tissue samples taken from the patient, wherein (i) a loss of one or more DDR biomarkers (e.g., compared to a reference value of at least one normal, non-proliferating tissue) indicates that the patient is likely to respond to the treatment, and / or (ii) normal or high levels of one or more DDR biomarkers indicate that the patient is unlikely to respond to the treatment.

[0104] In a further embodiment, the invention provides an assay comprising: (a) measuring or quantifying the level of one or more DDR biomarkers; (b) comparing the levels of said one or more DDR biomarkers (e.g., compared to control levels determined in healthy individuals) (e.g., compared to control levels determined in normal, non-proliferative tissue), and identifying a patient as suitable for treatment with an MDM2 antagonist if there is a loss in one or more DDR biomarkers (e.g., compared to control levels determined in normal, non-proliferative tissue).

[0105] In a further embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) contacting a biological sample obtained from a patient with an antibody (e.g., an antibody specific for one or more DDR biomarkers); (ii) washing the sample to remove unbound antibody; (iii) measuring the intensity of the signal from the bound antibody; (iv) comparing the measured signal intensity with a reference value, and if the measured intensity is higher than the reference value; (v) a biological sample obtained from the patient; a. primers (e.g., at least one oligonucleotide primer pair for any one or more DDR biomarkers); b. an antibody (e.g., an antibody specific for one or more DDR biomarkers), and / or c. Primers for genes or mutations that show loss of ≥1 DDR biomarker to come into contact with; (vi) subjecting the sample to PCR, RT-PCR or next-generation sequencing to amplify gene expression products / transcription products in the sample; (vii) determining the level of the expression product of at least one of said genes; and (viii) identifying the subject as likely to be suitable for treatment with an MDM2 antagonist; An assay comprising:

[0106] In a further embodiment, the invention provides a method of treating cancer comprising administering an MDM2 antagonist to a subject having loss of one or more DDR biomarkers in a tumor sample as determined by sequencing or immunoassay.

[0107] In a further embodiment, the present invention provides a method of administering an MDM2 antagonist to a patient in need thereof, comprising: (1) determining patient levels of one or more DDR biomarkers; (2) assigning a phenotype to the patient based on the levels of the genes and genotypes listed above in the tumor determined in (1), the phenotype being selected from low sensitivity (P), intermediate (I), and sensitivity (S), and the phenotype is assigned based on the gene levels in the tumor; and (3) administering MDM2 antagonists to patients with phenotype S; The method comprises:

[0108] In one embodiment, the homologous recombination deficiency (HRD) score from Myriad MyChoice CDx (an FDA-approved tumor test from Myriad Genetics Inc., Salt Lake City, UT, USA) is used to determine homologous recombination deficiency status by examining BRCA1 and BRCA2 mutations and assessing genomic instability using three biomarkers: loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions. HRD positivity is defined as an HRD score >= 42 and HRD negative is < 42.

[0109] The Myriad myChoice® CDx next-generation sequencing-based in vitro diagnostic test uses DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens to qualitatively detect and classify single-nucleotide variants, insertions and deletions, and large-scale rearrangement variants in the protein-coding regions and intron / exon boundaries of the BRCA1 and BRCA2 genes, and determines a genomic instability score (GIS), an algorithmic measure of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST).

[0110] The test results can be used to aid in identifying cancer patients with positive homologous recombination deficiency (HRD) status who may be eligible for treatment with an MDM2 antagonist (if approved in accordance with the approved therapeutic product label) because of a positive test result for a deleterious or suspected deleterious mutation in the BRCA1 or BRCA2 gene, or who may be eligible because of a positive test result for a deleterious or suspected deleterious mutation in the BRCA1 or BRCA2 gene, or a positive genomic instability score.

[0111] In one embodiment, MSI testing is performed, which can be performed on fresh frozen or paraffin-embedded tumor tissue using a PCR-based assay for the detection of instability.

[0112] A National Cancer Institute workshop agreed on five microsatellite markers necessary for determining MSI, including two mononucleotide markers (BAT25 / 26) and three dinucleotide markers (D2S123, D5S346, and D17S250). Profile interpretation requires comparison with each patient's normal DNA. To avoid matching analysis with normal DNA, an alternative molecular method based solely on quasi-monomorphic single-nucleotide markers was developed. This method has proven more specific and sensitive than the original NCI panel (J Clin Oncol. 2006 Jan 10;24(2):241-51).

[0113] Based on MSI status, cancers such as CRC are classified into three groups: MSI-H, when two or more of the five microsatellite markers show instability; MSI-L (low-level MSI), when only one of the five markers shows instability; and microsatellite stable (MSS), when none of the markers show instability (Cancer Res. 1998 Nov 15; 58(22):5248-57.).

[0114] IHC is a widely available alternative test that does not require a molecular laboratory and has the advantage of identifying affected genes by detecting defects in their protein products. Another advantage of IHC testing is that defects in specific mismatched gene products (MLH1, MSH2, MSH6, and PMS2) can direct germline testing to those specific genes, aiding in patient identification. Deficient mismatch repair (dMMR) is associated with high-frequency microsatellite instability (H-MSI), and dMMR testing can also be performed via IHC.

[0115] Cancer Cell Int 20, 16 (2020) provides an overview of microsatellite instability detection methods, including NGS (next-generation sequencing), PCR (polymerase chain reaction), CE (capillary electrophoresis), IHC (immunohistochemistry), and smMIPs (single molecule molecular inversion probes).

[0116] [Table 1]

[0117] In a further embodiment, the invention provides the use of an MDM2 antagonist in the manufacture of a medicament for use in treating cancer in a patient whose cancer tumor has lost one or more DDR biomarkers.

[0118] In a further embodiment, the invention provides the use of an MDM2 antagonist in the manufacture of a medicament for use in treating cancer in a patient identified according to the methods described herein as likely to respond to treatment with an MDM2 antagonist.

[0119] In a further embodiment, the invention provides an article of manufacture comprising, packaged together, an MDM2 antagonist drug in a pharmaceutically acceptable carrier and a package insert indicating that the cancer (e.g., mesothelioma, kidney tumor, or glioblastoma) agent is for treating a cancer patient based on the level of one or more biomarkers identified herein as determined by the assay method used to measure the level.

[0120] In a further embodiment, the present invention provides a method of advertising an MDM2 antagonist drug, comprising encouraging a target audience to use the MDM2 antagonist drug to treat cancer patients who have loss of one or more DDR biomarkers.

[0121] In a further embodiment, the present invention provides an apparatus configured to identify tumors (e.g., mesothelioma) in a cancer patient as likely to benefit from treatment with an MDM2-targeted therapeutic agent or combination of therapeutic agents, or as not likely to benefit from treatment with a therapeutic agent or combination of therapeutic agents. The apparatus may comprise a storage device that stores sequencing or immunoassay data from a tumor or blood-based sample regarding the levels of one or more DDR biomarker genes and / or loss of one or more DDR biomarkers, to identify patients as either likely or not likely to benefit from treatment with an MDM2-targeted therapeutic agent or combination of therapeutic agents.

[0122] In one embodiment of the methods described herein, the patient is administered an MDM2 antagonist if the level of one or more DDR biomarkers is low or absent (eg, missing).

[0123] In another embodiment of the methods described herein, if the level of one or more DDR biomarkers is elevated (or present), the patient is not administered an MDM2 antagonist.

[0124] In certain embodiments, an MDM2 antagonist can be administered to a patient in combination with an additional cancer therapeutic agent that is not an MDM2 antagonist. In one embodiment, at least one biomarker of the invention can be used to select patients for treatment with a combination of an MDM2 antagonist and an agent described in (i)-(xlix) below.

[0125] In certain embodiments, an MDM2 antagonist can be administered to a patient in combination with an agent to induce sensitivity to the MDM2 antagonist, e.g., to reduce the level of one or more genes or gene products in the DNA damage repair (DDR) pathway.

[0126] In certain embodiments, the method of treating cancer in a patient comprises: (a) selecting a patient having normal or elevated levels of a DDR pathway gene or gene product in a biological sample obtained from said patient; and (b) administering to the patient selected in step (a) a therapeutically effective amount of an MDM2 antagonist, e.g., an agent for inducing sensitivity to an MDM2 antagonist by reducing the level of one or more genes or gene products in the DNA damage repair (DDR) pathway. The compound comprises:

[0127] In one embodiment, the agent for decreasing the level of one or more DDR pathway gene products is an inhibitor of a DDR pathway gene or gene product.

[0128] In one embodiment, the agent for reducing the level of one or more DDR pathway gene products is an inhibitor of BRCA1, BRCA2, ATM and / or ATRX. [Brief explanation of the drawings]

[0129] [Figure 1] Figure 1: Dual-function loss-of-function CRISPR screen identifies DDR as the primary sensitizing pathway to compound 1. (A) Horizon CRISPR data: Network analysis of CRISPR hits showed enrichment for the Fanconi anemia pathway (Horizon bioinformatics analysis). (B) Gene set enrichment analysis (GSEA) showed enrichment for other DDR pathways (in-house bioinformatics analysis). (C) Replication stress gene expression signature in mesothelioma apoptotic cell lines. [Figure 2]Figure 2: ATM mutations are associated with increased sensitivity to compound 1. (A) ATM mutant cancer cell lines are significantly more dependent on MDM2 compared to ATM wild-type cell lines based on published DepMAP RNAi data (version 20Q4). (B) ATM mutation status in apoptotic and non-apoptotic mesothelioma cell lines. (C) Reduced proliferation in ATM mutant cell lines. (D) Apoptosis induction in ATM mutant cell lines. (E) Modulation of DDR signaling in ATM mutant cell lines. (F) Table summarizing reduced proliferation in BRCA1, BRCA2, and ATM mutant patient-derived organoids. (G) Reduced proliferation in breast cancer-derived BRCA2 mutant compared to BRCA2 wild-type patient-derived organoids. [Figure 3] Figure 3: ATRX is an additional hit from the cell panel analysis. Bioinformatics analysis of cell panel data using all indications combined, including only P53 wild-type cell lines. ANOVA was used to examine the association of genomic features with sensitivity to compound 1. ATRX deficiency was predicted to be significantly associated with sensitivity. (A) Volcano plot (ANOVA results for predictive biomarkers of sensitivity / resistance). (B) Boxplot of the area of ​​activity in ATRX-deficient cell lines and ATRX wild-type cell lines. [Figure 4] Figure 4: Microsatellite instability (MSI) status and susceptibility. (A) Table listing active regions and tumor mutation burden of MSI-H cancer cell lines in a cell panel. (B) Reduced proliferation in MSI-H cancer cell lines from various indications. (C) Reduced proliferation in patient-derived organoids from six MSI-H colorectal cancers. (D) In ​​vivo efficacy study showing reduced tumor growth in an MSI-H xenograft model of colorectal cancer. [Figure 5] Figure 5: Summary of major DDR pathways adapted from Razqallah Hakem, EMBO J (2008) 27:589-605. Boxes highlight DNA repair pathways for which the inventors identified specific biomarkers as sensitive markers for MDM2 antagonism. [Figure 6]Figure 6: X-ray powder diffraction pattern of (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid. [Figure 7] Figure 7: DSC scan of (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid. Specific Description of the Invention

[0130] definition The terms "MDM2 inhibitor" and "MDM2 antagonist" are used synonymously and define an MDM2 compound or an analog of an MDM2 compound as described herein, including ions, salts, solvates, isomers, tautomers, N-oxides, esters, prodrugs, isotopes, and protected forms thereof (preferably, a salt or tautomer or isomer or N-oxide or solvate thereof, more preferably, a salt or tautomer or N-oxide or solvate thereof) as described herein and as described above.

[0131] "MDM2 antagonist" refers to an antagonist of one or more MDM2 family members, particularly MDM2 and MDM4 (also referred to as MDMx). The term "antagonist" refers to an agent that blocks or reduces a biological response mediated by a receptor ligand or agonist. Antagonists have affinity for their cognate receptors but lack antagonistic potency; binding prevents interaction and inhibits the function of any ligand (e.g., endogenous ligand or substrate, agonist, or inverse agonist) at the receptor. This antagonism can occur directly or indirectly and can be mediated by any mechanism and at any physiological level. Consequently, ligand antagonism can manifest itself functionally differently under different circumstances. Antagonists may mediate their effects by binding to the receptor's active or allosteric site, or they may interact with a unique binding site not normally involved in the biological regulation of receptor activity. The activity of antagonists may be reversible or irreversible depending on the lifetime of the antagonist-receptor complex and thus on the nature of antagonist receptor binding.

[0132] "Potency" is a measure of drug activity expressed as the amount required to produce an effect of a certain strength. A drug with high potency will produce a greater response at a lower concentration. Potency is proportional to affinity and efficacy. Affinity is the ability of a drug to bind to a receptor. Efficacy is the relationship between receptor occupancy and the ability to elicit a response at the molecular, cellular, tissue, or system level.

[0133] As used herein, the term "via" when used with, for example, MDM2 / p53 as described herein (and applied, for example, to various physiological processes, diseases, conditions, pathologies, therapies, treatments, or interventions) is intended to limit the scope of the various processes, diseases, conditions, pathologies, treatments, and interventions to which the term is applied, to those in which the protein plays a biological role. When the term is applied to a disease, condition, or pathology, the biological role played by the protein may be direct or indirect and may be necessary and / or sufficient for the manifestation of symptoms of the disease, condition, or pathology (or the etiology or progression of the disease, condition, or pathology). Thus, the function of a protein (particularly an abnormal level of function, such as overexpression or underexpression) is not necessarily the proximate cause of the disease, condition, or pathology, and it is intended that intervening diseases, conditions, or pathologies include those with multifactorial etiologies and complex progressions in which the subject protein is only partially involved. When the term is applied to treatment, prevention, or intervention, the role played by the protein may be direct or indirect and may be necessary and / or sufficient for the outcome of the treatment, prevention, or intervention. Protein-mediated pathological conditions or conditions therefore include the development of resistance to particular anti-cancer drugs or treatments.

[0134] The term "treatment" as used herein in the context of treating a condition, i.e., a condition, disorder, or disease, generally relates to treatments and therapies, whether for humans or animals (e.g., in veterinary applications), that achieve some desired therapeutic effect, e.g., inhibiting the progression of the condition, including slowing the rate of progression, halting the rate of progression, ameliorating the condition, reducing or alleviating at least one symptom associated with or caused by the condition being treated, and curing the condition. For example, treatment can be the alleviation of one or more symptoms of a disorder, or the complete eradication of a disorder.

[0135] The term "prevention" as used herein in the context of treating a pathology, i.e., a condition, disorder, or disease (i.e., use of a compound as a prophylactic measure) generally relates to prevention or avoidance, whereby some desired prophylactic effect is achieved, e.g., prevention of the occurrence of a disease or protection from a disease, whether for humans or animals (e.g., in veterinary applications). Prevention includes completely and totally arresting all symptoms of a disorder indefinitely, simply delaying the onset of one or more symptoms of a disease, or making a disease less likely to occur.

[0136] References to the prevention or treatment of a disease state or condition such as cancer include, for example, alleviating or reducing the incidence of cancer.

[0137] The combinations of the present invention may provide a therapeutically beneficial effect over the therapeutic effect of the individual compounds / agents when administered separately.

[0138] The term "effective" includes beneficial effects such as additivity, synergy, reduced side effects, reduced toxicity, prolonged time to disease progression, prolonged survival, sensitization or resensitization of one agent to another, or improved response rate. Advantageously, an effective effect allows a patient to receive a reduced dose of each or any of the components, thereby reducing the toxicity of chemotherapy while achieving and / or maintaining the same therapeutic effect. A "synergistic" effect in this context refers to a therapeutic effect produced by a combination that is greater than the sum of the therapeutic effects of the combined agents when presented individually. An "additive" effect in this context refers to a therapeutic effect produced by a combination that is greater than the therapeutic effect of either of the combined agents when presented individually. The term "response rate," as used herein, refers to the degree of tumor size reduction at a given time point, e.g., 12 weeks, in the case of solid tumors. Thus, for example, a 50% response rate means a 50% reduction in tumor size. As used herein, the term "clinical response" refers to a response rate of 50% or greater. As used herein, a "partial response" is defined as a response rate of less than 50%.

[0139] As used herein, the term "combination" as applied to two or more compounds and / or agents is intended to define a material in which the two or more agents are associated. The terms "combination" and "combining" in this context should be interpreted accordingly.

[0140] The association of two or more compounds / agents in a combination can be physical or non-physical. Examples of physically associated combination compounds / agents include: a composition (e.g., a unit dosage form) comprising two or more compounds / agents admixed together (e.g., in the same unit dose); Compositions comprising materials in which two or more compounds / drugs are chemically / physicochemically linked (e.g., by cross-linking, molecular aggregation or binding to a common vehicle moiety); Compositions comprising materials in which two or more compounds / agents are chemically / physicochemically co-packaged (e.g., disposed on or within lipid vesicles, particles (e.g., microparticles or nanoparticles) or emulsion droplets); Pharmaceutical kits, pharmaceutical packs or patient packs in which two or more compounds / drugs are co-packaged or co-presented (e.g., as part of a unit dose sequence).

[0141] Examples of non-physical association mixed compounds / drugs include: · a material comprising at least one of two or more compounds / agents accompanied by instructions for the immediate association of at least one compound to form a physical association of the two or more compounds / agents (e.g., a non-unit dosage form); · Material comprising at least one of two or more compounds / drugs accompanied by instructions for combination therapy with the two or more compounds / drugs (e.g., non-unit dosage forms); a material comprising at least one of two or more compounds / drugs accompanied by instructions for administration to a patient population to which the others of the two or more compounds / drugs have been or are being administered; A material comprising at least one of two or more compounds / agents in an amount or form specifically adapted for use in combination with the other(s) of the two or more compounds / agents.

[0142] As used herein, the term "combination therapy" is intended to define a therapy comprising the use of a combination of two or more compounds / agents (as defined above). Accordingly, references in this application to "combination therapy," "combination," and "combined use" of compounds / agents may refer to compounds / agents administered as part of the same overall treatment regimen. As such, the pharmacokinetics of each of the two or more compounds / agents may be different, and each may be administered simultaneously or at different times. It will be recognized, therefore, that the compounds / agents of a combination may be administered sequentially (e.g., before or after) or simultaneously in the same pharmaceutical formulation (i.e., together) or in different pharmaceutical formulations (i.e., separately). Simultaneously in the same formulation is a unitary formulation, and simultaneously in different pharmaceutical formulations is a non-unitary formulation. The pharmacokinetics of each of the two or more compounds / agents in a combination therapy may also differ with respect to the route of administration.

[0143] As used herein, the term "pharmaceutical kit" defines an arrangement of one or more unit doses of a pharmaceutical composition together with an administration means (e.g., a metering device) and / or a delivery means (e.g., an inhaler or syringe), optionally all contained within a common outer package. In a pharmaceutical kit comprising a combination of two or more compounds / agents, the individual compounds / agents may be in unit or non-unit dosage forms. The unit doses may be contained within a blister pack. The pharmaceutical kit may optionally further comprise instructions for use.

[0144] As used herein, the term "pharmaceutical pack" defines an array of one or more unit doses of a pharmaceutical composition, optionally contained within a common outer package. In a pharmaceutical pack comprising a combination of two or more compounds / drugs, each compound / drug may be a unit or non-unit formulation. The unit doses may be contained within a blister pack. The pharmaceutical pack may optionally further comprise instructions for use.

[0145] The term "optionally substituted," as used herein, refers to a group that may be unsubstituted or substituted with substituents, as defined herein.

[0146] Detailed Description of the Invention The present invention is based on the identification of biomarkers that allow the identification of cancer patients who are likely to respond to MDM2 antagonist therapy, thereby providing for precise cancer therapy using MDM2 antagonists.

[0147] In certain embodiments, the present invention provides a companion diagnostic for the treatment of cancer with an MDM2 antagonist. As used herein, the term companion diagnostic refers to both tests required to determine whether a patient will respond to a drug (i.e., a required companion diagnostic) and tests intended to identify whether a patient will respond favorably or optimally (sometimes referred to as an adjunct diagnostic). In certain embodiments, biomarkers identify patients who are likely to respond, thus distinguishing responders from non-responders. In other embodiments, biomarkers identify patients who are likely to respond optimally, which then allows a physician to select the optimal treatment for that patient.

[0148] In some embodiments, the present invention provides assays for determining the expression or activity level of one, two, three, four, five, six, seven, eight, nine, ten, twenty, twenty-five, or more of the biomarkers identified herein. This can be determined directly or indirectly as described above. The assay may or may not include a step of predicting a prognostic outcome. The assays are generally in vitro assays performed on patient-derived samples, such as cancer biopsies or blood samples (whether or not the cancer is a blood cancer).

[0149] Biomarkers for effective cancer treatment The present disclosure provides biomarkers that indicate enhanced sensitivity of cancer cells to treatment with MDM2 antagonists, and thus, identification of one or more of the specified biomarkers allows for the selection of cancer patients for MDM2 antagonist treatment.

[0150] These biomarkers are DNA damage response (DDR) pathway genes. Optionally, the one or more DDR biomarker genes are from one or more of the following pathways: HR, NHEJ, MMR, FA, and / or BER. Optionally, the one or more DDR biomarkers measured comprise BRCA1. Optionally, the one or more DDR biomarkers measured comprise BRCA2. Optionally, the one or more DDR biomarkers measured comprise ATM. Optionally, the one or more DDR biomarkers measured comprise ATRX. Optionally, one or more of the DDR biomarkers are in the FA pathway. Optionally, when one or more of the DDR biomarkers are in the MMR pathway, depletion of said one or more biomarkers is identified by MSI (generally MSI-High) and / or high tumor mutation burden. Optionally, the one or more DDR biomarkers measured comprise MSH2, MSH3, MSH6, MLH1, PMS2 MLH3, POLE, and / or POLD1. Optionally, the cancer comprises a mutational signature SBS6 and / or SBS26 associated with deficient DNA mismatch repair, and / or a POLD1 mutational signature SBS20.

[0151] DNA damage response (DDR) Cells are constantly exposed to various genotoxic attacks and can repair diverse DNA damage through several DNA repair mechanisms, many of which involve p53. DNA repair errors can lead to mutations and chromosomal abnormalities, which alter the function of tumor suppressor genes or oncogenes, ultimately leading to cancer development.

[0152] As a central tumor suppressor, p53 protects the genome by orchestrating various DNA damage response (DDR) mechanisms (as highlighted in Williams and Schumacher, p53 in the DNA-Damage-Repair Process, Cold Spring Harbor Perspectives in Medicine, 2016, 1-15). Importantly, the p53 response is crucial in determining whether cells undergo apoptosis or cell cycle arrest after DNA damage.

[0153] Base excision repair (BER) targets oxidative base modifications, whereas nucleotide excision repair (NER) removes various helix-distorting lesions commonly induced by UV irradiation. Mismatch repair (MMR) corrects erroneously inserted nucleotides during replication. Alternatively, DNA double-strand breaks commonly induced by radiation are repaired by either nonhomologous end joining (NHEJ) or homologous recombination (HR).

[0154] Brown et al. Targeting DNA repair in cancer: beyond PARP inhibitors, Cancer Discovery, 2017, 20-37, reviews the DDR pathway. The NER pathway removes helix-distorting lesions from DNA, particularly photolesions induced by ultraviolet light (Brown et al. 2017). This involves the removal of short oligonucleotides containing the damaged lesions using structure-specific endonucleases, followed by DNA polymerase restoration of the DNA sequence. Genes involved in the NER pathway include XPC, DDB2, CSA, XPA, RPA, XPG, ERCC1, POLE, POLD1, LIG1, and LIG3.

[0155] DNA glycosylases in the BER pathway recognize and remove damaged bases, directing them to basic sites where they are processed by APE1. The BER pathway results in single-strand breaks (SSBs), which are repaired using the SSB repair pathway. PARP1 is a DNA damage sensor protein for DNA strand breaks in the SSB repair pathway. PARP1 localizes to DNA damage sites and generates extensive poly (ADP-ribose) chains. Ribosylated PARP1 promotes the recruitment of SSB repair proteins to DNA damage sites. Genes involved in the BER and SSB repair pathways include DNA glycosylases, APE1, PARP, XRCC1, PNKP, POLβ, FEN1, TDP1, aprataxin, LIG1, and LIG3A.

[0156] MSH2, MSH3, and MSH6 recognize base mismatches and insertion / deletion loops (Brown et al. 2017). MSH2, MSH3, and MSH6 recruit MLH1 and PMS2 to the damage site. Through the cooperative action of MMR proteins, EXO1 removes the mismatch, and then POLD and LIG1 fill the gap and seal the nick, respectively. Genes involved in MMR include MSH2, MSH3, MSH6, MLH1, PMS2, EXO1, POLD, and LIG1. Deficient mismatch repair (dMMR) is associated with high-frequency microsatellite instability (H-MSI), and dMMR testing can be performed via IHC.

[0157] Classical NHEJ (c-NHEJ) is the predominant DNA DSB repair pathway in human cells and functions throughout the cell cycle (Brown et al. 2017). It involves relatively rapid joining of broken DNA ends via the core NHEJ complex, which includes DNA-PK, XRCC4, LIG4, XLF, and PAXX. DNA end processing and DNA polymerase action may be required before joining occurs, making NHEJ inherently error-prone. NHEJ maintains genome stability by rapidly repairing DSBs in conditions where recombination events are likely to cause large-scale chromosomal rearrangements, such as non-cycling or G1 cells. Genes involved in NHEJ include Ku70 / Ku80, DNA-PK, XRCC4, XLF, LIG4, APLF, Artemis, PAXX, WRN, and ATRX.

[0158] Alternative NHEJ (Alt-NHEJ or MMEJ) is a DSB ligation pathway when c-NHEJ is genetically impaired (Brown et al. 2017). It occurs after limited DNA end resection and contributes to the excessive genomic deletions and chromosomal translocations seen in tumors. It may also provide a backup repair pathway in HR-deficient cells. Genes involved in Alt-NHEJ include PARP, XRCC1, LIG3, LIG1, CtIP, and POLQ.

[0159] HR is generally slow and limited to late S phase / G2 because it relies on homologous sister chromatid DNA strands for repair (Brown et al. 2017). Extensive DNA end resection by helicases and exonucleases, such as DNA2, BLM, WRN, and EXO1, results in 3'-ssDNA overhangs, which are then repaired by HR. Replication protein A (RPA), with the assistance of BRCA1 and PALB2, loads RAD51 onto RPA-coated ssDNA, triggering strand invasion. Several factors, including POLQ, PARI, RECQL5, FANCJ, and BLM, negatively regulate this process to prevent excessive recombination. Genes involved in HR include MRN, ATM, ATR, MK2, CtlP, BRCA1, BARD1, BRCA2, PALB2, RPA, RAD51, MUS81 / EME1, SLX1 / SLX4, RTEL1, BLM, TOPOIII, POLQ, PARI, RECQL5, FANCJ, and BLM.

[0160] The Fanconi anemia (FA) pathway is involved in the repair of interstrand crosslinks. Molecular details of the FA pathway are described in Niraj et al. The Fanconi anemia pathway in cancer, Annu. Rev. Cancer Biol. 2019. 3:457-78). The FA core complex comprises FANCA, FANCG, FANCB, FANCL, UBE2T (FANCT), FANCF, FANCC, FANCE, FANCM, REV1, REV7, and REV3. Also included are several Fanconi anemia-associated proteins (FAAPs). Mutations or deletions in the core complex genes or FAAPs are within the scope of the present invention.

[0161] DDR defects are found in approximately 13% of all cancers and are more frequent in certain tumor types, including pancreatic cancer (>35%), bladder cancer (35%), prostate cancer (33%), ovarian cancer (24%), and triple-negative breast cancer (16%).

[0162] Loss-of-function CRISPR screen identifies DDR as the primary sensitization pathway to MDM2 antagonists The following example describes a dual CRISPR screen in a panel of three P53 wild-type lung cancer cell lines in the presence or absence of Compound 1 to identify novel predictive biomarkers of MDM2 antagonist sensitivity.

[0163] Several DNA damage response (DDR)-related genes were identified as top hits (Figure 1A-B). Interestingly, these genes are involved in several DDR pathways, such as the homologous recombination pathway, Fanconi anemia (FA) pathway, base excision repair pathway, and replication stress pathway. Notably, the replication stress pathway is an indicator of genomic instability and is caused by multiple defects in the DDR pathway, leading to high levels of DNA damage (which affects the process of DNA replication). These data suggest that tumors with defects in the DDR mechanism are generally more sensitive to MDM2 antagonist treatment.

[0164] To confirm this result, we identified transcriptomic signatures differentially expressed in apoptotic and nonapoptotic samples using early-passage human mesothelioma cell lines previously characterized for their sensitivity to Compound 1. The "replicative stress" signature was strongly enriched in mesothelioma apoptotic cell lines, confirming the association between Compound 1 sensitivity and activated DDR pathways (Figure 1C).

[0165] The association between MDM2 antagonists and defects in multiple DDR pathways was identified through a dual CRISPR screen. Following the CRISPR screen, bioinformatics and wet-lab analysis of multiple systems facilitated the identification of specific biomarkers within the DDR. Further validation of these results confirms DDR defects that are sensitive to MDM2 antagonist treatment.

[0166] As a visual summary, the major DDR pathways are shown in Figure 5. Highlighted by boxes are DNA repair pathways for which specific biomarkers were identified by the inventors as markers of sensitivity to treatment with MDM2 antagonists.

[0167] HR pathway: BRCA1, BRCA2, and ATM alterations Genes involved in the homologous recombination pathway have been identified in CRISPR screens. Homologous recombination (HR) is an error-free DSB repair pathway that is largely restricted to the S and G2 phases of the cell cycle. The critical importance of the HR pathway for genome maintenance has been demonstrated by the identification of several cancer-disabling mutations in BRCA1, BRCA2, ATM, CHEK2, RAD50, and RAD51C in many cancers.

[0168] One of the central components of HR is the serine-threonine kinase Ataxia Telangiectasia Mutated protein (ATM), which phosphorylates multiple key players in various branches of the DDR. Somatic mutations or deletions of ATM are commonly found in lymphoid malignancies as well as some solid tumors, leading to reduced protein expression and impaired DNA double-strand break repair in the genome.

[0169] Bioinformatics analysis of the publicly available DepMAP RNAi data (version 20Q4) for MDM2 predicted that ATM mutant cell lines were significantly more dependent on MDM2 than ATM wild-type cell lines (Fig. 2A). Furthermore, we detected a strong enrichment of ATM mutations in apoptotic patient-derived mesothelioma lines (6 / 9) compared with nonapoptotic lines, all of which were ATM wild-type (6 / 6) (Fig. 2B).

[0170] Furthermore, in vitro validation of four ATM mutant cell lines from different indications (HCC1500-breast, LNCap-prostate, HT-144-melanoma, and HepG2-liver) demonstrated sensitivity to Compound 1 as measured by decreased cell proliferation (Figure 2C), while LNCap-prostate and HepG2-liver data demonstrated sensitivity to Compound 1 as measured by increased apoptosis (Figure 2D). Furthermore, Western blot analysis demonstrated clear modulation of the DDR signaling pathway upon Compound 1 treatment (Figure 2E).

[0171] Along with the identification of ATM mutations as a biomarker for MDM2 antagonist sensitivity, additional bioinformatics analysis indicated that defects or mutations in other HR pathway genes could serve as biomarkers for MDM2 antagonist sensitivity, including, but not limited to, BRCA1 and / or BRCA2.

[0172] In one embodiment, the homologous recombination repair deficiency comprises the loss of BRCA1 or BRCA2. In one embodiment, the cancer is BRCA-like. Tumors that have lost canonical HRR other than BRCA1 / 2 loss are BRCA-like (Trends in Cell Biology, September 2019, Vol. 29, No. 9, pg 740).

[0173] To confirm whether ATM, BRCA1, and / or BRCA2 mutations (or loss of expression) are associated with sensitivity to MDM2 antagonists, further in vitro validation was performed on patient-derived organoids (PDOs). Four PDOs from various indications harboring mutations in either ATM, BRCA1, and / or BRCA2 demonstrated sensitivity to Compound 1 as measured by reduced cell proliferation (Figure 2F-G). Of note, four additional PDOs from the same indications without alterations in ATM, BRCA1, and / or BRCA2 were resistant to Compound 1.

[0174] FA route FA pathway genes also serve as biomarkers for MDM2 antagonist therapy.

[0175] Genes identified by dual CRISPR screen (CRISPR knockout and CRISPRi) are involved in the Fanconi anemia (FA) pathway. Figure 1A shows the enrichment of the Fanconi anemia pathway in CRISPR hits.

[0176] CRISPR screen data indicate that tumors with defects in the FA pathway are generally sensitive to Compound 1 treatment.

[0177] These data therefore demonstrate an association between MDM2 antagonist sensitivity and Fanconi anemia pathway defects, and therefore loss of function in the FA pathway is a biomarker for MDM2 antagonist sensitivity.

[0178] NHEJ pathway: ATRX loss Furthermore, bioinformatics analysis of cell panel data predicted that ATRX deficiency is a key biomarker for sensitivity to MDM2 compound 1 (Figure 3). ATRX was also implicated in regulating DDR through both non-homologous end joining (NHEJ) and homologous recombination repair (HRR).

[0179] The identification and bioinformatics analysis of ATRX loss as a biomarker for MDM2 antagonist therapy suggests that losses or mutations in other NHEJ or HRR pathway genes may serve as biomarkers for MDM2 antagonist sensitivity.

[0180] MMR pathway: microsatellite instability (MSI) Microsatellites are regions containing multiple repeats of 1- to 5-base pair motifs and are widely distributed throughout the human genome. In normal cells, the number of microsatellite repeats is confirmed by mismatch repair (MMR) and maintained during cell division. A failure of the MMR system prevents cells from adjusting the length of microsatellites during cell division, a condition called microsatellite instability (MSI). MSI is frequently observed in several types of cancer (colorectal adenocarcinoma, endometrial adenocarcinoma, and gastric adenocarcinoma), and MSI-high colorectal tumors have been shown to be more sensitive to immune-enhancing therapies.

[0181] Information on the microsatellite stability and tumor mutation burden of the cell lines was obtained from the Sanger Cell Models Passport database. We found that MSI-H cell lines exhibited high tumor mutation burden (mutations / Mb) and were enriched for mutations associated with the DNA mismatch repair pathway (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, and PMS2). Furthermore, MSI-H cell lines showed strong enrichment for mutational signatures associated with DNA mismatch repair deficiency and POLD1 and / or POLE mutations (Figure 4A). Collectively, these findings were consistent, suggesting that MSI tumors, such as those from the colon, endometrium, and stomach, are sensitive to MDM2 antagonists. Furthermore, in vitro validation of eight MSI-H cell lines derived from different indications demonstrated sensitivity to compound 1, as measured by reduced cell proliferation (Figure 4B). To confirm whether MSI-H status correlates with MDM2 antagonist sensitivity, in vitro validation was further performed on patient-derived organoids (PDOs). Six MSI-H colorectal cancer PDOs demonstrated sensitivity to compound 1, as measured by reduced cell proliferation (Figure 4C). In vivo efficacy data confirmed that compound 1 significantly inhibited tumor growth in a xenograft model of MSI-H colorectal cancer (HCT-116) (Figure 4D).

[0182] Base excision repair (BER) pathway CRISPR screen analysis identified a strong correlation between the BER pathway and sensitivity to MDM2 antagonists (Figure 1). DDR biomarkers can comprise one or more BER pathway genes.

[0183] Biomarkers and Combinations For reference, the biomarkers of the present disclosure can be divided into five groups.

[0184] a. HR pathway, e.g., BRCA1, BRCA2, and / or ATM defects b. Deficiency in the NHEJ pathway, e.g., ATRX c. MMR pathway, e.g., MSI-H (microsatellite unstable tumors, characterized by high tumor mutation load) d.FA pathway e.BER path

[0185] In some embodiments, one biomarker is determined, which may be from any of groups a), b), c), d), or e).

[0186] In some embodiments, multiple biomarkers are determined, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biomarkers. These may comprise or consist of multiple biomarkers from a single group (i.e., group b) or group c)), or may be from different groups, e.g., 0, 1, 2 or more biomarkers from group a); and 0, 1, 2 or more biomarkers from group b); and 0, 1, 2 or more biomarkers from group c); and with or without group d) or group e); or comprising 0, 1, 2 or more biomarkers from group b); and 0, 1, 2 or more biomarkers from group c), and group d; or two or more biomarkers from group b); two or more biomarkers from group c); with or without group d; or 0, 1, 2 or more biomarkers from group a); 0, 1, 2 or more biomarkers from group b); 0, 1, 2 or more biomarkers from group c), 0, 1, 2 or more biomarkers from group d); and 0, 1, 2 or more biomarkers from group e). It may comprise or consist of:

[0187] When multiple biomarkers are determined, the combination of biomarkers may be referred to as a biomarker panel. A biomarker panel may comprise or consist of the identified biomarkers.

[0188] In addition to the biomarkers of the present invention, other biomarkers and / or data, such as demographic data (e.g., age, sex), can be included in the dataset applied to determine the suitability of MDM2 inhibition. Optionally, when other biomarkers are included, the total number of biomarkers (i.e., the biomarker panel of the present invention plus other biomarkers) can be 3, 4, 5, 6, or more. In some embodiments, a predictive biomarker panel with fewer components can simplify testing requirements.

[0189] The terms "loss" and "reduction" as used herein have their ordinary meanings. The terms "increase" and "enhancement" as used herein have their ordinary meanings.

[0190] Biomarkers can be determined by any suitable technique known to those skilled in the art. Biomarkers can be determined by direct or indirect techniques. Gene expression can be detected by detecting mRNA transcripts. Protein biomarkers can be detected by immunohistochemistry.

[0191] In some embodiments, depletion of one or more of the biomarkers of the invention can be determined by assessing the function of one or more biomarkers. The level of biomarker expression can be directly proportional to the level of function. The function of one or more biomarkers can be determined directly or indirectly.

[0192] In some embodiments, the expression or activity level can be compared to a threshold that also reflects an expression or activity level known to be associated with susceptibility to treatment to assess whether the test value is indicative of susceptibility to MDM2 inhibitor treatment in the patient.

[0193] Patients evaluated according to the present disclosure are known to have or are suspected of having cancer. The sample to be tested may be known to contain or be suspected of containing cancer cells. In a typical embodiment, the sample to be tested is a biopsy of cancerous tissue. The biopsy may be a liquid biopsy or a solid tissue (e.g., solid tumor) biopsy.

[0194] Biomarker levels The present invention provides low levels of one or more biomarkers. Generally, the comparison is made to normal healthy individuals, or more generally to non-cancerous cells of the same type as the cancer cells.

[0195] The reduction in biomarker levels could be due to depletion of the gene itself, for example, due to large-scale chromosomal rearrangements or other genetic abnormalities that result in the loss of DDR pathway genes in the genome of cancer cells, which of course also depletes the gene product and activity.

[0196] A decrease in a biomarker level can be a decrease in expression of a gene product.

[0197] A decrease in biomarker level may also be a decrease in activity caused, for example, by a loss-of-function mutation, which can be viewed as a depletion of the wild-type gene product.

[0198] In some embodiments, an increase or decrease in biomarker level is determined relative to non-cancerous cells from the same individual, generally non-cancerous cells of the same type from the same individual.

[0199] In a further embodiment, an increase or decrease in a biomarker level is determined relative to a laboratory standard and a value based on known normal population values. Generally, the known levels are obtained from non-cancerous cells.

[0200] In other embodiments, the increase or decrease in biomarker level is relative to a known value from a normal (non-cancer) individual, for example, GTEx is a data resource of gene expression in normal healthy individuals from 44 different tissues, as discussed elsewhere herein.

[0201] In some other embodiments, the increase or decrease in biomarker levels is assessed relative to levels determined in cancer samples from MDM2 inhibitor non-responders, or in cancer samples from MDM2 inhibitor non-responders, which can be particularly useful for one or more IFN signature biomarkers.

[0202] In one embodiment, the RNA level of one or more DDR biomarkers is decreased relative to the amount of said RNA in a control sample obtained from a normal subject not afflicted with cancer.

[0203] In another embodiment, the RNA level of one or more DDR biomarkers is decreased relative to the amount of said RNA in an earlier sample obtained from the same patient when the patient did not have cancer.

[0204] In one embodiment, it is decreased compared to normal levels (eg, the "upper limit of normal" or ULN).

[0205] In one embodiment, the level of at least one of the biomarkers relative to (a) the level of at least one of the biomarkers in a sample from a tissue or person that does not have cancer, or (b) the level of one or more control proteins in a sample from the subject, has an area under the curve (AUC) greater than 0.5 (for an elevated biomarker) or less than 0.5 (for a decreased biomarker) in the cancer sample relative to the control sample. Optionally, the AUC is less than or greater than 0.6, 0.7, 0.8, 0.9, 0.95, 0.975, or 0.99.

[0206] In some embodiments, the level of at least one of the biomarkers is at least one standard deviation from a control relative to (a) the level of one or more biomarkers in a sample from a tissue or person that does not have cancer, or (b) the level of one or more control proteins in a sample from a subject with cancer.

[0207] In some embodiments, the control for comparison is a sample obtained from a healthy patient or a non-cancerous tissue sample obtained from a patient diagnosed with cancer, e.g., a non-cancerous tissue sample from the same organ as the tumor (e.g., non-cancerous colon tissue can serve as a control for colon cancer). In some embodiments, the control is a historical control or standard value (i.e., a previously tested control sample or group of samples that represent a baseline or normal value).

[0208] Control or standard samples for comparison with samples to determine differential expression include samples that are considered normal (in the sense that they are not altered with respect to the desired characteristics, such as samples from subjects without colon cancer), as well as laboratory values ​​(although they may be arbitrarily set). Laboratory standards or laboratory values ​​can be set based on known or determined population values ​​and can be provided in the form of graphs or tables that allow comparison of measured, experimentally determined values.

[0209] In such embodiments, the reference score for one or more biomarkers is based on normal, healthy individuals.

[0210] cancer Cancers that exhibit one or more of the identified biomarkers exhibit an increased likelihood of successful treatment with an MDM2 antagonist. The cancer to be treated is not particularly limited, as long as it exhibits one or more of the biomarkers.

[0211] Cancer is generally p53 wild type.As recognized in the art, p53 wild type cancer cells express the tumor suppressor p53 at wild type level and wild type function.Wild type p53 cells do not contain the mutation of p53 gene that leads to the reduction of p53 tumor suppressor function.

[0212] The data provided in the examples was generated from a variety of cancer tissues, including colon, blood, breast, lung, prostate, liver, skin, ovary, and pancreas. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is colon cancer. In another embodiment, the cancer is blood cancer. In a further embodiment, the cancer is breast cancer. In another embodiment, the cancer is lung cancer. In yet another embodiment, the cancer is skin cancer, e.g., melanoma or carcinoma. In another embodiment, the cancer is ovarian cancer. In a different embodiment, the cancer is pancreatic cancer. In certain embodiments, the cancer is brain cancer, clear cell renal cell carcinoma (ccRCC), esophageal cancer, or melanoma.

[0213] Particular cancers that can be evaluated for treatment according to the present invention include, but are not limited to, mesothelioma, non-small cell lung cancer (NSCLC), glioblastoma (eg, GBM), and renal cancer (eg, KIRC).

[0214] Particular cancers that can be evaluated for treatment according to the present invention include, but are not limited to, acute myeloid leukemia (AML), squamous cell carcinoma, or tumors of the head and neck, skin, digestive system, or reproductive tract.

[0215] Particular cancers that can be evaluated for treatment according to the present invention include, but are not limited to, prostate cancer, ovarian cancer, breast cancer, and gynecological cancers.

[0216] Particular cancers that can be evaluated for treatment according to the present invention include, but are not limited to, colorectal, gastric and gynecological cancers.

[0217] Particular cancers that can be evaluated for treatment according to the present invention include, but are not limited to, breast cancer, ovarian cancer, prostate cancer and pancreatic cancer, particularly DDR-deficient breast cancer, ovarian cancer, prostate cancer and pancreatic cancer.

[0218] In certain embodiments, cancer cell proliferation is inhibited by an IC in the nanomolar range. 50 In certain embodiments, the MDM2 antagonist has an IC 50 In some embodiments, the IC value is less than 500 nM, less than 400 nM, less than 300 nM, or less than 200 nM. 50 The IC value is less than 100 nM. 50 Values ​​can be calculated using, for example, GraphPad Prism software, as shown in the Examples.

[0219] In certain embodiments, MDM2 antagonists induce apoptosis in cancer cells. Apoptosis is generally mediated by activated caspase-3. Induction of apoptosis can be determined by detecting cells positive for activated caspase-3 after 72 hours of treatment with 1 μM MDM2 antagonist. As will be apparent to those skilled in the art, other assay concentrations and / or treatment lengths may be used, such as 48 hours at 1 μM or 48 hours at 5 μM MDM2 antagonist. In certain embodiments, at least 10%, at least 20%, or at least 30% of cells staining positive for activated caspase-3 are indicative of apoptosis induction. In certain embodiments, 40% is a reliable level for identifying strong apoptosis induction, and apoptosis can be considered when more than 40% of cells in a population stain positive for activated caspase-3. Those skilled in the art will appreciate that other levels may be used, such as 10%, 20%, 30%, 50%, 60%, 70%, 75%, or more, as appropriate for the cells and assay. Active caspase-3 staining kits are commercially available, such as the "Cleaved Caspase-3 Staining Kit (Red)" available from Abcam (Cambridge, UK) under catalog number ab65617. Invitrogen Cell Event dye (C10423) can also be used.

[0220] Annexin V dye can also be used to detect apoptosis, which is used in the examples and is well known in the art as a useful dye for detecting apoptosis.

[0221] MDM2 antagonists The transformation-related protein 53 (TP53) gene encodes the 53-kDa protein p53. The tumor suppressor protein p53 acts as a signaling node in diverse pathways that are activated in response to cellular stresses, such as hypoxia, DNA damage, and oncogenic activation, through several post-translational modifications, including phosphorylation, acetylation, and methylation. p53 also plays additional roles in other physiological processes, including autophagy, cell adhesion, cell metabolism, fertility, and stem cell senescence and development. Phosphorylation of p53, resulting from activation of kinases including ATM, CHK1 and 2, and DNA-PK, leads to protein stabilization and transcriptional activation, resulting in the production of various gene products. Responses to p53 activation include apoptosis, survival, cell cycle arrest, DNA repair, angiogenesis, invasion, and autoregulation. These specific combinations, in concert with the cell's genetic background, result in the observed cellular effects, namely apoptosis, cell cycle arrest, and senescence. In tumor cells, apoptotic pathways may be favored due to oncogenic stress coupled with loss of control of tumor suppressor proteins and associated cell cycle checkpoints.

[0222] It is known that the intracellular level of p53 protein increases under stress conditions such as hypoxia and DNA damage. p53 is known to initiate the transcription of many genes that regulate cell cycle progression, initiation of DNA repair, and programmed cell death. This provides a mechanism for the tumor suppressor role of p53, as evidenced by genetic studies.

[0223] p53 activity is tightly negatively regulated by its binding interaction with MDM2 protein, and MDM2 transcription is itself directly regulated by p53. p53 is inactivated upon binding of MDM2 protein to its transactivation domain. Upon inactivation, p53 function is suppressed and the p53-MDM2 complex becomes a target for ubiquitination.

[0224] In normal cells, the balance between active and inactive MDM2-bound p53 is maintained by an autoregulatory negative feedback loop: p53 can activate MDM2 expression, which in turn suppresses p53.

[0225] Mutational inactivation of p53 has been found to be common in approximately half of all common adult sporadic cancers. Furthermore, in approximately 10% of tumors, loss of functional p53 due to gene amplification and overexpression of MDM2 leads to malignant transformation and uncontrolled tumor growth.

[0226] p53 inactivation by various mechanisms is a frequent causative event in cancer development and progression. These include mutational inactivation, targeting by oncogenic viruses, and, in a significant proportion, amplification and / or increased transcription of the MDM2 gene, which leads to overexpression or enhanced activation of the MDM2 protein. MDM2 gene amplification, leading to overexpression of the MDM2 protein, has been observed in tumor samples from common sporadic cancers. Overall, approximately 10% of tumors harbor MDM2 amplification, with the highest incidence observed in hepatocellular carcinoma (44%), lung (15%), sarcoma and osteosarcoma (28%), and Hodgkin's disease (67%) (Danovi et al., Mol. Cell. Biol. 2004, 24, 5835-5843; Toledo et al., Nat Rev Cancer 2006, 6, 909-923; Gembarska et al., Nat Med 2012, 18, 1239-1247). Normally, transcriptional activation of MDM2 by activated p53 leads to increased MDM2 protein levels, forming a negative feedback loop. The essential nature of p53 regulation by MDM2 and MDMX has been demonstrated in gene knockout mouse models. MDM2- / - knockout mice are embryonic lethal around the time of implantation. Double knockout of MDM2 and TP53 rescues lethality. MDM2 directly inhibits p53 activity by binding to and occluding the p53 transcriptional activation domain and by promoting the proteosomal destruction of the complex via its E3 ubiquitin ligase activity. Furthermore, because MDM2 is a transcriptional target of p53, these two proteins are linked in an autoregulatory feedback loop, ensuring that p53 activation is transient.

[0227] MDMX shares strong amino acid sequence and structural homology with MDM2, but neither protein can substitute for the deficiency of the other. MDMX-deficient mice die in utero, and MDM2 knockout mice are lethal during early embryonic development. However, both can be rescued by p53 knockout, demonstrating lethal p53 dependence. MDMX also binds to p53 and inhibits p53-dependent transcription; however, unlike MDM2, it is not activated by p53, thus not forming the same autoregulatory loop. Furthermore, MDMX lacks E3 ubiquitin ligase activity or a nuclear localization signal, but it is thought to form heterodimers with MDM2, contributing to the stabilization of MDM2 and thereby contributing to p53 degradation.

[0228] The therapeutic rationale for MDM2-p53 inhibition is that potent antagonists of this protein-protein interaction would liberate p53 from MDM2's inhibitory control and activate p53-mediated cell death in tumors. In tumors, selectivity is postulated to result from p53 sensing pre-existing DNA damage or oncogenic activating signals that were previously blocked by the action of normal or overexpressed levels of MDM2. In normal cells, p53 activation would be expected to result in the activation of non-apoptotic pathways, resulting in a rather protective growth-suppressive response. Furthermore, the mechanism of action of MDM2-p53 antagonists is non-genotoxic, making them particularly suitable for the treatment of pediatric cancers. MDM4 is also an important negative regulator of p53.

[0229] Approximately 50% of cancers harbor cells in which TP53, the gene that encodes p53, has been mutated, resulting in the loss of the protein's tumor-suppressing function and sometimes even in p53 protein variants that acquire new oncogenic functions.

[0230] Cancers in which high levels of MDM2 amplification are present include liposarcoma (88%), soft tissue sarcoma (20%), osteosarcoma (16%), esophageal cancer (13%), and certain pediatric malignancies, including B-cell malignancies.

[0231] Examples of MDM2 antagonists In one embodiment, the MDM2 antagonist is an agent that regulates MDM2, such as a small molecule, antisense nucleic acid, antibody, or nucleic acid that inhibits the expression of MDM2. In one embodiment, the MDM2 agent is a small molecule. In one embodiment, the MDM2 agent is a small molecule as described herein.

[0232] Roche's MDM2 small molecule antagonist, idasanutlin (RG-7388), is reportedly in Phase I-III clinical trials for solid tumors and hematological malignancies, AML, diffuse large B-cell lymphoma, essential thrombocythemia, polycythemia vera, and follicular lymphoma. Idasanutlin (RG-7388) has the following structure: [ka]

[0233] Idasanutlin (RG-7388) is commercially available or can be prepared, for example, as described in PCT patent application WO2014 / 128094, or by methods analogous thereto.

[0234] HDM-201 (NVP-HDM201, siremadrine) is being developed by Novartis in Phase I / II clinical trials for advanced / metastatic solid tumors characterized by wild-type TP53, ALL, AML, MS, metastatic uveal melanoma, and hematological malignancies including dedifferentiated and well-differentiated liposarcoma. The antagonist HDM-201 (NVP-HDM201) has the following chemical structure: [ka]

[0235] HDM-201 (NVP-HDM201) is commercially available or can be prepared, for example, as described in PCT patent application WO2013 / 111105, or by methods analogous thereto.

[0236] KRT-232 (AMG-232, nabtemadrine), a small molecule antagonist of MDM2, is being developed by NCI / Amgen / GSK in Phase I-I / II clinical trials for tumors, soft tissue sarcomas, e.g., liposarcoma, recurrent or newly diagnosed glioblastoma, metastatic breast cancer, refractory MM, metastatic cutaneous melanoma, and recurrent / refractory AML. KRT-232 (AMG-232) has the following chemical structure: [ka]

[0237] KRT-232 (AMG-232) is commercially available or can be prepared, for example, as described in PCT patent application WO2011 / 153509 or by methods analogous thereto.

[0238] ALRN-6924 (SP-315), a peptide dual antagonist of MDM2 and MDM4, is being developed by Aileron Therapeutics and Roche in Phase II clinical trials for the intravenous treatment of solid tumors, small cell lung cancer, and pediatric tumors including lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, retinoblastoma, hepatoblastoma, brain tumors, liposarcoma, and metastatic breast cancer. ALRN-6924 (SP-315) is a synthetic peptide developed based on stapled peptide technology, which locks peptides into a specific folded (biologically active) conformation that is resistant to proteases. ALRN-6924 (SP-315) has the following structure: [ka]

[0239] ALRN-6924 (SP-315) is commercially available or can be prepared, for example, as described in PCT patent application WO2017205786, or by methods analogous thereto.

[0240] CGM-097 (NVP-CGM-097), a small molecule MDM2 antagonist, is being developed by Novartis in Phase I clinical trials for advanced solid tumors and acute lymphoblastic leukemia (B-ALL). CGM-097 (NVP-CGM-097) has the following chemical structure: [ka]

[0241] CGM-097 (NVP-CGM-097) is commercially available or can be prepared, for example, as described in PCT patent application WO2011076786, or by methods analogous thereto.

[0242] Milademetan tosylate (DS-3032), a small molecule antagonist of MDM2 licensed to Rain Therapeutics and renamed under the study code RAIN-32, is being developed by Daiichi Sankyo in Phase I clinical trials for advanced solid tumors, lymphoma, melanoma, refractory or relapsed AML, ALL, multiple myeloma, blast-phase CML, or higher-risk MDS and diffuse large B-cell lymphoma. Milademetan tosylate (DS-3032) has the following chemical structure: [ka]

[0243] Milademethane tosylate (DS-3032) is commercially available or can be prepared, for example, as described in PCT patent application WO2015 / 033974, or by methods analogous thereto.

[0244] APG-115 (AAA-115; allizomadrine, NCT-02935907), a small molecule antagonist of MDM2, is being developed by Ascentage Pharma in Phase I clinical trials for the treatment of solid tumors and lymphomas, AML, and adenoid cystic carcinoma (ACC). APG-115 (AAA-115; NCT-02935907) has the following chemical structure: [ka]

[0245] APG-115 (AAA-115; NCT-02935907) is commercially available or can be prepared, for example, as described in PCT patent application WO2015 / 161032, or by methods analogous thereto.

[0246] BI-907828, an MDM2 antagonist, is being developed by BI in Phase I clinical trials for the treatment of GBM, metastatic brain tumors, NSCLC, soft tissue sarcoma, and transitional cell carcinoma (urothelial cell carcinoma).

[0247] BI-907828 is commercially available or can be prepared, for example, as described in PCT patent application WO2015 / 161032, or by methods analogous thereto.

[0248] The University of Michigan is developing LE-004, a PROTAC conjugate of MI-1061 and thalidomide, which has been shown to effectively inhibit the growth of a mouse model of human leukemia by inducing MDM2 degradation. Its structure is as follows and can be prepared, for example, as described in PCT patent application WO2017 / 176957 or WO2017 / 176958, or by methods analogous thereto. LE-004 has the following chemical structure: [ka]

[0249] MI-773 (SAR405838) is a highly potent and selective MDM2 inhibitor that binds to MDM2 with greater specificity than other proteins and potently inhibits cell growth in cancer cell lines. SAR405838 effectively induces apoptosis, inhibits cell growth, and induces dose-dependent apoptosis. It is currently being investigated in clinical trials. Its structure is as follows: [ka]

[0250] SAR405838 can be prepared, for example, as described in WO-A-2011 / 060049.

[0251] DS-5272 is an MDM2 antagonist currently being developed by Daiichi Sankyo for oral administration. Its structure is as follows: [ka]

[0252] DS-5272 can be prepared for example as described in PCT patent application WO2015 / 033974, or by processes analogous thereto.

[0253] SJ-0211 is an MDM2 antagonist being developed by the University of Tennessee, the University of Kentucky, and St. Jude Children's Research Hospital for the treatment of retinal diseases. Its structure is a Nutlin-3 analog.

[0254] BI-0252 is an MDM2 antagonist currently being developed by BI for oral administration. BI-0252 inhibits the interaction between MDM2 and p53. Its structure is as follows: [ka]

[0255] AM-7209 is an MDM2 antagonist and is being developed by Amgen as a backup for AMG-232. Its structure is as follows: [ka]

[0256] AM-7209 can be prepared, for example, as described in PCT patent application WO2014 / 200937, or by processes analogous thereto.

[0257] SP-141 (JapA) is a direct MDM2 antagonist under development at Texas Tech University. Its structure is as follows: [ka]

[0258] SCH-1450206 is an MDM2 antagonist being developed by Schering-Plough & Merck for oral administration. An example structure is: [ka]

[0259] Cytarabine, also known as MK-8242 and SCH-900242, is a cytidine antimetabolite analog with an altered sugar moiety (arabinose instead of ribose). The HDM2 inhibitor MK-8242, an orally available inhibitor of human homolog of double minute 2 (HDM2), has potential antineoplastic activity upon oral administration. It inhibits the binding of HDM2 protein to the transcriptional activation domain of the tumor suppressor protein p53. Preventing this HDM2-p53 interaction inhibits p53 degradation, which may result in the restoration of p53 signaling, inducing p53-mediated tumor cell apoptosis.

[0260] Nutlin-3a is an antagonist or inhibitor of MDM2 (human homolog of murine double minute 2) and disrupts its interaction with p53, resulting in the stabilization and activation of p53. Its structure is as follows: [ka]

[0261] NXN-6 (NXN-7; NXN-552; NXN-561; NXN-11) is an MDM2 antagonist currently being developed by Nexus, Priaxon, and BI for oral administration. An example structure is shown below. [ka]

[0262] ADO-21 is an MDM2 antagonist being developed by the Adamed Group.

[0263] CTX-50-CTX-1 is a small molecule MDM2 antagonist being developed by MiRx Pharmaceuticals, CRC.

[0264] ISA-27 is a small molecule MDM2 antagonist being developed by the Universities of Napoli and Salerno. Its structure is: [ka]

[0265] RG-7112 (RO5045337) is the first clinically available, orally active, potent, selective MDM2-p53 inhibitor that crosses the blood-brain barrier. The structure is: [ka] RO-8994 is a small molecule MDM2 antagonist being developed by Roche. RO-8994 has been shown to inhibit tumor growth and induce the mitochondrial effects of p53. Its structure is: [ka]

[0266] RO-8994 is commercially available or can be prepared, for example, as described in PCT patent application WO2011 / 067185, or by methods analogous thereto.

[0267] RO-6839921 (RG-7775) is a small molecule MDM2 antagonist being developed by Roche for IV administration. Its structure is: [ka]

[0268] RO-6839921 (RG-7775) can be prepared, for example, as described in PCT patent application WO2014 / 206866, or by processes analogous thereto.

[0269] JNJ26854165 (celdemethane), with the following structure, is an oral HDM2 inhibitor (or antagonist) that exhibits potent activity against multiple myeloma (MM) cells in vitro and ex vivo, restores p53 function, and may potentially impact other HDM2-dependent pathways. [ka]

[0270] ATSP-7041 (SP-154), a stapled synthetic peptide dual antagonist of MDM2 and MDM4, is being developed by Aileron Therapeutics and Roche and is in preclinical development. ATSP-7041 (SP-154) has the following structure: [ka]

[0271] SAH-p53-8 is a stapled synthetic peptide antagonist of MDM4, Hdm2, and caspase 3 that is being developed by Harvard College and Dana-Faber and is in preclinical development. SAH-p53-8 has the following structure: [ka]

[0272] PM-2 (sMTide-02) is a stapled synthetic peptide antagonist of MDM4, Hdm2, and caspase-3, currently in preclinical development by Harvard College and Dana-Faber. PM-2 (sMTide-02) has the following structure: [ka]

[0273] K-178 is a small molecule MDM4 antagonist currently in preclinical development at Kansai Medical University. K-178 has the following chemical structure: [ka]

[0274] MMRi-64 is a discovery-stage small molecule antagonist of MDM2 and MDM4 being developed by Roswell Park Cancer Institute. MMRi-64 has the following chemical structure: [ka] Small molecule antagonists of MDM2 and MDM4 are also under development by Jagiellonian University and Second Military Medical University. One example has the following structure: [ka]

[0275] Small molecule antagonists of MDM2 and MDM4 are being developed by Emory and Georgia State University and are in preclinical development for the treatment of acute lymphoblastic leukemia.

[0276] Small molecule antagonists of MDM2 and MDM4 are being developed by Adamed and are at the discovery stage.

[0277] In one embodiment of the invention, the MDM2 antagonist is selected from the group consisting of idasanutlin, HDM-201, KRT-232, ALRN-6924, CGM-097, milademethane tosylate, APG-115, BI-907828, LE-004, DS-5272, SJ-0211, BI-0252, AM-7209, SP-141, SCH-1450206, NXN-6, ADO-21, CTX-50-CTX-1, ISA-27, RO-8994, RO-6839921, ATSP-7041, SAH-p53-8, PM-2, K-178, MMRi-64, and [ka] or a tautomer or solvate or a pharmaceutically acceptable salt thereof.

[0278] In one embodiment of the invention, the MDM2 antagonist is selected from the group consisting of idasanutlin, HDM-201, KRT-232 (AMG-232), ALRN-6924, CGM-097, milademethane tosylate (DS-3032b), APG-115, BI-907828, LE-004, DS-5272, SJ-0211, APG-155, RG-7112, RG7388 (idasanutlin), S AR405939, cytarabine (also known as MK-8242 and SCH-900242), BI-0252, AM-7209, SP-141, SCH-1450206, NXN-6, ADO-21, CTX-50-CTX-1, ISA-27, RO-8994, RO-6839921, ATSP-7041, SAH-p53-8, PM-2, K-178, MMRi-64, and [ka] or a tautomer or solvate or a pharmaceutically acceptable salt thereof.

[0279] In one embodiment of the invention, the MDM2 antagonist is selected from the group consisting of idasanutlin, HDM-201, KRT-232 (AMG-232), ALRN-6924, CGM-097, milademethane tosylate (DS-3032b), APG-115, BI-907828, LE-004, DS-5272, SJ-0211, BI-0252, AM-7209, SP-141, SCH-1450206, NXN-6, ADO-21, CTX-50-CTX-1, ISA-27, RO-8994, RO-6839921, ATSP-7041, SAH-p53-8, PM-2, K-178, MMRi-64, and [ka] or a tautomer or solvate or a pharmaceutically acceptable salt thereof.

[0280] In one embodiment of the invention, the MDM2 antagonist is selected from the group consisting of idasanutlin (RG-7388), HDM-201, KRT-232 (AMG-232), ALRN-6924, MI-773 (SAR405838), milademetan (DS-3032b), APG-115, BI-907828, or a tautomer or solvate or pharmaceutically acceptable salt thereof.

[0281] In one embodiment of the invention, the MDM2 antagonist is idasanutlin (RG-7388), HDM-201, KRT-232 (AMG-232), ALRN-6924, MI-773 (SAR405838), milademetan (DS-3032b), APG-115, BI-907828, or a compound represented by formula I o or a tautomer or solvate or a pharmaceutically acceptable salt thereof.

[0282] Formula I o Compounds of Particular MDM2 antagonists are the isoindoline compounds disclosed in the inventors' earlier international patent applications PCT / GB2016 / 053042 and PCT / GB2016 / 053041, filed September 29, 2016, which claim priority to UK patent applications Nos. 1517216.6 and 1517217.4, filed September 29, 2015, the contents of all of which are incorporated herein by reference in their entireties. In particular, the compound (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid ("Compound 1") is disclosed in our earlier international patent application PCT / GB2016 / 053042.

[0283] In one embodiment, the MDM2 antagonist has the formula I o Compounds of: [ka] wherein cyc is phenyl or a heterocyclic group Het, i.e. pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl, or an N-oxide thereof; R 1 is hydroxy, halogen, nitro, nitrile, C 1-4 Alkyl, HaloC 1-4 Alkyl, Hydroxy C 1-4 Alkyl, C 2-6 Alkenyl, C 1-4 Alkoxy, HaloC 1-4 Alkoxy, C 2-4 Alkynyl, -O 0,1 -(CR x R y ) v -CO2H, -(CR x R y ) v -CO2C 1-4 Alkyl, -(CR x R y ) v -CON(C 1-4 alkyl)2, -P(=O)(R x )2, -S(O) d -R x , -S(O) having 3 to 6 ring members d -heterocyclic groups, and -S(O) d -N(R 8 )2 independently selected from cyc and Het, then R 1 is bonded to a carbon atom; R 2 is hydrogen, C 1-4 Alkyl, C 2-6 Alkenyl, Hydroxy C 1-4 Alkyl, -(CR x R y ) u -CO2H, -(CR x R y ) u -CO2C 1-4 Alkyl, and -(CR x R y ) u -CONR x R y Selected from; s is selected from 0 and 1; R 3 is hydrogen or -(A) t -(CR x R y ) q -X; t is selected from 0 and 1; q is selected from 0, 1 and 2; R 3 Ga-(A) t -(CR x R y ) q -X, then (i) at least one of s, t, and q is non-zero, and (ii) if t is 0, then s is 1 and q is non-zero; A is C 3-6 a cycloalkyl group or a heterocyclic group having 3 to 6 ring members, said heterocyclic group comprising one or more (e.g., 1, 2, or 3) heteroatoms selected from N, O, S, and oxidized forms thereof; X is hydrogen, halogen, -CN, -OR 9 , -(CH2) v -CO2H, -(CH2) v -CO2C 1-4 Alkyl, -S(O) d -R x , -C(=O)-C 1-4 Alkyl, -S(O) d -N(H) e (C 1-4 alkyl) 2-e , -NR x R y , -NHSO2R x , -NR x COR y , and -C(=O)NR x R y Selected from; R 4 and R 5 is halogen, nitrile, C 1-4 Alkyl, HaloC 1-4 Alkyl, C 1-4 Alkoxy and HaloC 1-4 independently selected from alkoxy; R6 and R 7 is hydrogen, C 1-6 Alkyl, HaloC 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, Hydroxy, HydroxyC 1-6 Alkyl, -COOC 1-6 Alkyl, -(CH2) j -OC 1-6 Alkyl, -(CH2) j -O-(hydroxy C 1-6 alkyl), -C 1-6 Alkyl-NR x R y , -(CR x R y ) p -CONR x R y , -(CR x R y ) p -NR x COR y , -(CR x R y ) p -O-CH2-CONR x R y , heterocyclic groups having 3 to 7 ring members, -CH2-heterocyclic groups having 3 to 7 ring members, -CH2-O-heterocyclic groups having 3 to 7 ring members, -CH2-NH-heterocyclic groups having 3 to 7 ring members, -CH2-N(C 1-6 alkyl)-heterocyclic groups, -C(=O)NH-heterocyclic groups having 3 to 7 ring members, C 3-8 Cycloalkyl, -CH2-C 3-8 Cycloalkyl, -CH2-OC 3-8 Cycloalkyl, and C 3-8 cycloalkenyl, wherein said cycloalkyl, cycloalkenyl or heterocyclic group is independently selected from one or more R z groups, in each case the heterocyclic group comprises one or more (e.g., 1, 2, or 3) heteroatoms selected from N, O, S, and oxidized forms thereof; Or R 6 and R 7The groups, together with the carbon atoms to which they are attached, are C 3-6 A cycloalkyl or heterocyclyl group can be formed, said heterocyclic group comprising one or more (e.g., 1, 2, or 3) heteroatoms selected from N, O, S, and oxidized forms thereof, and said C 3-6 Cycloalkyl and heterocyclyl groups may optionally include one or more R z may be substituted with a group; R 8 and R 9 is hydrogen, C 1-6 Alkyl, HaloC 1-6 Alkyl, Hydroxy C 1-6 Alkyl, -(CH2) k -OC 1-6 Alkyl, -(CH2) k -O-(hydroxy C 1-6 alkyl), hydroxy C 1-6 Alkoxy, -(CH2) k -CO2C 1-6 Alkyl, -(CH2) k -CO2H, -C 1-6 Alkyl-N(H) e (C 1-4 alkyl) 2-e , -(CH2) j -C 3-8 Cycloalkyl and -(CH2) j -C 3-8 cycloalkenyl; R x and R y is hydrogen, halogen, nitro, nitrile, C 1-6 Alkyl, HaloC 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, Hydroxy, HydroxyC 1-6 Alkyl, C 1-6 Alkoxy, -(CH2) k -OC 1-6 Alkyl, Hydroxy C 1-6 Alkoxy, -COOC 1-6 Alkyl, -N(H) e (C 1-4 alkyl) 2-e, -C 1-6 Alkyl-N(H) e (C 1-4 alkyl) 2-e , -(CH2) k -C(=O)N(H) e (C 1-4 alkyl) 2-e , C 3-8 Cycloalkyl and C 3-8 independently selected from cycloalkenyl; Or R x and R y The groups, together with the carbon or nitrogen atom to which they are attached, form a C 3-6 It can form a cycloalkyl or saturated heterocyclyl group having 3 to 6 ring members, which may optionally be fused with an aromatic heterocyclyl group having 3 to 5 ring members; Or on a carbon atom, R x Group and R y The groups can be linked together to form a =CH2 group; R z is halogen, nitro, nitrile, C 1-6 Alkyl, HaloC 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, =O, Hydroxy, HydroxyC 1-6 Alkyl, C 1-6 Alkoxy, -(CH2) k -OC 1-6 Alkyl, Hydroxy C 1-6 Alkoxy, -C(=O)C 1-6 Alkyl, -C(=O)C 1-6 Alkyl-OH, -C(=O)C 1-6 Alkyl-N(H) e (C 1-4 alkyl) 2-e , -C(=O)N(H) e (C 1-4 alkyl) 2-e , -(CH2) r -CO2C 1-6 Alkyl, -(CH2) r -COH, -N(H) e (C 1-4 alkyl)2-e , -C 1-6 Alkyl-N(H) e (C 1-4 alkyl) 2-e , heterocyclyl groups having 3 to 6 ring members, -C(=O)C 1-4 Alkyl-substituted heterocyclyl groups having 3 to 6 ring members, -C(=O)OC 1-4 Alkyl-substituted heterocyclyl groups having 3 to 6 ring members, -C(=O)N(H) e (C 1-4 alkyl) 2-e heterocyclyl groups having 3 to 6 ring members substituted with -C(=O)heterocyclyl groups having 3 to 6 ring members, C 3-8 Cycloalkyl and C 3-8 cycloalkenyl; R 7 is pyridine, R z is other than -NH2; a, j, d, e, n, r and p are independently selected from 0, 1 and 2; k and m are independently selected from 1 and 2; u is selected from 0, 1, 2 and 3; and v is independently selected from 0 and 1. or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0284] Formula (I o ) compounds are referred to as * " has a chiral center indicated by ". [ka]

[0285] Formula (I o The compound of formula (I) contains a stereocenter at the indicated position (referred to herein as (3)) and is chiral and non-racemic. o ) has the stereochemistry shown by the dashed wedge bond and the solid wedge bond, and this stereoisomer predominates.

[0286] Generally, the compound of formula (Io In one general embodiment, at least 55% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compounds of formula (I) are present as the depicted stereoisomer. o ) may be present as a single stereoisomer, with 97% (eg, 99%) or more (eg, substantially all) of the total amount of compounds present.

[0287] These compounds may also contain one or more additional chiral centers (e.g., —CR 6 R 7 OH groups and / or R 3 group and / or -CHR 2 (in the case of bases).

[0288] Generally, the compound of formula (I o ) has an enantiomeric excess of at least 10% (e.g., at least 20%, 40%, 60%, 80%, 85%, 90%, or 95%). In one general embodiment, compounds of formula (I o ) has an enantiomeric excess of 97% (e.g., 99%) or greater.

[0289] For the purposes of this section, the isoindolin-1-one ring is numbered as follows: [ka]

[0290] Compounds are named according to protocols used by chemical naming software packages.

[0291] Formula (I) wherein cyc is phenyl o ) compounds Formula (I) wherein cyc is phenyl o) are disclosed in our earlier international patent application PCT / GB2016 / 053042, published as WO2017 / 055860 on April 6, 2017. The compounds, subformulas, and substituents disclosed in WO2017 / 055860 (e.g., Formulas (I), I(e), I(f), I(g), I(g'), I(h), I(i), I(j), I(k), I(L), I(m), I(m'), I(n), I(o), I(o'), I(o''), I(p), I(p'), I(q), I(q'), I(q''), I(q'''), I(q''''), I(r), I(s), I(t), I(t), I(t) A cross-reference is made to I(u), I(v), I(v'), I(w), I(x), I(x'), I(y), (II), (IIa), (IIb), (IIIa), (IIIb), (IVa), (IVb), (V), (VI), (Via), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIId'), (VIIe), (VIIe'), (a), (b), (ba), (bb), (bc) or (c)). Thus, by virtue of this cross-reference, the compounds, subformulas, and substituents of WO2017 / 055860 are directly and unambiguously disclosed by this application.

[0292] Formula (I) wherein cyc is phenyl o Specific subformulas, embodiments and compounds of include:

[0293] In one embodiment, R 1 is chloro or nitrile, especially chloro.

[0294] R 2 is other than hydrogen, the formula (I o ) can exist as at least two diastereoisomers. [ka]

[0295] For the avoidance of doubt, the general formula (I o ) and all subformulas are intended to represent the individual diastereoisomers and -CHR 2In one embodiment, the compound of formula (I) includes both a mixture of diastereoisomers related epimerically at the - group. o In one embodiment, the compound of formula (I) is diastereoisomer 1A or a tautomer or solvate or pharmaceutically acceptable salt thereof. o ) is diastereoisomer 1B or a tautomer or solvate or a pharmaceutically acceptable salt thereof.

[0296] In one embodiment, R 2 is hydrogen and -(CR x R y ) u -CO2H (e.g., selected from -COOH, -CH2COOH, -CH2CH2-CO2H, -(CH(CH3))-CO2H and -(C(CH3)2)-CO2H).

[0297] In one embodiment, a is 1 and the substituent R 4 is at the 4-position of isoindolin-1-one, and has the formula (I o ) is a compound of formula (Ir) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka] R 4 is halogen, nitrile, C 1-4 Alkyl, HaloC 1-4 Alkyl, C 1-4 Alkoxy and HaloC 1-4 alkoxy.

[0298] In one embodiment, R 4 is halogen. In one embodiment, R 4 is fluoro or chloro. In another embodiment, R 4 is fluoro.

[0299] In one embodiment, a is 1 and the substituent R 4 is at the 4-position of isoindolin-1-one, and R 4 is F, and the formula (Io ) is a compound of formula (Is) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0300] R 6 and R 7 are different, the formula (I o ) can exist as at least two diastereoisomers. [ka]

[0301] For the avoidance of doubt, the general formula (I o ) and all subformulas are the individual diastereoisomers and -CR 6 R 7 It includes both mixtures of diastereoisomers related as epimers at the OH group.

[0302] In one embodiment, R 6 is C 1-6 alkyl (methyl or ethyl, e.g., methyl), and R 7 is oxanyl, and the formula (I o ) is a compound of formula (Iw). [ka]

[0303] In one embodiment of Formula (Iw), R z is hydrogen or fluorine.

[0304] subexpression In one embodiment, R 6 is methyl or ethyl, and o ) is a compound of formula (IIIb) or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 7 , a, m and s are as defined herein.

[0305] In one embodiment, s is 0 and the compound of formula (I o ) is a compound of formula (IVb) or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 7 , a, m and s are as defined herein.

[0306] In one embodiment, m is 1 and the substituent R 4 is at the 4-position of the phenyl group, and o ) is a compound of formula (VI) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0307] In one embodiment, R 5 is chloro and the compound of formula (VI) is a compound of formula (VIa) or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0308] In one embodiment, R 3is methyl and the compound of formula (VI) is a compound of formula (VIIf) or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0309] In one embodiment of Formula (VIIf), R 6 is ethyl.

[0310] In one embodiment of the compound of Formula (VIIf), R 7 is selected from methyl, oxanyl, pyrazolyl, imidazolyl, piperidinyl, and cyclohexyl, and said cycloalkyl and heterocyclic groups are each selected from one or more R z It may be optionally substituted with groups such as methyl, fluorine, or hydroxy.

[0311] In one embodiment of the compound of Formula (VIIf), R 7 is selected from oxanyl and methyl.

[0312] In one embodiment of the compound of Formula (VIIf), R 7 is an R greater than or equal to 1. z piperidinyl optionally substituted with a group such as methyl, fluorine, or hydroxy.

[0313] In another embodiment of the above subformula, R 2 is selected from —(CH(CH3))—CO2H and —(C(CH3)2—CO2H).

[0314] In one embodiment, the MDM2 antagonist has the formula (I o or a tautomer or solvate or a pharmaceutically acceptable salt thereof, wherein R 1 is a halogen (e.g. Cl), nitrile, O 0,1 (CR x R y ) vCOOH (e.g., -COOH, -CHCOOH, -OCHCOOH, or -C(CH)COOH); n is 1 or 2; R 2 is hydrogen and -(CR x R y ) u -COH (e.g., selected from -COOH, -CHCOOH, -CHCH-COH, -(CH(CH))-COH, and -(C(CH))-COH); R 3 is hydrogen and s is 1; R 4 is a halogen (e.g., F); R 5 is a halogen (e.g., Cl); m is 1; R 6 is hydrogen or C 1-6 alkyl (e.g., -CH3 or -CH2CH3); R 7 is C 1-4 Alkyl (e.g., methyl), hydroxyl C 1-4 Alkyl (e.g., hydroxymethyl), methoxy C 1-4 alkyl (e.g., methoxymethyl), heterocyclic groups having 5 or 6 ring members (e.g., piperidinyl, oxanyl, imidazolyl, or pyrazolyl); The heterocyclic group having 5 or 6 ring members is C 1-4 one or two R independently selected from alkyl (e.g., methyl) z It may be optionally substituted with a group.

[0315] In one embodiment, the MDM2 antagonist is one of Examples 1-137 or a compound of formula (I) selected from Examples 1-137. o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof as described in the first set of examples defined herein, i.e., a compound wherein cyc is phenyl, as also described in WO2017 / 055860.

[0316] In one embodiment, the MDM2 antagonist is one of Examples 1-97 (Examples where cyc is phenyl) or a compound of formula (I) selected from Examples 1-97 (Examples where cyc is phenyl). o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof as described in the first set of examples defined herein, i.e., a compound wherein cyc is phenyl, as also described in WO2017 / 055860.

[0317] In one embodiment, the MDM2 antagonist is the following compound: 4-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-1-{[1-(hydroxymethyl)cyclopropyl]methoxy}-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}benzonitrile, for example [ka] ; and (3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-hydroxy-1-(oxan-4-yl)ethyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]propanoic acid, for example [ka] Formula (I) o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0318] In one embodiment, the MDM2 antagonist is the following compound: 4-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-1-{[1-(hydroxymethyl)cyclopropyl]methoxy}-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}benzonitrile; and (3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-hydroxy-1-(oxan-4-yl)ethyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]propanoic acid Formula (I) o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0319] In one embodiment, the MDM2 antagonist is diastereoisomer 2B, the following compound: 4-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-1-{[1-(hydroxymethyl)cyclopropyl]methoxy}-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}benzonitrile; and (3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-hydroxy-1-(oxan-4-yl)ethyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]propanoic acid Formula (I) o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0320] In one embodiment, the compound of formula (I o) is 2-(5-chloro-2-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}phenyl)-2-methylpropanoic acid, or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof. For example, [ka]

[0321] In one embodiment, the MDM2 antagonist is (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid ("Compound 1"), Formula (I o or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof. For example, [ka]

[0322] For the avoidance of doubt, it is to be understood that each general and specific embodiment and example of one substituent may be combined with one or more, and in particular all, general and specific embodiments and examples of other substituents as defined herein, and all such embodiments are encompassed by this application.

[0323] Formula (I) wherein cyc is a heterocyclic group o ) compounds Formula (I) wherein cyc is a heterocyclic group o) is disclosed in our earlier international patent application PCT / GB2016 / 053041, published as WO2017 / 055859 on April 6, 2017. The compounds, subformulas, and substituents disclosed in WO2017 / 055859 (e.g., Formulas (I), I(a), I(a'), I(b), I(c), I(d), I(e), I(f), I(g), I(g'), I(h), I(i), I(j), I(k), I(L), I(m), I(m'), I(n), I(o), I(o'), I(o''), I(p), I(p'), I(q), I(q'), I(q''), I(q'''), I(q''''), I(r), I(s), I(t) , I(u), I(v), I(v'), I(w), I(x), I(x'), I(y), (II), (IIa), (IIb), (IIIa), (IIIb), (IVa), (IVb), (V), (VI), (VIa), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIId'), (VIIe), (VIIe'), (a), (b), (ba), (bb), (bc), or (c)) and examples thereof as defined herein. Thus, by virtue of this cross-reference, the compounds, subformulas, and substituents of WO2017 / 055859 are directly and unambiguously disclosed by this application.

[0324] Formula (I) wherein cyc is a heterocyclic group o Specific subformulas, embodiments and compounds of include:

[0325] In another embodiment, R 2 is hydrogen, and o ) is a compound of formula (Ie) or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka] R 2 is other than hydrogen, the formula (I o ) can exist as at least two diastereoisomers. [ka]

[0326] For the avoidance of doubt, the general formula (I o ) and all subformulas are intended to represent the individual diastereoisomers and -CHR 2 -group. In one embodiment, the compound of formula I is diastereoisomer 1A, or a tautomer, solvate, or pharmaceutically acceptable salt thereof. In one embodiment, the compound of formula I is diastereoisomer 1B, or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0327] In one embodiment, A is C 3-6 is a cycloalkyl group (i.e., g is 1, 2, or 3), t is 1, and s is 0 or 1; o ) is a compound of formula (If), or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0328] In one embodiment, A is C 3-6 is a cycloalkyl group (i.e., g is 1, 2, or 3), t is 1, s is 1, and is a group represented by the formula (I o ) is a compound of formula (Ig) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0329] In one embodiment, A is C 3-6 is a cycloalkyl group (i.e., g is 1, 2, or 3), t is 1, s is 1, and the cycloalkyl group is geminally disubstituted (i.e., -(CR x R y ) qthe -X group and the -CH2-O-isoindolinone group are both attached to the same atom of the cycloalkyl group), o ) is a compound of formula (Ih) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0330] In one embodiment, A is a cyclopropyl group (i.e., g is 1), t is 1, and s is 1. Thus, the cycloalkyl group is a cyclopropyl group and has the formula (I o ), is a compound of formula (Ii) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0331] In one embodiment, A is C 3-6 is a cycloalkyl group (i.e., g is 1, 2, or 3), t is 1, s is 1, X is —CN, and the formula (I o ) is a compound of formula (Ik') or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0332] In another embodiment, A is C 3-6 is a cycloalkyl group (i.e., g is 1, 2, or 3), t is 1, s is 1, and R x and R y is hydrogen ( 1 H and 2 H), and o ) is a compound of formula (IL) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0333] In one embodiment, A is a C3-cycloalkyl group (i.e., g is 1), t is 1, s is 1, X is -CN, and the compound of formula (I o ) is a compound of formula (In') or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka] wherein q is 0 or 1. In one embodiment of compound (In), q is 0.

[0334] In one embodiment, R 3 Ha-(CR x R y ) q -X, s is 1, t is 0, q is 1 or 2, o ) is a compound of formula (Ip). [ka]

[0335] In one embodiment, A is C 3-6 a cycloalkyl group or a saturated heterocyclic group having 3 to 6 ring members, wherein t is 1, s is 1, Y is independently selected from —CH—, O, or SO, i is 0 or 1, g is 1, 2, 3, or 4, and i+g is 1, 2, 3, or 4, and is a group of formula (I o ) is a compound of formula (Iq) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0336] In one embodiment, i is 1 and Y is O or SO, particularly O. In one embodiment, the compound of formula (Iq) is a compound of formula (Iq''") or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0337] In one embodiment, s is 0, t is 1, A is tetrahydofuranyl, q is 0, and X is hydrogen. 3 is tetrahydrofuranyl and s is 0.

[0338] In one embodiment, a is 1 and the substituent R 4 is at the 4-position of isoindolin-1-one, and has the formula (I o ) is a compound of formula (Ir) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0339] R 4 is halogen, nitrile, C 1-4 Alkyl, HaloC 1-4 Alkyl, C 1-4 Alkoxy and HaloC 1-4 alkoxy.

[0340] In one embodiment, R 4 is halogen. In one embodiment, R 4 is fluoro or chloro. In another embodiment, R 4 is fluoro.

[0341] In one embodiment, a is 1 and the substituent R 4 is at the 4-position of isoindolin-1-one, and R 4 is F, and the formula (I o ) is a compound of formula (Is) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0342] R 6 and R 7 are different, the formula (I o ) can exist as at least two diastereoisomers. [ka]

[0343] For the avoidance of doubt, the general formula (I o ) and all subformulas are the individual diastereoisomers and -CR 6 R 7 It includes both mixtures of diastereoisomers related as epimers at the OH group.

[0344] In one embodiment, R 7 is 4-fluoro-1-methylpiperidin-4-yl, and has the formula (I o ) is a compound of formula (Ix″) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka]

[0345] subexpression In one embodiment, the compound of formula (I o ) is a compound of formula (II) or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka] where L is CR 1 , CH or N, and R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7, a, m, and s are as defined herein. In one embodiment, L is CH. In one embodiment, L is N. In one embodiment, L is C—OH or C-hydroxyC 1-4 CR such as alkyl (e.g., C-OH or C-CHOH) 1 is.

[0346] In another embodiment, R 1 is chloro or nitrile, and the compound of formula (II) is a compound of formula (IIa) or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka]

[0347] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 7 , m and s are as defined herein.

[0348] In one embodiment, R 6 is ethyl, and the compound of formula (II) is a compound of formula (IIIb) or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 7 , a, m and s are as defined herein.

[0349] In one embodiment, s is 0 and the compound of Formula (II) is a compound of Formula (IVb) or a tautomer or solvate or pharmaceutically acceptable salt thereof: [ka] In the formula, R1 , R 2 , R 3 , R 4 , R 5 , R 7 , m and s are as defined herein.

[0350] In one embodiment, R 4 is F, and the formula (I o ) is a compound of formula (V) or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka] In the formula, R 1 , R 2 , R 3 , R 5 , R 7 , m and s are as defined herein.

[0351] In one embodiment, m is 1 and the substituent R 4 is at the 4-position of the phenyl group, and the compound of formula (II) is a compound of formula (VI) or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0352] In one embodiment, R 5 is chloro and the compound of formula (VI) is a compound of formula (VIa) or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0353] In one embodiment, A is C 3-6 is a cycloalkyl group (g is 1, 2 or 3), t is 1, and the compound of formula (VI) is a compound of formula (VII) or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0354] In one embodiment, A is C 3-6 is a cycloalkyl group (g is 1, 2, or 3), t is 1, and the cycloalkyl group is geminally disubstituted (i.e., -(CR x R y )—X group and CH group (when s is 1) or oxygen atom (when s is 0) are both attached to the same atom of the cycloalkyl group, and the compound of formula (VII) is a compound of formula (VIIa) or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0355] In one embodiment, g is 1, and therefore the cycloalkyl group is a cyclopropyl group, and the compound of formula (VIIa) is a compound of formula (VIIb) or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0356] In one embodiment, s is 1 and the compound of formula (VIIb) is a compound of formula (VIIc) or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0357] In one embodiment, X is —CN and the compound of formula (VlId) is a compound of formula (VIle″) or a tautomer or solvate or a pharmaceutically acceptable salt thereof: [ka] wherein q is 0 or 1, in particular q is 0.

[0358] In one embodiment, R 3 is methyl and the compound of formula (VI) is a compound of formula (VIIf) or a tautomer or solvate or a pharmaceutically acceptable salt thereof. [ka]

[0359] In one embodiment of the compound of formula (a), R 7 is C 1-6 Piperidinyl or piperazinyl optionally substituted with alkyl (eg methyl) and / or halo (eg fluoro).

[0360] In one embodiment of the compound of formula (a'), R 7 is C 1-6 and piperidinyl optionally substituted with alkyl (eg, methyl) and / or halo (eg, fluoro).

[0361] In one embodiment, A is a heterocyclyl group having 3 to 6 ring members, which heterocyclic group comprises one or more (e.g., 1, 2, or 3) heteroatoms selected from N, O, S, and oxidized forms thereof (t is 1; g is 1, 2, 3, or 4; Z represents N, O, S, and oxidized forms thereof; i is 1, 2, or 3; i+g=2, 3, 4, or 5), and the compound of formula (VI) is a compound of formula (b) or a tautomer or solvate or pharmaceutically acceptable salt thereof. [ka]

[0362] In one embodiment, s is 0, g is 2, q is 0, X is hydrogen, and the compound of formula (b) is a compound of formula (bb) or a tautomer or solvate or pharmaceutically acceptable salt thereof. [ka]

[0363] In another embodiment, a compound of formula (I o ) is a compound of formula (c) or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka] In the formula, R 1 is chloro or nitrile, s is 1 and X is hydroxyl, or s is 0 and X is -C(=O)NH2.

[0364] In another embodiment, a compound of formula (I o ) is a compound of formula (c') or a tautomer or solvate or a pharmaceutically acceptable salt thereof, [ka] In the formula, R 1 is chloro or nitrile, s is 1 and X is hydroxyl, or s is 0 and X is —CN.

[0365] In one embodiment, the MDM2 antagonist has the formula (I o or a tautomer or solvate or a pharmaceutically acceptable salt thereof, wherein Het is pyridinyl or pyrimidinyl, R 1 is bonded to a carbon atom and is hydroxy, halogen, nitro, nitrile and C 1-4 independently selected from alkyl; R 2 is hydrogen, C 1-4 Alkyl, C 2-6 Alkenyl, Hydroxy C 1-4 selected from alkyl and -CH2CO2H; R 3 is hydrogen or -(A) t -(CR x R y ) q -X; s and t are independently selected from 0 and 1; q is selected from 0, 1 and 2; R 3 Ga-(A) t -(CR x R y ) q -X, then (i) at least one of s, t, and q is non-zero, and (ii) if t is 0, then s is 1 and q is non-zero; A is a heterocyclic group having 3 to 6 ring members, said heterocyclic group comprising one or more (e.g., 1, 2, or 3) heteroatoms selected from N, O, S, and oxidized forms thereof; X is hydrogen, halogen, -CN and -OR 9 Selected from; R 4 and R 5 is halogen, nitrile and C 1-4 independently selected from alkyl; R 6 is hydrogen and C 1-6 alkyl; R 7 represents a heterocyclic group having 3 to 7 ring members, a —CH2-heterocyclic group having 3 to 7 ring members, C 3-8 Cycloalkyl, and -CH2-C 3-8 cycloalkyl, wherein said cycloalkyl or heterocyclic group is selected from one or more R z groups, in each case the heterocyclic group comprises one or more (e.g., 1, 2, or 3) heteroatoms selected from N, O, S, and oxidized forms thereof; R 9 is hydrogen and C 1-6 alkyl; R x and R y is hydrogen and C 1-6 independently selected from alkyl; R z is halogen, nitro, nitrile, C 1-6 Alkyl, HaloC 1-6 Alkyl, C 2-6 Alkenyl, Hydroxy, HydroxyC1-6 Alkyl, C 1-6 Alkoxy, -C(=O)C 1-6 Alkyl, and -N(H) e (C 1-4 alkyl) 2-e are independently selected from; n and e are independently selected from 0, 1 and 2; m is selected from 1 and 2; and a is selected from 0 and 1.

[0366] In one embodiment, the MDM2 antagonist has the formula (I o or a tautomer or solvate or a pharmaceutically acceptable salt thereof, wherein Het is pyridinyl or pyrimidinyl, R 1 are attached to a carbon atom and are independently selected from halogen, hydroxy, and nitrile; R 2 is hydrogen, C 1-4 selected from alkyl and -CH2CO2H; R 3 is hydrogen or -(A) t -(CR x R y ) q -X; A is a heterocyclic group having 3 to 6 ring members, said heterocyclic group comprising one or more (e.g., 1, 2, or 3) heteroatoms selected from N, O, S, and oxidized forms thereof; s and t are independently selected from 0 and 1; q is selected from 0, 1 and 2; R 3 Ga-(A) t -(CR x R y ) q -X, then (i) at least one of s, t, and q is non-zero, and (ii) if t is 0, then s is 1 and q is non-zero; X is hydrogen, halogen or -OR 9 Selected from; R4 and R 5 are independently selected from halogens; R 6 is hydrogen and C 1-6 alkyl; R 7 represents a heterocyclic group having 3 to 7 ring members, a —CH2-heterocyclic group having 3 to 7 ring members, C 3-8 Cycloalkyl, and -CH2-C 3-8 cycloalkyl, wherein said cycloalkyl, cycloalkenyl or heterocyclic group is selected from one or more R z groups, in each case the heterocyclic group comprises one or more (e.g., 1, 2, or 3) heteroatoms selected from N, O, S, and oxidized forms thereof; R 9 is hydrogen and C 1-6 alkyl; R x and R y is hydrogen and C 1-6 independently selected from alkyl; R z is halogen, nitro, nitrile, and C 1-6 independently selected from alkyl; n is 1 and m is 1; and a is selected from 0 and 1.

[0367] In one embodiment, the MDM2 antagonist has the formula (I o or a tautomer or solvate or a pharmaceutically acceptable salt thereof, wherein Het is pyridinyl or pyrimidinyl, R 1 are attached to a carbon atom and are independently selected from halogen, hydroxy, and nitrile; R 2 is hydrogen, C 1-4 selected from alkyl and -CH2CO2H; R 3 is -(A) t -(CR x R y )q -X; A is a heterocyclic group having 3 to 6 ring members, said heterocyclic group comprising one or more (e.g., 1, 2, or 3) heteroatoms selected from N, O, S, and oxidized forms thereof; s and t are independently selected from 0 and 1; q is selected from 0, 1 and 2; (i) at least one of s, t, and q is non-zero, and (ii) if t is 0, then s is 1 and q is non-zero; X is hydrogen, halogen and -OR 9 Selected from; R 4 and R 5 are independently selected from halogens; R 6 is hydrogen and C 1-6 alkyl; R 7 is one or more R z a heterocyclic group having 3 to 7 ring members optionally substituted with a group; R 9 is hydrogen and C 1-6 alkyl; R x and R y is hydrogen and C 1-6 independently selected from alkyl; R z is halogen and C 1-6 independently selected from alkyl; n is 1, m is 1, and a is 1.

[0368] In one embodiment, the MDM2 antagonist is one of Examples 1-580 (Examples in which cyc is a heterocyclic group) or a compound of Formula (I) selected from Examples 1-580. o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof (as defined herein in the second set of examples) oi.e., compounds in which cyc is Het, as also described in WO2017 / 055859.

[0369] In one embodiment, the MDM2 antagonist is one of Examples 1-460 or a compound of Formula (I) selected from Examples 1-460. o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof (as defined herein in the second set of examples) o i.e., compounds in which cyc is Het, as also described in WO2017 / 055859.

[0370] In one embodiment, the MDM2 antagonist is one of Examples 1-459 or a compound of Formula (I) selected from Examples 1-459. o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof (as defined herein in the second set of examples) o i.e., compounds in which cyc is Het, as also described in WO2017 / 055859.

[0371] In one embodiment, the MDM2 antagonist is a compound of formula (I) selected from the following compounds: o or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof: (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-{1-hydroxy-1-[trans-4-hydroxycyclohexyl]ethyl}-3-{[1-(hydroxymethyl)cyclopropyl]methoxy}-2,3-dihydro-1H-isoindol-1-one; for example, [ka] 2-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-oxo-1-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-2-yl]methyl}pyrimidine-5-carbonitrile; for example, [ka] (3R)-2-[(5-chloro-3-hydroxypyridin-2-yl)methyl]-3-(4-chlorophenyl)-4-fluoro-6-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-(2-hydroxyethoxy)-2,3-dihydro-1H-isoindol-1-one; for example, [ka] 6-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-(4-fluorooxan-4-yl)-1-hydroxypropyl]-3-oxo-1-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carbonitrile; for example, [ka] 6-{[(1R)-1-(4-chlorophenyl)-7-fluoro-1-[(3-fluorooxetan-3-yl)methoxy]-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carbonitrile; for example, [ka] 6-{[(1R)-1-(4-chlorophenyl)-7-fluoro-1-({1-[hydroxy( 2 H2) methyl]cyclopropyl}( 2H2) methoxy)-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carbonitrile; for example, [ka] and (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[1-hydroxy-1-(1-methylpiperidin-4-yl)propyl]-3-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-1-one for example, [ka]

[0372] In one embodiment, the MDM2 antagonist is diastereoisomer 2A, selected from the following compounds of formula (I) o or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof: (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-{1-hydroxy-1-[trans-4-hydroxycyclohexyl]ethyl}-3-{[1-(hydroxymethyl)cyclopropyl]methoxy}-2,3-dihydro-1H-isoindol-1-one; 2-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-oxo-1-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-2-yl]methyl}pyrimidine-5-carbonitrile; (3R)-2-[(5-chloro-3-hydroxypyridin-2-yl)methyl]-3-(4-chlorophenyl)-4-fluoro-6-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-(2-hydroxyethoxy)-2,3-dihydro-1H-isoindol-1-one; 6-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-(4-fluorooxan-4-yl)-1-hydroxypropyl]-3-oxo-1-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carbonitrile; 6-{[(1R)-1-(4-chlorophenyl)-7-fluoro-1-[(3-fluorooxetan-3-yl)methoxy]-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carbonitrile; 6-{[(1R)-1-(4-chlorophenyl)-7-fluoro-1-({1-[hydroxy( 2 H2) methyl]cyclopropyl}( 2 H2) methoxy)-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carbonitrile; and (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[1-hydroxy-1-(1-methylpiperidin-4-yl)propyl]-3-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-1-one.

[0373] In one embodiment, the MDM2 antagonist is diastereoisomer 2B and is selected from the following compounds of formula (I) o or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof: (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-{1-hydroxy-1-[trans-4-hydroxycyclohexyl]ethyl}-3-{[1-(hydroxymethyl)cyclopropyl]methoxy}-2,3-dihydro-1H-isoindol-1-one; 2-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-oxo-1-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-2-yl]methyl}pyrimidine-5-carbonitrile; (3R)-2-[(5-chloro-3-hydroxypyridin-2-yl)methyl]-3-(4-chlorophenyl)-4-fluoro-6-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-(2-hydroxyethoxy)-2,3-dihydro-1H-isoindol-1-one; 6-{[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[1-(4-fluorooxan-4-yl)-1-hydroxypropyl]-3-oxo-1-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carbonitrile; 6-{[(1R)-1-(4-chlorophenyl)-7-fluoro-1-[(3-fluorooxetan-3-yl)methoxy]-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carbonitrile; 6-{[(1R)-1-(4-chlorophenyl)-7-fluoro-1-({1-[hydroxy( 2 H2) methyl]cyclopropyl}( 2 H2) methoxy)-5-[1-hydroxy-1-(1-methyl-1H-imidazol-4-yl)propyl]-3-oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carbonitrile; and (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[1-hydroxy-1-(1-methylpiperidin-4-yl)propyl]-3-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-1-one.

[0374] In one embodiment, the MDM2 antagonist is a compound of formula (I) selected from the following compounds: o or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof: (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[2-hydroxy-1-(4-methylpiperazin-1-yl)butan-2-yl]-3-[(3S)-oxolan-3-yloxy]-2,3-dihydro-1H-isoindol-1-one; for example, [ka] (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-methoxy-2,3-dihydro-1H-isoindol-1-one; for example, [ka] 1-({[(1R)-1-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-7-fluoro-5-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-oxo-2,3-dihydro-1H-isoindol-1-yl]oxy}methyl)cyclopropane-1-carbonitrile; for example, [ka] (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-[cis-3-hydroxycyclobutoxy]-2,3-dihydro-1H-isoindol-1-one; for example, [ka] and (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-[(2R)-2-hydroxypropoxy]-2,3-dihydro-1H-isoindol-1-one for example, [ka]

[0375] In one embodiment, the MDM2 antagonist is a compound represented by formula (I), which is 1-({[(1R)-1-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-7-fluoro-5-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-oxo-2,3-dihydro-1H-isoindol-1-yl]oxy}methyl)cyclopropane-1-carbonitrile. o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0376] In one embodiment, the MDM2 antagonist is a compound of formula (I), which is (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-methoxy-2,3-dihydro-1H-isoindol-1-one. o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0377] In one embodiment, the MDM2 antagonist is diastereoisomer 2A, which is 1-({[(1R)-1-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-7-fluoro-5-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-oxo-2,3-dihydro-1H-isoindol-1-yl]oxy}methyl)cyclopropane-1-carbonitrile, Formula (I o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0378] In one embodiment, the MDM2 antagonist is diastereoisomer 2A, which is (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-methoxy-2,3-dihydro-1H-isoindol-1-one, Formula (I o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0379] In one embodiment, the MDM2 antagonist is diastereoisomer 2B, which is 1-({[(1R)-1-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-7-fluoro-5-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-oxo-2,3-dihydro-1H-isoindol-1-yl]oxy}methyl)cyclopropane-1-carbonitrile, Formula (I o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0380] In one embodiment, the MDM2 antagonist is diastereoisomer 2B, (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-methoxy-2,3-dihydro-1H-isoindol-1-one, Formula (I o ) or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0381] In one embodiment, the MDM2 antagonist is (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[(1S)-1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-methoxy-2,3-dihydro-1H-isoindol-1-one, or a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof.

[0382] In one embodiment, the MDM2 antagonist is (3R)-3-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-4-fluoro-6-[(1R)-1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-methoxy-2,3-dihydro-1H-isoindol-1-one, or a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof.

[0383] In one embodiment, the MDM2 antagonist is 1-({[(1R)-1-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-7-fluoro-5-[(1S)-1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-oxo-2,3-dihydro-1H-isoindol-1-yl]oxy}methyl)cyclopropane-1-carbonitrile, or a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof.

[0384] In one embodiment, the MDM2 antagonist is 1-({[(1R)-1-(4-chlorophenyl)-2-[(5-chloropyrimidin-2-yl)methyl]-7-fluoro-5-[(1R)-1-(4-fluoro-1-methylpiperidin-4-yl)-1-hydroxypropyl]-3-oxo-2,3-dihydro-1H-isoindol-1-yl]oxy}methyl)cyclopropane-1-carbonitrile, or a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof.

[0385] For the avoidance of doubt, it is to be understood that each general and specific embodiment and example of one substituent may be combined with one or more, and in particular all, general and specific embodiments and examples of other substituents as defined herein, and all such embodiments are encompassed by this application.

[0386] Specific compounds The uses and methods of the present invention comprise a compound of Formula I as described herein. o i.e., MDM2 antagonists are compounds of formula I o or any specific compound described herein, or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof.

[0387] In one embodiment, the MDM2 antagonist is a compound of Formula I selected from Examples 1-134 as set forth in the first set of examples defined herein. o (i.e., a compound in which cyc is phenyl, as also described in WO2017 / 055860).

[0388] In one embodiment, the MDM2 antagonist is a compound of Formula I selected from Examples 1-580 as set forth in the second set of examples defined herein. o (i.e., compounds where cyc is Het, as also described in WO2017 / 055859).

[0389] In one particular embodiment of the present invention, the MDM2 antagonist is of formula (I) as defined herein. o ) or a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof, i.e., (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid.

[0390] (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid is referred to herein as "Compound 1." for example, [ka]

[0391] (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid is disclosed as Example 124 in International Patent Application PCT / GB2016 / 053042, published as WO2017 / 055860 on April 6, 2017.

[0392] A method for preparing Compound 1 can be found in International Patent Application PCT / GB2018 / 050845, published as WO2018 / 178691 on October 4, 2018.

[0393] In one embodiment, the MDM2 antagonist is the free acid form of Compound 1. In another embodiment, the MDM2 antagonist is a pharmaceutically acceptable salt of Compound 1.

[0394] Overview Other MDM2 antagonists can be prepared in a conventional manner, for example by processes similar to those described.

[0395] The posology of MDM2 antagonists is known to those skilled in the art. It will be recognized that the preferred administration method and dosage and administration regimen of each MDM2 antagonist will depend on the specific tumor to be treated and the specific host to be treated. The optimal method, administration schedule, dosage and regimen can be easily determined by those skilled in the art using routine methods and taking into account the information provided herein.

[0396] Salts, solvates, tautomers, isomers, N-oxides, esters, prodrugs and isotopes Reference herein to any compound also includes ionic forms, salts, solvates, isomers (including geometric and stereochemical isomers unless specified), tautomers, N-oxides, esters, prodrugs, isotopes, and protected forms thereof, e.g., as described below; particularly, salts or tautomers or isomers or N-oxides or solvates thereof; more particularly, salts or tautomers or N-oxides or solvates thereof. In one embodiment, a reference to a compound also includes salts or tautomers or solvates thereof.

[0397] salt These compounds may exist as salts, such as acid addition salts, or in certain cases, salts of organic and inorganic bases, such as carboxylates, sulfonates, and phosphates. All such salts are within the scope of the present invention and are within the scope of the formula (I). o ) includes salt forms of that compound.

[0398] N-oxide Compounds containing an amine function may form N-oxides, and references herein to compounds containing an amine function also include N-oxides.

[0399] Geometric Isomers and Tautomers These compounds may exist in several different geometric isomeric and tautomeric forms and are represented by the formula (I o ) includes all such forms. For the avoidance of doubt, compounds may exist in one of several geometric isomeric or tautomeric forms and although only one may be specifically described or shown, all others are also encompassed for use in the present invention.

[0400] For example, certain heteroaryl rings may exist in two tautomeric forms, such as A and B shown below. For simplicity, the formula may only show one form, but the formula should be considered to encompass both tautomeric forms.

[0401] stereoisomer Unless otherwise stated or indicated, the chemical designation of a compound represents all possible stereochemically isomeric forms of the compound.

[0402] Formula (I o ) compounds Stereocenters are indicated in the usual manner using "dashed" or "solid" wedges, for example: [ka]

[0403] When a compound is described as a mixture of two diastereoisomers / epimers, the configuration of the stereocenters is not specified but is represented by a straight line.

[0404] Where a compound contains one or more chiral centers and can exist in two or more optically isomeric forms, a reference to the compound includes all optically isomeric forms thereof (e.g., enantiomers, epimers and diastereoisomers), either as individual optical isomers or mixtures (e.g., racemic or scalenic mixtures) or two or more optical isomers, unless the context requires otherwise.

[0405] Of particular interest are stereochemically pure compounds. For example, if a compound is designated as R, this means that the compound is substantially free of the S isomer. For example, if a compound is designated as E, this means that the compound is substantially free of the Z isomer. The terms cis, trans, R, S, E, and Z are well known to those skilled in the art.

[0406] isotopic variants The present invention includes the use of all pharmaceutically acceptable isotopically labeled compounds, i.e., compounds in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.

[0407] Solvates and Crystal Forms Also included within the present compounds are any polymorphic forms of the compounds, and solvates, such as hydrates, alcoholates, and the like.

[0408] In one embodiment, the MDM2 antagonist is a crystalline form of the free acid of (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid.

[0409] In one embodiment, the MDM2 antagonist is (a) an X-ray powder diffraction pattern characterized by peaks at diffraction angles 15.1, 15.5, 15.8, and 22.3° 2θ (±0.2° 2θ); or (b) Planar spacings of 3.99, 5.62, 5.71, and 5.87 Å The crystalline form of (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid has the formula:

[0410] In particular, the crystalline form of (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid is (a) an X-ray powder diffraction pattern characterized by peaks at diffraction angles 11.3, 15.1, 15.5, 15.8, 17.2, 20.8, 22.3, and 28.6° 2θ (±0.2° 2θ); or (b) Interplanar spacings at 3.12, 3.99, 4.27, 5.17, 5.62, 5.71, 5.87, and 7.85 Å It has.

[0411] In particular, the crystalline form of (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid has an X-ray powder diffraction pattern characterized by the presence, interplanar spacings (d) and intensities of major peaks at diffraction angles (2θ) set forth in Table 6 herein.

[0412] In particular, the crystalline form of (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid has an X-ray powder diffraction pattern that exhibits peaks at the same diffraction angles as the X-ray powder diffraction pattern shown in FIG. 6, and preferably these peaks have the same relative intensities as the peaks in FIG. 6.

[0413] In particular, the crystalline form of (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid has an X-ray powder diffraction pattern substantially as shown in FIG.

[0414] In one embodiment, the crystalline form of (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid exhibits an exothermic peak at 266-267°C (e.g., 266.61°C) when subjected to DSC (Figure 7).

[0415] The crystalline form may be substantially crystalline, meaning that one single crystalline form may predominate, although other crystalline forms may be present in minor, preferably negligible, amounts.

[0416] For example, a crystalline form may contain up to 5% by weight of any other crystalline form.

[0417] complex Compounds also include within their scope complexes (e.g., inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals). Inclusion complexes, clathrates, and metal complexes can be formed by methods well known to those skilled in the art.

[0418] Prodrug Also encompassed by the term "compound" is any prodrug of a compound. "Prodrug" refers, for example, to any compound that is converted in vivo into a biologically active compound.

[0419] Methods for preparing compounds used in the present invention Formula (I o ) compounds In this section, as in all other sections of this application, unless the context indicates otherwise, the term "compounds of formula I" is used. o where "a" is used, it also includes all other sub-formulas and embodiments thereof as defined herein, unless the context indicates otherwise.

[0420] Formula (I o Compounds of formula (I) can be prepared according to synthetic methods well known to those skilled in the art.

[0421] The necessary intermediates are either commercially available, known in the literature, prepared by methods analogous to those in the literature, or prepared by methods analogous to those described in the experimental procedure examples below. Other compounds can be prepared by functional interconversion of groups using methods well known in the art.

[0422] General procedures for preparing, isolating, and purifying compounds where cyc is phenyl can be found in International Patent Application PCT / GB2016 / 053042, published as WO2017 / 055860 on April 6, 2017.

[0423] General procedures for preparing, isolating, and purifying compounds where cyc is Het can be found in International Patent Application PCT / GB2016 / 053041, published as WO2017 / 055859 on April 6, 2017.

[0424] Biomarker detection In some embodiments, a sample of patient tissue is tested, which may comprise one or more cancer cells, or may comprise nucleic acid, generally DNA, from cancer cells, such as circulating tumor DNA (ctDNA) obtained from the blood.

[0425] In some embodiments, the sample is placed in an in vitro diagnostic device that measures the relative expression or activity of one or more biomarkers of interest.

[0426] When the present invention is practiced to determine whether a treatment is likely to be effective, the patient is generally known or suspected to have cancer. Thus, in certain embodiments, the method is for assessing whether a human patient known or suspected to have cancer can be treated using an MDM2 antagonist.

[0427] The methods of the present invention generally comprise detecting one or more of the identified biomarkers, and optionally additional biomarkers, using one or more detection reagents and / or detection techniques. Detection is generally performed ex vivo, e.g., in vitro, on a sample from a patient. In one embodiment, the biomarker is measured directly. In another embodiment, a biomarker substrate can be measured to indirectly assess biomarker levels.

[0428] "Detection" refers to measuring, quantifying, scoring, or assaying the expression or activity level of a biomarker. Methods for evaluating biological compounds, including biomarker proteins, genes, or mRNA transcripts, are known in the art. It is recognized that methods for detecting biomarkers include direct and indirect measurements. Those skilled in the art will be able to select an appropriate method for assaying a particular biomarker.

[0429] A "detection reagent" is an agent or compound that specifically (or selectively) binds to, interacts with, or detects a biomarker of interest. Such detection reagents may include, but are not limited to, antibodies, polyclonal antibodies, or monoclonal antibodies that preferentially bind to protein biomarkers, or oligonucleotides that are complementary to mRNA or DNA biomarkers and generally selectively bind under stringent hybridization conditions.

[0430] The phrases "specifically (or selectively) bind" or "specifically (or selectively) immunoreact" when referring to a detection reagent refer to a binding reaction that determines the presence of a biomarker in a heterogeneous population of biological molecules. For example, under specified immunoassay conditions, a particular detection reagent (e.g., an antibody) will bind to a particular protein at least twice as much as background and will not bind substantially in significant amounts to other proteins present in the sample. Specific binding under such conditions may require an antibody selected for its specificity for a particular protein. Various immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays (enzyme-linked immunosorbent assays) are routinely used to select antibodies that specifically immunoreact with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction will be at least twice as much as background signal or noise, more commonly 10-100 times or more above background.

[0431] Techniques such as in situ hybridization (ISH), quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry (IHC) have traditionally been used to diagnose or detect disease biomarkers. However, the emergence of high-throughput, highly sensitive approaches such as next-generation sequencing, single-molecule real-time sequencing, digital pathology, and quantitative histopathology has led to a shift toward technology platforms that enable companion diagnostics or CDx. Quantitative histopathology and digital pathology are both medical image-based diagnostic approaches that provide localization and measurement of protein biomarkers in tissue samples. Tissue markers are identified and quantified using automated, fluorescence-based imaging platforms.

[0432] When the biomarker to be detected is a protein, the detection method includes antibody-based assay, protein array assay, mass spectrometry (MS)-based assay, and (near) infrared spectroscopy-based assay. For example, immunoassays include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blot, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, and fluorescent immunoassay. Such assays are conventional and well known in the art.

[0433] "Analyzing" includes determining a set of values ​​associated with a sample by measuring a marker in the sample (e.g., the presence or absence of a marker, or the expression or activity level of a component, etc.) and comparing the measurements to measurements of a sample or set of samples from the same subject or other control subjects. The markers of the present teachings can be analyzed by any of a variety of conventional methods known in the art. "Analyzing" can include, for example, performing a statistical analysis to determine whether a subject is a responder or non-responder to a therapy (e.g., an MDM2 antagonist treatment as described herein).

[0434] In the context of the present teachings, a "sample" refers to any biological sample isolated from a subject, such as a blood sample or biopsy. Samples can include, but are not limited to, single or multiple cells, cell fragments, aliquots of bodily fluids, whole blood, platelets, serum, plasma, red blood cells, white blood cells, endothelial cells, tissue biopsies, synovial fluid, lymphatic fluid, ascites, and interstitial or extracellular fluid. The term "sample" also encompasses fluids found in the intercellular spaces, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or other bodily fluids. A "blood sample" can refer to whole blood or any fraction thereof, including blood cells, red blood cells, white blood cells, platelets, serum, and plasma. Samples can be obtained from a subject by means including, but not limited to, venipuncture, voiding, ejaculation, massage, biopsy, needle aspiration, lavage, scraping, surgical incision or intervention, or other means known in the art.

[0435] analysis technology Prior to administration of an MDM2 antagonist, the patient can be screened to determine whether the disease or condition the patient has or may have is one that is susceptible to treatment with a compound that inhibits MDM2 / p53. The term "patient" includes animal subjects, such as humans and primates, particularly human patients.

[0436] For example, a biological sample taken from a patient can be analyzed to determine whether a condition or disease, such as cancer, that the patient has or may have is characterized by a genetic abnormality or aberrant protein expression that results in upregulation of MDM2 levels or upregulation of biochemical pathways downstream of MDM2 / p53. Further, a biological sample taken from a patient can be analyzed to determine whether a condition or disease, such as cancer, that the patient has or may have is characterized by a biomarker of the invention.

[0437] Examples of such abnormalities that lead to MDM2 activation or sensitization include the loss or inhibition of regulatory pathways that affect MDM2 expression, the upregulation of receptors or their ligands, cytogenetic abnormalities, or the presence of receptor or ligand mutants.Tumors that show MDM2 / p53 upregulation, especially MDM2 overexpression or wild-type p53, may be particularly sensitive to MDM2 / p53 inhibitors.For example, as discussed herein, the amplification of MDM2 and / or the deletion of its negative regulator, such as p14ARF, have been identified in various cancers.

[0438] The terms "elevation" and "increase" include upregulation or overexpression of expression, including gene amplification (i.e., multiple gene copies), cytogenetic abnormalities, and increased expression due to transcriptional or post-translational effects. Thus, patients can be subjected to diagnostic tests to detect appropriate proteins or markers characteristic of upregulation of the biomarkers of the invention. The term diagnosis also includes screening.

[0439] The term "marker" or "biomarker" includes, for example, measurements of DNA composition to identify the presence of p53 mutations, MDM2 amplification, or p14ARF deletions (deletions), or generally genetic markers, including the biomarkers of the present invention discussed extensively herein. The term marker also includes markers characteristic of MDM2 / p53 upregulation, or upregulation or downregulation of the biomarkers outlined herein, including protein levels, protein status, and mRNA levels of the aforementioned proteins. Gene amplification includes those with more than seven copies, as well as gains of 2 to 7 copies.

[0440] The terms "reduction," "depletion," or "decrease" include decreased or diminished expression, including downregulation (i.e., fewer gene copies), decreased expression due to cytogenetic abnormalities, and transcriptional effects. Thus, patients can be subjected to diagnostic tests to detect lower levels of the biomarkers of the invention.

[0441] Diagnostic tests and screens are generally performed on biological samples (i.e., body tissues or fluids) selected from tumor biopsy samples, blood samples (isolation and enrichment of free tumor cells or isolation of circulating tumor DNA), cerebrospinal fluid, plasma, serum, saliva, fecal biopsy, sputum, chromosome analysis, pleural fluid, peritoneal fluid, buccal spears, skin biopsy, or urine.

[0442] Additionally, liquid biopsies, such as blood-based (systematic) circulating tumor DNA (ctDNA) testing or NGS-based liquid biopsy testing, can also be used, particularly for cancer detection or mutation identification. Liquid-based biopsies, including next-generation sequencing (NGS), complement traditional detection methods of PCR and tumor biopsy, for example, by whole-genome sequencing of circulating tumor cells (CTCs) or massively parallel sequencing of circulating tumor DNA (ctDNA).

[0443] In one embodiment, the sample obtained is a blood sample, such as a plasma or serum sample, particularly a serum sample. In one embodiment, the sample obtained is a tumor biopsy sample.

[0444] In one embodiment, blood collected, typically in serum separator tubes, is analyzed in a medical laboratory or at the point of care. In a second embodiment, tumors are analyzed by biopsy and analyzed in a medical laboratory.

[0445] Screening methods may include, but are not limited to, standard methods such as reverse transcriptase polymerase chain reaction (RT-PCR), protein analysis or in-situ hybridization such as fluorescent in situ hybridization (FISH).

[0446] Methods for identifying and analyzing cytogenetic abnormalities, gene amplification, protein deletion, downregulation, mutation, and upregulation are known to those skilled in the art. Screening methods can include standard methods, such as, but not limited to, DNA sequencing using conventional Sanger or next-generation sequencing, reverse transcriptase polymerase chain reaction (RT-PCR), RNA sequencing (RNAseq), nanostring hybridization proximity RNA nCounter assay, or in-situ hybridization such as fluorescent in situ hybridization (FISH), or allele-specific polymerase chain reaction (PCR). Additionally, methods for assessing protein levels include immunohistochemistry or other immunoassays. Thus, in one embodiment, protein expression is analyzed in patient samples. In another embodiment, gene expression, such as genetic abnormalities, is analyzed in patient samples using techniques such as FISH. Methods for assessing gene copy alterations include techniques routinely used in cytogenetic laboratories, such as MLPA (multiplex ligation-dependent probe amplification), multiplex PCR to detect copy number abnormalities, or other PCR techniques capable of detecting gene amplifications, gains, and deletions.

[0447] In RT-PCR screening, the level of mRNA in tumor is evaluated by making a cDNA copy of mRNA, and then amplifying the cDNA by PCR.The method of PCR amplification, the selection of primers, and the conditions for amplification are known to those skilled in the art.Nucleic acid manipulation and PCR are carried out by standard methods, such as those described in Ausubel, FM et al. (eds.) (2004) Current Protocols in Molecular Biology, John Wiley & Sons Inc., or Innis, MA et al. (eds.) (1990) PCR Protocols: a guide to methods and applications, Academic Press, San Diego.Reactions and manipulations involving nucleic acid technology are also described in Sambrook et al., (2001), 3rd Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Alternatively, commercially available RT-PCR kits (such as Roche Molecular Biochemicals) can be used, or the methodology described in U.S. Patent No. 4,666,828; U.S. Patent No. 4,683,202; U.S. Patent No. 4,801,531; U.S. Patent No. 5,192,659; U.S. Patent No. 5,272,057; U.S. Patent No. 5,882,864; and U.S. Patent No. 6,218,529, which are incorporated herein by reference, can be used.For example, the mutation in the gene outlined herein can be determined by PCR.In one embodiment, specific primer pairs are commercially available or described in literature.

[0448] An example of an in-situ hybridization technique for assessing mRNA expression is fluorescent in-situ hybridization (FISH) (see Angerer (1987) Meth. Enzymol., 152:649).

[0449] Next generation sequencing (NGS), DNA sequencing or nanostringing can be performed.

[0450] Generally, in situ hybridization involves the following major steps: (1) fixation of the tissue to be analyzed, (2) prehybridization treatment of the sample to increase the accessibility of the target nucleic acid and reduce nonspecific binding, (3) hybridization of the nucleic acid mixture to nucleic acids in the biological structure or tissue, (4) posthybridization washes to remove unbound nucleic acid fragments, and (5) detection of the hybridized nucleic acid fragments. Probes used in such applications are typically labeled, for example, with radioisotopes or fluorescent reporters. Certain probes are sufficiently long, e.g., from about 50, 100, or 200 nucleotides to about 1,000 or more nucleotides, to allow specific hybridization with the target nucleic acid under stringent conditions. Standard methods for performing FISH are described in Ausubel, FM et al. (eds.) (2004) Current Protocols in Molecular Biology, John Wiley & Sons Inc and Fluorescence In Situ Hybridization: Technical Overview by John MS Bartlett in Molecular Diagnosis of Cancer, Methods and Protocols, 2nd Edition; ISBN: 1-59259-760-2; March 2004, pp. 077-088; Series: Methods in Molecular Medicine.

[0451] The method for gene expression profiling has been described in (DePrimo et al. (2003), BMC Cancer, 3:3). Briefly, the protocol is as follows: double-stranded cDNA is synthesized from total RNA using (dT)24 oligomers to prime first-strand cDNA synthesis, followed by second-strand cDNA synthesis using random hexamer primers. The double-stranded cDNA is used as a template for in vitro transcription of cRNA using biotinylated ribonucleotides. The cRNA is chemically fragmented according to the protocol described by Affymetrix (Santa Clara, CA, USA) and then hybridized overnight to a human genome array. Alternatively, single nucleotide polymorphism (SNP) arrays, a type of DNA microarray, can be used to detect polymorphisms within a population.

[0452] Additionally, the test kit may use nanostring technology or ddPCR.

[0453] Alternatively, protein products expressed from mRNA may be assayed by immunohistochemistry (or other immunoassays) of tumor samples, solid-phase immunoassays using microtiter plates, Western blotting, two-dimensional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry, and other methods known in the art for detecting specific proteins, such as capillary electrophoresis. Detection methods include the use of site-specific antibodies. Those skilled in the art will recognize that all such well-known techniques for detecting upregulation of MDM2 and p53, detecting MDM2 or p53 mutants or variants, or detecting loss of negative regulators of MDM2 (e.g., p14ARF) or the genes described herein are applicable to the present case. In particular, the levels of the genes described herein can be measured using immunohistochemistry. Cytoplasmic expression can be assessed by staining of tumor cells. In some embodiments, one or both of the protein biomarkers of the present invention are assayed using these techniques. In some embodiments, one or more biomarker substrates are assayed using these techniques.

[0454] Protein level, particularly the increase, decrease or abnormal level of protein can be measured using standard protein assay.Increase or decrease in level, or under- or over-expression can also be detected by measuring protein level in tissue sample, for example, tumor tissue, using assay such as Chemicon International.Immunoprecipitation of target protein from sample lysate and measuring its level.

[0455] In embodiments in which the gene is a DDR biomarker, it will be appreciated that a variety of assays are available for measurement, such as ELISA, immunoturbidimetry, rapid immunodiffusion, and visual agglutination.

[0456] In embodiments where gene expression is examined, for example, for IFN signature biomarkers, it will be recognized that there are a variety of assays available for measurement.

[0457] In one embodiment comprising detecting the loss of one or more DDR biomarkers, such detection can generally be performed on biopsies using clinically validated assays at the DNA level (i.e., DNA sequencing), RNA level (i.e., qPCR, gene array, exome sequencing, etc.), or protein level (i.e., immunohistochemistry). In another embodiment, detecting the loss of one or more DDR biomarkers comprises one or more of reverse phase protein array, Western blotting, semi-quantitative or quantitative IHC.

[0458] Immunohistochemistry (IHC) is an important technique for biomarker detection. First, IHC allows direct visualization of biomarker expression in histologically relevant regions of the examined cancer tissue. Second, IHC is performed on FFPE tissue sections processed using standard methods, ensuring that biomarker assays can be performed on clinically available specimens. Third, validated IHC assays can be easily implemented into clinical practice. For example, there are multiple validated IHC assays in clinical use, such as assays for detecting PD-L1, HER2, and ALK (https: / / www.fda.gov / medical-devices / vitro-diagnostics / list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools). Traditionally, pathologists manually scored IHC data. For example, the HSCORE is calculated by multiplying the sum of the percentage of area stained at each intensity level by a staining intensity weighting (e.g., 1, 2, or 3; 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) [McCarty et al: Cancer Res 1986, 46:4244s-4248s]. For assay validation purposes, these analyses are often performed on specimens arrayed on stained TMA sections, ensuring sufficient specimen representation for statistically rigorous testing. Because tissue specimens are adequately represented with only a few tissue cores on a slide, IHC costs and tissue usage are minimized, facilitating intraobserver, interobserver, and interlaboratory studies. Computer-assisted methods for image classification of regions of interest (e.g., cancerous areas in a tissue specimen) and quantifying IHC staining intensity within those regions are also available for data generation.

[0459] Such techniques are equally applicable to the detection of other genes described herein. In some embodiments, detecting increased levels of the genes described herein comprises a polymerase chain reaction (PCR) assay, or direct nucleic acid sequencing, or hybridization with a nucleic acid probe specific for the gene.

[0460] Thus, all of these techniques can be used to identify tumors that are particularly suitable for treatment with MDM2 antagonists.

[0461] Ex-vivo functional assays, such as circulating leukemia cells in cancer patients to assess response to MDM2 / p53 inhibitor administration, can also be used where applicable.

[0462] Thus, a further aspect of the invention includes the use of an MDM2 antagonist for the manufacture of a medicament for the treatment or prevention of a disease state or condition in a patient who has been screened and determined to be suffering from, or at risk of suffering from, a disease or condition that may be susceptible to treatment with an MDM2 / p53 inhibitor.

[0463] Another aspect of the invention includes an MDM2 antagonist for use in preventing or treating cancer in patients selected from a subpopulation having loss of one or more DDR biomarkers.

[0464] Another aspect of the invention includes an MDM2 antagonist for use in preventing or treating cancer in patients selected from the subpopulation having wild-type p53 and loss of one or more DDR biomarkers.

[0465] Another aspect of the invention includes an MDM2 antagonist for use in the prevention or treatment of cancer in patients with loss of an MDM2 negative regulator, such as p14ARF, and loss of one or more DDR biomarkers.

[0466] MRI determination of vascular normalization in combination with circulating biomarkers (e.g., using MRI gradient echo, spin echo, and contrast to measure blood volume, relative vessel size, and vascular permeability) also can be performed using the formula (I o ) can be used to identify patients suitable for treatment with the compound.

[0467] Thus, a further aspect of the present invention is a method for the diagnosis and treatment of a disease state or condition mediated by MDM2 / p53, comprising (i) screening a patient to determine whether the disease or condition from which the patient is suffering or likely to suffer is one that is susceptible to treatment with an MDM2 / p53 inhibitor; and (ii) if the patient is shown to have such a susceptible disease or condition, then administering to the patient an MDM2 antagonist, and subgroups or examples thereof, as defined herein.

[0468] In one embodiment, the method of the invention further comprises the step of screening patients for overexpression of one or more MDM family members (eg, MDM2 and / or MDMx).

[0469] In one embodiment, the method of the invention further comprises a step of screening patients with cytogenetic abnormalities that result in overexpression of MDM2, e.g., patients selected as having loss of the negative regulator p14ARF.

[0470] In one embodiment, a sample obtained from a patient is contacted with a primer, antibody, substrate, or probe to determine the level of a gene described herein.

[0471] In one embodiment, the method comprises (i) contacting a patient sample with a primer, antibody, substrate, or probe, and (ii) determining the level of a gene described herein.

[0472] Basal levels can be analyzed, for example, by performing intracellular staining of untreated cells with an antibody conjugated to a fluorescent probe. Antibodies against the biomarkers described herein are commercially available from various suppliers. In particular, the antibodies used can be part of an FDA-approved in vitro diagnostic kit (IVD).

[0473] In one embodiment, the method comprises (i) contacting a patient sample with an antibody, and (ii) determining the level of one or more biomarkers described herein. In another embodiment, step (i) of the method comprises contacting the patient sample with one or more PCR primers for one or more biomarker substrates.

[0474] In one embodiment, the method comprises (i) contacting a patient sample with an antibody, and (ii) determining the level of nuclear localization to assess the level of one or more biomarkers described herein. In another embodiment, step (i) of the method comprises contacting the patient sample with a biomarker substrate antibody.

[0475] Where applicable, the level of nuclear localization can be determined using immunohistochemistry or immunofluorescence with antibodies.

[0476] Mutations that result in the loss of DDR biomarkers can be detected using reverse-phase protein arrays, Western blotting, semi-quantitative or quantitative IHC, or DNA sequencing. In one embodiment, this method comprises (i) contacting a patient sample with an anti-mutant antibody, and (ii) determining that the patient's tumor has the DDR biomarker loss. In one embodiment, this method comprises (i) contacting a patient sample with an anti-mutant antibody, and (ii) determining the level (or loss) of one or more DDR biomarkers.

[0477] Detection of DDR biomarker deletions and mutations can be performed by extracting DNA from patient samples, such as tumor biopsies, PCR amplification using appropriate primers, and DNA sequencing. PCR primers can be designed or are commercially available. Mutation array kits are also commercially available.

[0478] In one embodiment, the method comprises (i) contacting the patient sample with one or more DDR biomarker PCR primers, and (ii) determining the presence or absence of a DDR biomarker mutation or deletion. In another embodiment, step (i) of the method comprises contacting the patient sample with one or more PCR primers for one or more biomarker substrates.

[0479] In one embodiment, the method comprises (i) contacting a patient sample with a DDR biomarker antibody and (ii) determining the presence or absence of a mutation or deletion in the DDR biomarker. In another embodiment, step (i) of the method comprises contacting the patient sample with a biomarker substrate antibody.

[0480] Protein levels can be determined using ELISA kits. ELISA kits for use with patient samples can be used to evaluate blood chemistry in clinical settings. These use antibodies specific to the protein, such as anti-biomarker antibodies, such as anti-ATM or anti-ATRX, or binding antibodies. In particular, the antibodies used are part of FDA-approved in vitro diagnostic kits. In one embodiment, levels are determined using tests that comply with the standards defined by the Association for Clinical Biochemistry (ACB).

[0481] In one embodiment, the method comprises (i) contacting a patient sample with an antibody, and (ii) determining the level of a protein derived from a gene described herein.

[0482] In particular, the sample is contacted under conditions for quantifying the level.

[0483] For example, in the contacting step described above, the sample is contacted with a primer, a probe, a substrate, or an antibody, generally in the presence of a buffer. The substrate can be, for example, a fluorescent probe.

[0484] Patient selection It will be appreciated that patients selected for treatment with an MDM2 antagonist according to the present invention will be tested or measured for one or more DDR biomarkers according to the methodology described in the previous section.

[0485] For example, such selected patients have reduced or low expression or activity of one or more DDR biomarkers.

[0486] In one embodiment, the selected patient exhibits or displays at least one symptom of cancer, in particular a TP53 wild-type tumor.

[0487] In one embodiment, the selected cancer patient has not previously been treated with an MDM2 antagonist, hi one embodiment, the selected patient has not previously responded to therapy with an MDM2 antagonist.

[0488] In some embodiments, nucleic acid expression profiles (e.g., IFN gene signatures) are determined by PCR, HTG EdgeSeq, or quantitative gene expression assays such as NanoString nCounter. In some embodiments, protein expression profiles (e.g., DDR pathway gene products, or, for example, BAP1 and / or CDKN2A) are determined by immunoassays.

[0489] Optional Interferon Gene Signature (IFN) Increased DNA damage can induce an interferon response, which can be identified by an "interferon signature." Expression of these proteins is generally determined by measuring mRNA transcripts.

[0490] In one embodiment, the patient or sample comprises: Decreased or low expression or activity of one or more DDR biomarkers; and Elevated expression of one, two, three, four, five or more interferon signature genes It has.

[0491] In one embodiment, elevated expression of one, two, three, four, five or more interferon signature genes is selected from the group consisting of CXCL10, CXCL11, RSAD2, MX1, BATF2, IFI44L, IFITM1, ISG15, CMPK2, IFI27, CD74, IFIH1, CCRL2, IFI44, HERC6, ISG20, IFIT3, HLA-C, OAS1, IFI35, IRF9, EPSTI1, USP18, BST2, CSF1 Elevated or high expression of one, two, three, four, five or more of the following: C1S, DHX58, TRIM14, OASL, IRF7, LGALS3BP, DDX60, LAP3, LAMP3, PARP12, PARP9, SP110, PLSCR1, WARS, STAT1, IRF3, IRF5, MSC, JUN, SPI1, IRF1, COMMD3-BMI1, STAT2, RUNX3, SREBF1, and / or FLI1.

[0492] In one embodiment, CXCL10, CXCL11, RSAD2, MX1, BATF2, IFI44L, IFITM1, ISG15, CMPK2, IFI27, CD74, IFIH1, CCRL2, IFI44, HERC6, ISG20, IFIT3, HLA-C, OAS1, IFI35, IRF9, EPSTI1, USP18, BST2, CSF1, C1S, DHX58, TRIM14, OASL, IRF7, LGALS The RNA level of one or more of 3BP, DDX60, LAP3, LAMP3, PARP12, PARP9, SP110, PLSCR1, WARS, STAT1, IRF3, IRF5, MSC, JUN, SPI1, IRF1, COMMD3-BMI1, STAT2, RUNX3, SREBF1, and / or FLI1 is elevated compared to the amount of said RNA in a control sample obtained from a normal subject not afflicted with cancer.

[0493] In another embodiment, CXCL10, CXCL11, RSAD2, MX1, BATF2, IFI44L, IFITM1, ISG15, CMPK2, IFI27, CD74, IFIH1, CCRL2, IFI44, HERC6, ISG20, IFIT3, HLA-C, OAS1, IFI35, IRF9, EPSTI1, USP18, BST2, CSF1, C1S, DHX58, TRIM14, OASL, IRF7, LG The RNA levels of ALS3BP, DDX60, LAP3, LAMP3, PARP12, PARP9, SP110, PLSCR1, WARS, STAT1, IRF3, IRF5, MSC, JUN, SPI1, IRF1, COMMD3-BMI1, STAT2, RUNX3, SREBF1, and / or FLI1 are elevated in tumors compared to the amount of said RNA in non-tumor samples obtained from the same patient.

[0494] In one embodiment, the cancer exhibits elevated expression of CXCL10 or CXCL11.

[0495] In another embodiment, the cancer exhibits elevated expression of IRF7, IFITM1, IRF9, MX1, or IFI35.

[0496] In another embodiment, the cancer exhibits elevated expression of one or more, such as two or more, of IRF7, IFITM1, IRF9, MX1, IFI35, CXCL10, or CXCL11.

[0497] In one embodiment, the cancer exhibits elevated expression of one, two, three, four, five or more of CXCL10, CXCL11, RSAD2, MX1, BATF2, IFI44L, IFITM1, ISG15, CMPK2, IFI27, CD74, IFIH1, CCRL2, IFI44, HERC6, ISG20, IFIT3, HLA-C, OAS1, IFI35, IRF9, EPSTI1, USP18, BST2, CSF1, C1S, DHX58, TRIM14, OASL, IRF7, LGALS3BP, DDX60, LAP3, LAMP3, PARP12, PARP9, SP110, PLSCR1 and WARS.

[0498] In one embodiment, the cancer exhibits elevated expression of one, two, three, four, five or more of IRF7, STAT1, IRF3, IRF5, MSC, JUN, SPI1, IRF1, COMMD3-BMI1, STAT2, RUNX3, SREBF1, IRF9, and FLI1.

[0499] In another embodiment, the cancer exhibits elevated expression of IRF7, IFITM1, IRF9, MX1, or IFI35.

[0500] In another embodiment, the cancer exhibits elevated expression of one or more, eg, two or more, of IRF7, IFITM1, IRF9, MX1, IFI35, CXCL10, or CXCL11.

[0501] In some embodiments, the elevated level is relative to the amount of RNA determined in a sample from an MDM2 inhibitor non-responder subject.

[0502] In one embodiment, it is an elevation or increase compared to normal levels.

[0503] The upper limit of normal (ULN) refers to the level at 95% of the total range. It is the set of values ​​within which 95 percent of the normal population falls (i.e., the 95% prediction interval).

[0504] In one embodiment, the elevated level is greater than 1-fold relative to a control sample, the upper limit of normal (ULN), or a sample taken from said patient, e.g., 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100-fold, or any range therebetween. In one embodiment, the elevated level is 1-50-fold relative to a control sample or the ULN. In one embodiment, the elevated level is very high, e.g., greater than a 10-fold difference relative to a control sample, the ULN, or a sample taken from the patient, e.g., a 10-, 10.5-, 11-, 11.5-, 12-, 12.5-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100-, 1000-fold difference, or any range therebetween. In one embodiment, the elevated level is a 10- to 1000-fold difference relative to a control sample or the ULN. In one embodiment, the elevated level is a 2- to 10-fold (e.g., 5-fold) difference relative to a control sample.

[0505] The fold difference between diseased individuals and normal individuals (reference value or control sample) can be determined. This reference value can be calculated from normal individuals or can be based on a pool of samples excluding the sample type being tested (e.g., TP53 wild-type and DDR biomarker loss). In one embodiment, the difference between interferon gene expression in normal tissues (Source: GTEx; Nat Biotechnol. 2017 Apr 11;35(4):314-316) and patient mesothelioma samples (Source: TCGA) is greater than 5-fold to 0.05-fold (log2 scale), specifically, an average of 1.5-fold (log2 scale) increase across a set of genes.

[0506] In one embodiment, the concentration of RNA is determined by RT-PCR and / or microarray and / or nanostring.Each assay method typically has an "upper limit of normal" (ULN) associated with the specific assay method.This ULN is usually determined from a sufficient sample size of normal, healthy subjects using a specific assay method for measuring RNA concentration.Then, the ULN is usually determined as the highest RNA concentration that is still considered to be within the normal range (e.g., within 2 standard deviations of the mean value).This ULN value varies depending on the specific assay method used to measure the concentration, so each specific assay method has its own ULN value associated with the assay method.

[0507] As shown herein, concentrations can be used to predict whether a cancer patient is likely to benefit from MDM2 antagonist treatment.

[0508] DDR biomarker assays In one embodiment, the protein level of one or more of the DDR biomarkers is decreased relative to the amount of said protein in a control sample obtained from a normal subject not afflicted with cancer.

[0509] In another embodiment, the protein level of one or more DDR biomarkers is decreased relative to the amount of said protein in an earlier sample obtained from the same patient.

[0510] In one embodiment, it is decreased or reduced compared to normal levels.

[0511] The upper limit of normal (ULN) refers to the level at 95% of the total range. It is the set of values ​​within which 95 percent of the normal population falls (i.e., the 95% prediction interval).

[0512] In one embodiment, the decrease in level is less than 1-fold difference from the control sample, the upper limit of normal (ULN), or the sample taken from the patient, e.g., 0.75, 0.5, 0.4, 0.3, 0.2, 0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01-fold, or any range therebetween. In one embodiment, the decrease in level is 1-0.01-fold difference from the control sample or ULN. In one embodiment, the decrease in level is very small, e.g., more than 0.01-fold difference from the control sample, ULN, or the sample taken from the patient, e.g., 0.001-fold difference, or any range therebetween. In one embodiment, the decrease in level is 0, i.e., not present at all.

[0513] In another embodiment, the level of one or more DDR biomarkers is determined by immunohistochemistry.

[0514] Proteins, protein complexes, or proteomic markers can be specifically identified and / or quantified by a variety of methods known in the art, which can be used alone or in combination. Immunological or antibody-based techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), Western blotting, immunofluorescence, microarrays, several chromatographic techniques (i.e., immunoaffinity chromatography), flow cytometry, immunoprecipitation, and the like. Such methods are based on the specificity of one or more antibodies for a particular epitope or combination of epitopes associated with the protein or protein complex of interest. Non-immunological methods include those based on the physical characteristics of the protein or protein complex itself. Examples of such methods include electrophoresis, several chromatographic techniques (e.g., high-performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), affinity chromatography, ion-exchange chromatography, size-exclusion chromatography, etc.), mass spectrometry, sequencing, protease digests, and the like. Such methods are based on the mass, charge, hydrophobicity or hydrophilicity derived from the amino acid complement of a protein or protein complex, and the specific sequence of amino acids.

[0515] In one embodiment, there is no expression of one or more DDR biomarkers. Samples with low levels of one or more DDR biomarkers may be identified as DDR biomarker negative, e.g., DDR biomarker missing.

[0516] In one embodiment, loss of one or more DDR biomarkers is assessed by mutation analysis, eg, DNA sequencing.

[0517] Cytoplasmic and nuclear expression levels of one or more DDR biomarkers can also be measured. Nuclear localization of proteins is a marker for intracellular expression. The level of nuclear expression can be scored using histology, with a score (ranging from 0 to 100) representing the percentage of positive cells obtained after treatment with an antibody (e.g., a monoclonal anti-human antibody against the biomarker). An immunostaining expression score can be generated.

[0518] The level of one or more DDR biomarkers in the cytoplasm can also be measured using immunohistochemistry or immunofluorescence.

[0519] In one embodiment, the level of one or more of the DDR biomarkers is decreased relative to the amount of said protein in a control sample obtained from a normal subject not afflicted with cancer.

[0520] In one embodiment, the level of one or more of the DDR biomarkers is decreased in the tumor relative to the amount of said protein in a non-tumor sample obtained from the same patient.

[0521] In one embodiment, the expression level of one or more of the DDR biomarkers is reduced by 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. A 100% reduction in expression is a complete reduction, i.e., total loss. In some embodiments, a reduction of at least 50% is exhibited. In some embodiments, a reduction of at least 75% is exhibited.

[0522] In some embodiments, a reduction of at least 80% is exhibited.

[0523] In some embodiments, a reduction of at least 95%, such as at least 99%, is exhibited.

[0524] Quantification methods The present invention relates to identifying patients for treatment with MDM2 antagonists. In some embodiments, these methods include: (a) contacting a sample from a patient with an antibody to one or more DDR biomarkers (or one or more DDR biomarker substrates); (b) performing an ELISA or immunohistochemistry assay on said sample; (c) determining the level of one or more DDR biomarkers; and (d) identifying the patient as a candidate for treatment with an MDM2 antagonist if (i) the level of one or more DD biomarkers is lower than the upper limit of normal (ULN); or (ii) one or more DDR biomarkers is absent; or (iii) the level of one or more DDR biomarkers is lower than the upper limit of normal (ULN). The present invention comprises at least the following:

[0525] In other embodiments, the method for identifying a patient for treatment with an MDM2 antagonist comprises: (a) contacting a sample from the patient with an antibody to one or more DDR biomarkers (and / or one or more DDR biomarker substrates) to determine the level of protein expression; and / or (b) contacting a sample from the subject with an antibody to one or more DDR biomarkers (and / or one or more DDR biomarker substrates) different from (a) to determine the level of protein expression; (c) treating patients with an MDM2 antagonist if the levels of one or more DDR biomarkers are decreased compared to the upper limit of normal (ULN); The compound comprises:

[0526] Also described is a method for identifying or selecting a patient for treatment with an MDM2 antagonist, the method comprising: (a) contacting a sample from a patient with an antibody to one or more DDR biomarkers to determine the level of protein expression; and / or (b) contacting a sample from the patient with an antibody to one or more DDR biomarkers to determine the level of protein expression; and / or (c) contacting the sample from the patient with a plurality of oligonucleotide primers, said plurality of primers comprising at least one pair of oligonucleotide primers for any one or more DDR biomarkers; (d) treating patients with an MDM2 antagonist if the levels of one or more DDR biomarkers are decreased compared to the upper limit of normal (ULN); The compound comprises:

[0527] The patient selected is typically a cancer patient. The patient is typically selected if the level of one or more DDR biomarkers in a sample derived from the patient's biological sample is below a predetermined value (or is absent).

[0528] A method for predicting the efficacy of an MDM2 antagonist against cancer in a patient comprises determining the level of one or more DDR biomarkers in a sample from a biological patient, where efficacy for the patient is indicated if the level of the one or more DDR biomarkers in the biological sample is lower than a predetermined value.

[0529] System for carrying out the method The methods described herein may utilize a system to aid in patient assessment or prognosis. The system may be a single device that integrates various device components (units) therein. The system may also have its various components, or some of these components, as separate devices. The components may comprise a measuring device, a graphical user interface, and a computer processing unit.

[0530] The system generally comprises a data connection to an interface, which may itself be part of the system, or may be a remote interface. The latter refers to the possibility of using a separate device, preferably a portable device such as a smartphone or tablet computer, to provide the actual interface. The data connection in such cases preferably involves wireless data transfer, such as Wi-Fi or Bluetooth, or other technologies or standards.

[0531] In certain embodiments, the measurement device is configured to receive a tissue sample, for example, by placing one or more cancer cells or a drop of blood on a cartridge, which can be inserted into the device. The device can be an existing device that can determine the level of one or more biomarkers from the same sample. The processing unit can receive the protein concentration value from the measurement device. The processing unit typically includes software (usually built-in software) that allows it to calculate a score based on the input data.

[0532] In another embodiment, a system for assessing whether a human cancer patient is suitable for treatment with an MDM2 antagonist comprises: (a) A detection means capable of and adapted to detect one or more biomarkers of the invention in a sample from a human patient. Such means are known and readily available to those skilled in the art. Generally, a container is provided for receiving a sample of interest therein, the container comprising the detection means; (b) a processor capable of and adapted to determine from the determined concentration of said protein an indication of the likelihood that the patient will be treated with an MDM2 antagonist.

[0533] Optionally, the system comprises a user interface (or a data connection to a remote interface) through which information can be presented, particularly a graphical user interface (GUI); a GUI is a type of user interface that allows a user to interact with an electronic device through visual indicators such as graphical icons and secondary notations instead of text-based user interfaces, typed command labels, or text navigation (the present invention does not exclude any such interface types); GUIs are commonly known and are commonly used in handheld mobile devices such as MP3 players, portable media players, gaming devices, smartphones, and small home, office, and industrial control devices; as mentioned above, the interface may optionally also be selected to allow for input of information about the patient, etc.

[0534] In one embodiment, a system for determining the suitability of a human cancer patient for treatment with an MDM2 antagonist includes: a storage memory for storing data associated with a sample from the patient, the data comprising data associated with a biomarker panel indicative of biomarker expression levels in the sample from the subject; a biomarker panel comprising one or more biomarkers of the invention; and and a processor communicatively connected to the storage memory for classifying the patient.

[0535] kit The present invention also provides kits for detecting one or more biomarkers of the invention to assess a patient's likelihood of responding to MDM2 inhibition for cancer treatment, either separately or as part of the aforementioned system. The kits generally comprise one or more detection reagents for detecting one or more of the biomarkers of the invention. These reagents may be for direct or indirect detection of the biomarker, e.g., detection of a correlation substrate.

[0536] Generally, the kit comprises two or more, or three or more detection reagents, each directed to a different biomarker of the invention.

[0537] As described above with respect to the methods of the invention, the kit may comprise more detection reagents, such as for other proteins. In preferred embodiments, the detection reagents available in the kit consist of detection reagents for the detection of two, three, or four proteins that make up the biomarker panel of the invention, as described above.

[0538] The kit may comprise a solid support, such as a chip, microtiter plate or beads or a resin, comprising said detection reagent. In some embodiments, the kit comprises a mass spectrometry probe.

[0539] The kit may also provide unbound detection reagents or wash solutions and / or detection reagents specific for any of the biomarkers (sandwich type assays).

[0540] Such kits suitably comprise a biosensor for the detection and / or quantification of one or more biomarkers of the invention, and optionally instructions for use of the kit according to the methodology as described herein.

[0541] There are well-established genetic and biochemical means to characterize the status of one or more biomarkers of the invention, as well as well-established biochemical means to characterize protein abundance in blood, such as a serum sample.

[0542] In one embodiment, the present invention includes a packaged cancer therapeutic. The packaged therapeutic comprises a composition of the present invention packaged together with instructions for using an effective amount of the composition for intended use in a patient selected using the present invention. In another embodiment, the present invention provides the use of any of the compositions of the present invention for the manufacture of a medicament for treating cancer in a subject.

[0543] In one embodiment, the invention provides a kit or panel or array for determining the level of one or more of the biomarkers of the invention from a single patient sample.

[0544] Biological effects The compounds described herein, their subgroups and examples have been shown to inhibit the interaction between p53 and MDM2.This inhibition leads to cell growth arrest and cell death (generally apoptosis), which can be useful for the prevention or treatment of the disease states or pathological conditions described herein, such as the diseases and pathological conditions described below, and the above-mentioned diseases and pathological conditions in which p53 and MDM2 play a role.Therefore, for example, it is envisioned that the compounds used in the present invention can be useful for alleviating or reducing the occurrence of cancer.

[0545] The compounds described herein may be useful in treating the adult population. The compounds of the invention may be useful in treating the pediatric population.

[0546] The compounds described herein have been shown to be good antagonists of the formation of the MDM2-p53 complex. The compounds described herein can bind to MDM2 and exhibit potency against MDM2. The potency of the compounds of the present invention has been determined against MDM2 / p53 using the assay protocols described herein and other methods known in the art. More specifically, the compounds of formula (I) o ) and its subgroups have affinity for MDM2 / p53.

[0547] Particular compounds for use in the present invention have an IC of less than 0.1 μM, in particular less than 0.01 or 0.001 μM. 50 It has value.

[0548] The function of MDM2 / p53 has been implicated in many diseases due to its role in various processes, such as vascular remodeling and angiogenesis, regulation of metabolic pathways, and carcinogenesis. As a result of its affinity for MDM2, this compound has been shown to be effective in treating autoimmune diseases; diabetes mellitus; chronic inflammatory diseases, such as lupus nephritis, systemic lupus erythematosus (SLE), autoimmune-mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, autoimmune diabetes mellitus, eczema hypersensitivity reactions, asthma, COPD, rhinitis, and upper respiratory tract diseases; hyperkeratotic diseases, such as autosomal recessive congenital ichthyosis (ARCI); glomerular disorders, chronic kidney disease (CKD), renal inflammation, podocyte depletion, and glomerular sclerosis. It is expected that the compounds may be useful in treating or preventing a variety of diseases or conditions, including kidney diseases, including glaucoma, proteinuria, and progressive kidney disease; cardiovascular diseases, such as cardiac hypertrophy, restenosis, arrhythmias, and atherosclerosis; ischemic injuries associated with myocardial infarction, vascular damage, stroke, and reperfusion injury; vascular proliferative diseases; ocular diseases, such as age-related macular degeneration, particularly wet age-related macular degeneration, ischemic proliferative retinopathies, e.g., retinopathy of prematurity (ROP) and diabetic retinopathy; and hemangiomas.

[0549] As a result of their affinity for MDM2, it is expected that these compounds may be useful in the treatment or prevention of proliferative disorders such as cancer.

[0550] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include, but are not limited to, cancers of the bladder and urinary tract, breast, gastrointestinal tract (including esophagus, stomach, small intestine, colon, intestines, large intestine, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney (e.g., renal cell carcinoma), lung (e.g., adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g., cancers of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovary, fallopian tube, peritoneum, vagina, vulva, penis, testicle, cervix, myometrium, and intrauterine Tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other cancers), such as cancers of the membranes, thyroid gland (e.g., follicular thyroid carcinoma), brain, adrenal gland, prostate, skin, and adnexa (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematological malignancies (e.g., leukemia, lymphoma), premalignant hematological disorders, and borderline malignant neoplastic disorders, e.g., hematological malignancies of the lymphatic system and related conditions (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], diffuse large B-cell lymphoma, etc.); B-cell lymphomas such as DLBCL, follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), as well as hematological malignancies and related diseases of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders (e.g., tumors of mesenchymal origin, for example, sarcomas of soft tissue, bone, or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and fibrosarcomas; tumors of the central or peripheral nervous system (for example, astrocytomas (e.g., gliomas), schwannomas and glioblastomas, meningiomas, ependymomas, pineal tumors, and schwannomas);Endocrine tumors (e.g., pituitary tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinoma); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratoma, seminoma, ectodermal tumor, hydatidiform mole, and choriocarcinoma); and pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor); or syndromes, congenital or otherwise, that predispose a patient to malignancies (e.g., xeroderma pigmentosum);

[0551] Cell proliferation is a tightly regulated function. Cancer, a pathological condition of abnormal cell proliferation, occurs when cells replicate (increase in number) in an uncontrolled manner, proliferate (grow larger) uncontrollably, and / or undergo reduced cell death by apoptosis (programmed cell death), necrosis, or anoikis. In one embodiment, the abnormal cell proliferation is selected from uncontrolled cell proliferation, excessive cell proliferation, or reduced programmed cell death. In particular, the pathological condition or disease of abnormal cell proliferation is cancer.

[0552] Thus, in the pharmaceutical compositions, uses or methods of the present invention for treating a disease or condition comprising abnormal cell proliferation (i.e., uncontrolled and / or rapid cell growth), the disease or condition comprising abnormal cell proliferation is, in one embodiment, cancer.

[0553] Many diseases are characterized by persistent and uncontrolled angiogenesis.Chronic proliferative diseases are often accompanied by significant angiogenesis, which promotes or maintains inflammatory and / or proliferative conditions, or leads to tissue destruction through the invasive proliferation of blood vessels.It has been found that tumor growth and metastasis depend on angiogenesis.Therefore, the compound used in the present invention can be useful in preventing and inhibiting the onset of tumor angiogenesis.

[0554] Angiogenesis is commonly used to refer to the development of new or replacement blood vessels, or neovascularization. Angiogenesis is a necessary, physiologically normal process by which the vascular system is established in the embryo. Angiogenesis does not generally occur in most normal adult tissues, except at sites of ovulation, menstruation, and wound healing. However, many diseases are characterized by persistent, uncontrolled angiogenesis. For example, in arthritis, new capillaries invade joints and destroy cartilage. In diabetes (as well as in many different eye diseases), new blood vessels invade the macula or retina or other eye structures, which can cause blindness. The process of atherosclerosis has been linked to angiogenesis. Tumor growth and metastasis have been found to be dependent on angiogenesis. The compounds may be useful in the treatment of diseases such as cancer and metastasis, eye diseases, arthritis, and hemangiomas.

[0555] Therefore, the compounds for use in the present invention may be useful for treating metastasis and metastatic cancer.Metastasis or metastatic disease is the spread of disease from one organ or part to another non-adjacent organ or part.Cancers that can be treated with the compounds of the present invention include primary tumors (i.e., cancer cells at the site of origin), local invasion (cancer cells that penetrate and infiltrate surrounding normal tissues locally), and metastatic (or secondary) tumors, i.e., tumors formed from malignant cells that circulate to other parts and tissues in the body via the bloodstream (hematogenous metastasis), or via lymphatic vessels, or across body cavities (transosmotic metastasis).In particular, the compounds for use in the present invention may be useful for treating metastasis and metastatic cancer.

[0556] In one embodiment, the hematological malignancy is leukemia. In another embodiment, the hematological malignancy is lymphoma. In one embodiment, the cancer is AML. In another embodiment, the cancer is CLL.

[0557] In one embodiment, the compounds used in the present invention are for use in the prevention or treatment of leukemia, such as acute or chronic leukemia, particularly acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or chronic myelogenous leukemia (CML). In one embodiment, the compounds used in the present invention are for use in the prevention or treatment of lymphoma, such as acute or chronic lymphoma, particularly Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or diffuse large B-cell lymphoma.

[0558] In one embodiment, the compounds used in the present invention are for use in the prevention or treatment of acute myeloid leukemia (AML) or acute lymphocytic leukemia (ALL).

[0559] In one embodiment, the compounds used in the present invention are directed to the treatment of hematological malignancies (i.e., leukemias, lymphomas) and borderline neoplastic disorders, including pre-malignant hematological disorders and hematological malignancies of the lymphatic system and related conditions (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer [NK] cell lymphoma, Hodgkin's lymphoma, and the like). lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematological malignancies and related conditions of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndromes, myeloproliferative disorders (e.g., polycythemia vera, essential thrombocythemia, and primary myelofibrosis), myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia.

[0560] One embodiment includes a compound used in the invention for use in the prevention or treatment of cancer in patients selected from the subpopulation with cancer that is p53 wild-type or has MDM2 amplification.

[0561] The cancer may be a cancer that is susceptible to treatment with an MDM2 antagonist. The cancer may be a cancer that overexpresses MDM2. The cancer may be a p53 wild-type cancer.

[0562] Particular cancers include those with MDM2 amplification and / or MDM2 overexpression, such as hepatocellular carcinoma, lung, sarcoma, osteosarcoma, and Hodgkin's disease.

[0563] Specific cancers include those with wild-type p53. Specific cancers include, but are not limited to, cancer cells with wild-type p53 when MDM2 is highly expressed.

[0564] In one embodiment, the cancer is a p53-functional tumor. In one embodiment, the disease to be treated is a p53-functional solid and hematological malignant tumor. In another embodiment, the patient to be treated has a p53-mutated tumor, for example, an AML patient has a p53-mutated tumor.

[0565] In one embodiment, the cancer is a brain tumor, for example, a glioma or a neuroblastoma.

[0566] In one embodiment, the cancer is a cancer of the skin, for example, melanoma.

[0567] In one embodiment, the cancer is a lung cancer, such as NSCLC or mesothelioma. In one embodiment, the cancer is a lung cancer, such as mesothelioma. In one embodiment, the mesothelioma is malignant peritoneal mesothelioma or malignant pleural mesothelioma.

[0568] In one embodiment, the cancer is of the digestive tract, eg, GIST, gastric, colorectal or gastric cancer.

[0569] In one embodiment, the cancer is osteosarcoma.

[0570] In one embodiment, the cancer is liposarcoma.

[0571] In one embodiment, the cancer is Ewing's sarcoma.

[0572] In one embodiment, the cancer is a particular pediatric malignancy, including liposarcoma, soft tissue sarcoma, osteosarcoma, esophageal cancer, and B-cell malignancies.

[0573] In one embodiment, the cancer is colorectal cancer, breast cancer, lung cancer and brain cancer.

[0574] In one embodiment, the cancer is a childhood cancer.

[0575] In one embodiment, the cancer is p53 wild-type.

[0576] In one embodiment, the cancer is a cancer of the lung, for example NSCLC or mesothelioma, a brain cancer such as kidney cancer, for example KIRC or glioblastoma.

[0577] Whether a particular cancer is sensitive to an MDM2 antagonist can be determined by methods such as those set forth in the section headed "Diagnostic Methods."

[0578] A further aspect provides the use of a compound for the manufacture of a medicament for the treatment of a disease or condition as described herein, in particular cancer.

[0579] Patients with Fanconi anemia are at increased risk of developing acute myeloid leukemia (AML), squamous cell carcinoma, or tumors of the head, neck, skin, digestive system, or reproductive tract.

[0580] Homologous recombination deficiency (HRD) is common in prostate cancer, ovarian cancer, breast cancer, and gynecological cancer. Thus, in one embodiment, the cancer is prostate, ovarian, breast, or gynecological cancer. In one embodiment, the HRR pathway deficient (HRD) cancer is prostate cancer, ovarian, breast, or gynecological cancer.

[0581] MSI-H is common in colorectal, gastric and gynecological cancers. Thus, in one embodiment, the cancer is colorectal, gastric and gynecological cancer.

[0582] Certain cancers are resistant to treatment with specific drugs. This can be due to the type of tumor (most common epithelial malignancies are inherently resistant to chemotherapy, while the prostate is relatively resistant to currently available chemotherapy or radiotherapy regimens), or resistance can occur naturally as a result of disease progression or treatment. In this regard, reference to prostate includes prostate resistant to antiandrogen therapy, particularly abiraterone or enzalutamide, or castration-resistant prostate. Similarly, reference to multiple myeloma includes bortezomib-sensitive or refractory multiple myeloma, and reference to chronic myelogenous leukemia includes imitanib-sensitive and refractory chronic myelogenous leukemia. In this regard, reference to mesothelioma includes mesothelioma resistant to topoisomerase poisons, alkylating agents, antitubulin agents, antifolate agents, platinum compounds, and radiotherapy, particularly cisplatin-resistant mesothelioma.

[0583] These compounds may also be useful in treating tumor growth, pathogenesis, resistance to chemotherapy and radiotherapy by sensitizing cells to chemotherapy, and as anti-metastatic agents.

[0584] Anticancer therapeutic interventions of any kind inevitably increase stress on target tumor cells. MDM2 / p53 antagonists are a class of chemotherapeutic agents with the potential to: (i) sensitize malignant cells to anticancer drugs and / or treatments; (ii) mitigate or reduce the development of resistance to anticancer drugs and / or treatments; (iii) reverse resistance to anticancer drugs and / or treatments; (iv) enhance the activity of anticancer drugs and / or treatments; and (v) delay or prevent the development of resistance to anticancer drugs and / or treatments.

[0585] In one embodiment, the present invention provides a compound for use in treating an MDM2-mediated disease or condition. In a further embodiment, the MDM2-mediated disease or condition is a cancer characterized by overexpression and / or increased activity of MDM2, or high copy numbers of MDM2 and / or wild-type p53.

[0586] A further aspect provides the use of a compound for the manufacture of a medicament for the treatment of a disease or condition described herein, in particular cancer.

[0587] In one embodiment, compounds are provided for use in the prevention or treatment of MDM2 / p53-mediated diseases or conditions. In one embodiment, compounds are provided for inhibiting the interaction between MDM2 protein and p53.

[0588] In one embodiment, a pharmaceutical composition is provided comprising an effective amount of at least one compound as defined.

[0589] In one embodiment, a method is provided for the prevention or treatment of cancer comprising administering to a mammal a medicament comprising at least one compound as defined.

[0590] Pharmaceutical preparations While it is possible for the active compound to be administered alone, it will generally be presented as a pharmaceutical composition (eg, a formulation).

[0591] The present invention therefore further provides a pharmaceutical composition as defined above, o ) (and subgroups thereof as defined herein), together with (e.g., in admixture with) one or more pharmaceutically acceptable excipients and optionally other therapeutic or prophylactic agents as described herein.

[0592] The pharmaceutically acceptable excipient can be selected from, for example, carriers (e.g., solid, liquid, or semi-solid carriers), adjuvants, diluents, fillers or bulking agents, granulating agents, coating agents, release-controlling agents, binders, disintegrants, lubricants, preservatives, antioxidants, buffers, suspending agents, thickeners, flavoring agents, sweeteners, flavoring agents, stabilizers, or any other excipient conventionally used in pharmaceutical compositions. Examples of excipients for various types of pharmaceutical compositions are provided in more detail below.

[0593] The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each excipient must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0594] Formula (I o Pharmaceutical compositions containing MDM2 antagonists, including compounds of formula (I), can be formulated according to known techniques, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

[0595] The pharmaceutical composition can be in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, sublingual, ophthalmic, otorhinologic, rectal, vaginal, or transdermal administration. If the composition is intended for parenteral administration, it can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration, or direct delivery to the target organ or tissue by injection, infusion, or other delivery means. Delivery can be by bolus injection, short-term infusion, or long-term infusion, and can be by passive delivery or by using a suitable infusion pump or syringe driver.

[0596] Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, cosolvents, surfactants, organic solvent mixtures, cyclodextrin complexing agents, emulsifiers (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gelling polymers for forming polymeric gels, lyoprotectants, and combinations of agents, among others, to stabilize the active ingredient in a soluble form and to render the formulation isotonic with the blood of the intended recipient. Pharmaceutical formulations for parenteral administration may also take the form of aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents (RG Strickly, Solubilizing Excipients in Oral and Injectable Formulations, Pharmaceutical Research, Vol. 21(2) 2004, p. 201-230).

[0597] The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules, vials, and pre-filled syringes, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injection, immediately prior to use. In one embodiment, the formulation is presented as the active pharmaceutical ingredient in a bottle for subsequent reconstitution with a suitable diluent.

[0598] The pharmaceutical formulation may comprise a compound of formula (I o The MDM2 antagonists, including the compounds of the formula (I) or subgroups thereof, can be prepared by lyophilization. Lyophilization refers to the procedure of freeze-drying a composition. Therefore, freeze-drying and lyophilization are used synonymously herein.

[0599] Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0600] The pharmaceutical composition of the present invention for parenteral injection can also comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use.Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., sunflower oil, safflower oil, corn oil or olive oil), and injectable organic esters, such as ethyl oleate.Proper fluidity can be maintained by using thickeners such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants.

[0601] The compositions of the present invention may contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial activity can be ensured by incorporating various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. In addition, it may be desirable to incorporate agents for adjusting tonicity, such as sugars and sodium chloride. The absorption of injectable pharmaceutical preparations can be prolonged by incorporating agents that delay absorption, such as aluminum monostearate and gelatin.

[0602] In one exemplary embodiment of the present invention, the pharmaceutical composition is in a form suitable for intravenous administration, for example, by injection or infusion. For intravenous administration, the solution can be administered as is or can be injected into an infusion bag (containing a pharmaceutically acceptable excipient, such as 0.9% saline or 5% dextrose) before administration.

[0603] In another exemplary embodiment, the pharmaceutical composition is in a form suitable for subcutaneous (sc) administration.

[0604] Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard or soft shell), caplets, pills, lozenges, syrups, liquids, powders, granules, elixirs, suspensions, and patches such as sublingual tablets, wafers, or buccal patches.

[0605] Thus, tablet compositions may contain a unit dose of active compound together with an inert diluent or carrier, such as a sugar or sugar alcohol, e.g., lactose, sucrose, sorbitol, or mannitol; and / or a non-sugar-derived diluent, such as sodium carbonate, calcium phosphate, calcium carbonate, or cellulose or its derivatives, e.g., microcrystalline cellulose (MCC), methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and starch, such as corn starch. Tablets may also contain such standard ingredients as binders and granulating agents, such as polyvinylpyrrolidone, disintegrants (e.g., swellable cross-linked polymers, such as cross-linked carboxymethylcellulose), lubricants (e.g., stearates), preservatives (e.g., parabens), antioxidants (e.g., BHT), buffers (e.g., phosphate buffers or citrate buffers), and effervescent agents, such as citrate / bicarbonate mixtures. Such excipients are well known and need not be described in detail here.

[0606] Tablets may be designed to release the drug upon contact with gastric fluids (immediate-release tablets) or may be designed to release the drug in a controlled manner over an extended period of time or in specific areas of the gastrointestinal tract (controlled-release tablets).

[0607] Capsule formulations may be of the hard or soft gelatin variety and may contain the active ingredient in solid, semi-solid, or liquid form. Gelatin capsules may be formed from animal gelatin or equivalents of synthetic or vegetable origin.

[0608] Solid dosage forms (e.g., tablets, capsules, etc.) can be coated or uncoated. Coatings can serve as a protective film (e.g., polymers, waxes, or varnishes), as a mechanism for controlling drug release, or for aesthetic or identification purposes. Coatings (e.g., Eudragit™-type polymers) can be designed to release the active ingredient at a desired location in the gastrointestinal tract. Thus, coatings can be selected to degrade under specific pH conditions in the gastrointestinal tract, thereby selectively releasing the compound in the stomach or the ileum, duodenum, jejunum, or colon.

[0609] Instead of or in addition to a coating, the drug can be provided in a solid matrix comprising a release control agent, e.g., a release retardant, which can be adapted to release the compound in a controlled manner in the gastrointestinal tract. Alternatively, the drug can be provided in a polymer coating, e.g., a polymethacrylate polymer coating, which can be adapted to selectively release the compound under conditions of changing acidity or alkalinity in the gastrointestinal tract. Alternatively, the matrix material or release-retarding coating can take the form of an erodible polymer (e.g., maleic anhydride polymer) that is substantially continuously eroded as the dosage form passes through the gastrointestinal tract. Alternatively, the coating can be designed to disintegrate under microbial action in the intestine. As a further option, the active compound can be incorporated into a delivery system that provides osmotic control of the release of the compound. Osmotic release and other delayed-release or sustained-release formulations (e.g., formulations based on ion exchange resins) can be prepared according to methods well known to those skilled in the art.

[0610] Formula (I oMDM2 antagonists, including compounds of the formula (I), may be formulated with a carrier and administered in the form of nanoparticles, where the increased surface area of ​​the nanoparticles aids their absorption. Furthermore, nanoparticles offer the possibility of direct penetration into cells. Nanoparticle drug delivery systems are described in "Nanoparticle Technology for Drug Delivery," edited by Ram B. Gupta and Uday B. Kompella, Informa Healthcare, ISBN 9781574448573, published March 13, 2006. Nanoparticles for drug delivery are also described in J. Control. Release, 2003, 91 (1-2), 167-172, and Sinha et al., Mol. Cancer Ther. August 1, (2006) 5, 1909.

[0611] Pharmaceutical compositions generally comprise approximately 1% (w / w) to approximately 95% of the active ingredient and 99% (w / w) to 5% (w / w) of a pharmaceutically acceptable excipient or combination of excipients. Generally, compositions comprise approximately 20% (w / w) to approximately 90% (w / w) of the active ingredient and 80% (w / w) to 10% of a pharmaceutically acceptable excipient or combination of excipients. Pharmaceutical compositions comprise approximately 1% to approximately 95%, typically approximately 20% to approximately 90%, of the active ingredient. Pharmaceutical compositions according to the present invention may be in unit dose form, for example, as ampoules, vials, suppositories, pre-filled syringes, dragees, tablets, or capsules.

[0612] Pharmaceutically acceptable excipients can be selected depending on the desired physical form of the formulation and can be selected from, for example, diluents (e.g., solid diluents such as fillers or bulking agents; and liquid diluents such as solvents and co-solvents), disintegrants, buffers, lubricants, flow aids, release-controlling agents (e.g., release-retarding or delaying polymers or waxes), binders, granulating agents, pigments, plasticizers, antioxidants, preservatives, flavors, flavoring agents, tonicity adjusting agents, and coating agents.

[0613] Those skilled in the art will have the expertise to select the appropriate amounts of ingredients to be used in a formulation. For example, tablets and capsules typically contain 0-20% disintegrant, 0-5% lubricant, 0-5% flow aid, and / or 0-99% (w / w) filler or bulking agent (depending on the drug dose). They may also contain 0-10% (w / w) polymer binder, 0-5% (w / w) antioxidant, and 0-5% (w / w) pigment. Sustained-release tablets further contain 0-99% (w / w) polymer (depending on the dose). The film coat of a tablet or capsule typically contains 0-10% (w / w) release-controlling (e.g., retarding) polymer, 0-3% (w / w) pigment, and / or 0-2% (w / w) plasticizer.

[0614] Parenteral formulations generally contain 0-20% (w / w) buffer, 0-50% (w / w) cosolvent, and / or 0-99% (w / w) water for injection (WFI) (depending on dose and whether lyophilized). Intramuscular depot formulations may contain 0-99% (w / w) oil.

[0615] Pharmaceutical compositions for oral administration can be prepared by combining the active ingredient with a solid carrier, optionally granulating the resulting mixture, adding suitable excipients as desired or necessary, and then processing the mixture into tablets, dragee cores, or capsules. The active ingredient can also be incorporated into a polymeric or waxy matrix that allows it to diffuse or release measured amounts.

[0616] The compounds used in the present invention can also be formulated as solid dispersions. A solid dispersion is a homogeneous, extremely finely dispersed phase of two or more solids. Solid solutions (molecular dispersions), a type of solid dispersion, are well known for use in pharmaceutical technology (see Chiou and Riegelman, J. Pharm. Sci., 60, 1281-1300 (1971)), and are useful for increasing the dissolution rate and bioavailability of poorly water-soluble drugs.

[0617] The present invention also provides solid dosage forms comprising the solid solutions described herein. Solid dosage forms include tablets, capsules, chewable tablets, and dispersible or effervescent tablets. The solid solutions can be combined with known excipients to provide the desired dosage form. For example, capsules can contain a solid solution containing (a) a disintegrant and a lubricant, or (b) a disintegrant, a lubricant, and a surfactant. Additionally, capsules can contain a filler such as lactose or microcrystalline cellulose. Tablets can contain a solid solution containing at least one disintegrant, a lubricant, a surfactant, a filler, and a lubricant. Chewable tablets can contain a solid solution containing a filler, a lubricant, and optionally additional sweeteners (such as artificial sweeteners) and suitable flavors. Solid solutions can also be formed by spraying a solution of the drug and a suitable polymer onto the surface of an inert carrier, such as sugar beads ("nonpareils"). These beads can then be filled into capsules or compressed into tablets.

[0618] Pharmaceutical preparations are sometimes provided to patients in "patient packs" that contain an entire course of treatment in a single package, usually a blister pack. Patient packs have the advantage over traditional prescriptions, where a pharmacist portions out the patient's medication from a bulk supply, that the patient always has access to the package insert included in the patient pack, which is typically missing from prescriptions. The inclusion of the package insert has been shown to improve patient compliance with physician instructions.

[0619] Compositions for topical use and nasal delivery include ointments, creams, sprays, patches, gels, liquid drops, and inserts (e.g., intraocular inserts). Such compositions can be formulated according to known methods.

[0620] Examples of preparations for rectal or vaginal administration include pessaries and suppositories, which may be formed, for example, from a moldable or waxy material containing the active compound. Solutions of the active compound may also be used for rectal administration.

[0621] The composition for administration by inhalation may take the form of an inhalable powder composition or a liquid or powder spray, and can be administered in standard form using a powder inhaler or aerosol delivery device. Such devices are well known. When administered by inhalation, the powder formulation usually comprises the active compound together with an inert solid powder diluent such as lactose.

[0622] Formula (I o MDM2 antagonists, including compounds of the formula (I), are generally provided in unit dosage form and, thus, generally contain sufficient compound to provide a desired level of biological activity. For example, a formulation may contain 1 nanogram to 2 grams of active ingredient, e.g., 1 nanogram to 2 milligrams of active ingredient. Within these ranges, particular subranges of compound are 0.1 milligram to 2 grams of active ingredient (more usually 10 milligrams to 1 gram, e.g., 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams (e.g., 1 microgram to 10 milligrams, e.g., 0.1 milligram to 2 milligrams of active ingredient).

[0623] For oral compositions, a unit dosage form may contain from 1 milligram to 2 grams, more usually 10 milligrams to 1 gram, for example, 50 milligrams to 1 gram, for example, 100 milligrams to 1 gram, of active compound.

[0624] The active compounds are administered to a patient in need thereof (eg, a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect.

[0625] Combination with other anticancer drugs MDM2 antagonists as defined herein may be useful in the prevention or treatment of a variety of MDM2 / p53-mediated disease states or conditions, examples of which are set out above.

[0626] These compounds are generally administered to a subject in need of such administration, for example a human or animal patient, generally a human.

[0627] These compounds are generally therapeutically or prophylactically useful and are generally administered in non-toxic amounts. However, in some situations (e.g., in the case of life-threatening diseases), the compounds used in the present invention (e.g., compounds of formula (I)) may be administered in amounts that are not toxic to the patient. o The benefits of administering a compound of formula (I) may outweigh the drawbacks of toxic or side effects, in which case it may be desirable to administer an amount of the compound that is associated with some toxicity.

[0628] The compounds may be administered over an extended period to maintain beneficial therapeutic effect, or for a shorter period, or may be administered continuously or in an intermittent manner (e.g., pulsed administration).

[0629] A typical daily dose of an MDM2 antagonist may range from 100 picograms to 100 milligrams per kilogram of body weight, more usually from 5 nanograms to 25 milligrams per kilogram of body weight, and more usually from 10 nanograms to 15 milligrams per kilogram of body weight (e.g., from 10 nanograms to 10 milligrams per kilogram of body weight, more usually from 1 microgram to 20 milligrams, e.g., from 1 microgram to 10 micrograms per kilogram of body weight), although higher or lower doses may be administered as needed. o The compound of formula (I) can be administered daily or repeatedly, for example, every 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days.

[0630] Dose is also defined as the amount of drug administered relative to the patient's body surface area (mg / m 2 A typical daily dose of an MDM2 antagonist is 3700 pg / m 2 ~3700mg / m 2 , more commonly 185 ng / m 2 ~925mg / m 2 , more usually 370 ng / m 2 ~555mg / m 2(e.g., 370 ng / m 2 ~370mg / m 2 , more commonly 37 mg / m 2 ~740mg / m 2 , e.g., 37 mg / m 2 ~370mg / m 2 ), although higher or lower doses may be administered as needed. o The compound of formula (I) can be administered daily or repeatedly, for example, every 2, or 3, or 4, or 5, or 6, or 7, or 10, or 14, or 21, or 28 days.

[0631] The compounds of the present invention may be orally administered in doses ranging from, for example, 0.1 to 5000 mg, or 1 to 1500 mg, 2 to 800 mg, or 5 to 500 mg, e.g., 2 to 200 mg or 10 to 1000 mg; specific dose examples include 10, 20, 50, and 80 mg. The compounds may be administered once or more times daily. The compounds may be administered continuously (i.e., every day without a break for the duration of the treatment regimen). Alternatively, the compounds may be administered intermittently (i.e., administered continuously for a predetermined period, such as one week, followed by a cessation of administration for a period, such as one week, followed by continued administration for another period, such as one week, throughout the duration of the treatment regimen). Examples of treatment regimens involving intermittent administration include regimens in which administration is performed for one week on and one week off; or two weeks on and one week off; or three weeks on and one week off; or two weeks on and two weeks off; or four weeks on and two weeks off; or one week on and three weeks off, with one or more cycles, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cycles. This discontinuous treatment can also be based on days rather than weeks. For example, treatment can comprise daily administration for one to six days, followed by no administration for one to six days, with this pattern repeated throughout the treatment protocol. The number of days (or weeks) during which the compound used in the present invention is not administered does not necessarily equal the number of days (or weeks) during which the compound used in the present invention is administered.

[0632] In one embodiment, the compounds used in the present invention are administered at a dose of 3 mg / m daily. 2 ~125mg / m 2 The amount of the compound can be administered in an amount of 0.01 to 0.01 mg / kg. Treatment can be by continuous daily administration or, more usually, can consist of multiple treatment cycles with spaced treatment intervals. An example of one treatment cycle would be five consecutive daily doses followed by three weeks without treatment.

[0633] One particular dosing regimen is once daily (e.g., orally) for one week (e.g., five days) of treatment followed by one, two, or three weeks of rest. Another dosing regimen is once weekly (e.g., orally) for one, two, three, or four weeks.

[0634] In certain dosing schedules, patients receive o The compound of formula (I) is administered by infusion for one hour daily for up to 10 days, particularly up to 5 days, for one week, with the treatment repeated at desired intervals, such as every 2-4 weeks, particularly every 3 weeks.

[0635] More particularly, the patient is administered a compound of formula (I o ) compound will be administered as a daily 1-hour infusion for 5 days, with the treatment repeated every 3 weeks.

[0636] In another particular dosing schedule, the patient is given an infusion of 30 minutes to 1 hour, followed by a maintenance infusion of varying duration, for example, 1 to 5 hours, eg, 3 hours.

[0637] The compounds used in the present invention can also be administered by bolus or continuous infusion.The compounds used in the present invention can be administered daily to once a week, or once every two weeks, or once every three weeks, or once every four weeks during a treatment cycle.When administered daily during a treatment cycle, this daily administration can be discontinuous over the number of weeks in the treatment cycle, for example, administered for one week (or several days) and not administered for one week (or several days), and this pattern is repeated during the treatment cycle.

[0638] A more particular administration schedule involves administering a continuous infusion to a patient for a period of 12 hours to 5 days, particularly a continuous infusion of 24 hours to 72 hours.

[0639] Ultimately, however, the amount of compound administered and the type of composition used will be commensurate with the nature of the disease or physiological condition being treated and will be at the discretion of the physician.

[0640] It may be beneficial to use the compounds used in the present invention as single agents or to combine them with other agents that act by different mechanisms to regulate cell proliferation to treat two hallmarks of cancer development. Combination studies can be performed, for example, as described in Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regulat 1984;22:27-55.

[0641] The compounds as defined herein can be administered as a single therapeutic agent, or can be administered in combination with one or more other compounds (or therapies) for the treatment of certain conditions, for example, neoplastic diseases such as cancer as defined above.For the treatment of the above conditions, the compounds used in the present invention can be advantageously used in combination with one or more other pharmaceutical agents, more particularly, other anti-cancer agents or adjuvants in cancer therapy.Examples of other therapeutic agents or treatments that can be administered together with MDM2 antagonists (simultaneously or at different time intervals) include, but are not limited to: Topoisomerase I inhibitors ·Antimetabolites Tubulin-targeting agents DNA binders and topoisomerase II inhibitors Alkylating agents Monoclonal antibodies Antihormonal Signal transduction inhibitors Proteasome inhibitors DNA methyltransferase inhibitors Cytokines and retinoids Chromatin-targeted therapy Radiation therapy, and Other therapeutic or preventative medications

[0642] Specific examples of anti-cancer agents or adjuvants (or salts thereof) include, but are not limited to, any agent selected from the following groups (i) to (xlviii), and optionally group (xlix):

[0643] (i) platinum compounds, such as cisplatin (optionally in combination with amifostine), carboplatin, or oxaliplatin; (ii) a taxane compound, such as paclitaxel, paclitaxel protein-bound particles (Abraxane™), docetaxel, cabazitaxel, or larotaxel; (iii) a topoisomerase I inhibitor, e.g., a camptothecin compound, e.g., camptothecin, irinotecan (CPT11), SN-38, or topotecan; (iv) topoisomerase II inhibitors, such as antitumor epipodophyllotoxins or podophyllotoxin derivatives, such as etoposide or teniposide; (v) Vinca alkaloids, such as vinblastine, vincristine, liposomal vincristine (Onco-TCS), vinorelbine, vindesine, vinflunine, or vinbecil; (vi) nucleoside derivatives, such as 5-fluorouracil (5-FU, optionally in combination with leucovorin), gemcitabine, capecitabine, tegafur, UFT, S1, cladribine, cytarabine (Ara-C, cytosine arabinoside), fludarabine, clofarabine, or nelarabine; (vii) antimetabolites, e.g., clofarabine, aminopterin, or methotrexate, azacitidine, cytarabine, floxuridine, pentostatin, thioguanine, thiopurine, 6-mercaptopurine, or hydroxyurea (hydroxycarbamide); (viii) alkylating agents, e.g., nitrogen mustards or nitrosoureas, e.g., cyclophosphamide, chlorambucil, carmustine (BCNU), bendamustine, thiotepa, melphalan, treosulfan, lomustine (CCNU), altretamine, busulfan, dacarbazine, estramustine, fotemustine, ifosfamide (optionally in combination with mesna), pipobroman, procarbazine, streptozocin, temozolomide, uracil, mechlorethamine, methylcyclohexylchloroethylnitrosourea, or nimustine (ACNU); (ix) anthracyclines, anthracenediones and related drugs, such as daunorubicin, doxorubicin (optionally in combination with dexrazoxane), liposomal formulations of doxorubicin (e.g., Keryx™, Myocet™, Doxil™), idarubicin, mitoxantrone, epirubicin, amsacrine, or valrubicin; (x) Epothilones, such as ixabepilone, patupilone, BMS-310705, KOS-862 and ZK-EPO, epothilone A, epothilone B, desoxyepothilone B (also known as epothilone D or KOS-862), aza-epothilone B (also known as BMS-247550), laulimalide, isolaulimalide, or lueterobin; (xi) a DNA methyltransferase inhibitor, e.g., temozolomide, azacitidine, or decitabine; (xii) a folate antagonist, e.g., methotrexate, pemetrexed disodium, or raltitrexed; (xiii) cytotoxic antibiotics, such as antinomycin D, bleomycin, mitomycin C, dactinomycin, carminomycin, daunomycin, levamisole, plicamycin, or mithramycin; (xiv) tubulin-binding agents, such as combrestatin, colchicine, or nocodazole; (xv) signal transduction inhibitors, for example, kinase inhibitors, for example, receptor tyrosine kinase inhibitors (e.g., EGFR (epidermal growth factor receptor) inhibitors, VEGFR (vascular endothelial growth factor receptor) inhibitors, PDGFR (platelet-derived growth factor receptor) inhibitors, Axl inhibitors, MTKI (multiple-targeted kinase inhibitors), Raf inhibitors, ROCK inhibitors, mTOR inhibitors, MEK inhibitors or PI3K inhibitors), for example, imatinib mesylate, erlotinib, gefitinib, dasatinib, lapatinib, dovotinib, axitinib, nilotinib, vandetanib, vatalanib, pazopanib, sorafenib, sunitinib, temsirolimus, everolimus (RAD 001), vemurafenib (PLX4032 or RG7204), dabrafenib, encorafenib, selumetinib (AZD6244), trametinib (GSK121120212), dactolisib (BEZ235), buparlisib (BKM-120; NVP-BKM-120), BYL719, copanlisib (BAY-80-6946), ZSTK-474, CUDC-907, apitolisib (GDC-0980; RG-7422), pictilisib (pictilisib, GDC-0941, RG -7321), GDC-0032, GDC-0068, GSK-2636771, idelalisib (formerly CAL-101, GS1101, GS-1101), MLN1117 (INK1117), MLN0128 (INK128), IPI-145 (INK1197), LY-3023414, ipatasertib, afuresertib, MK-2206, MK-8156, LY-3023414, LY294002, SF1126 or PI-103, sonolisib (PX-866), or AT13148; (xvi) Aurora kinase inhibitors, such as AT9283, barasertib (AZD1152), TAK-901, MK0457 (VX680), cenisertib (R-763), danusertib (PHA-739358), alisertib (MLN-8237), or MP-470; (xvii) CDK inhibitors, including, for example, AT7519, roscovitine, seliciclib, alvocidib (flavopiridol), dinaciclib (SCH-727965), 7-hydroxy-staurosporine (UCN-01), JNJ-7706621, BMS-387032 (also known as SNS-032), PHA533533, ZK-304709, or AZD-5438 and CDK4 inhibitors such as palbociclib (PD332991) and ribociclib (LEE-011); (xviii) PKA / B inhibitors and PKB(akt) pathway inhibitors, such as AT13148, AZ-5363, Semaphore, SF1126 and MTOR inhibitors, such as rapamycin analogues, AP23841 and AP23573, calmodulin inhibitors (forkhead translocation inhibitors), API-2 / TCN (triciribine), RX-0201, enzastaurin HCl (LY317615), NL-71-101, SR-13668, PX-316, or KRX-0401 (Perifosine / NSC 639966); (xix) Hsp90 inhibitors, such as onarespi (AT13387), rubimycin, geldanamycin (GA), 17-allylamino-17-desmethoxygeldanamycin (17-AAG), such as NSC-330507, Kos-953, and CNF-1010, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), such as NSC-707545 and Kos-1022, NVP-AUY922 (VER-52296), NVP-BEP800, CNF-2024 (BIIB-021 oral purine), ganetespib (STA-9090), SNX-5422 (SC-102112), or IPI-504; (xx) Monoclonal antibodies (unconjugated or conjugated with radioisotopes, toxins or other drugs), antibody derivatives and related drugs, for example, anti-CD, anti-VEGFR, anti-HER2 or anti-EGFR antibodies, for example, rituximab (CD20), ofatumumab (CD20), ibritumomab tiuxetan (CD20), GA101 (CD20), tositumomab (CD20), epratuzumab (CD22), lintuzumab (CD33), gemtuzumab Ozogamicin (CD33), alemtuzumab (CD52), galiximab (CD80), trastuzumab (HER2 antibody), pertuzumab (HER2), trastuzumab-DM1 (HER2), ertumaxomab (HER2 and CD3), cetuximab (EGFR), panitumumab (EGFR), necitumumab (EGFR), nimotuzumab (EGFR), bevacizumab (VEGF), catumaxomab (EpCAM and CD3), abagovomab (CA125), farletuzumab (folate receptor), elotuzumab (CS1), denosumab (RANK ligand), figitumumab (IGF1R), CP751,871 (IGF1R), mapatumumab (TRAIL receptor), metMAB (met), mitumomab (GD3 ganglioside), naptumomab estafenatox (5T4), or siltuximab (IL6) or immunomodulatory agents, e.g., CTLA-4 blockade anti-PD-1 and / or PD-L1 and / or PD-L2 antibodies, such as ipilimumab (CTLA4), MK-3475 (pembrolizumab, formerly lambrolizumab, anti-PD-1), nivolumab (anti-PD-1), BMS-936559 (anti-PD-L1), MPDL320A, AMP-514 or MEDI4736 (anti-PD-L1), or tremelimumab (formerly ticilimumab, CP-675,206, anti-CTLA-4); (xxi) estrogen receptor antagonists or selective estrogen receptor modulators (SERMs) or estrogen synthesis inhibitors, such as tamoxifen, fulvestrant, toremifene, droloxifene, faslodex, or raloxifene; (xxii) aromatase inhibitors and related drugs, such as exemestane, anastrozole, letrazole, testolactone aminoglutethimide, mitotane or vorozole; (xxiii) antiandrogens (i.e., androgen receptor antagonists) and related agents, such as bicalutamide, nilutamide, flutamide, cyproterone, or ketoconazole; (xxv) Hormones and their analogues, such as medroxyprogesterone, diethylstilbestrol (also known as diethylstilboestrol or octreotide); (xxvi) steroids, such as dromostanolone propionate, megestrol acetate, nandrolone (decanoate, phenpropionate), fluoxime estrone or gossypol; (xxvii) steroid cytochrome P450 17α-hydroxylase-17,20-lylase inhibitors (CYP17), for example, abiraterone; (xxvii) gonadotropin-releasing hormone agonists or antagonists (GnRAs), such as abarelix, goserelin acetate, histrelin acetate, leuprolide acetate, triptorelin, buserelin, or deslorelin; (xxviii) Glucocorticoids, such as prednisone, prednisolone, dexamethasone; (xxix) differentiation agents, such as retinoids, rexinoids, vitamin D or retinoic acid and retinoic acid metabolism blockers (RAMBAs), such as Accutane, alitretinoin, bexarotene, or tretinoin; (xxx) farnesyltransferase inhibitors, for example, tipifarnib; (xxxi) chromatin-targeted therapy, for example, histone deacetylase (HDAC) inhibitors, such as sodium butyrate, suberoylanilide hydroxamic acid (SAHA), depsipeptide (FR901228), dacinostat (NVP-LAQ824), R306465 / JNJ-16241199, JNJ-26481585, trichostatin A, vorinostat, chlamydocin, A-173, JNJ-MGCD-0103, PXD-101, or apicidin; (xxxii) Ubiquitin-proteasome pathway targeting drugs, for example, proteasome inhibitors, for example, bortezomib, carfilzomib, CEP-18770, MLN-9708, or ONX-0912; NEDD8 inhibitors; HDM2 antagonists and deubiquitinases (DUBs); (xxxiii) photodynamic drugs, such as porfimer sodium or temoporfin; (xxxiv) anticancer agents derived from marine organisms, for example, trabectidin; (xxxv) Radiolabeled drugs for radioimmunotherapy, for example with beta particle-emitting isotopes (e.g., iodine-131, yttrium-90) or alpha particle-emitting isotopes (e.g., bismuth-213 or actinium-225), such as ibritumomab or iodine tositumomab or alpha radium-223; (xxxvi) telomerase inhibitors, for example, telomestatin; (xxxvii) matrix metalloproteinase inhibitors, such as batimastat, marimastat, prinostat, or metastat; (xxxviii) recombinant interferons (e.g., interferon-gamma and interferon-alpha) and interleukins (e.g., interleukin-2), such as aldesleukin, denileukin diftitox, interferon-alpha-2a, interferon-alpha-2b, or pegylated interferon-alpha-2b; (xxxix) selective immune response modifiers, such as thalidomide or lenalidomide; (xl) therapeutic vaccines, e.g., sipuleucel-T (Provenge) or OncoVex; (xli) cytokine activators, such as picibanil, romurtide, sizofiran, virulidin, or thymosin; (xlii) arsenic trioxide; (xliii) G protein-coupled receptor (GPCR) inhibitors, for example, atrasentan; (xliv) an enzyme such as L-asparaginase, pegaspargase, rasburicase, or pegademase; (xlv) a DNA repair inhibitor, e.g., a PARP inhibitor, e.g., olaparib, veliparib, iniparib, INO-1001, AG-014699, or ONO-2231; (xlvi) Agonists of death receptors (e.g., TNF-related apoptosis-inducing ligand (TRAIL) receptors), such as mapatumumab (forme...

Claims

1. A pharmaceutical composition for treating cancer comprising an MDM2 antagonist, wherein the cancer has a depletion of one or more genes or gene products in one or more DNA damage repair (DDR) pathways or wherein the cancer has at least one loss-of-function mutation in at least one DDR pathway gene, and the one or more DDR pathway genes or gene products include BRCA1 and / or BRCA2.

2. The one or more DDR paths are a. the homology directed repair (HRR) pathway; b. Non-homologous end joining (NHEJ) pathway; c. the mismatch repair (MMR) pathway; d. the Fanconi Anemia (FA) pathway; and / or e. Base excision repair (BER) pathway The pharmaceutical composition of claim 1 ,

3. the one or more genes or gene products comprise or consist of an HRR pathway gene or gene product other than ATM; or The one or more genes or gene products comprise or consist of BRCA1 and / or BRCA2, and ATM; The pharmaceutical composition according to claim 1 or 2. (i) the one or more genes or gene products comprise ATRX; and / or (ii) the one or more genes or gene products comprise MSH2, MSH3, MSH6, MLH1, MLH3, PMS2, POLE and / or POLD1; or the cancer comprises a mutational signature SBS6 or SBS26 associated with defective DNA mismatch repair, and / or a POLD1 mutational signature SBS20; and / or (iii) the one or more genes or gene products comprise FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCO, FANCP, FANCQ, FANCR, FANCS, FANCT, FANCU, FANCV and / or FANCW; The pharmaceutical composition according to any one of claims 1 to 3.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein depletion or mutation of the DDR gene is detected by assessing the microsatellite instability status and / or tumor mutation burden of the cancer, said cancer optionally being MSI-high.

6. a sample of patient tissue is tested to determine a cancer expression profile prior to treatment, and optionally the patient is selected for treatment based on the determined expression profile; Optionally, the sample comprises cancer DNA, ctDNA, or cancer cells; and / or the test comprises an assay for detecting protein, mRNA and / or ctDNA; 6. The pharmaceutical composition of any one of claims 1 to 5, optionally wherein (i) proteins are detected using immunoassays, protein-binding assays, antibody-based assays, antigen-binding protein-based assays, protein-based arrays, enzyme-linked immunosorbent assays (ELISA), flow cytometry, protein arrays, blots, Western blots, nephelometry, turbidimetry, chromatography, mass spectrometry, enzyme activity, radioimmunoassays, immunofluorescence, immunochemiluminescence, immunoelectrochemiluminescence, immunoelectrophoresis, competitive immunoassays, or immunoprecipitation; and / or (ii) mRNA is detected using RT-PCR or quantitative gene expression assays; and / or (iii) DNA or RNA is detected by Next Generation Sequencing; and / or (iv) proteins are detected by immunohistochemistry.

7. (i) The cancer is acute myeloid leukemia (AML), squamous cell carcinoma or tumors of the head, neck, skin, digestive system or reproductive tract; or prostate, ovarian, breast or gynecological cancer; or Colorectal, gastric or gynecologic cancer and / or (ii) the cancer is p53 wild-type; and / or (iii) the cancer cells undergo apoptosis following the treatment step; and / or (iv) activated caspase-3 is induced by the MDM2 antagonist in at least a proportion of cancer cells; optionally (v) Activated caspase-3 is induced by the MDM2 antagonist in at least 40% of cancer cells or at least 60% of cancer cells.

8. The cancer is Decreased expression of one, two or three of CDKN2A, BAP1 and SKP2 compared to a control; and / or Increased expression of one, two, three, four, five or more interferon signature genes compared to a control and optionally, The interferon signature genes are CXCL10, CXCL11, RSAD2, MX1, BATF2, IFI44L, IFITM1, ISG15, CMPK2, IFI27, CD74, IFIH1, CCRL2, IFI44, HERC6, ISG20, IFIT3, HLA-C, OAS1, IFI35, IRF9, EPSTI1, USP18, BST2, CS F1, C1S, DHX58, TRIM14, OASL, IRF7, LGALS3BP, DDX60, LAP3, LAMP3, PARP12, PARP9, SP110, PLSCR1, WARS, STAT1, IRF3, IRF5, MSC, JUN, SPI1, IRF1, COMMD3-BMI1, STAT2, RUNX3, SREBF1 and FLI1; or the cancer exhibits elevated expression of CXCL10 or CXCL11; The pharmaceutical composition according to any one of claims 1 to 7.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the cancer exhibits elevated expression of one, two, three, four, five or more of IRF7, STAT1, IRF3, IRF5, MSC, JUN, SPI1, IRF1, COMMD3-BMI1, STAT2, RUNX3, SREBF1, IRF9 and FLI1.

10. The MDM2 antagonist has the formula (I) as defined herein. ｏ or a tautomer, N-oxide, pharma- ceutically acceptable salt or solvate thereof, such as (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2]-yl-2-methylpropanoic acid, or a tautomer, N-oxide, pharma- ceutically acceptable salt or solvate thereof; or The MDM2 antagonist is selected from the group consisting of Compound 1, idasanutlin (RG-7388), HDM-201, KRT-232 (AMG-232), ALRN-6924, MI-773 (SAR405838), CGM-097, milademethane tosylate, APG-115, BI-907828, LE-004, and DS-52. 72, SJ-0211, BI-0252, AM-7209, SP-141, SCH-1450206, NXN-6, ADO-21, CTX-50-CTX -1, ISA-27, RO-8994, RO-6839921, ATSP-7041, SAH-p53-8, PM-2, K-178, MMRi-64 and 【Chemistry 1】 The pharmaceutical composition according to any one of claims 1 to 9, wherein the compound is selected from the group consisting of: or a tautomer or solvate or a pharma- ceutically acceptable salt thereof.

11. The use of expression or activity levels of one or more of one or more DDR pathway genes or gene products in a cancer cell sample from a human patient as one or more biomarkers for assessing whether a cancer is susceptible to treatment with an MDM2 antagonist, wherein the one or more DDR pathway genes or gene products include BRCA1 and / or BRCA2, e.g., the MDM2 antagonist is a compound represented by formula (I) as defined herein. ｏ or a tautomer, N-oxide, pharma- ceutically acceptable salt or solvate thereof, for example, (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid, or a tautomer, N-oxide, pharma- ceutically acceptable salt or solvate thereof.

12. 1. A method for prognosing or assessing the responsiveness of a human cancer patient to treatment with an MDM2 antagonist, comprising assessing the expression or activity level of one or more DDR pathway genes, including BRCA1 and / or BRCA2, in a sample from a cancer patient, and determining whether the expression or activity level examined indicates that the cancer should be treated with an MDM2 antagonist, and optionally the evaluating step comprises comparing the expression or activity level with (i) an expression or activity level associated with responsiveness or non-responsiveness to treatment with an MDM2 antagonist, or (ii) an expression or activity level in a healthy, non-cancerous cell of the same type; and / or The patient is classified into groups based on a biomarker profile, and optionally the groups include: (i) Responders and non-responders; or (ii) Strong Responders and / or a patient is identified as being particularly suitable for the treatment if one, two, three, four, five, six, seven, eight, nine, ten or more DDR pathway genes are expressed at a lower level than in patients identified as not suitable for the treatment; and / or The method, wherein the patient is identified for treatment with an MDM2 antagonist if (i) an expression level associated with non-responsiveness to treatment with an MDM2 antagonist, or (ii) a decrease in expression of one or more DDR pathway genes is detected compared to the expression levels of healthy, non-cancerous cells of the same type.

13. detecting the expression or activity level of said biomarker in a cancer cell sample from said human patient, and optionally The method of claim 12, wherein the detection is performed using an in vitro detection assay.

14. A method for determining the sensitivity of a human cancer patient to treatment with an MDM2 antagonist, comprising detecting expression or activity of one or more DDR pathway genes, including BRCA1 and / or BRCA2, in a cancer cell sample from the patient, and assessing whether the patient's cancer is likely to respond to treatment with an MDM2 antagonist based on the expression or activity level of a biomarker in the sample.

15. 1. An in vitro method for detecting the expression or activity level of one or more DDR pathway genes in a human patient suffering from cancer, wherein the one or more DDR genes include BRCA1 and / or BRCA2, and optionally the method comprises: (a) obtaining a cancer cell sample from a human patient; and (b) detecting whether said one or more biomarkers are expressed in the sampled cancer cells by contacting said sample with one or more reagents for detecting expression of said one or more biomarkers. The method comprising:

16. The MDM2 antagonist has the formula (I) as defined herein. ｏ or a tautomer, N-oxide, pharma- ceutically acceptable salt or solvate thereof, such as (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid, or a tautomer, N-oxide, pharma- ceutically acceptable salt or solvate thereof; or The MDM2 antagonist is selected from the group consisting of Compound 1, idasanutlin, HDM-201, KRT-232, ALRN-6924, ALRN-6924, CGM-097, milademethane tosylate, APG-115, BI-907828, LE-004, DS-5272, SJ-0211, BI-0252, AM-7209, SP-141, SCH-1450206, NXN-6, ADO-21, CTX-50-CTX-1, ISA-27, RO-8994, RO-6839921, ATSP-7041, SAH-p53-8, PM-2, K-178, MMRi-64, and 【Chemistry 2】 or a tautomer or solvate or a pharma- ceutically acceptable salt thereof; and / or providing a pharmaceutical composition for the treatment of cancer comprising an MDM2 antagonist, optionally comprising: The method of any one of claims 12 to 15, wherein the treatment is selected for the patient based on the results of the method.

17. A kit or device for detecting the expression or activity level of at least one biomarker for sensitivity to MDM2 inhibition in a sample from a human patient, comprising detection reagents for detecting one or more DDR pathway genes or gene products, wherein the one or more DDR pathway genes or gene products include BRCA1 and / or BRCA2.

18. 1. A system for determining suitability of a human cancer patient for treatment with an MDM2 antagonist, comprising: a storage memory for storing data relating to the patient-derived sample, the data comprising data relating to a panel of biomarkers indicative of expression or activity levels of the biomarkers in the subject-derived sample; and a processor communicatively connected to the storage memory for classifying the patient, The system, wherein the biomarker panel comprises one or more DDR pathway genes or gene products, and the one or more DDR pathway genes or gene products include BRCA1 and / or BRCA2.

19. The pharmaceutical composition, use, method, kit or system of any one of claims 1 to 18, wherein the cancer exhibits loss of one or more DDR pathway genes, gene products or activities, and the one or more DDR pathway genes, gene products or activities include BRCA1 and / or BRCA2.

20. 20. The pharmaceutical composition, use or method of any one of claims 1 to 16 or 19, wherein the MDM2 antagonist is part of a combination therapy with a second therapeutic agent, optionally a PARP inhibitor.

21. A pharmaceutical composition comprising an MDM2 antagonist for use in a method of treating a cancer patient, comprising: (i) the MDM2 antagonist is used in a method of treating cancer in combination with an agent for inducing sensitivity to the MDM2 antagonist, e.g., for reducing the level of one or more genes or gene products in the DNA damage repair (DDR) pathway; the cancer has normal or elevated levels of one or more genes or gene products in one or more DNA damage repair (DDR) pathways, where the one or more DDR genes or gene products comprise BRCA1 and / or BRCA2, or the cancer has no detectable loss-of-function mutations in any DDR pathway genes; or (ii) the process comprises (a) selecting a patient having normal or elevated levels of a DDR pathway gene or gene product in a biological sample obtained from said patient, wherein said one or more DDR pathway genes or gene products include BRCA1 and / or BRCA2; and (b) administering to said patient selected in step (a) a therapeutically effective amount of an MDM2 antagonist and an agent for inducing sensitivity to the MDM2 antagonist, e.g., by reducing the level of one or more genes or gene products in the DNA damage repair (DDR) pathway. and optionally, The pharmaceutical composition, wherein the agent for inducing sensitivity to an MDM2 antagonist is a DNA damaging agent or a DNA repair inhibitor.

22. A pharmaceutical composition comprising an MDM2 inhibitor for use in the treatment of cancer as defined in any one of claims 1 to 5 in a patient, wherein the MDM2 inhibitor is of formula (I ｏ or a tautomer, N-oxide, pharma- ceutically acceptable salt, or solvate thereof, such as (2S,3S)-3-(4-chlorophenyl)-3-[(1R)-1-(4-chlorophenyl)-7-fluoro-5-[(1S)-1-hydroxy-1-(oxan-4-yl)propyl]-1-methoxy-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-2-methylpropanoic acid, or a tautomer, N-oxide, pharma- ceutically acceptable salt, or solvate thereof.

23. A pharmaceutical composition comprising an MDM2 antagonist for use in a method of treating a cancer patient, comprising: (a) the method comprises: (i) determining that a patient sample is deficient in one or more genes or gene products in one or more DNA damage repair (DDR) pathways or that the cancer has at least one loss-of-function mutation in a DDR pathway gene, wherein the one or more DDR pathway genes or gene products include BRCA1 and / or BRCA2; (ii) administering to said patient an effective amount of an MDM2 antagonist. or (b) an MDM2 antagonist is used in combination with an anti-cancer agent, e.g., a DNA damaging agent or a DNA repair inhibitor, in a method of treating cancer, wherein the cancer has low levels of one or more genes or gene products in one or more DNA damage repair (DDR) pathways, or the cancer has a detectable loss-of-function mutation in any DDR pathway gene, wherein the one or more DDR pathway genes or gene products include BRCA1 and / or BRCA2; or (c) the method comprising: (i) selecting a patient having a low level of a DDR pathway gene or gene product in a biological sample obtained from said patient, wherein said DDR pathway genes or gene products include BRCA1 and / or BRCA2; and (ii) administering to the patient selected in step (a) a therapeutically effective amount of an MDM2 antagonist and an anti-cancer agent, e.g., a DNA damaging agent or a DNA repair inhibitor. or (d) the cancer is deficient in one or more genes or gene products in one or more DNA damage repair (DDR) pathways, or the cancer has at least one loss-of-function mutation in at least one DDR pathway gene, and the one or more DDR genes or gene products do not consist of ATM and / or ATR; or the cancer is deficient in two or more genes or gene products in one or more DNA damage repair (DDR) pathways, or the cancer has at least one loss-of-function mutation in at least two DDR pathway genes, and optionally The DDR pathway is a. the homology directed repair (HRR) pathway, provided that said DDR gene or gene product does not consist of ATM and / or ATR; b. Non-homologous end joining (NHEJ) pathway; c. the mismatch repair (MMR) pathway; d. the Fanconi Anemia (FA) pathway; and / or e. Base excision repair (BER) pathway or (e) the method comprises administering to the subject an MDM2 antagonist, wherein the cancer is deficient in one or more genes or gene products in one or more DNA damage repair (DDR) pathways, or the cancer has at least one loss-of-function mutation in at least one DDR pathway gene, and the one or more DDR pathway genes or gene products include BRCA1 and / or BRCA2; or (f) the cancer exhibits loss of one or more DDR pathway genes, gene products or activities, and the method comprises administering to the patient an MDM2 antagonist, wherein the one or more DDR pathway genes include BRCA1 and / or BRCA2; or (g) the cancer has normal or elevated levels of one or more genes or gene products in one or more DNA damage repair (DDR) pathways, or the cancer has no detectable loss-of-function mutations in any DDR pathway genes; or the method comprises administering to the patient an MDM2 antagonist in combination with an agent for inducing sensitivity to the MDM2 antagonist; (h) the patient has a cancer susceptible to MDM2 antagonist treatment, and the method comprises: (i) determining that a sample from a patient is deficient in one or more genes or gene products in one or more DNA damage repair (DDR) pathways or that the cancer has at least one loss-of-function mutation in a DDR pathway gene, wherein the one or more DDR pathway genes or gene products include BRCA1 and / or BRCA2; (ii) administering to said patient an effective amount of an MDM2 antagonist.

23. A pharmaceutical composition comprising:

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