Methods of treating cancer using chk1 inhibitors

Abstract

To provide methods of treating cancer in a subject.SOLUTION: A method comprises administering a therapeutically effective amount of a Checkpoint kinase 1 (Chk1) inhibitor to a subject identified as having cancer cells with a genetic abnormality in one or more genes selected from cell cycle regulation genes, replication stress genes, DNA damage response and repair network genes, and oncogenic driver genes.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 847,810, filed May 14, 2019, and U.S. Provisional Application No. 62 / 855,910, filed May 31, 2019, both of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Introduction Cells activate signaling pathways when DNA is damaged. The signal activates the cell cycle machinery to induce DNA repair and / or cell death and reduce proliferation. Checkpoint kinase 1 (Chk1) serves as an important bridge within the cell when DNA damage is detected. See Cancer Biology & Therapy (2004) 3:3, 305 - 313, which is hereby incorporated by reference. Chk1 plays a role in regulating a number of broad - ranging cellular functions, including immune and inflammatory responses, spindle formation, DNA damage signaling, and generally cell apoptosis. Chk1 inhibitors prevent DNA damage - induced cell cycle arrest in the S phase and / or G2 / M phase. Currently, there are no Chk1 inhibitors approved for the treatment of inhibiting tumor growth.

Summary of the Invention

[0003] The present disclosure provides methods of using a checkpoint kinase (Chk1) inhibitor in the treatment of cancer in a subject having at least intermediate tumor mutational burden (TMB) or having a genetic abnormality in one or more specific genes associated with replication stress. Accordingly, methods of treating cancer in a subject having at least intermediate tumor mutational burden (TMB-I) are provided. Also provided are methods of treating cancer in a subject having a genetic abnormality in one or more specific genes selected from cell cycle regulatory genes, replication stress genes, oncogenic driver mutations, and DNA damage response genes and repair network genes. Methods of selecting a subject for Chk1 inhibitor therapy are provided. The methods can include administering to the subject an effective amount of the SRA737 compound, in combination in some cases with a low dose of gemcitabine.

[0004] These and other features, aspects, and advantages of the present disclosure will become better understood with reference to the following description and the accompanying drawings.

Brief Description of the Drawings

[0005]

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DETAILED DESCRIPTION OF THE INVENTION

[0006] The present disclosure provides a method of treating cancer in a subject having at least an intermediate amount of tumor gene mutations (TMB) or having a genetic abnormality in one or more specific genes associated with replication stress, using a checkpoint kinase 1 (Chk1) inhibitor. The genetic abnormality may be found in one or more genes selected from the class of cell cycle regulatory genes, replication stress genes, oncogenic driver mutations, and / or DNA damage response network genes and repair network genes. Also provided is a method of selecting a subject for Chk1 inhibition therapy. The method may in some cases include administering an effective amount of an SRA737 compound to the subject in combination with a low dose of gemcitabine.

[0007] Before describing the present invention in more detail, it is to be understood that the invention is not limited to the specific embodiments described, and accordingly, it goes without saying that it can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention is defined only by the appended claims.

[0008] Where a range of values is provided, each intervening value, to one tenth of the unit of the lower limit value, between the upper and lower limit values of that range, as well as any other stated value or intervening value in the stated range, is understood to be encompassed by the present invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, and are also included herein, subject to any specific exclusion limits set forth within the stated range. Where the stated range includes one or both of the limiting values, ranges excluding one or both of those included limiting values are also included in the present invention.

[0009] Certain ranges are presented herein with the term "about" preceding the numerical value. The term "about" is used herein to provide a precise backing for the exact number preceding the term "about", and for a number that is near or approximate to the number preceding the term "about". In determining whether a number is near or approximate to a clearly recited number, a number that is not described as near or approximate may, in the context in which it is presented, be a number that provides a substantially equivalent value to the clearly recited number.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, representative and exemplary methods and materials are described herein.

[0011] All publications and patents cited in this specification are hereby incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials related to the cited publications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention has no right to antedate such publication by virtue of prior invention. Further, the dates of the publications provided may be different from the actual publication dates which may need to be independently confirmed.

[0012] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude optional elements. Accordingly, this description is intended to serve as a basis for use of exclusive terminology such as "solely", "only", etc. or the use of "negative" limitations in the recitation of claim elements.

[0013] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from, or combined with, the features of any of several other embodiments without departing from the scope or spirit of the invention. Any recited method can be performed in the order of recited events or in any other order that is logically possible.

[0014] The apparatus and method are described, or are to be described, for reasons of grammatical fluidity with functional explanations, but unless expressly stated under 35 U.S.C. § 112, the claims should not be construed as necessarily being limited absolutely by the construction of "means" or "step" limitations, and the meaning of the definition provided by the claims and the full scope of equivalents should be recognized under the judicially created doctrine of equivalents, and it should be expressly understood that where the claims are expressly stated under 35 U.S.C. § 112, full statutory equivalents should be recognized under 35 U.S.C. § 112.

[0015] Method of treating cancer Aspects of the present disclosure include methods of treating cancer using a Chk1 inhibitor in a subject identified as having at least intermediate TMB, or genetic abnormalities in one or more specific genes associated with replication stress, wherein the biomarker can indicate sensitivity to Chk1 inhibitor therapy. The methods of the subject may optionally include administration of a Chk1 inhibitor such as SRA737 in combination therapy with low-dose gemcitabine (LDG). Also provided are methods of selecting a subject who would benefit from Chk1 inhibition (Chk1i) therapy.

[0016] The present disclosure presents the results of first-in-human clinical trials of Chk1 inhibitor therapy, namely SRA737 monotherapy (Study SRA737-01), and SRA737 combination therapy with low-dose gemcitabine (Studies SRA737-02, SRA737+LDG). Multiple solid tumor indications were evaluated based on the prevalence of Chk1i sensitivity and / or replication stress (RS)-associated tumor genomics. Tumors carrying RS-related genetic changes and / or subjects with other histories related to Chk1i sensitivity were evaluated. Clinical study response data and tumor genomics of individual patients were analyzed to identify genomic signatures specific to indications showing enhanced response to SRA737 therapy. Accordingly, the present disclosure provides methods of treating cancer in a subject having specific genetic abnormalities using Chk1 inhibitor therapy such as SRA737 in combination with low-dose gemcitabine.

[0017] Replication stress driver gene This disclosure provides genetic abnormalities in replication stress (RS) driver genes that can exhibit responsiveness to Chk1 inhibition therapy. Aspects of the disclosure include methods of treating cancer with a Chk1 inhibitor in a subject identified as having one or more such genetic abnormalities in a gene that exhibits sensitivity to Chk1 inhibition (e.g., as described herein).

[0018] Chk1 is a master regulator of replication stress (RS). Chk1 is a serine / threonine protein kinase within the DNA damage response (DDR) network that can reduce elevated replication stress in certain tumor cells. Replication stress (RS) manifests by deceleration and stalling of replication forks, resulting in vulnerable single-stranded DNA that is susceptible to damage. An increase in RS leads to genomic instability and confers certain growth and survival advantages to tumor cells, but if not properly managed, can result in extensive DNA damage and cell death. Consequently, tumor cells enhance their dependence on Chk1 to manage elevated endogenous RS. Cancer cells with higher RS may be more sensitive to Chk1 inhibitor therapy.

[0019] Drivers of RS that can be used in the selection of a subject for treatment according to the subject methods include genetic abnormalities in tumor suppressor genes, oncogenic drivers, and / or DNA damage response network genes and repair network genes. Tumors harboring defects in these functional gene networks may have higher levels of endogenous RS due to dysregulation of cell cycle control, increased proliferative demands, and / or increased genomic instability. These RS driver genes can be divided into several functional categories including G1 / S tumor suppressor genes, oncogenic drivers, and defective DNA damage response genes and repair genes.

[0020] The present disclosure provides RS driver genes that may lead to an increased dependence on Chk1 within target tumor cells. A subject identified as having such tumor cells can be treated with Chk1 therapy according to the methods described herein. In some cases, the Chk1 therapy utilized in the subject methods is a combination therapy of a Chk1 inhibitor and an exogenous RS inducer that depletes replication building blocks such as low-dose gemcitabine.

[0021] Accordingly, a patient selected for treatment according to the subject methods may have one or more genetic abnormalities in one or more of the RS-inducing genes described herein.

[0022] Classes of endogenous RS-inducing genes of interest include, but are not limited to, cell cycle dysregulation mutations (such as p53 pathway subclasses or G1 / S subclasses), oncogenic driver mutations (such as CCNE subclasses, MYC subclasses, and / or PI3K / AKT subclasses), and mutations in DNA damage response and repair networks (such as HR / NHEJ subclasses, FANC / BRCA replication fork subclasses, chromatin subclasses, and / or mismatch repair subclasses). See, for example, FIGS. 10 and 18.

[0023] RS-inducing genes include a class of cell cycle dysregulation. Cell cycle dysregulation genes of interest include, but are not limited to, p53 pathway subclasses and G1 / S subclasses.

[0024] The p53 tumor suppressor protein can function as a transcription factor to activate or suppress various target genes. Targets downstream of p53 regulate the pathways of cell cycle arrest, apoptosis, and DNA repair to maintain a dynamic balance between cell growth and arrest in response to factors including DNA damage, hypoxia (oxygen deficiency), and deficiencies in growth factors or nutrients. Specific target genes of a particular subclass of p53 cell cycle dysregulation that may increase tumor cell dependence on Chk1 include, but are not limited to, MDM2 (MDM2 proto-oncogene) and TP53 (tumor protein p53).

[0025] The G1 / S transition is a stage of the cell cycle between the G1 growth phase and the S phase of DNA replication. It is regulated by cell cycle checkpoints to ensure the integrity of the cell cycle, and the subsequent S phase may arrest in response to inappropriately or partially replicated DNA. Specific target genes of a particular subclass of G1 / S cell cycle dysregulation that may increase tumor cell dependence on Chk1 include, but are not limited to, RB1 (RB transcriptional corepressor 1), CDKN1A / B (cyclin-dependent kinase inhibitor 1A / 1B), and CDKN2A / B / C (cyclin-dependent kinase inhibitor 2A / 2B / 2C).

[0026] RS-induced genes include a class of oncogenic drivers. The oncogenic driver genes of interest include, but are not limited to, the CCNE (cyclin E) subclass, the MYC subclass, and / or the PI3K / AKT subclass. Cyclin E forms a complex with cyclin-dependent kinase (CDK2). Cyclin E / CDK2 regulates multiple cell processes by phosphorylating a number of downstream proteins and plays a role in the G1 and G1-S phase transitions. Overexpression of cyclin E (CCNE) may be associated with tumorigenesis. Myc (or MYC) is a group of regulatory genes and proto-oncogenes encoding transcription factors and includes three related human genes, c-myc, l-myc, and n-myc.

[0027] Protein kinase B, also known as PKB or AKT, is a serine / threonine-specific protein kinase that plays an important role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. The AKT signaling cascade is activated by receptor tyrosine kinases, integrins, B-cell and T-cell receptors, cytokine receptors, G-protein-coupled receptors, and other stimuli that induce the production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K). Phosphoinositide 3-kinase (PI3K) is a group of related intracellular signaling enzymes. Dysregulation of the PI3K / AKT pathway is associated with several human diseases including cancer.

[0028] Endogenous RS-inducing genes include classes of mutations in DNA damage response networks and repair networks. The DNA damage response network genes and repair network genes of interest include, but are not limited to, the HR / NHEJ subclass, the FA / BRCA (Fanconi anemia / breast cancer susceptibility protein) replication fork subclass, the chromatin subclass, and the mismatch repair subclass. In some cases, it should be understood that a particular gene of interest may be considered part of two subclasses where there is an intersection of two gene networks (e.g., the HR / NHEJ subclass and the FA / BRCA subclass as shown in FIG. 18).

[0029] The two major pathways for repairing DNA double-strand breaks are homologous recombination (HR) and non-homologous end joining (NHEJ). Several genes are present in higher eukaryotes to regulate both pathways. The HR / NHEJ subclass mutations of interest include, but are not limited to, PALB2, ATM, BRCA1 / 2, RAD51B, RAD51C, and PRKDC, as well as ATR.

[0030] The FA / BRCA (Fanconi anemia / breast cancer susceptibility protein) replication fork subclass of genes of interest includes, but is not limited to, PRKDC, ATR, BRCA1 / 2, CDK12, the FANC genes include FANC A, D2, E, G, I, or M, and the RAD genes include RAD52, RAD50, RAD51B, RAD51C, and RAD54L.

[0031] The chromatin subclass of genes of interest includes, but is not limited to, MLL2, ARID1A, and ARID1B. The mismatch repair subclass of genes of interest includes, but is not limited to, MLH1, MSH2, MSH6, and PMS2. The DNA polymerase (DNA pol) subclass of genes of interest includes, but is not limited to, POLD1 and POLE.

[0032] Any convenient genetic abnormality of any of the target genes disclosed herein can be observed and considered a desired marker of sensitivity. The genetic abnormality of interest may be a change, amplification, overexpression, or underexpression of the target gene. Various genetic abnormalities within the RS-inducing genes can be targeted. In some cases, the genetic abnormality is a gene change, such as a mutation.

[0033] In some embodiments of the method, one or more genes are selected from cell cycle regulatory genes associated with the G1 / S checkpoint and / or the p53 pathway. In certain examples, one or more genes are selected from MDM2, TP53, RB1, CDKN1A / B, and CDKN2A / B / C.

[0034] In some embodiments of the method, one or more genes are selected from replication stress genes related to Chk1 pathway sensitivity. In certain examples, one or more genes are selected from Chk1 and ATR.

[0035] In some embodiments of the method, one or more genes are selected from DNA damage response genes and repair genes associated with homologous recombination (HR), non-homologous end joining (NHEJ), Fanconi anemia (FA), or mismatch repair, and genes encoding chromatin or DNA polymerase. In certain examples, one or more genes are selected from PALB2, ATM, BRCA1 / A2, RAD51B, RAD51C, PRKDC, CDK12, FANCA, FANCD2, FANCE, FANCG, FANCI, FANCM, RAD52, RAD50, RAD51C, RAD54L, MLL2, ARID1A, ARID1B, MLH1, MSH2, MSH6, PMS2, POLD1, and POLE.

[0036] In some embodiments of the method, one or more genes are oncogenic driver genes of the following subclasses, namely CCNE, MYC, and PI3K / AKT. In certain examples, one or more genes are of the CCNE subclass and are selected from CCNE1, FBXW7, and PARK2.

[0037] In some embodiments of the method, one or more genes are of the PI3K / AKT subclass and are selected from PIK3CA, PTEN, AKT1, AKT2, and AKT3.

[0038] In some embodiments of the method, one or more genes are of the MYC subclass and are selected from MYC, MYCN, and MYCL1.

[0039] In some embodiments of the method, the subject is identified as having cancer cells with genetic abnormalities in one or more genes (e.g., one, two, or more genes) selected from DNA damage response genes and repair genes of the FA / BRCA replication fork subclass. Figure 10 reveals that a subject receiving SRA737 monotherapy, whose tumor harbored FA / BRCA network mutations, showed favorable outcomes (DCR = 71%, DOS = 3.8 cycles). In certain examples, one or more genes include ATR or PRKDC.

[0040] In some embodiments of the method, the subject is identified as having cancer cells with genetic abnormalities in two or more genes selected from oncogenic driver genes of the PI3K / AKT subclass and DNA damage response genes and repair genes of the FA / BRCA replication fork subclass. FIG. 18 shows a summary of responses and disease control rates (DCR) that vary across the investigated gene network. This table shows that the DCR and response rates to SRA737+LDG therapy for subjects with genetic abnormalities in the PI3K / ARK subclass were 75% and 13%, respectively, while those for subjects with genetic abnormalities in the FA / BRCA replication fork subclass were 81% and 25%, respectively. See also FIGS. 19-20 showing waterfall plots of SRA737+LDG therapy for various subjects with such genetic abnormalities. In certain embodiments of the method, the two or more genes are selected from AKT, PIK3CA, PTEN, ATR, PRKDC, BRCA1, BRCA2, CDK12, FANCA, FANCD2, FANCE, FANCG, FANCI, FANCM, RAD52, RAD50, RAD51C, and RAD54L.

[0041] In some embodiments of the method, the subject is further identified as having cancer that is positive for human papillomavirus (HPV). In some embodiments of the method, the subject is further identified as having at least an intermediate tumor mutational burden (TMB) (e.g., as described herein).

[0042] Aspects of the present disclosure include determining the presence or absence of genetic abnormalities within the RAS gene (e.g., KRAS) in a sample obtained from a subject. A subject having wild-type RAS can be selected for treatment according to the methods of the present disclosure. In some cases, subjects having cancer with a genetic abnormality (e.g., a mutation) in the RAS gene are excluded from treatment. In some embodiments, tumor cells of a subject treated according to the methods of the subject matter are identified as having wild-type RAS. Thus, a subject selected for treatment may be a subject having tumor cells lacking any mutations in the KRAS gene, NRAS gene, and / or HRAS gene. In some cases, the KRAS mutation of interest is at G12, G13, G34, G35, G37, G38, Q61, K117, or A146. In certain cases, the KRAS mutation of interest is G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, G34T, G34A, G34C, G35T, G35C, G35A, G37T, G37C, G37A, G48T, G38A, G38A, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T or A146V.

[0043] Genetic abnormalities can be evaluated in a sample of a subject using any convenient method. Various assays can be adapted for use in determining whether a change, amplification, overexpression, or underexpression of one or more genes is present in the cancer of the subject. For example, detection of gene substitutions, insertions-deletions (indels), and copy number alterations (CNA) shown in Table 1 can be achieved using the FoundationOne CDx assay. The methods of interest include the methods described in US2019 / 0085403, the disclosure of which is incorporated herein by reference.

[0044] In some examples, a sample is obtained from a subject to assess or determine genetic abnormalities in one or more genes of interest. The sample can be selected from a tissue sample, a whole blood sample, a plasma sample, and a serum sample. In some cases, the sample includes tissue obtained from the subject. In a specific example, the sample includes tumor cells. In some cases, the sample obtained from the subject includes at least 20% tumor cells.

[0045] Intermediate tumor gene mutation amount A biomarker of genetic abnormalities that may show responsiveness to Chk1 inhibition therapy is the tumor mutational burden (TMB). The TMB of a subject's cancer is based on the number of somatic mutations identified within the cancer genome. TMB values vary across a population of cancer subjects and can be characterized according to categories of low TMB levels, intermediate TMB levels, and high TMB levels. Based on the results of the clinical studies described herein, subjects with intermediate or higher levels of TMB were determined to have better clinical outcomes when treated with Chk1 inhibition therapy than subjects with low TMB levels.

[0046] Any convenient method for assessing TMB can be used in conjunction with the subject method. The methods of interest include the methods described in US2019 / 0085403, the disclosure of which is incorporated herein by reference. It should be understood that the absolute value of TMB may vary depending on the method used to assess the TMB of the cancer cells of an individual cancer subject. In some cases, the entire sequence of the cancer genome can be determined to identify the total number of somatic mutations. In certain cases, a subset of genes of interest within the cancer genome is targeted for assessment of somatic mutations. For example, described in the following experimental section is the FoundationOne CDx™ (F1CDx) assay, which is a next-generation sequencing-based in vitro diagnosis for genomic signatures including tumor mutational burden (TMB) that detects changes (indels) due to substitutions, insertions, and deletions in up to 324 or more genes, as well as copy number alterations (CNAs), and uses DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens.

[0047] Chalmers et al ("Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden Genome Medicine, 2017, 9:34), which is incorporated herein by reference, compares the TMB values determined by a targeted comprehensive genomic profiling assay (FoundationOne assay) with the TMB values measured by whole exome sequencing (WES). Chalmers has shown that a CGP assay targeting approximately 1.1 Mb of the coding genome provides an accurate assessment of TMB comparable to whole exome sequencing.

[0048] It is retrospectively determined that subjects with intermediate levels of TMB and subjects with high levels of TMB may also have better clinical outcomes when treated with the Chk1 inhibition therapy described herein. Thus, in some embodiments, the subjects treated according to the methods of the present disclosure are subjects identified as having an intermediate level or more of tumor gene mutation amount (TMB-I), such as, for example, intermediate TMB or high TMB (TMB-I / H). Thus, TMB-I may be, for example, the level of somatic mutations in the cancer cells of a subject that exhibits responsiveness to Chk1 inhibitor therapy, as set forth explicitly herein.

[0049] Figures 27A - 27B show waterfall plots of the percent change in tumor diameter from baseline during treatment with SRA737 in combination with low - dose gemcitabine (LDG) in individual patients with various cancers (Figures 27A and 27B, SRA737 - 02). In the plots, the TMB status of individual patients is shown, for example, as the TMB in patient samples determined using the FoundationOne CDx™ (F1CDx) assay as described in the experimental section. For example, subjects with anal cancer having intermediate or high TMB levels showed improved responsiveness to treatment compared to subjects with low TMB. This data indicates that subjects with various cancers having intermediate TMB (TMB - I) or high TMB (TMB - H) can respond to treatment with SRA737 in combination with LDG. In some cases, high TMB corresponds to somatic mutations of about 20 or more per megabase pair (mutations / Mb). In some cases, intermediate TMB corresponds to about 6 - about 19 mutations / Mb. In a specific example, low TMB corresponds to about 5 or fewer mutations / Mb.

[0050] Determining whether a subject will benefit from Chk1 inhibitor therapy can be achieved, for example, by comparing the subject's TMB value to a reference TMB value, such as a cutoff value representing the intermediate TMB level versus the low TMB level. The reference TMB value may be based on a cutoff value that separates a first subset of subjects in a reference population from a second subset of subjects in the reference population based on a significant difference in the responsiveness of the subjects to treatment with a Chk1 inhibitor (as described, for example, herein). The first subset of subjects can be characterized as having low TMB and not responding to treatment. The second subset of subjects can be characterized as having intermediate or high TMB and responding to treatment. The reference TMB value represents the cutoff TMB value of subjects having intermediate TMB rather than low TMB, and this distinguishes the first and second subsets of subjects in the reference population. It should be understood that the reference TMB value may vary depending on the method used to measure TMB in the subject's cancer cells.

[0051] In some cases, the reference TMB value is about 6 somatic mutations per megabase (mutations / Mb), as determined using the FoundationOne assay (e.g., as described herein).

[0052] In certain embodiments, the method further comprises obtaining a sample (e.g., a tumor sample or a sample derived from a tumor) from a subject, either directly or indirectly, and evaluating the sample for mutational burden or TMB, as described herein. In some cases, the TMB is based on somatic mutations in a predetermined gene set.

[0053] In certain embodiments, determining the level of somatic mutations in the predetermined gene set described in Table 1 includes determining the level of somatic mutations in about 25 or more genes, such as about 50 or more, about 100 or more, about 150 or more, about 200 or more, about 250 or more, about 260 or more, about 270 or more, about 280 or more, about 290 or more, about 300 or more, about 310 or more, or all of the genes described in Table 1. In certain embodiments, the predetermined gene set evaluated (e.g., by the FoundationOne assay) is 500 or less, such as 450 or less, 400 or less, or 350 or less.

[0054] In some embodiments, determining the level of somatic mutations in the predetermined gene set described in Table 1 includes determining the number of somatic mutations per preselected unit, such as per megabase in the coding region of the predetermined gene set, for example, within the coding region of the predetermined gene set whose sequence has been determined.

[0055]

Table 1-1

[0056]

Table 1-2

[0057]

Table 1-3

[0058]

Table 1-4

[0059] In certain embodiments, determining that the number of somatic mutations in a given gene set is, for example, about 5 or less (such as 4.5 or less, 4 or less, 3.5 or less, 3 or less) somatic mutations per megabase pair within the coding region of the given gene set described in Table 1 indicates that the subject is a partial responder or non-responder to therapy, or has the potential to be a partial responder or non-responder to therapy.

[0060] In certain embodiments, determining that the number of somatic mutations in a given gene set is, for example, between about 6 and about 19 somatic mutations per megabase pair within the coding region of a given gene set selected from the genes described in Table 1, such as between about 7 and about 19, between about 8 and about 19, or between about 10 and about 19, indicates that the subject is a partial responder to therapy, or has the potential to be a partial responder (or will respond partially, or likely will respond partially).

[0061] In some embodiments of the method, the intermediate TMB is determined by comparing the TMB value determined from a subject's sample to a reference TMB value indicative of responsiveness to Chk1 inhibitor therapy. In some cases, the reference TMB value is about 5 or more (such as about 5.5 or more, about 6 or more, about 6.5 or more, about 7 or more, about 8 or more, about 9 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, or about 50 or more) somatic mutations per megabase pair (Mb) within the coding region of a given gene set. In certain examples, the reference TMB value is about 6 somatic mutations per megabase pair (Mb) of the coding sequence.

[0062] In some embodiments of the method, the reference TMB value is a reference range of TMB values representing tumor cells of a plurality of subjects having cancer, and a subject is classified as a subject who benefits from Chk1 inhibitor therapy when the TMB value determined from the sample is at least the 35th percentile (e.g., at least the 40th percentile, at least the 45th percentile, at least the 50th percentile) of the reference range of TMB values. In certain examples, the three categories of low, medium, and high TMB are determined based on the distribution of TMB values across the population of subjects of interest. In some cases, the cutoff between low TMB and medium TMB is determined to be at approximately the 33rd percentile of the distribution of TMB values. In some cases, the cutoff between medium and high TMB is determined to be at approximately the 66th percentile of the distribution of TMB values. In certain examples, subjects having an intermediate TMB value that is the median or mean TMB of the distribution, or less, are selected for treatment according to the methods of the subject matter. Thus, the method may further comprise determining a reference range or distribution of TMB values from a plurality of tumor cell samples obtained from a plurality of subjects having cancer.

[0063] Method Disclosed herein is a method of inhibiting tumor growth in a subject, such as a human, by administration of the Chk1 inhibitor SRA737. Detailed descriptions of the compounds, kits containing the compounds, and methods of using them are provided below.

[0064] Tumor inhibition The present disclosure is directed to methods of using an effective amount of the compound SRA737 to inhibit tumor progression, reduce the size of tumor aggregates, decrease the volume of tumors, and / or otherwise inhibit tumor growth. Also provided herein are methods of treating a underlying disease, such as cancer, and extending the survival of a subject.

[0065] In some embodiments, provided is a method of inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of SRA737. In some embodiments, the present disclosure provides a method of administering to a subject an effective amount of SRA737 to inhibit tumor growth, wherein the tumor growth is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% as measured by tumor volume. In some embodiments, the present disclosure provides a method of administering to a subject an effective amount of SRA737 to inhibit tumor growth, wherein the tumor growth is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% as measured by the absolute size of the tumor. In some embodiments, the present disclosure provides a method of administering to a subject an effective amount of SRA737 to inhibit tumor growth, wherein the tumor growth is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% as measured by the level of expression of a tumor marker of that type of tumor.

[0066] In some embodiments, a method of treating cancer is provided that includes administering to a subject having cancer an effective amount of an SRA737 compound. In some embodiments, a method of treating cancer is provided that includes administering to a subject having cancer an effective amount of an SRA737 compound, whereby tumor regression occurs. Regression is generally determined relative to a baseline measurement. Regression may be partial or complete. Regression can generally be measured by any assay useful for quantifying tumor size, volume, and / or growth, such as, for example, medical imaging techniques known in the art. Regression can be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% regression as measured by tumor volume. Regression can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% regression as measured by the absolute size of the tumor.Regression may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% regression as measured by the expression level of a tumor marker of that type of tumor. Regression may be 30% regression. Regression may be 30% regression as measured by any assay useful for quantifying the size, volume, and / or growth of a tumor, such as, for example, medical imaging techniques known in the art.

[0067] Also, the present disclosure is directed to methods of using an effective amount of compound SRA737 and a second effective amount of a further therapeutic agent to inhibit tumor progression, reduce the size of tumor aggregates, decrease tumor volume, and / or otherwise inhibit tumor growth. Also provided herein are methods of treating underlying diseases such as cancer, for example, and extending the survival of a subject. In some embodiments, a method of inhibiting tumor growth in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of SRA737 and a second effective amount of a further therapeutic agent. In some embodiments, the present disclosure provides a method of administering to a subject an effective amount of SRA737 and a second effective amount of a further therapeutic agent to inhibit tumor growth, wherein the tumor growth is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100%, as measured by tumor volume. In some embodiments, the present disclosure provides a method of administering to a subject an effective amount of SRA737 and a second effective amount of a further therapeutic agent to inhibit tumor growth, wherein the tumor growth is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100%, as measured by the absolute size of the tumor.In some embodiments, the present disclosure provides a method of administering to a subject an effective amount of SRA737 and a second effective amount of a further therapeutic agent to inhibit tumor growth, wherein the tumor growth is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% as measured by the expression level of a tumor marker of that type of tumor.

[0068] In some embodiments, provided is a method of treating cancer comprising administering to a subject having cancer an effective amount of an SRA737 compound and a second effective amount of a further therapeutic agent. In some embodiments, provided is a method of treating cancer comprising administering to a subject having cancer an effective amount of an SRA737 compound and a second effective amount of a further therapeutic agent, wherein tumor regression occurs. Regression is generally determined in comparison to a baseline measurement. Regression can be partial regression or complete regression. Regression can generally be measured by an assay useful for quantifying the size, volume, and / or growth of a tumor, such as, for example, medical imaging techniques known in the art. Regression can be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% regression as measured by tumor volume. Regression can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% regression as measured by the absolute size of the tumor.Regression may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% regression as measured by the expression level of a tumor marker of that type of tumor. Regression may be 30% regression. Regression may be 30% regression as measured by any assay useful for quantifying the size, volume, and / or growth of a tumor, such as, for example, medical imaging techniques known in the art.

[0069] Type of tumor In some aspects, the present disclosure provides a method of inhibiting tumor growth, wherein the tumor is colorectal cancer, ovarian cancer, high-grade serous ovarian cancer (HGSOC), non-small cell lung cancer (NSCLC), small cell lung cancer, lung adenocarcinoma, prostate cancer, castration-resistant prostate cancer, bile duct cancer, cholangiocarcinoma, melanoma, uterine cancer, thyroid cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, endometrial cancer, hepatocellular carcinoma, leukemia, lymphoma, non-Hodgkin lymphoma, myeloma, brain cancer, neuroblastoma, squamous cell carcinoma, head and neck squamous cell carcinoma (HNSCC), and anal squamous cell carcinoma (SCCA), anal genital cancer (e.g., anal cancer), rectal cancer, pancreatic cancer, urothelial cancer, sarcoma and soft tissue sarcoma, metastatic colorectal cancer (CRC), platinum-resistant or -intolerant HGSOC, advanced NSCLC, and metastatic castration-resistant prostate cancer (mCRPC), triple-negative breast cancer, invasive breast cancer, metastatic breast cancer, HER2-positive breast cancer and inflammatory breast cancer.

[0070] Accordingly, the present disclosure also provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of SRA737. In some embodiments, methods for treating cancer are disclosed, and the cancer is colorectal cancer, ovarian cancer, high-grade serous ovarian cancer (HGSOC), non-small cell lung cancer (NSCLC), small cell lung cancer, lung adenocarcinoma, prostate cancer, castration-resistant prostate cancer, cholangiocarcinoma, bile duct cancer, melanoma, uterine cancer, thyroid cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, endometrial cancer, hepatocellular carcinoma, leukemia, lymphoma, non-Hodgkin lymphoma, myeloma, brain cancer, neuroblastoma, squamous cell carcinoma, head and neck squamous cell carcinoma (HNSCC), and anal squamous cell carcinoma (SCCA), anal-genital cancer (e.g., anal cancer), rectal cancer, pancreatic cancer, urothelial cancer, sarcoma and soft tissue sarcoma, metastatic colorectal cancer (CRC), platinum-resistant or -intolerant HGSOC, advanced NSCLC, and metastatic castration-resistant prostate cancer (mCRPC), triple-negative breast cancer, invasive breast cancer, metastatic breast cancer, HER2-positive breast cancer and inflammatory breast cancer.

[0071] In certain embodiments of the subject methods, the cancer is selected from colon, colorectal, endometrial, esophageal, lung, mesothelioma, and prostate.

[0072] In certain embodiments of the subject methods, the cancer is squamous cell carcinoma. In certain embodiments of the subject methods, the cancer is selected from progressive / metastatic squamous cell carcinoma of the anus, penis, vagina, and vulva. In certain embodiments of the subject methods, the cancer is selected from anal-genital, rectal, ovarian, and cervical.

[0073] In some embodiments of the subject methods, the cancer is anal-genital cancer. In certain examples, the cancer is of the anus. In some cases, the cancer is of the rectum. In some cases, the cancer is of the cervix. In some cases, the cancer is of squamous cervical.

[0074] In some embodiments of the subject methods, the cancer is ovarian. In some cases, the ovarian cancer is high-grade serous ovarian cancer (HGSOC).

[0075] In one specific example of any of the above cancers, the cancer is positive for human papillomavirus (HPV).

[0076] Clinical evaluation items Provided herein is a method for inhibiting tumor and / or cell proliferation in a subject, wherein the conditions of the method are that clinically relevant evaluation items are generated by the method.

[0077] Tumor growth occurs when one or more somatic cells grow and divide much more rapidly and increase the number of cells compared to the normal and healthy process of cell division. This phenomenon indicates that the cells are in a diseased state such as cancer or pre-cancer. Furthermore, tumor growth often occurs in individual stages before the aggregated cells form a tumor.

[0078] There are several methods that a skilled person can use to measure the cell replication rate. The overall metabolic activity inside the cell can be measured via labeled biological agents. For example, there are several commercially available dyes (e.g., MTT) that can penetrate cells and interact with specific enzymes and other factors to produce detectable products. Also, cell biomarkers can be measured inside the cell. For example, the BrdU assay can incorporate a thymidine derivative into cell DNA and detect it with an antibody. Proliferating cell nuclear antigen (PCNA) is another such biomarker for detection. In addition to tagging techniques, a person skilled in the art can also use, for example, microscopy or flow cytometry to enable the counting of cells.

[0079] In one aspect, cell replication is measured by clinical evaluation items including quality of life (QOL) score, duration of response (DOR), clinical benefit rate (CBR), patient-reported outcome (PRO), objective response rate (ORR) score, disease-free survival (DFS) or progression-free survival (PFS), time to progression (TTP), overall survival (OS), time to treatment failure (TTF), RECIST criteria, and / or complete remission. Clinical evaluation items can be determined using methods well known to those skilled in the art.

[0080] In some embodiments, the present disclosure provides a method in which, after administration of an effective amount of SRA737, tumor growth is reduced by 5, 10, 20, 40, 50, 60, 80, 90, 95, 97, 99, or 99.9% or less.

[0081] In some embodiments, the present disclosure provides a method in which the percentage reduction is calculated based on the measured value(s) of one or more clinical evaluation items.

[0082] In some embodiments, the present disclosure provides a method in which, after administration of an effective amount of SRA737, tumor growth is measured by an increase or decrease in the total cell number in an MTT assay or a change in the gene profile measured by a ctDNA assay and does not exceed or is reduced by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99, or 99.9%.

[0083] In some embodiments, the present disclosure provides a method in which, after administration of an effective amount of SRA737, tumor growth is reduced by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99, or 99.9%. In some embodiments, the present disclosure provides a method in which, after administration of an effective amount of SRA737, tumor growth is measured by an increase or decrease in the total cell number in an MTT assay or a change in the gene profile measured by a ctDNA assay and is reduced by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99, or 99.9%.

[0084] In some embodiments, as a result of administration, the present disclosure provides a method in which the value of IC 50 is less than 10 μM and / or the value of GI 50 is less than 1 μM. In some embodiments, as a result of administration, the present disclosure provides a method in which, at twenty-four (24) hours after administration, the value of IC 50 is less than 10 μM and / or the value of GI 50 is less than 1 μM. In some embodiments, as a result of administration, the present disclosure provides a method in which, at forty-eight (48) hours after administration, the value of IC 50A method is provided in which the value of 50 is less than 10 μM and / or the value of GI

[0085] In some embodiments, the present disclosure provides a method in which the AUC as a result of administration is at least 1, 10, 25, 50, 100, 200, 400, 600, 800, or 1000.

[0086] In some embodiments, the present disclosure provides a method in which, as a result of administration, the value of IC 50 is 0.001, 0.005, 0.01, 0.05, 0.1, 1, 3, 5, 10, 20, 40, 50, 60, 80, 90, 100, 200, 250, 300, 350, or 400 μM or less.

[0087] In some embodiments, the present disclosure provides a method in which, as a result of administration, the value of EC 50 is at least 0.01, 0.1, 1, 3, 5, 10, 20, 40, 50, 60, 80, 90, 100, 200, 250, 300, 350, or 400 μM.

[0088] In some embodiments, the present disclosure provides a method in which, as a result of administration, the therapeutic index (TI) value is in the range of about 1.001:1 to about 50:1, about 1.1:1 to about 15:1, about 1.2:1 to about 12:1, about 1.2:1 to about 10:1, about 1.2:1 to about 5:1, or about 1.2:1 to about 3:1.

[0089] In some embodiments, the present disclosure provides a method in which, as a result of administration, the value of GI 50 is at least 0.1 μM, 0.3 μM, 0.5 μM, 0.7 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, or 10 μM.

[0090] In some embodiments, the present disclosure provides a method in which, as a result of administration, the maximum observed response (maximum response) value is 0.1, 0.5, 1, 2 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, or 10 μM or less.

[0091] Tumor growth can be represented by the total tumor volume or total tumor size. Generally, and even specific to certain tumor models, there are formulas that can be used to calculate tumor volume based on the assumption by those skilled in the art that solid tumors are more or less spherical. In this regard, those skilled in the art can use ultrasonic images, manual or digital calipers, ultrasound examinations, computed tomography (CT), microCT, etc. to measure tumor volume. 18Experimental tools such as F-FDG-microPET or magnetic resonance imaging (MRI) can be used. For example, see Monga SP, Wadleigh R, Sharma A, et al. Intratumoral therapy of cisplatin / epinephrine injectable gel for palliation in patients with obstructive esophageal cancer. Am. J. Clin. Oncol. 2000;23(4):386-392, Mary M. Tomayko C., Patrick Reynolds, 1989. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemotherapy and Pharmacology, Volume 24, Issue 3, pp 148-154, E Richtig, G Langmann, K Mullner, G Richtig and J Smolle, 2004. Calculated tumour volume as a prognostic parameter for survival in choroidal melanomas. Eye (2004) 18, 619-623, Jensen et al. BMC Medical Imaging 2008. 8:16, Tomayko et al. Cancer Chemotherapy and Pharmacology September 1989, Volume 24, Issue 3, pp 148-154, and Faustino-Rocha et al. Lab Anim (NY). 2013 Jun;42(6):217-24, each of which is incorporated herein by reference in its entirety. In an exemplary example, tumor growth and / or tumor size can be measured as the sum of the diameters of all target lesions (longest for non-nodal lesions and short axis for nodal lesions), and generally can be calculated and reported as the baseline sum diameter.The baseline total diameter can generally be used as a criterion to further characterize any objective tumor regression in a measurable dimension of the disease.

[0092] In some embodiments, the present disclosure provides methods that lead to a reduction in tumor size of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99, or 99.9% upon administration of an effective amount of SRA737. In some embodiments, the present disclosure provides methods that lead to a reduction in tumor size of at least 30% upon administration of an effective amount of SRA737. In some embodiments, the present disclosure provides methods that lead to a decrease in tumor volume of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99, or 99.9% upon administration of an effective amount of SRA737. In some embodiments, the present disclosure provides methods that lead to a decrease in tumor volume of at least 30% upon administration of an effective amount of SRA737. In some embodiments, the present disclosure provides methods that lead to a decrease in tumor volume or tumor size one (1), two (2), three (3), four (4), six (6), eight (8), twelve (12), sixteen (16), twenty (20), twenty-four (24), thirty-six (36), or fifty-two (52) weeks after administration. In some embodiments, the present disclosure provides methods that lead to a decrease in tumor volume or tumor size of at least 30% one (1), two (2), three (3), four (4), six (6), eight (8), twelve (12), sixteen (16), twenty (20), twenty-four (24), thirty-six (36), or fifty-two (52) weeks after administration. The decrease in tumor volume or tumor size can be measured by medical imaging techniques. The decrease in tumor volume or tumor size is generally determined in comparison to a baseline measurement.

[0093] Subject The present disclosure provides for administering an effective amount of SRA737 to a subject in need thereof, including a subject identified as having a genetic abnormality or biomarker of interest (e.g., as described herein). The present disclosure provides for administering an effective amount of SRA737 to a subject in need thereof in combination therapy with an additional therapeutic agent. In some embodiments, the subject's tumor is tested by genetic testing and / or sequencing prior to administration. In some embodiments, the subject's tumor is tested by genetic testing and / or sequencing after administration. In some embodiments, the subject's tumor is tested both after and prior to administration. In some embodiments, the subject's healthy cells are tested by genetic testing and / or sequencing before, after, or both. In some embodiments, the subject's tumor is tested by other biopsies or assays to determine the expression levels of specific biomarkers. In some embodiments, the subject's tumor is tested by both genetic testing and / or sequencing and other biomarker tests or assays.

[0094] In some embodiments, the present disclosure provides a method wherein the subject is a mammal. In some embodiments, the present disclosure provides a method wherein the subject is a primate.

[0095] In some embodiments, the present disclosure provides a method wherein the subject is a mouse.

[0096] In some embodiments, the present disclosure provides a method wherein the subject is a human.

[0097] In some embodiments, the present disclosure provides a method wherein the subject is a human having a tumor with a gene mutation in one or more of the following genes, namely, a tumor suppressor gene, a DNA damage repair gene, a replication stress gene, or an oncogenic driver gene. In some embodiments, the present disclosure provides a method wherein the subject has a cancer in which the cancer cells have a gene mutation in one or more of the following genes, namely, a tumor suppressor gene, a DNA damage repair gene, a replication stress gene, or an oncogenic driver gene.

[0098] In some embodiments, the present disclosure provides a method for a human suffering from a cancer selected from the group consisting of colorectal cancer, ovarian cancer, high-grade serous ovarian cancer (HGSOC), non-small cell lung cancer (NSCLC), small cell lung cancer, lung adenocarcinoma, prostate cancer, castration-resistant prostate cancer, cholangiocarcinoma, bile duct cancer, melanoma, uterine cancer, thyroid cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, endometrial cancer, hepatocellular carcinoma, leukemia, lymphoma, non-Hodgkin lymphoma, myeloma, brain cancer, neuroblastoma, squamous cell carcinoma, head and neck squamous cell carcinoma (HNSCC), and anal squamous cell carcinoma (SCCA), anal genital cancer (e.g., anal cancer), rectal cancer, pancreatic cancer, urothelial cancer, sarcoma and soft tissue sarcoma, metastatic colorectal cancer (CRC), platinum-resistant or -intolerant HGSOC, advanced NSCLC, and metastatic castration-resistant prostate cancer (mCRPC), triple-negative breast cancer, invasive breast cancer, metastatic breast cancer, HER2-positive breast cancer and inflammatory breast cancer. In certain embodiments, the tumor is in a human suffering from a cancer selected from the colon, colorectal, endometrial, esophageal, lung, mesothelioma, and prostate.

[0099] In certain embodiments, the tumor is in a human suffering from a cancer that is squamous cell carcinoma. In certain embodiments, the tumor is in a human suffering from a cancer selected from progressive / metastatic squamous cell carcinoma of the anus, penis, vagina, and vulva. In certain embodiments, the tumor is in a human suffering from a cancer selected from those of the anogenital, rectal, ovarian, and cervical. In some embodiments, the tumor is in a human suffering from anogenital cancer. In a specific example, the cancer is of the anus. In some cases, the cancer is of the rectum. In some cases, the cancer is of the cervix. In some cases, the cancer is of the squamous cervix. In some embodiments, the tumor is in a human suffering from a cancer that is of the ovary. In some cases, the ovarian cancer is high-grade serous ovarian cancer (HGSOC).

[0100] In some embodiments, the subject has the following. a. A histologically or cytologically proven progressive malignant tumor of the following type, for which no other conventional therapy is considered appropriate i. High-grade serous ovarian cancer (HGSOC) 1. Histologically confirmed high-grade serous ovarian cancer, fallopian tube cancer, or primary peritoneal cancer 2. Platinum-resistant or refractory disease, or if the subject is intolerant to platinum therapy ii. Small cell lung cancer 1. Unless otherwise approved by the sponsor of the clinical trial, generally, the subject has received at least one but no more than three prior treatments for progressive disease. iii. Soft tissue sarcoma 1. Includes undifferentiated pleomorphic sarcoma / malignant fibrous histiocytoma (MFH) (including high-grade spindle cell sarcoma / polymorphic liposarcoma), leiomyosarcoma, and dedifferentiated liposarcoma. Other types of STS may be eligible with the approval of the sponsor of the clinical trial. 2. Unless otherwise approved by the sponsor of the clinical trial, generally, the subject has received at least one but no more than three prior treatments for progressive disease. iv. Cervical cancer / anal genital cancer 1. Includes all cervical cancers and progressive / metastatic squamous cell carcinomas of the anus, penis, vagina, and vulva 2. Unless otherwise approved by the sponsor of the clinical trial, generally, the subject has received at least one but no more than three prior treatments for progressive disease. v. Urothelial cancer 1. Histologically confirmed locally advanced and unresectable or metastatic urothelial cancer of the bladder, upper ureter, or urethra 2. Generally, the subject has received at least one but no more than three prior treatments for progressive disease. b. Measurable disease according to RECIST v1.1 (see below) c. The subject has predicted sensitivity to Chk1 inhibition based on factors including gene profiling of tumor tissue or ctDNA, HPV status, and germline BRCA1 and BRCA2 gene status. All subjects have gene profiling from tumor tissue or ctDNA, which is performed prospectively if profiling is required to assess Chk1 sensitivity or retrospectively otherwise. i. For subjects with HGSOC, a documented description of the wild-type status of somatic or germline BRCA1 and BRCA2 confers eligibility without the need for prospective gene profiling. If a description of the BRCA status is not available, gene profiling may be performed prospectively to determine eligibility. ii. Subjects with SCLC are eligible without the need for prospective gene profiling based on the very high prevalence of cancer-related changes in tumor suppressor genes (e.g., TP53 and RB1) in this population. iii. For subjects with STS and all other subjects for whom gene profiling is performed prospectively, eligibility is determined by review by the trial sponsor of genetic abnormalities detected in genes in the following categories. Key tumor suppressor genes that regulate G1 cell cycle progression / arrest, such as RB1 and TP53. For related cancers, the status of positive human papillomavirus (HPV) is also considered for eligibility. a. DNA damage response pathways, including ATM, BRCA1, and BRCA2, mismatch repair gene alterations, and / or high microsatellite instability b. Genetic indicators of replication stress, such as gain of function / amplification of Chk1 or ATR or other related genes c. Oncogenic drivers, such as MYC and CCNE1 iv. For subjects with anal-genital cancer, a known HPV-positive status confers eligibility without the need for prospective gene profiling. If the HPV status is unknown or negative, gene profiling (or an HPV test if appropriate) may be performed prospectively to determine eligibility. Subjects with cervical cancer or anal squamous cell carcinoma are eligible without the need for prospective gene profiling based on the very high prevalence of HPV positivity in these populations.

[0101] In some embodiments, the subject has one of the above-described histologically or cytologically proven progressive malignancies, and it is demonstrated that the tumor tissue or ctDNA harbors one or more mutations that are expected to confer sensitivity of the tumor to Chk1 inhibition. Eligibility can be determined by review by the sponsor of the genetic abnormalities detected in genes in the following categories. a. Key tumor suppressor genes that regulate the progression / arrest of the G1 cell cycle, such as RB1, TP53. In the case of related cancers, the positive human papillomavirus (HPV) status is also considered for eligibility. b. DNA damage response pathways including ATM, BRCA1, BRCA2, mismatch repair gene changes, and / or high microsatellite instability c. Genetic indicators of replication stress such as gain of function / amplification of Chk1 or ATR or other related genes d. Oncogenic drivers such as MYC, KRAS

[0102] In some embodiments, the subject is excluded based on the following criteria. a. Prior to administration of SRA737, received the following prior or current anticancer therapies within the indicated time frame and recovered from the toxicity. i. Radiation therapy, chemotherapy, PARP inhibitor, other targeted therapy, or other IMP within 2 weeks ii. Nitrosourea or mitomycin C within 6 weeks iii. Any prior treatment with a Chk1 inhibitor at any time point or prior treatment with an ATR inhibitor within 6 months b. Three or fewer prior treatment regimens for progressive disease (not applicable to the HGSOC expanded cohort) c. Other malignancies within the past 2 years, excluding appropriately treated tumors d. If, in the opinion of the study physician, the subject is very likely to experience clinically significant myelosuppression e. Onset of toxic symptoms during continuation of prior treatment greater than NCI-CTCAE grade 1 f. History of allergy to gemcitabine g. New or ongoing brain metastases. Subjects with brain metastases that are asymptomatic, stable on X-ray, and not treated with steroids over an 8-week period may be included with the approval of the sponsor of the clinical trial. h. High medical risk due to non-malignant systemic diseases i. Serologically positive for hepatitis B, hepatitis C, or HIV j. Severe heart disease, baseline left ventricular ejection fraction < 45%, history of myocardial ischemia within the past 6 months, or history of cardiac arrhythmia requiring treatment, unless approved by the sponsor of the clinical trial k. Previous bone marrow transplantation or extensive radiotherapy to the bone marrow exceeding 25% within the past 8 weeks l. Peanut allergy m. QTcF > 450 msec in adult males and > 470 msec in adult females n. Digestive tract (GI) dysfunction or GI diseases that may significantly modify the absorption of SRA737 o. Inability to swallow the capsule without chewing or crushing p. A participant in, or scheduled to participate in, another interventional clinical trial q. Any other condition that, in the opinion of the responsible investigator of the clinical trial, would make the subject an inappropriate candidate

[0103] Administration As disclosed herein, the method of the present invention comprises the administration of an effective amount of SRA737. In one embodiment, the effective amount of SRA737 is administered as monotherapy.

[0104] Also, as disclosed herein, the methods of the invention include combination therapies that administer an effective amount of SRA737 and coadminister a second effective amount of an additional therapeutic agent. Additional therapies include administering a chemotherapeutic agent, administering an antibody or antibody fragment (such as an immune checkpoint inhibitor), administering a radiotherapeutic agent, administering an exogenous inducer of replication stress, and administering combinations thereof, but are not limited thereto. Also, additional therapies include administering any one of gemcitabine, olaparib, niraparib, rucaparib, talazoparib, cisplatin, a ribonucleotide reductase inhibitor, etoposide, SN-38 / CPT-11, mitomycin C, and combinations thereof, but are not limited thereto. Coadministration includes methods in which SRA737 and the additional therapeutic agent are administered simultaneously, methods in which SRA737 and the additional therapeutic agent are administered sequentially, and methods in which one or both of SRA737 and the additional therapeutic agent are administered intermittently or continuously, or simultaneously, sequentially, intermittently, and / or continuously in any combination. Intermittent administration includes a first administration of a drug and then a later, separate administration of exactly the same drug, such that one of ordinary skill in the art will recognize that intermittent administration is not necessarily the same as continuous administration. Further, intermittent administration includes interrupting the first administration of a drug with the administration of another drug before the first drug is administered again, such that one of ordinary skill in the art will understand that intermittent administration includes continuous administration in some aspects. Further, one of ordinary skill in the art will also know that continuous administration can be achieved by several routes, including intravenous infusion or a feeding tube.

[0105] Further, and more generally, the term "coadministered" includes any and all ways in which the individual administrations of SRA737 and the additional therapeutic agent to a subject overlap during any time frame.

[0106] In one aspect, the frequency of administration of SRA737 and additional therapeutic agents to the subject includes, but is not limited to, Q1d, Q2d, Q3d, Q4d, Q5d, Q6d, Q7d, Q8d, Q9d, Q10d, Q14d, Q21d, Q28d, Q30d, Q90d, Q120d, Q240d, or Q365d. The term "QnD or qnd" refers to drug administration once every "n" days. For example, QD (or qd) refers to once daily or daily dosing, Q2D (or q2d) refers to dosing once every two days, Q7D refers to dosing once every seven days or weekly, Q5D refers to dosing once every five days, and so on. In one aspect, SRA737 and additional therapeutic agents are administered on different schedules.

[0107] In another aspect, the frequency of administration of SRA737 or additional therapeutic agents to the subject includes, but is not limited to, dosing for five days per week followed by two days of non-dosing, daily dosing for one week followed by one, two, or three weeks of non-dosing, daily dosing for two or three weeks followed by one or two weeks of non-dosing, twice-daily dosing, or dosing on the second and third days of a weekly cycle. In one aspect, SRA737 and additional therapeutic agents are administered on different schedules.

[0108] In one aspect, the present disclosure provides a method in which either or both of SRA737 and / or a further therapeutic agent, or any combination thereof, are administered intermittently. In one aspect, the present disclosure provides a method of administering to a subject either or both of SRA737 or a further therapeutic agent, or any combination thereof, having an administration interval with a delay of at least ten (10) minutes, fifteen (15) minutes, twenty (20) minutes, thirty (30) minutes, forty (40) minutes, sixty (60) minutes, two (2) hours, three (3) hours, four (4) hours, six (6) hours, eight (8) hours, ten (10) hours, twelve (12) hours, fourteen (14) hours, eighteen (18) hours, twenty-four (24) hours, thirty-six (36) hours, forty-eight (48) hours, three (3) days, four (4) days, five (5) days, six (6) days, seven (7) days, eight (8) days, nine (9) days, ten (10) days, eleven (11) days, twelve (12) days, thirteen (13) days, fourteen (14) days, three (3) weeks, or four (4) weeks. In such an aspect, the delayed administration follows a pattern in which either or both of SRA737 and / or a further therapeutic agent, or any combination thereof, are continuously administered for a given period of from about ten (10) minutes to about three hundred and sixty-five (365) days and then not administered for a given period of from about ten (10) minutes to about thirty (30) days. In one aspect, the present disclosure provides a method in which either SRA737 or any combination thereof is administered intermittently while the other is being continuously administered.

[0109] In one aspect, the present disclosure provides a method in which a combination of an effective amount of SRA737 and a further therapeutic agent of a second effective amount is administered continuously.

[0110] In one aspect, the present disclosure provides a method in which SRA737 and a further therapeutic agent are administered simultaneously. In one aspect, the present disclosure provides a method in which a combination of an effective amount of SRA737 and a second effective amount of a further therapeutic agent is administered sequentially. In such an aspect, the combination is also said to be "administered simultaneously". This is because the term includes any and all ways in which the subject is exposed to both components of the combination. However, such an aspect is not limited to combinations that are administered only in one formulation or composition. In some cases, the specific concentrations of SRA737 and the further therapeutic agent may be more advantageous for delivery at specific intervals, and thus the effective amount of SRA737 and the second effective amount of the further therapeutic agent may vary depending on the formulation being administered.

[0111] In some aspects, the present disclosure provides a method in which SRA737 and a further therapeutic agent are administered simultaneously or sequentially. In some aspects, the present disclosure provides a method in which an effective amount of SRA737 is administered sequentially after a second effective amount of a further therapeutic agent. In some aspects, the present disclosure provides a method in which a second effective amount of a further therapeutic agent is administered sequentially after an effective amount of SRA737.

[0112] In some aspects, the present disclosure provides a method in which the combination is administered in one formulation. In some aspects, the present disclosure provides a method in which the combination is administered in two (2) compositions in which an effective amount of SRA737 is administered in a formulation separate from the formulation of the second effective amount of the further therapeutic agent.

[0113] In some embodiments, the present disclosure provides a method in which an effective amount of SRA737 is administered continuously after a second effective amount of an additional therapeutic agent. In some embodiments, the present disclosure provides a method in which a second effective amount of an additional therapeutic agent is administered continuously after an effective amount of SRA737. In some embodiments, SRA737 and an additional therapeutic agent are administered, and then both SRA737 and the additional therapeutic agent are administered at least intermittently every twenty-four (24) hours. In some embodiments, SRA737 and the additional therapeutic agent are administered on an alternating every-other-day schedule without overlap. In some embodiments, the additional therapeutic agent is administered on day 1, and SRA737 is administered on days 2 and 3 of a weekly schedule.

[0114] In some embodiments, the present disclosure provides a method in which an effective amount of SRA737 is administered more than four (4) hours after a second effective amount of an additional therapeutic agent. In one embodiment, the present disclosure provides a method in which an effective amount of SRA737 is administered more than ten (10) minutes, more than fifteen (15) minutes, more than twenty (20) minutes, more than thirty (30) minutes, more than forty (40) minutes, more than sixty (60) minutes, more than one (1) hour, more than two (2) hours, more than four (4) hours, more than six (6) hours, more than eight (8) hours, more than ten (10) hours, more than twelve (12) hours, more than twenty-four (24) hours, more than two (2) days, more than four (4) days, more than six (6) days, more than eight (8) days, more than ten (10) days, more than twelve (12) days, more than fourteen (14) days, more than twenty-one (21) days, or more than thirty (30) days after a second effective amount of an additional therapeutic agent. In one embodiment, the present disclosure provides a method in which a second effective amount of an additional therapeutic agent is administered more than ten (10) minutes, more than fifteen (15) minutes, more than twenty (20) minutes, more than thirty (30) minutes, more than forty (40) minutes, more than sixty (60) minutes, more than one (1) hour, more than two (2) hours, more than four (4) hours, more than six (6) hours, more than eight (8) hours, more than ten (10) hours, more than twelve (12) hours, more than twenty-four (24) hours, more than two (2) days, more than four (4) days, more than six (6) days, more than eight (8) days, more than ten (10) days, more than twelve (12) days, more than fourteen (14) days, more than twenty-one (21) days, or more than thirty (30) days after an effective amount of SRA737.

[0115] In some embodiments, the present disclosure provides a method in which either or both of SRA737 and / or a further therapeutic agent, or any combination thereof, are administered by a route selected from the group consisting of intravenous, subcutaneous, dermal, oral, intramuscular, and intraperitoneal. In some embodiments, the present disclosure provides a method in which either or both of SRA737 and / or a further therapeutic agent, or any combination thereof, are administered intravenously. In some embodiments, the present disclosure provides a method in which either or both of SRA737 and / or a further therapeutic agent, or any combination thereof, are administered orally.

[0116] It will be understood by those skilled in the art that the unit dosage forms of the present disclosure can be administered orally in the same or different physical forms, i.e., capsules or tablets, and / or in liquid form, etc., via intravenous infusion, etc. Further, the unit dosage form for each administration may vary depending on the particular route of administration. There may be various dosage forms for either or both of SRA737 and the further therapeutic agent. Since different medical conditions may warrant different routes of administration, the same components of the combination of SRA737 and the further therapeutic agent described herein may be completely similar in composition and physical form, but may need to be administered in different ways and perhaps at different times to alleviate the medical condition. For example, medical conditions such as persistent nausea, especially with vomiting, may make it difficult to use an oral dosage form. In such cases, another unit dosage form, i.e., perhaps the same unit dosage form as other dosage forms used previously or subsequently, needs to be administered by an inhalation, buccal, sublingual, or suppository route instead of or in addition to the oral route. Due to issues involving various factors such as chemical stability or pharmacokinetics, a particular dosage form may be a requirement for a particular combination of SRA737 and the further therapeutic agent.

[0117] Therapeutically Effective Amounts and Unit Dosage Forms The present disclosure provides a method of treatment in which an effective amount of SRA737 is administered to a subject. The terms "effective amount" or "therapeutically effective amount" refer to an amount that is effective for improving the symptoms of a disease, such as an amount that is effective for inhibiting tumor growth. In some embodiments, the effective amount of SRA737 is below the maximum tolerated dose (MTD), below the highest non-severe toxic dose (HNSTD), and below the no-observed-adverse-effect level (NOAEL). In some embodiments, the effective amount of SRA737 is less than 2000 mg / day orally. In some embodiments, the effective amount of SRA737 is less than 1500 mg / day orally. In some embodiments, the effective amount of SRA737 is less than 1300 mg / day orally. In some embodiments, the effective amount of SRA737 is more than 600 mg / day orally. In some embodiments, the effective amount of SRA737 is from 600 to 2000 mg / day orally. In some embodiments, the effective amount of SRA737 is from 600 to 1500 mg / day orally. In some embodiments, the effective amount of SRA737 is from 600 to 1300 mg / day orally. In some embodiments, the effective amount of SRA737 is from 600 to 1000 mg / day orally. In some embodiments, the effective amount of SRA737 is 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, 1000 mg / day, 1100 mg / day, 1200 mg / day, 1300 mg / day, 1500 mg / day, or 2000 mg / day orally.

[0118] In certain embodiments of the present invention, an effective amount of SRA737 is administered to a subject as monotherapy. In some aspects, the effective amount of SRA737 monotherapy is below the maximum tolerated dose (MTD), below the highest non-severe toxic dose (HNSTD), or below the no-observed-adverse-effect level (NOAEL). In some aspects, the effective amount of SRA737 monotherapy is less than 2000 mg / day orally. In some aspects, the effective amount of SRA737 monotherapy is less than 1500 mg / day orally. In some aspects, the effective amount of SRA737 monotherapy is less than 1300 mg / day orally. In some aspects, the effective amount of SRA737 monotherapy is more than 600 mg / day orally. In some aspects, the effective amount of SRA737 monotherapy is 600 to 2000 mg / day orally. In some aspects, the effective amount of SRA737 monotherapy is 600 to 1500 mg / day orally. In some aspects, the effective amount of SRA737 monotherapy is 600 to 1300 mg / day orally. In some aspects, the effective amount of SRA737 monotherapy is 600 to 1000 mg / day orally. In some aspects, the effective amount of SRA737 monotherapy is 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, 1000 mg / day, 1100 mg / day, 1200 mg / day, 1300 mg / day, 1500 mg / day, or 2000 mg / day orally. In some aspects, the effective amount of SRA737 monotherapy is 600 mg / day. In some aspects, the effective amount of SRA737 monotherapy is 700 mg / day. In some aspects, the effective amount of SRA737 monotherapy is 800 mg / day. In some aspects, the effective amount of SRA737 monotherapy is 900 mg / day. In some aspects, the effective amount of SRA737 monotherapy is 1000 mg / day. In some aspects, the effective amount of SRA737 monotherapy is 1100 mg / day. In some aspects, the effective amount of SRA737 monotherapy is 1200 mg / day. In some aspects, the effective amount of SRA737 monotherapy is 1300 mg / day. In some aspects, the effective amount of SRA737 monotherapy is 1500 mg / day. In some aspects, the effective amount of SRA737 monotherapy is 2000 mg / day.

[0119] In certain embodiments of the present invention, an effective amount of SRA737 is administered to a subject as combination therapy. In some aspects, the effective amount of the SRA737 combination therapy is below the maximum tolerated dose (MTD), below the highest non-severe toxic dose (HNSTD), or below the no-observed-adverse-effect level (NOAEL). In some aspects, the effective amount of the SRA737 combination therapy is less than the effective amount of SRA737 monotherapy. In some aspects, the effective amount of the SRA737 combination therapy is less than 2000 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is less than 1500 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is less than 1300 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is less than 600 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is at least 300 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is at least 100 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is at least 600 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is 100 - 2000 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is 300 - 2000 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is 600 - 2000 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is 300 - 1500 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is 300 - 1300 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is 300 - 1000 mg / day orally. In some aspects, the effective amount of the SRA737 combination therapy is 100 mg / day, 150 mg / day, 200 mg / day, 300 mg / day, 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, 1000 mg / day, 1100 mg / day, 1200 mg / day, 1300 mg / day, 1500 mg / day, or 2000 mg / day orally.In some embodiments, the effective amount of the SRA737 combination therapy is 300 mg / day, 400 mg / day, 500 mg / day, 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, 1000 mg / day, 1100 mg / day, 1200 mg / day, 1300 mg / day, 1500 mg / day, or 2000 mg / day orally.

[0120] In some embodiments, the effective amount of the SRA737 combination therapy is 300 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 400 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 500 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 600 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 700 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 800 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 900 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1000 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1100 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1200 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 300 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 400 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 500 mg / day orally. In some embodiments, the effective amount of the SRA737 combination therapy is at least 600 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 700 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 800 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 900 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1000 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1100 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1200 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 300 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 400 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 500 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 600 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 700 mg / day or less.In some embodiments, the effective amount of the SRA737 combination therapy is 800 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 900 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1000 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1100 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1200 mg / day or less.

[0121] In some embodiments, the effective amount of the SRA737 combination therapy is 350 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 450 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 550 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 650 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 750 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 850 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 950 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1050 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1150 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1250 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 350 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 450 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 550 mg / day orally. In some embodiments, the effective amount of the SRA737 combination therapy is at least 650 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 750 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 850 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 950 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1050 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1150 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1250 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 350 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 450 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 550 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 650 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 750 mg / day or less.In some embodiments, the effective amount of the SRA737 combination therapy is 850 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 950 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1050 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1150 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1250 mg / day or less.

[0122] In some embodiments, the effective amount of the SRA737 combination therapy is 325 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 425 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 525 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 625 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 725 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 825 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 925 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1025 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1125 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1225 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 325 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 425 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 525 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 625 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 725 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 825 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 925 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1025 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1125 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1225 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 325 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 425 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 525 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 625 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 725 mg / day or less.In some embodiments, the effective amount of the SRA737 combination therapy is 825 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 925 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1025 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1125 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1225 mg / day or less.

[0123] In some embodiments, the effective amount of the SRA737 combination therapy is 375 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 475 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 575 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 675 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 775 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 875 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 975 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1075 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1175 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 1275 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 375 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 475 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 575 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 675 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 775 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 875 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 975 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1075 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1175 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is at least 1275 mg / day. In some embodiments, the effective amount of the SRA737 combination therapy is 375 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 475 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 575 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 675 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 775 mg / day or less.In some embodiments, the effective amount of the SRA737 combination therapy is 875 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 975 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1075 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1175 mg / day or less. In some embodiments, the effective amount of the SRA737 combination therapy is 1275 mg / day or less.

[0124] In certain embodiments of the invention, the effective amount of SRA737 is administered to a subject as a combination therapy with a second effective amount of a further therapeutic agent. In some embodiments, the second effective amount is in an amount from about 0.001 mg / kg to about 15 mg / kg. In some embodiments, the second effective amount of the further therapeutic agent is 0.001, 0.005, 0.010, 0.020, 0.050, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, or 15.0 mg / kg. In some embodiments, the second effective amount of the further therapeutic agent is 10 - 2000 mg / m 2 / day. In some embodiments, the second effective amount of the further therapeutic agent is 50 - 1250 mg / m 2 / day. In some embodiments, the second effective amount of the further therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, 300 mg / m 2 / day, 350 mg / m 2 / day, 400 mg / m 2 / day, 450 mg / m 2 / day, 500 mg / m 2 / day, 550 mg / m 2 / day, 600 mg / m 2 / day, 650 mg / m 2 / day, 700 mg / m 2 / day, 750 mg / m 2 / day, 800 mg / m 2 / day, 850 mg / m 2 / day, 900 mg / m 2 / day, 950 mg / m 2 / day, 1000 mg / m 2 / day, 1050 mg / m 2 / day, 1100 mg / m 2 / day, 1150 mg / m 2 / day, 1200 mg / m 2 / day, or 1250 mg / m 2 / day.

[0125] Generally, the compounds of the present technology are administered in a therapeutically effective amount by any of the recognized modes of administration of agents that provide similar utility. The actual amount of the compound of the present technology, i.e., the active ingredient, will depend on a number of factors, such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors well known to those skilled in the art. The drug can be administered at least once a day, preferably once or twice a day.

[0126] In order to easily determine the most effective and convenient route of administration and the most appropriate formulation, the effective amount of such an agent can also be easily determined by routine experimentation. A variety of formulations and drug delivery systems are available in the art. See, for example, Gennaro, A.R., ed. (1995) Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Co.

[0127] The therapeutically effective dose can be initially estimated using various techniques well known in the art. The initial dose used in animal experiments can be based on the effective concentration established in cell culture assays. The appropriate dosage range for human subjects can be determined, for example, using data obtained from animal experiments and cell culture assays.

[0128] An agent, e.g., an effective amount or a therapeutically effective amount or dosage of a compound of the present technology, refers to the amount of an agent or compound that brings about alleviation of the symptoms of a subject or an extension of the survival period. The toxicity and therapeutic efficacy of such molecules can be determined by standard pharmacological procedures in cell cultures or experimental animals, e.g., the maximum tolerated dose (MTD), the highest non-severe toxic dose (HNSTD), the no-observed-adverse-effect level (NOAEL), or LD 50 (the 50% lethal dose of the population), and ED 50 (the dose that has a therapeutic effect in 50% of the population). The dose ratio of toxicity to therapeutic effect is the therapeutic index, which can be expressed as the ratio of MTD, HNSTD, NOAEL, or LD 50 to ED 50 . Agents with a high therapeutic index are preferred.

[0129] An effective amount or a therapeutically effective amount is the amount of a compound or pharmaceutical composition that elicits a biological or medical response in a tissue, system, animal, or human being that a researcher, veterinarian, physician, or other clinician is seeking. The dosage amount falls, in particular, within a blood concentration range that includes an ED 50 with little or no toxicity. The dosage amount may vary within this range depending on the dosage form being used and / or the route of administration being utilized. The exact formulation, route of administration, dosage amount, and dosing interval need to be selected in light of the details of the subject's medical condition according to methods known in the art.

[0130] The amount and interval of dosing can be adjusted individually to provide a plasma concentration of the active moiety that is sufficient to achieve the desired effect, i.e., the minimum effective concentration (MEC). The MEC varies for each compound but can be estimated, for example, from in vitro data and animal experiments. The dosage amount required to achieve the MEC will depend on the individual characteristics and route of administration. In the case of topical administration or selective uptake, the effective local concentration of the drug may not be related to the plasma concentration.

[0131] The amount of the agent or composition to be administered may depend on various factors including the sex, age, and weight of the subject to be treated, the severity of the pain, the mode of administration, and the judgment of the prescribing physician.

[0132] The therapeutically effective amount may be the same as or different from either or both of the effective amount of SRA737 and the second effective amount of the additional therapeutic agent. This is the case even when the present disclosure provides that neither the effective amount of SRA737 nor the second effective amount of the additional therapeutic agent should be an amount that alone improves the symptoms of the disease (for example, the amount of SRA737 and / or the additional therapeutic agent may be considered an "amount below the therapeutic amount" when administered as an individual therapy), in order to define that the methods described herein are effective. However, the present disclosure provides that a therapeutically effective amount of the combination must be provided, that is, the combination has at least an impact on the treatment of the symptoms of the disease.

[0133] Unit dosage forms are terms generally understood by those skilled in the art. A unit dosage form is a pharmaceutical preparation sold for a specific use. The preparation most frequently takes the form of the active ingredient(s) and any inert ingredient(s) including a pharmaceutically acceptable carrier or vehicle. It should be understood that multiple unit dosage forms are separate pharmaceutical preparations. Thus, one unit dosage form may be, for example, a combination of 250 mg of SRA737 and an additional therapeutic agent at a specific ratio of each component, while another completely separate unit dosage form may be, for example, a combination of 750 mg of SRA737 and an additional therapeutic agent at the same specific ratio of each component as referenced above. Thus, from one unit dosage to another, both the effective amount of SRA737 and the second effective amount of the additional therapeutic agent may remain the same. Needless to say, when either the effective amount of SRA737 or the second effective amount of the additional therapeutic agent changes, the unit dosage form is different.

[0134] In some embodiments, the effective amount is specific to the SRA737 compound. That is, the effective amount is different from the second effective amount of a further therapeutic agent. In some embodiments, the effective amount of SRA737 is an amount equivalent to a "therapeutically effective amount" or an amount that produces a therapeutic and / or beneficial effect. In some embodiments, the effective amount of SRA737 is a "therapeutically effective amount". In some embodiments, the second effective amount of a further therapeutic agent is a "therapeutically effective amount". In some embodiments, neither the effective amount of SRA737 nor the second effective amount of a further therapeutic agent is a "therapeutically effective amount". In some embodiments, the second effective amount is specific to the further therapeutic agent. That is, the second effective amount is a different amount for different further therapeutic agents.

[0135] In some embodiments, the combination of SRA737 and a further therapeutic agent is formulated in a single (1) unit dosage form. In some embodiments, the same unit dosage form is administered for at least four (4) hours, six (6) hours, eight (8) hours, twelve (12) hours, twenty-four (24) hours, one (1) day, two (2) days, three (3) days, seven (7) days, ten (10) days, fourteen (14) days, twenty-one (21) days, or thirty (30) days.

[0136] In some embodiments, the combination of SRA737 and a further therapeutic agent is formulated in at least two (2) individually different unit dosage forms. In some embodiments, the first effective amount is different between the first unit dosage form and the second unit dosage form. In some embodiments, the effective amount of SRA737 is the same in both the first unit dosage form and the second unit dosage form.

[0137] In some embodiments, the first unit dosage form is the same as the second unit dosage form. In some embodiments, the first unit dosage form is the same as the second unit dosage form and the third unit dosage form. In some embodiments, the first unit dosage form is the same as the second, third, and fourth unit dosage forms.

[0138] The compounds of the present invention In one aspect, the present disclosure provides a method of using the compound SRA737.

[0139] SRA737 The compound SRA737 is also identified by the chemical name 5-[[4-[[morpholin-2-yl]methylamino]-5-(trifluoromethyl)-2-pyridyl]amino]pyrazine-2-carbonitrile. Each of the enantiomers of SRA737 is useful in the compositions, methods, and kits disclosed herein.

[0140] SRA737 is a compound disclosed in International Patent Application No. PCT / GB2013 / 051233, which is incorporated herein by reference. One skilled in the art can find the method for synthesizing SRA737 in International Patent Application No. PCT / GB2013 / 051233.

[0141] In one aspect, the structure of SRA737 is as shown in the following table.

[0142]

Table 2

[0143] Combination therapy In another aspect, the present disclosure provides a method of using the compound SRA737 in combination therapy with additional therapeutic agents.

[0144] Additional therapies include, but are not limited to, administering a chemotherapeutic agent, administering an antibody or antibody fragment (such as an immune checkpoint inhibitor), administering a radiotherapeutic agent, administering an external inducer of replication stress, and administering combinations thereof.

[0145] The term "chemotherapy" refers to the administration of any genotoxic substance (e.g., a DNA-damaging agent), including conventional or non-conventional chemotherapeutic agents, for the treatment or prevention of cancer. Examples of chemotherapeutic agents include agents that have been modified (e.g., fused to an antibody or other targeted agent). Examples of chemotherapeutic agents include platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, nitrogen mustard, thiotepa, melphalan, busulfan, procarbazine, streptozocin, temozolomide, dacarbazine, bendamustine, mitomycin C), antitumor antibiotics (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, bleomycin, plicamycin, dactinomycin), taxanes (e.g., paclitaxel, nab-paclitaxel, and docetaxel), antimetabolites (e.g., 5-fluorouracil, cytarabine, pemetrexed, thioguanine, floxuridine, capecitabine, and methotrexate), nucleoside analogs (e.g., fludarabine, clofarabine, cladribine, pentostatin, nelarabine, gemcitabine, 5-fluorouracil), topoisomerase inhibitors (e.g., topotecan, irinotecan, SN-38, CPT-11), hypomethylating agents (e.g., azacitidine and decitabine), proteasome inhibitors (e.g., bortezomib), epipodophyllotoxins (e.g., etoposide and teniposide), DNA synthesis inhibitors (e.g., hydroxyurea), and vinca alkaloids (e.g., vincristine, vindesine, vinorelbine, and vinblastine), but are not limited to these. Chemotherapeutic agents include DNA intercalating agents (e.g., pyrrolobenzodiazepine).

[0146] The term "exogenous inducer of replication stress" refers to any agent that causes an increase in replication fork stalling, an increase in genomic instability, an increase in mutations and / or mutation rate, activation of DNA damage repair pathways, activation of the DNA damage response (DDR), activation or increased expression of replication stress gene(s), or a combination thereof. Examples of inducers of replication stress include, but are not limited to, genotoxic chemotherapeutic agents (e.g., gemcitabine and other nucleoside analogs, temozolomide, cisplatin, alkylating agents such as mitomycin C, topoisomerase inhibitors such as camptothecin and etoposide, and others). Exogenous inducers of cellular stress include agents that decrease the concentration of nucleotides in the cell (e.g., ribonucleotide reductase inhibitors, etc.). Exogenous inducers of cellular stress also include PARP inhibitors.

[0147] The term "DNA damage repair (DDR) gene" or "DNA damage repair pathway gene" refers to any gene that directly or indirectly promotes the repair of DNA mutations, breaks, or other DNA damage or structural changes. DNA damage repair genes include, but are not limited to, ATM, CDK12, BRCA1, BRCA2, MRE11A, ATR, and Rad50. Also, DDR genes include genes of the Fanconi anemia (FA) pathway. Genes of the FA pathway include, but are not limited to, Fanconi anemia complementation group (FANC) genes.

[0148] The term "immune checkpoint inhibitor" refers to a binding molecule that binds to one or more immune checkpoint molecules and blocks or inhibits the activity of one or more immune checkpoint molecules, or a drug that inhibits an immunosuppressive protein. Exemplary immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof that target one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, CGEN-15049, and IDO1.

[0149] The term "PARP inhibitor" or "PARPi" refers to an inhibitor of PARP. The PARPi may be a small molecule, an antibody or a nucleic acid. The PARPi may function to reduce the expression or activity of PARP in a cell, or a combination thereof. The PARPi includes inhibitors that modify or do not modify the binding of PARP to DNA. The PARPi may inhibit any element of the PARP group. The PARPi includes, but is not limited to, olaparib, rucaparib, veliparib, niraparib, iniparib, talazoparib, veliparib, fluzoparib, BGB-290, CEP-9722, BSI-201, EZ449, PF-01367338, AZD2281, INO-1001, MK-4827, SC10914, and 3-aminobenzylamine.

[0150] In certain embodiments, further treatment includes administering any one of gemcitabine, olaparib, niraparib, rucaparib, talazoparib, cisplatin, ribonucleotide reductase inhibitor, etoposide, SN-38 / CPT-11, mitomycin C, and combinations thereof, but is not limited thereto.

[0151] Pharmaceutical composition Methods for inhibiting tumor growth, methods for inhibiting cancer progression, or methods for treating cancer are described herein. The methods of the invention include administering an effective amount of SRA737 and a second effective amount of a further therapeutic agent. SRA737 and the further therapeutic agent can each be formulated in a pharmaceutical composition. These pharmaceutical compositions may include, in addition to the active compound(s), pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials need to be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material may depend on the route of administration, such as oral, intravenous, dermal or subcutaneous, nasal, intramuscular, intraperitoneal routes, etc.

[0152] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain solid carriers such as gelatin. Liquid pharmaceutical compositions generally contain liquid carriers such as water, or oils derived from petroleum, animals, plants or synthesis, for example, oils including peanut oil, soybean oil, mineral oil, sesame oil, etc. It may include physiological saline, dextrose or other saccharide solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.

[0153] In the case of intravenous, intradermal or subcutaneous injection, or injection at the site of pain, the active ingredient is in the form of a parenterally acceptable aqueous solution that does not contain pyrogens and has appropriate pH, isotonicity, and stability. A person skilled in the art can adequately prepare an appropriate solution using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as necessary.

[0154] The composition can be administered alone or in combination with other therapeutic agents, either simultaneously or sequentially, depending on the medical condition being treated.

[0155] The present technology is not limited to any particular composition or pharmaceutical carrier as such things vary. Generally, the compounds of the present technology are administered as pharmaceutical compositions by any one of the following routes, namely oral, systemic (e.g., transdermal, intranasal, or suppository), or parenteral (e.g., intramuscular, intravenous, or subcutaneous). The preferred method of administration is oral using a convenient daily dosing schedule that can be adjusted according to the degree of pain. The composition may take the form of tablets, pills, capsules, semi-solids, powders, sustained-release formulations, liquids, suspensions, elixirs, aerosols, or any other suitable composition. Another method suitable for administering the compounds of the present technology is inhalation.

[0156] The selection of a formulation depends on various factors such as the mode of drug administration and the bioavailability of the active ingredient. In the case of delivery via inhalation, the compound can be formulated as a solution, suspension, aerosol spray, or dry powder and filled into a dispenser appropriate for administration. There are several types of drug inhalation devices - nebulizer inhalers, metered-dose inhalers (MDIs), and dry powder inhalers (DPIs). Nebulizer devices generate a high-speed airflow that jets the therapeutic agent (formulated in liquid form) as a mist, which is carried to the target's airways. MDIs are typically formulations packaged with a compressed gas. Upon activation, a fixed amount of the therapeutic agent is expelled from the device by the compressed gas, thus providing a reliable method of administering a set dose of the agent. DPIs dispense the therapeutic agent in the form of a free-flowing powder, which can be dispersed by the device into the target's inspiratory airflow during breathing. To achieve a free-flowing powder, the therapeutic agent is formulated with an excipient such as lactose. A fixed amount of the therapeutic agent is stored in a capsule form and dispensed with each activation.

[0157] The pharmaceutical dosage forms of the compounds of the present technology can be manufactured by any of the methods well-known in the art, such as conventional mixing, sieving, dissolving, melting, granulating, coating, tableting, suspending, extruding, spray drying, triturating, emulsifying, (nano / micro) encapsulating, entrapping, or lyophilization processes. As described above, the compositions of the present technology may contain one or more physiologically acceptable inert components that facilitate the processing of the active molecule into a pharmaceutical formulation.

[0158] Recently, pharmaceutical formulations have been developed based on the principle that bioavailability can be enhanced by increasing the surface area, i.e., reducing the particle size, especially for drugs showing insufficient bioavailability. For example, U.S. Patent No. 4,107,288 describes a pharmaceutical formulation having particles in the size range of 10 to 1,000 nm with the active substance supported on a polymeric cross-linked matrix. U.S. Patent No. 5,145,684 describes the manufacture of a pharmaceutical formulation in which the drug substance is milled into nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to provide a pharmaceutical formulation showing significantly high bioavailability.

[0159] The composition generally consists of a compound of the present technology in combination with at least one pharmaceutically acceptable excipient. Acceptable excipients are non-toxic, assist in administration, and do not adversely affect the therapeutic effect of the claimed compound. Such excipients may generally be any solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous excipient that is available to one of ordinary skill in the art.

[0160] Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, skim milk powder, and the like. Liquid and semi-solid excipients may be selected from various oils including glycerol, propylene glycol, water, ethanol, and oils derived from petroleum, animal, vegetable, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Liquid carriers particularly suitable for injection solutions include water, physiological saline, dextrose solution, and glycols.

[0161] Compressed gas can be used to disperse the compounds of the present technology in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc. Other suitable pharmaceutical excipients and their formulations are described in Remington’s Pharmaceutical Sciences, edited by E.W. Martin (Mack Publishing Company, 18th ed., 1990).

[0162] In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to salts derived from various organic and inorganic counterions well-known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, etc. Suitable salts include those described in Stahl and Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use, 2002.

[0163] The composition can be provided in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient, if desired. Such a pack or device can include, for example, a metal or a plastic foil such as a blister pack, or glass, and a rubber stopper such as a vial. A pack or dispenser device may be accompanied by administration instructions. Also, a composition containing a compound of the present technology formulated with a compatible pharmaceutical carrier can be formulated, placed in a suitable container, and labeled for the treatment of the indication.

[0164] The amount of the compound in the complex can vary within the entire range adopted by those skilled in the art. Typically, the formulation will contain from about 0.01 to 99.99% by weight of the compound of the present technology, based on the total formulation, in units of weight percent (wt%), and the balance will be one or more suitable pharmaceutical excipients. Preferably, the compound is present at a level of about 1 to 80% by weight. Representative pharmaceutical formulations are described below.

[0165] Formulation Example The following are representative pharmaceutical formulations containing SRA737 alone or in combination with additional therapeutic agents.

[0166] The composition can be administered either simultaneously or sequentially, depending on the condition being treated, alone or in combination with other therapeutic agents.

[0167] Kit The present disclosure also provides a kit comprising a combination of SRA737 and an additional therapeutic agent, and instructions for use. The present disclosure further provides a kit comprising one or more pharmaceutical compositions wherein the pharmaceutical composition(s) comprise SRA737 and an additional therapeutic agent, and instructions for use, optionally wherein the combination comprises at least one pharmaceutically acceptable carrier or excipient.

[0168] The individual components of the kit can be packaged in separate containers, and a notice in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products can be associated with such containers, the notice reflecting approval by that agency for manufacture, use, or sale. The kit can optionally include instructions or directions outlining a method of use or dosing schedule for the antigen-binding construct.

[0169] In some aspects, the present disclosure provides a kit comprising a combination of SRA737 and an additional therapeutic agent, and at least one pharmaceutically acceptable carrier or excipient.

[0170] When one or more components of the kit are provided as a solution, such as an aqueous solution or a sterile aqueous solution, the container means itself may be an inhaler, syringe, pipette, drip apparatus, or other such device from which the solution can be administered to the subject, or applied to and mixed with other components of the kit.

[0171] Alternatively, the components of the kit may be provided in a dry or lyophilized form, and the kit may further include a suitable solvent for the reconstruction of the lyophilized components. Also, regardless of the number or type of containers, the kits described herein may include an instrument for assisting in the administration of the composition to a patient. Such an instrument may be an inhaler, nasal spray, syringe, pipette, forceps, measuring spoon, drip apparatus, or similar medically approved delivery means.

[0172] In another aspect of the invention, there is provided a manufactured article comprising a material useful for the treatment, prevention, and / or diagnosis of the disorders described herein, such as inhibition of tumor growth. The manufactured article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The container may be formed from a variety of materials such as glass or plastic. The container(s) may hold a composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing the disorder and may have a sterile access port (e.g., the container may be a vial having a stopper penetrable by an intravenous solution bag or a hypodermic needle).

[0173] The manufactured product of the present embodiment described in this specification may further include a label or package insert indicating that the composition can be used to treat a specific medical condition. Alternatively, or in addition, the manufactured product may further include a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0174] Polypeptides and Nucleic Acids Described herein are the polypeptide and nucleic acid sequences of genes useful in the present invention, such as, for example, the gene for CHK1. In some embodiments, the polypeptide and nucleic acid sequences useful in the present invention are at least 95, 96, 97, 98, or 99% identical to the sequences described herein or referenced herein by database accession number. In some embodiments, the polypeptide and nucleic acid sequences useful in the present invention are 100% identical to the sequences described herein or referenced herein by database accession number.

[0175] The term "percent identity" in the context of the sequences of two or more nucleic acids or polypeptides refers to the percentage of nucleotides or amino acid residues that are identical as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) for maximum correspondence when compared and aligned, or as visually determined. Depending on the application, percent "identity" may exist over regions of the sequences being compared, such as, for example, over functional domains, or instead may exist over the full lengths of the two sequences being compared. For sequence comparison, typically one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, sub-sequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’l Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (generally, see Ausubel et al.). An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0176] Definitions The terms used in the claims and the specification are defined as described herein, unless otherwise specified.

[0177] The practice of the present invention involves the use of conventional techniques in organic chemistry, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0178] In this application, several technical terms are referred to. For example, all numerical representations such as pH, temperature, time, concentration, and weight, including each range, are usually approximate values that may vary, if necessary, in increments of (+) or (-) 0.1, 1.0, or 10.0. It can be understood that the term "about" precedes all numerical representations. The reagents described herein are illustrative, and equivalents of such may be known in the art.

[0179] The compounds utilized in the present invention possess asymmetric carbon atoms (optical centers) or double bonds, and racemates, diastereomers, geometric isomers, positional isomers, and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention. Also, the compounds of the present invention may contain unnatural proportions of atomic isotopes in one or more of the atoms constituting such compounds. For example, the compounds may be radiolabeled using radioactive isotopes such as, but not limited to, tritium ( 3 H), iodine-125 ( 125 I), or carbon-14 ( 14 C). All isotopic modifications of the compounds of the present invention are intended to be encompassed within the scope of the present invention, whether radioactive or not.

[0180] The term "subject" refers to mammals including any mammal including humans, and animals of veterinary and research interest including monkeys, cattle, horses, dogs, cats, and rodents, but not limited to these. Animals such as mice and rats, as well as other mammals, can be used in drug screening, characterization, and evaluation. As used herein, the terms patient, subject, and individual are used interchangeably.

[0181] The term "administer" or "administration" of a drug and / or therapy to a subject (and grammatical equivalents of this phrase) may be direct administration or indirect administration, which may be by a medical professional to the subject or self - administration, and / or the act of prescribing a drug and / or therapy to the subject, or directing a person to prescribe a drug and / or therapy to the subject, referring to both direct and indirect administration.

[0182] The term "co - administration" refers to two or more compounds administered in such a way as to exert a pharmacological effect during the same period. Such co - administration can be achieved by any of simultaneous administration, contemporaneous administration, or sequential administration of the two or more compounds.

[0183] The term "treat" or "treatment" of a disorder or disease refers to taking steps, for example, to alleviate the symptoms of a disorder or disease such as tumor growth or cancer, or otherwise to obtain some beneficial or desired result for the subject, including clinical outcomes. Any beneficial or desirable clinical outcome can include, but is not limited to, alleviation or improvement of one or more symptoms of cancer, or conditional survival and reduction of tumor burden or tumor volume, reduction of the extent of the disease, delay or retardation of tumor or disease progression, improvement, alleviation, or stabilization of the state of the tumor and / or disease, or other beneficial results.

[0184] The term "ex vivo" or "in vitro" refers to a process that occurs in living cells that grow separately from the living body, for example, growing in tissue culture.

[0185] The term "in vivo" refers to a process that occurs in a living body.

[0186] The term "Chk1" or "CHEK1" or "checkpoint kinase 1" refers to a serine / threonine protein kinase encoded by the CKEK1 gene.

[0187] The term "effective amount" means an amount sufficient to produce a desired effect, such as an amount sufficient to inhibit the growth of a tumor.

[0188] The term "reduction" (and grammatical equivalents of this phrase) of one or more symptoms refers to a decrease in the severity or frequency of the symptom(s) or the elimination of the symptom(s).

[0189] Also, notwithstanding the appended claims, the present disclosure is defined by the following clauses.

[0190] Clause 1. A method of treating cancer, comprising administering to a subject having said cancer an effective amount of an SRA737 compound, wherein said effective amount is less than 2000 mg / day, said method of treating cancer.

[0191] Clause 2. The method according to clause

[0212] , wherein the SRA737 compound is administered orally.

[0192] Clause 3. The method according to any one of clauses

[0212] to 0, wherein the SRA737 compound is administered daily.

[0193] Clause 4. The method according to clause 0, wherein the SRA737 compound is administered continuously for at least 28 days.

[0194] Clause 5. The method according to clause 0, wherein the SRA737 compound is administered continuously for at least 7 days.

[0195] Clause 6. The method according to clause

[0212] or 0, wherein the SRA737 compound is administered intermittently.

[0196] The method according to clause 0, wherein the SRA737 compound is administered with a delay between administrations of at least ten (10) minutes, fifteen (15) minutes, twenty (20) minutes, thirty (30) minutes, forty (40) minutes, sixty (60) minutes, two (2) hours, three (3) hours, four (4) hours, six (6) hours, eight (8) hours, ten (10) hours, twelve (12) hours, fourteen (14) hours, eighteen (18) hours, twenty-four (24) hours, thirty-six (36) hours, forty-eight (48) hours, three (3) days, four (4) days, five (5) days, six (6) days, seven (7) days, eight (8) days, nine (9) days, ten (10) days, eleven (11) days, twelve (12) days, thirteen (13) days, fourteen (14) days, three (3) weeks, or four (4) weeks.

[0197] Clause 8. The method according to any one of clauses

[0212] to 0, wherein the SRA737 compound is administered over one or more 28-day cycles.

[0198] Clause 9. The method according to clause 0, wherein the SRA737 compound is administered on one or more days of the one or more 28-day cycles.

[0199] Clause 10. The method according to clause 0, wherein the SRA737 compound is administered on the 2nd, 3rd, 9th, 10th, 16th, and 17th days of the one or more 28-day cycles.

[0200] Clause 11. The method according to clauses 0 to 0, further comprising administering a first dose of the SRA737 compound before the first cycle of the one or more 28-day cycles.

[0201] Clause 12. The method according to clause 0, wherein the first dose is administered 4, 5, 6, or 7 days before the first cycle of the one or more 28-day cycles.

[0202] Clause 13. The method according to any one of clauses 0 to 0, wherein the one or more 28-day cycles include two, three, four, five, six or more 28-day cycles.

[0203] Clause 14. The method according to any one of Clauses [

[0212] ]~0, wherein the SRA737 compound is administered according to a dosing schedule selected from the group consisting of dosing for 5 days per week followed by 2 days of non-dosing, daily dosing for 1 week followed by 1, 2, or 3 weeks of non-dosing, daily dosing for 2 or 3 weeks followed by 1 or 2 weeks of non-dosing, and dosing on the 2nd and 3rd days of a weekly cycle.

[0204] Clause 15. The method according to any one of Clauses [

[0212] ]~0, wherein the effective amount is administered as a single dose once a day.

[0205] Clause 16. The method according to Clauses [

[0212] ]~0, wherein half of the effective amount is administered twice a day.

[0206] Clause 17. The method according to any one of Clauses [

[0212] ]~0, wherein the effective amount is less than 1500 mg / day.

[0207] Clause 18. The method according to any one of Clauses [

[0212] ]~0, wherein the effective amount is less than 1300 mg / day.

[0208] Clause 19. The method according to any one of Clauses [

[0212] ]~0, wherein the effective amount is 1000 mg / day or less.

[0209] Clause 20. The method according to any one of Clauses [

[0212] ]~0, wherein the effective amount is 900 mg / day or less.

[0210] Clause 21. The method according to any one of Clauses [

[0212] ]~0, wherein the effective amount is 800 mg / day or less.

[0211] Clause 22. The method according to any one of Clauses

[0212] ~0, wherein the effective amount is 700 mg / day or less.

[0212] Clause 23. The method according to any one of Clauses [

[0212] ]~0, wherein the effective amount is 600 mg / day or less.

[0213] Clause 24. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 500 mg / day or less.

[0214] Clause 25. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 400 mg / day or less.

[0215] Clause 26. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is between 600 mg / day and 1300 mg / day.

[0216] Clause 27. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is between 300 mg / day and 1300 mg / day.

[0217] Clause 28. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is between 300 mg / day and 1000 mg / day.

[0218] Clause 29. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is between 300 mg / day and 800 mg / day.

[0219] Clause 30. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is between 500 mg / day and 1300 mg / day.

[0220] Clause 31. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is between 500 mg / day and 1000 mg / day.

[0221] Clause 32. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is between 500 mg / day and 800 mg / day.

[0222] Clause 33. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is selected from the group consisting of 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, 1000 mg / day, 1100 mg / day, and 1200 mg / day.

[0223] Clause 34. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is selected from the group consisting of 40 mg / day, 80 mg / day, 300 mg / day, 500 mg / day, 600 mg / day, 700 mg / day, and 800 mg / day.

[0224] Clause 35. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 300 mg / day.

[0225] Clause 36. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 400 mg / day.

[0226] Clause 37. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 500 mg / day.

[0227] Clause 38. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 600 mg / day.

[0228] Clause 39. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 700 mg / day.

[0229] Clause 40. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 800 mg / day.

[0230] Clause 41. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 900 mg / day.

[0231] Clause 42. The method according to any one of Clauses

[0212] to 0, wherein the effective amount is 1000 mg / day.

[0232] Clause 43. The method according to any one of Clauses

[0212] to

[0253] , wherein the cancer is metastatic cancer.

[0233] Clause 44. The method according to any one of Clauses

[0212] to

[0253] , wherein the cancer is a disease or disorder selected from the group consisting of colorectal cancer, ovarian cancer, high-grade serous ovarian cancer (HGSOC), non-small cell lung cancer (NSCLC), small cell lung cancer, lung adenocarcinoma, prostate cancer, castration-resistant prostate cancer, cholangiocarcinoma, cholangiocarcinoma, melanoma, uterine cancer, thyroid cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, endometrial cancer, hepatocellular carcinoma, leukemia, lymphoma, non-Hodgkin lymphoma, multiple myeloma, brain cancer, neuroblastoma, squamous cell carcinoma, head and neck squamous cell carcinoma (HNSCC), and anal squamous cell carcinoma (SCCA), ano-genital cancer (e.g., anal cancer), rectal cancer, pancreatic cancer, urothelial cancer, sarcoma and soft tissue sarcoma, metastatic colorectal cancer (CRC), platinum-resistant or -intolerant HGSOC, advanced NSCLC, and metastatic castration-resistant prostate cancer (mCRPC), triple-negative breast cancer, invasive breast cancer, metastatic breast cancer, HER2-positive breast cancer and inflammatory breast cancer.

[0234] Clause 45. The method according to Clauses

[0212] to

[0253] , wherein the cancer is colorectal cancer.

[0235] Clause 46. The method according to Clause

[0256] , wherein the colorectal cancer is characterized by having microsatellite instability or a defect in mismatch repair (MMR).

[0236] Clause 47. The method according to Clauses

[0212] to

[0253] , wherein the cancer is non-small cell lung cancer.

[0237] Clause 48. The method according to Clauses

[0212] to

[0253] , wherein the cancer is HNSCC.

[0238] Clause 49. The method according to Clauses

[0212] to

[0253] , wherein the cancer is SCCA.

[0239] Clause 50. The method according to Clauses

[0212] to

[0253] , wherein the cancer is ano-genital cancer.

[0240] Clause 51. The method according to Clauses

[0212] to

[0253] , wherein the cancer is prostate cancer.

[0241] Clause 52. The method according to clause [

[0262] ], wherein the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).

[0242] Clause 53. The method according to clauses [

[0212] ] to [

[0253] ], wherein the cancer is ovarian cancer.

[0243] Clause 54. The method according to clause [

[0264] ], wherein the ovarian cancer is high-grade serous ovarian cancer (HGSOC).

[0244] Clause 55. The method according to clause [

[0265] ], wherein the tumor associated with the HGSOC is identified as having an increased expression of the cyclin E1 (CCNE) gene.

[0245] Clause 56. The method according to clause [

[0266] ], wherein the increased expression is the result of genetic amplification.

[0246] Clause 57. The method according to clause [

[0265] ], wherein the tumor is identified as having a somatic or germline wild-type state of BRCA1 and BRCA2.

[0247] Clause 58. The method according to any one of clauses [

[0212] ] to [

[0268] ], wherein the tumor associated with the cancer is identified as having a gain-of-function mutation, amplification, or overexpression of at least one oncogenic driver gene or another gene related to Chk1 pathway sensitivity.

[0248] Clause 59. The method according to clause [

[0269] ], wherein the oncogenic driver gene is selected from the group consisting of MYC, MYCN, KRAS, and CCNE1.

[0249] Clause 60. The method according to any one of clauses [

[0212] ] to [

[0270] ], wherein the tumor associated with the cancer is identified as having a loss-of-function mutation or a deleterious mutation in at least one DNA damage repair (DDR) pathway gene related to Chk1 pathway sensitivity. Or is identified as having a deleterious mutation.

[0250] Article 61. The method according to Article

[0271] , wherein the DDR pathway gene is selected from the group consisting of ATM, CDK12, BRCA1, BRCA2, MRE11A, ATR, and FA pathway genes.

[0251] Article 62. The method according to Article

[0271] or

[0272] , wherein the loss-of-function mutation or the deleterious mutation is determined by establishing microsatellite instability or a defect in mismatch repair (MMR).

[0252] Article 63. The method according to any one of Articles

[0212] to

[0273] , wherein the tumor associated with the cancer is identified as having a gain-of-function mutation or amplification of at least one replication stress gene related to Chk1 pathway sensitivity.

[0253] Article 64. The method according to Article

[0274] , wherein the replication stress gene is ATR or CHK1.

[0254] Article 65. The method according to any one of Articles

[0212] to

[0275] , wherein the tumor associated with the cancer is identified as having a deleterious mutation in a tumor suppressor (TS) gene related to Chk1 pathway sensitivity.

[0255] Article 66. The method according to Article

[0276] , wherein the tumor associated with the tumor suppressor gene is selected from the group consisting of RB1, TP53, ATM, RAD50, FBXW7, and PARK2.

[0256] Article 67. The method according to any one of Articles

[0212] to

[0277] , wherein the subject is positive for human papillomavirus (HPV).

[0257] Article 68. The method according to any one of Articles

[0212] to

[0278] , wherein the subject is human.

[0258] Clause 69. The method according to any one of Clauses

[0212] to

[0279] , further comprising administering a further therapeutic agent in a second effective amount, wherein the further therapeutic agent is selected from the group consisting of chemotherapeutic agents, antibodies or antibody fragments, radiotherapy agents, external inducers of replication stress, and combinations thereof.

[0259] Clause 70. The method according to Clause

[0280] , wherein the further therapeutic agent is selected from the group consisting of gemcitabine, olaparib, niraparib, rucaparib, talazoparib, cisplatin, ribonucleotide reductase inhibitors, etoposide, SN-38 / CPT-11, mitomycin C, and combinations thereof.

[0260] Clause 71. The method according to Clause

[0280] , wherein the further therapeutic agent comprises gemcitabine.

[0261] Clause 72. The method according to any one of Clauses

[0280] to

[0282] , wherein the further therapeutic agent is administered daily.

[0262] Clause 73. The method according to any one of Clauses

[0280] to

[0282] , wherein the further therapeutic agent is administered on the first day, and the SRA737 compound is administered on the second and third days of a weekly schedule.

[0263] Clause 74. The method according to any one of Clauses

[0280] to

[0282] , wherein the further therapeutic agent and the SRA737 compound are administered over one or more 28-day cycles.

[0264] Clause 75. The method according to Clause 0, wherein the further therapeutic agent is administered on the first, eighth, and fifteenth days of the one or more 28-day cycles, and the SRA737 compound is administered on the second, third, ninth, tenth, sixteenth, and seventeenth days of the one or more 28-day cycles.

[0265] Clause 76. The second effective amount of the further therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2 / day selected from the group consisting of, the method according to any one of clauses

[0282] ~0.

[0266] Clause 77. The second effective amount of the additional therapeutic agent is 600 mg / m 2 / day or less, the method according to any one of clauses

[0282] ~0.

[0267] Clause 78. The second effective amount of the additional therapeutic agent is between 50 and 600 mg / m 2 / day, the method according to any one of clauses

[0282] ~0.

[0268] Clause 79. The second effective amount of the additional therapeutic agent is between 50 and 300 mg / m 2 / day, the method according to any one of clauses

[0282] ~0.

[0269] Clause 80. The effective amount of the SRA737 compound is 80 mg / day, and the second effective amount of the additional therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2 / day selected from the group consisting of, the method according to any one of clauses

[0282] ~0.

[0270] Clause 81. The effective amount of the SRA737 compound is 150 mg / day, and the second effective amount of the additional therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2The method according to any one of clauses

[0282] to 0, selected from the group consisting of / day.

[0271] Clause 82. The effective amount of the SRA737 is 300 mg / day, and the second effective amount of the additional therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2 The method according to any one of clauses

[0282] to 0, selected from the group consisting of / day.

[0272] Clause 83. The effective amount of the SRA737 is 500 mg / day, and the second effective amount of the additional therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2 The method according to any one of clauses

[0282] to 0, selected from the group consisting of / day.

[0273] Clause 84. The effective amount of the SRA737 is 600 mg / day, and the second effective amount of the additional therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2 The method according to any one of clauses

[0282] to 0, selected from the group consisting of / day.

[0274] Clause 85. The effective amount of the SRA737 is 700 mg / day, and the second effective amount of the additional therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2 The method according to any one of clauses

[0282] to 0, selected from the group consisting of / day.

[0275] Clause 86. The effective amount of the SRA737 compound is 800 mg / day, and the second effective amount of the additional therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2 The method according to any one of clauses

[0282] to 0, selected from the group consisting of / day.

[0276] Clause 87. The effective amount of the SRA737 compound is 900 mg / day, and the second effective amount of the additional therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2 The method according to any one of clauses

[0282] to 0, selected from the group consisting of / day.

[0277] Clause 88. The effective amount of the SRA737 compound is 1000 mg / day, and the second effective amount of the additional therapeutic agent is 50 mg / m 2 / day, 100 mg / m 2 / day, 150 mg / m 2 / day, 200 mg / m 2 / day, 250 mg / m 2 / day, and 300 mg / m 2 The method according to any one of clauses

[0282] to 0, selected from the group consisting of / day.

[0278] Clause 89. The method according to clauses

[0280] to 0, wherein the cancer is urothelial carcinoma.

[0279] Clause 90. The method according to clause

[0300] , wherein the urothelial cancer is selected from the group consisting of (a) unresectable urothelial cancer of the bladder, upper urinary tract, or urethra, and (b) metastatic urothelial cancer of the bladder, upper urinary tract, or urethra.

[0280] Clause 91. The method according to any one of clauses

[0280] to 0, wherein the cancer is HGSOC.

[0281] Clause 92. The method according to clause

[0302] , wherein the tumor associated with the HGSOC is identified as having a somatic or germline wild-type state of BRCA1 and BRCA2.

[0282] Clause 93. The method according to any one of clauses

[0280] to 0, wherein the cancer is small cell lung cancer.

[0283] Clause 94. The method according to any one of clauses

[0280] to 0, wherein the cancer is soft tissue sarcoma.

[0284] Clause 95. The method according to clause

[0305] , wherein the soft tissue sarcoma is selected from the group consisting of undifferentiated pleomorphic sarcoma, malignant fibrous histiocytoma (MFH) / high-grade spindle cell sarcoma, pleomorphic liposarcoma, leiomyosarcoma, and dedifferentiated liposarcoma.

[0285] Clause 96. The method according to any one of clauses

[0280] to 0, wherein the cancer is cervical cancer or anal genital cancer.

[0286] Clause 97. The method according to clause

[0307] , wherein the cervical cancer or anal genital cancer is selected from the group consisting of progressive / metastatic squamous cell carcinoma of the anus, penis, vagina, and vulva.

[0287] Clause 98. The method according to any one of clauses

[0212] to

[0308] , wherein the method results in inhibition of the growth of the tumor associated with the cancer.

[0288] Clause 99. The method according to clause

[0309] , wherein the inhibition of the growth of the tumor associated with the cancer is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% minimum growth inhibition compared to the untreated tumor.

[0289] Clause 100. The method according to any one of clauses

[0212] to

[0310] , wherein the method results in tumor regression associated with the cancer as compared to the baseline measurement.

[0290] Clause 101. The method according to clause

[0311] , wherein the regression is a 30% regression of the tumor associated with the cancer as compared to the baseline measurement.

[0291] Clause 102. The method according to clause

[0311] , wherein the regression is a complete regression of the tumor associated with the cancer as compared to the baseline measurement.

[0292] Clause 103. The method according to any one of clauses

[0212] to

[0313] , wherein the method results in cytotoxicity of the tumor associated with the cancer.

[0293] Clause 104. The method according to any one of clauses

[0212] to

[0314] , wherein the method leads to partial remission, complete remission, or stable disease in the subject as compared to the baseline measurement.

[0294] Clause 105. The method according to any one of clauses

[0212] to

[0314] , wherein the method leads to partial remission in the subject as compared to the baseline measurement.

[0295] Clause 106. The method according to any one of clauses

[0212] to

[0314] , wherein the method leads to complete remission in the subject as compared to the baseline measurement.

[0296] Clause 107. The method according to any one of Clauses

[0212] to

[0314] , which leads to a disease that is stable in the subject as compared to the baseline measurement value.

[0297] Clause 108. The method according to any one of Clauses

[0212] to

[0318] , wherein the method results in a plasma C of the SRA737 compound of at least 100 ng / ml in the subject for at least 24 hours after administration. min thereof.

[0298] Clause 109. The method according to any one of Clauses

[0212] to

[0318] , wherein the method results in a plasma C of the SRA737 compound of at least 100 nM in the subject for at least 24 hours after administration. min thereof.

[0299] Clause 110. The method according to any one of Clauses

[0212] to

[0320] , wherein the method results in a plasma AUC of the SRA737 compound of at least 100 ng·h / mL, at least 300 ng·h / mL, at least 600 ng·h / mL, at least 800 ng·h / mL, at least 1000 ng·h / mL, at least 1600 ng·h / mL, at least 2300 ng·h / mL, at least 2500 ng·h / mL, at least 3000 ng·h / mL, at least 3500 ng·h / mL, at least 8000 ng·h / mL, at least 12000 ng·h / mL, at least 15000 ng·h / mL, at least 18000 ng·h / mL, at least 20000 ng·h / mL, at least 25000 ng·h / mL, or at least 29000 ng·h / mL in the subject after administration. 0-24 thereof.

[0300] Clause 111. The method provides a plasma AUC of the SRA737 compound of at least 400 ng·h / mL, at least 500 ng·h / mL, at least 600 ng·h / mL, at least 1600 ng·h / mL, at least 2600 ng·h / mL, at least 4500 ng / mL, at least 5000 ng·h / mL, at least 8000 ng·h / mL, at least 8000 ng·h / mL, at least 1000 ng·h / mL in the subject after administration. 0-12 The method according to any one of Clauses

[0212] to

[0320] .

[0301] Clause 112. The method provides a plasma C of the SRA737 compound of at least 500 ng / mL, at least 600 ng / mL, at least 800 ng / mL, at least 100 ng / mL, at least 150 ng / mL, at least 175 ng / mL, at least 350 ng / mL, at least 990 ng / mL, at least 1980 ng / mL, at least 2000 / mL, or at least 3228 ng / mL in the subject after administration. max The method according to any one of Clauses

[0212] to

[0322] .

[0302] Clause 113. The method provides a plasma C of the SRA737 compound of less than 500 ng / mL, less than 600 ng / mL, less than 800 ng / mL, less than 100 ng / mL, less than 150 ng / mL, less than 175 ng / mL, less than 350 ng / mL, less than 990 ng / mL, less than 1980 ng / mL, less than 2000 ng / mL, or less than 3228 ng / mL in the subject after administration. max The method according to any one of Clauses

[0212] to

[0322] .

[0303] Clause 114. The method provides a plasma C of the SRA737 compound between 500 and 3200 ng / mL in the subject after administration. max The method according to any one of Clauses

[0212] to

[0322] .

[0304] Clause 115. The method according to any one of Clauses

[0212] to

[0322] , which results in a plasma C of the SRA737 compound in the subject between 500 and 2400 ng / mL after administration. max

[0305] Clause 116. The method according to any one of Clauses

[0212] to

[0322] , which results in a plasma C of the SRA737 compound in the subject between 500 and 650 ng / mL after administration. max

[0306] Clause 117. The method according to any one of Clauses

[0212] to

[0322] , which results in a plasma C of the SRA737 compound in the subject between 500 and 550 ng / mL after administration. max

[0307] Clause 118. The method according to any one of Clauses

[0212] to

[0322] , which results in a plasma C of the SRA737 compound in the subject between 500 and 5500 ng / mL after administration. max

[0308] Clause 119. The method according to any one of Clauses

[0212] to

[0322] , which results in a plasma C of the SRA737 compound in the subject between 500 and 4000 ng / mL after administration. max

[0309] Clause 120. The method according to any one of Clauses

[0212] to

[0330] , wherein the subject is fasted before administering the effective amount of the SRA737 compound.

[0310] Clause 121. The method according to Clause

[0331] , wherein the subject is fasted for 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, or 4 hours or more before administering the effective amount of the SRA737 compound.

[0311] Clause 122. The method according to Clause

[0331] , wherein the subject is fasted for 2 hours or more before administering the effective amount of the SRA737 compound.​​​​​

[0312] Clause 123. The method according to any one of Clauses

[0212] to

[0333] , further comprising fasting the subject after administering the effective amount of the SRA737 compound.

[0313] Clause 124. The method according to Clause

[0334] , wherein the subject fasts for 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, or 4 hours or more after administering the effective amount of the SRA737 compound.

[0314] Clause 125. The method according to Clause

[0334] , wherein the subject fasts for 1 hour or more after administering the effective amount of the SRA737 compound.

Examples

[0315] The following are examples of specific embodiments for carrying out the present invention. The examples are presented for illustrative purposes only and are in no way intended to limit the scope of the present invention. Although efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), it goes without saying that some experimental errors and deviations should be taken into account.

[0316] In the practice of the present invention, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are utilized within the skill of the art. Such techniques are fully explained in the references. For example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition), Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989), Methods in Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.), Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990), Carey and Sundberg Advanced Organic Chemistry 3 rd See (Plenum Press) Vols A and B (1992).

[0317] Abbreviations

Table 3

[0318] Example 1: Summary of Non-Clinical Pharmacology For example, as described in detail by Walton et al. (Oncotarget. 2016 Jan 19;7(3):2329-2342), which is incorporated herein by reference in its entirety for all teachings, SRA737 has previously been found to be a potent and selective inhibitor of Chk1 with restricted off-target activity against other kinases. In vitro, SRA737 potently inhibited Chk1 autophosphorylation induced by genotoxic chemotherapy and prevented downstream signaling (data not shown). This Chk1 inhibition resulted in a dose-dependent inhibition of genotoxin-induced checkpoint arrest, as well as a dose-dependent enhancement of the cytotoxicity of genotoxic chemotherapeutic agents and targeted drugs by SRA737.

[0319] A program of in vivo efficacy studies was conducted to evaluate the activity of SRA737 in combination with genotoxic chemotherapeutic agents and targeted drugs, and as monotherapy.

[0320] Significant dose-dependent antitumor activity of SRA737 in combination with standard-dose gemcitabine was observed in multiple cancer xenograft models, including HT29 human colon cancer, SJSA-1 human osteosarcoma, SW620 mouse colon cancer, Calu6 human (NSCLC), KPC-1 pancreatic cancer, and patient-derived bladder cancer (data not shown). Synergistic effects with low-dose gemcitabine were also observed in HT29 (Figure 5), OVCAR3, and SJSA-1 CDX models, not only in the TNBC PDX model, synergistic effects with gemcitabine and carboplatin in the Calu6 model, and synergistic effects with irinotecan in the HT29 model (data not shown). Significant antitumor activity was also observed in three syngeneic mouse models (mTmG, MC38, and Pan02) in combination with PD1 / PDL-1 inhibitors (data not shown). Significant antitumor activity of SRA737 presented as a single agent was observed in several HGSOC PDX models harboring CCNE1 amplification with TP53 mutations (data not shown). One of these models carried MYCN amplification. A fourth HGSOC PDX model that was partially resistant to PARPi was also sensitive to high-dose SRA737 monotherapy. SRA737 also demonstrated single-agent efficacy in the HGSOC OVCAR3 model, the Eμ-Myc model of B-cell lymphoma, the MOLM-13 model of AML, the TH-MYC model of neuroblastoma, the MDA-MB-231 model of TNBC, and two syngeneic models of renal and lung cancer (RENCA and LL / 2, respectively) (data not shown).

[0321] The effect of SRA737 on gemcitabine-induced CHK1 S296 autophosphorylation was evaluated as described in Walton et al. (Oncotarget. 2016 Jan 19;7(3): 2329-2342), which is incorporated herein by reference in its entirety. Briefly, mice bearing HT29 tumor xenografts were administered (i) vehicle control or (ii) gemcitabine (100 mg / kg in saline, IV) or (iii) a combination of SRA737 (12.5, 25, 50 or 100 mg / kg in DTPW, oral) and gemcitabine (100 mg / kg), and SRA737 was administered 24 hours after gemcitabine administration (n = 3 for each treatment time point). Inhibition of pS296 Chk1 was observed at SRA737 doses of 12.5 mg / kg and above (Figure 1), which corresponded to a minimum (total) plasma concentration of approximately 100 nM (actual value 78 ± 27 nM, approximately 40 ng / mL) at 24 hours (Figure 2 and Table 2). Exposure within the tumor was 10-fold higher than in plasma. The circulating plasma concentration at this 24-hour time point corresponded to a free drug concentration of approximately 6 nM (2 ng / mL) based on 94% plasma protein binding in mice.

[0322] PK / PD data indicated that relatively low plasma concentrations of SRA737 that persisted above the effective concentration (e.g., SRA737 was above 100 nM plasma concentration for 24 hours) elicited significant anti-tumor activity in mice and provided a PK / PD benchmark for use in the clinical setting.

[0323]

Table 4

[0324] SRA737 + LDG is a novel drug combination in which low-dose gemcitabine (LDG), which is non-cytotoxic, functions as a potent exogenous inducer of replication stress that enhances the anti-tumor activity of SRA737. Preclinical models have demonstrated that only sub-therapeutic levels of gemcitabine are required to enhance the anti-tumor effect of SRA737.

[0325] Example 2: Summary of Pharmacokinetics (PK) Studies Several studies have been conducted to evaluate the PK properties of SRA737, including absorption (in vitro permeability assay and in vivo PK following intravenous and oral administration), distribution (in vivo tissue distribution and in vitro plasma protein binding), and metabolism (in vitro hepatocyte and studies of CYP inhibition and induction).

[0326] The PK of SRA737 was determined in mice, rats, dogs, and monkeys following oral and intravenous administration (Table 3). Very favorable absolute oral bioavailability (F%) was observed, particularly in mice (105%) and monkeys (90 - 104%), which was consistent with moderate metabolism and favorable permeability observed in in vitro models. Also, acceptable elimination t 1 / 2 was observed in various species. Furthermore, the effect of diet on the PK of the SRA737 clinical formulation capsule presentation was evaluated in dogs. There was no significant effect of diet on oral bioavailability (Error! Reference source not found. 4). The plasma protein binding of SRA737 at 1 and 10 μM was examined in mouse, minipig, monkey, and human plasma using ultracentrifugation, and in dog plasma (10 μM) using rapid equilibrium dialysis. High plasma protein binding (about 94%) was observed in mice, while moderate plasma protein binding was observed in humans (about 87%) and non - rodent toxicology species (about 80% and 87% for minipigs and monkeys, respectively) (Table 5).

[0327] [Table 5]

[0328] [Table 6]

[0329] [Table 7]

[0330] The membrane permeability of SRA737 was evaluated by parallel artificial membrane permeability assay (PAMPA) and Caco-2 assay. The permeability in the PAMPA assay was classified as low. At 10 μM, the permeability in the Caco-2 assay had an efflux ratio (A>B / B>A) of 0.8 and was 20.7±9.1×10 -6 cm / sec, indicating that SRA737 had relatively high passive permeability and low efflux potential.

[0331] The formation of SRA737-related metabolites was measured in cryopreserved hepatocytes from human, mouse, rat, minipig, and monkey samples after incubation with SRA737 at a nominal concentration of 10 μM for up to 4 hours. The rank order of stability from highest to lowest across species was rat≈mouse>monkey≈human>>dog>>minipig. In the preparation of human hepatocytes, approximately 67% of the parent remained after 4 hours of incubation, compared to 75% and 7% in the preparations of rat and minipig, respectively. Eight human SRA737 metabolites were observed. Also, all SRA737 metabolites formed by human hepatocytes were also formed by monkey hepatocytes. In monkeys, six metabolites were present in equal or greater abundance. No human-specific metabolites were observed, although two of the human metabolites were not formed in any other species in equal or greater abundance.

[0332] The excretion of SRA737 was studied in mice and rats administered SRA737 either intravenously at 5 mg / kg or orally at 10 mg / kg. Urine and feces were collected over a 24-hour period after dosing. In mice, renal excretion of intact SRA737 was consistently low (less than 10% of the dose) after both oral and intravenous administration. After intravenous administration of SRA737, less than 8% of the dose was recovered as intact drug in feces. In contrast, after oral administration, this figure was less than 3%. Excretion of intact SRA737 was lower in rats than in mice, with less than 1% of the dose being excreted renally over 24 hours and less than 1.5% of the dose being excreted in feces over 24 hours.

[0333] At the highest concentration of SRA737 investigated (IC 50 > 10 - 50 μM), no significant inhibition of major CYP enzymes (1A2, 2A6, 2C9, 2C19, 2D6, and 3A4) was observed, suggesting that the compound has a low potential to mediate significant metabolic drug-drug interactions. A minimal concentration-dependent induction of CYP1A2 (< 10% of positive control, omeprazole) was observed in vitro, suggesting that SRA737 may have a minimal impact on the metabolism of co-administered drugs that are mainly metabolized by CYP1A2.

[0334] Collectively, the permeability, metabolic stability, and demonstrable oral bioavailability observed in preclinical species suggest favorable oral absorption in humans.

[0335] Example 3: Summary of Toxicology Study Results The toxicity of SRA737 was evaluated in mice, minipigs, and monkeys in a 28-day repeated oral dose study under Good Laboratory Practice (GLP), as well as in a 3-cycle combination study in mice using gemcitabine and cisplatin.

[0336] Toxicokinetic data for SRA737 administered as monotherapy are summarized in Error! Reference source not found6. The pattern of toxicology study results observed in the pivotal studies in mice, minipigs, and monkeys was generally similar and consistent with the mechanism of action of SRA737, but generally, monkeys appeared to be the least sensitive toxicology species. Data from the studies in monkeys suggest that higher exposures than would be predicted from the data in mice and minipigs are likely to be tolerated in humans. Based on the similarity of monkey and human data for plasma protein binding, hepatocyte stability, and other ADMET data, monkeys were identified as the most suitable non-clinical model for the determination of potential human toxicity.

[0337] Dose-dependent toxicology study results related to myelotoxicity, including various decreased red blood cell and white blood cell parameters associated with increased bone marrow or extramedullary hematopoiesis, and atrophy of lymphoid organs including the thymus, were observed in key studies in mice and minipigs. These study results were reversible upon discontinuation of drug administration. Also, toxicology study results in the gastrointestinal tract were observed in minipigs and in early mouse and monkey studies, and changes in reproductive organs, particularly the testes, were observed in minipigs and mice but not in monkeys. These latter changes were not reversible in mice, although their relevance to adult cancer patients from these study results in sexually immature animals appears limited.

[0338] The MTD was 75 / mg / kg / day (225mg / m 2 / day) in mice, and the HNSTD was 10mg / kg / day (350mg / m 2 / day) in minipigs. Since no toxicology study results were observed in the key monkey toxicity study, the NOAEL of 20mg / kg / day (240mg / m 2 / day) was the highest dose tested.

[0339] Study results from triple combination studies in mice (SRA737 administered in combination with IV gemcitabine and cisplatin on an intermittent schedule over 18 days) reproduced the study results observed in single-agent toxicology studies in this species. However, reversible intestinal epithelial degeneration was also observed in the high-dose triple combination group. Only reversible myelotoxicity, resulting splenomegaly, and high-dose intestinal observations were considered to be exacerbated by the administration of SRA737 compared to those observed following administration of cisplatin / gemcitabine control group alone. Thus, in some cases, the clinical combination of SRA737 and gemcitabine caused exacerbation of hematologic toxicity and gastrointestinal toxicity.

[0340]

Table 8

[0341] Example 4: Phase 1 Clinical Study to Establish the Maximum Tolerated Dose and Plasma Concentration with SRA737 Monotherapy The Phase 1 clinical study was conducted in "all comers" to establish stability, tolerability, and pharmacokinetics, i.e., no genetic selection was performed ("dose escalation phase"). Initially, a cohort consisting of a single patient was administered escalating doses of SRA737, starting with cohort 1, and 20 mg / day was orally administered on a continuous daily dosing schedule with a 28-day cycle. The dose was escalated until the maximum tolerated dose (MTD) was identified.

[0342] In the dose escalation phase, 18 subjects were administered SRA737 at 9 dose level cohorts from 20 to 1300 mg QD, and the median treatment period was 62.5 days (range 1 - 226). The dose level cohorts up to 1000 mg of SRA737 were completed without dose-limiting toxicity (DLT). Two of the three subjects experienced DLT at a once-daily dose of 1300 mg, and each was unable to receive 75% of the planned SRA737 due to GI intolerance, and the individual Gi effects were mild. Thus, 1300 mg exceeded the maximum tolerated dose on a once-daily dosing schedule. Assuming a half-life of SRA737 of approximately 10 hours, a cohort administered 500 mg twice daily was added to determine if a twice-daily dosing schedule could improve GI tolerability. One of the six subjects experienced DLT in the cohort administered 500 mg twice daily. This was due to grade 4 thrombocytopenia with grade 3 neutropenia and anemia, and 75% of the planned SRA737 could not be administered. Based on overall tolerability and GI events (nausea, vomiting, and diarrhea), subjects were enrolled at a once-daily dose level of 800 mg and were overall more tolerable than 1000 mg (subjects required fewer dose reductions, experienced fewer severe (G3 / G4) AEs and significantly fewer fatigue AEs). The maximum tolerated dose (MTD) was established at 1000 mg QD or 500 mg BID.

[0343] The pharmacokinetic parameters of the monotherapy cohorts were monitored and summarized in Table 7. C at 1000 mg QDmax and AUC 0-24 were 2391 ng / mL and 26795 ng·h / mL, respectively. C max was calculated at 1000 mg QD (411 ng / mL), exceeding that determined to be effective in the preclinical model. Also, doses ≥ 300 mg QD also exceeded the preclinical model and were effective.

[0344]

Table 9

[0345] Example 5: Phase 1 Clinical Study to Establish the Maximum Tolerated Dose and Plasma Concentrations with SRA737 Combination Therapy The Phase 1 clinical study was conducted in "all comers" to establish the stability, tolerability, and pharmacokinetics of SRA737 administered in combination with gemcitabine, i.e., no genetic selection was performed ("dose escalation phase"). First, a cohort consisting of a single patient was administered escalating doses of SRA737, starting with cohort 1, with 40 mg / day orally administered on days 2, 3, 9, 10, 16, and 17 of each 28-day cycle. Also, the cohorts were administered various doses of gemcitabine, starting with cohort 1, with 300 mg / m 2 / day administered intravenously over 30 minutes on days 1, 8, and 15 of each 28-day cycle.

[0346] Figure 3A presents a summary of the dosing of the cohorts tested. During the dose escalation phase, a total of 55 subjects were administered SRA737 at doses of 40 - 600 mg of SRA737 combined with LDG doses of 50 - 300 mg / m 2 in 13 dose escalation cohorts. No dose-limiting toxicity (DLT) as defined by the protocol was observed.

[0347] The pharmacokinetic parameters of the single-agent therapy cohorts were monitored and summarized in Table 8. The pharmacokinetic profile of SRA737 was an AUC of 3550 ng·h / mL and C 0-24 of 548 ng / mL with 150 mg of SRA737max was revealed. At this dose, C min (52 ng / mL) exceeded what was determined to be effective in the preclinical model.

[0348]

Table 10

[0349] Additional cohorts are monitored and the dose of SRA737 is escalated until the maximum tolerated dose (MTD) is identified and to optimize combination dosing with gemcitabine. All enrolled subjects who receive at least one dose of SRA737 and provide at least one evaluable PK concentration or have evaluable data for each specific PDn assessment are evaluable for PK and PDn, respectively. Collection of serious adverse events (SAEs) is initiated on the date of informed consent. Radiological evaluations are performed within 4 weeks of the first dose of SRA737 (or gemcitabine if PK SRA737 dosing is omitted) and repeated every 6 weeks in stage 1. In stage 2, evaluations are performed every 8 weeks and at long-term follow-up evaluations every 16 weeks. Evaluations are performed more frequently if clinically necessary. Cardiac evaluations (echocardiogram [ECHO] and electrocardiogram [ECG]) are performed. Optional triplet tumor biopsies are collected within 28 days prior to administration of the first dose of SRA737 in some cases. Within 7 days of the first dose of SRA737 (or gemcitabine if PK SRA737 dosing is omitted), the following evaluations are completed: a complete physical examination, clinical disease assessment, SAEs, and concomitant medications, WHO performance status, and laboratory (for hematology, biochemistry, and pregnancy testing) blood evaluations. During the single-dose PK lead-in period at the visit from day -7 to day -4, concomitant medications, vital signs (including temperature, blood pressure, and pulse), height, weight, body surface area (BSA), and WHO performance status are collected. Blood samples are obtained pre-dose for hematology, biochemistry, pregnancy testing, troponin I or T, and for tumor markers and tumor profiling. Adverse events (AEs) are collected starting at the time of SRA737 administration. Archival tissue is submitted for tumor profiling. PK samples are collected at up to 10 time points over a 48-hour period from day -7 to day -4 (first dose of SRA737 for PK). The sponsor may, in some cases, reduce the PK sampling requirements, including changes or exclusions to the visit from day -7 to day -4, when sufficient data have been collected and analyzed to evaluate the single-dose PK of SRA737. Dosing starts on day 1 and the following procedures are performed at regular intervals. · Adverse events and concomitant medications: Continuously · Physical examination centered on symptoms (if medically necessary): On day 1 of each cycle · Evaluation of radiation disease: Every 6 weeks after day 1 of stage 1, and every 8 weeks after day 1 of stage 2 · Clinical disease evaluation: In stage 1, every 6 weeks after day 1, and in stage 2, every 4 weeks after day 1 · Tumor markers (serum or urine) (if applicable): In stage 1, every 6 weeks from day 1 of cycle 1, and in stage 2, every 4 weeks from day 1 of cycle 1 · WHO performance status and weight / BSA: On day 1 of each cycle · Vital signs: On day 1 and day 8 in stage 1, and on day 1, day 8, and day 15 in stage 2 · Evaluation of blood (hematology, biochemistry, troponin I or T if applicable) and urine (urinalysis) by the testing facility - See section 7.2.2 for the detailed schedule. · Echocardiogram: On day 1 of cycle 2 · ECG: On day 1 of cycles 1 and 2, then on day 1 before dosing every third cycle thereafter, and on day 1 of any cycle with dose escalation within the subject · PK pharmacokinetic samples are taken at the following time points in cycle 1, namely, before dosing on day 1, before dosing on day 10, and up to 6 time points after dosing on day 10. Only in the case of stage 2, additional samples are taken before dosing on day 8 and before dosing on day 15. Compliance review of the subject diary card.

[0350] Example 6: A phase 1 / 2 clinical study to confirm the efficacy of SRA737 monotherapy in selected tumors with genetic changes conferring Chk1 sensitivity Clinical trials for administration to humans are conducted to confirm the effectiveness of the treatment method and patient selection strategy of the SRA737 monotherapy disclosed herein for prospectively selected, genetically defined subjects having tumor types known to have a high prevalence of genomic changes that are expected to sensitize tumors to Chk1 inhibition ("cohort expansion phase"). The cohort expansion phase consists of six indication-specific expansion cohorts of approximately 20 prospectively selected, genetically defined subjects each. The cohorts are subjects with previously treated metastatic colorectal cancer [CRC], high-grade serous ovarian cancer [HGSOC] without CCNE1 gene amplification, HGSOC with CCNE1 gene amplification (or alternative gene changes with similar functional effects), metastatic castration-resistant prostate cancer [mCRPC], advanced non-small cell lung cancer [NSCLC], and head and neck squamous cell carcinoma [HNSCC], or anal squamous cell carcinoma [SCCA]. Subjects are first administered SRA737 according to the dosing regimen established in Example 4. The dosing regimen may change during the course of the clinical trial in some cases.

[0351] The subject has evidence of tumor tissue or ctDNA that their tumor harbors a combination of mutations predicted to confer sensitivity to Chk1 inhibition. Subjects are selected based on prospective tumor tissue gene profiling using NGS.

[0352] Expansion cohort subjects have tumors harboring genomic changes predicted to confer sensitivity to Chk1 inhibition in at least two of the following categories (a) - (c). a. Important tumor suppressor genes that regulate the progression / arrest of the G1 cell cycle, such as RB1, TP53. In the case of patients with NHSCC or SCCA, the HPV-positive status is also considered for eligibility. b. The DDR pathway, including ATM, CDK12, BRCA1, and BRCA2. In the case of patients with CRC, mismatch repair (MMR) gene changes and / or high microsatellite instability are also considered for eligibility. c. Genetic indicators of replication stress such as gain-of-function / amplification of Chk1, ATR, or other related genes d. Oncogenic drivers such as MYC, KRAS e. In the HGSOC cohort specific to CCNE1 gene amplification, CCNE1 gene amplification (or alternative gene changes with similar functional effects) is required.

[0353] In some embodiments, the subject meets one of the following criteria (a - e). a. Metastatic CRC i. Histologically and / or cytologically confirmed CRC ii. Generally, has received at least one prior treatment for a progressive / metastatic disease. b. HGSOC i. Histologically confirmed high-grade serous ovarian cancer, fallopian tube cancer, or primary peritoneal cancer ii. Platinum-intolerant recurrence subjects, or subjects with platinum-resistant disease defined as radiological evidence of disease progression within 6 months of receiving prior platinum-based chemotherapy. Patients with platinum-refractory disease (as defined by the European Society for Medical Oncology [ESMO] guidelines) are ineligible. c. Progressive NSCLC i. Locally advanced, recurrent, or metastatic histologically confirmed NSCLC ii. Generally, has received at least one prior treatment for a progressive / metastatic disease. d. mCPRC i. Adenocarcinoma of the prostate, histologically or cytologically confirmed, that has progressed after androgen blockade therapy e. HNSCC or SCCA i. Histologically confirmed HNSCC from any primary site or SCCA ii. In the case of HNSCC: Locally advanced disease (i.e., disease that persists or progresses after radiotherapy for treatment purposes and is not a candidate for surgical relief due to incurable or morbid conditions), or metastatic disease iii. For SCCA: Locally advanced or metastatic disease for which no curative therapy is available iv. The subject has received at least one prior treatment for a progressive / metastatic disease.

[0354] The subject generally has a measurable disease (according to Response Evaluation Criteria in Solid Tumors, version 1.1 [RECIST v1.1]), or in the case of mCRPC, a disease evaluable according to any of the following, i.e., a measurable disease according to RECIST v1.1, an increase in prostate specific antigen (PS), or a circulating tumor cell (CTC) count of 5 or more cells per 7.5 ml of blood.

[0355] Enrollment into the expanded cohort occurs, in some cases, in parallel with the dose escalation phase (see Example 5). Subjects eligible for the cohort expansion phase are enrolled into the escalating cohort whenever possible. Any such subject is considered to be enrolled in both phases simultaneously.

[0356] The disease is measured according to RECIST v1.1 criteria for subjects with solid tumors, according to the revised IWG criteria (Cheson 2007) for subjects with NHL, and for patients with mCRPC, according to any one of the following complexes: A) measurable disease according to RECIST v1.1, B) an increase in PSA, or C) a CTC count of 5 or more cells per 7.5 ml of blood.

[0357] Baseline evaluation includes radiation measurements of the lesion appropriate to the nature of the malignant tumor. In some cases, this includes CT scans, liver CT scans, abdominal CT scans, MRIs, X-rays, bone scans, and / or other radiation measurements clinically required or clinical measurements as needed (e.g., evaluation of palpable lesions or measurement of tumor markers). All areas of disease present are documented (even if a particular lesion is not followed for response), and the dimensions of all measurable lesions are clearly recorded in the scan report. Any non-measurable lesions are noted as present. In the case of clinical measurements, documentation by color photography including a ruler to estimate the size of the lesion is strongly recommended as this will aid in an independent external review of the response.

[0358] Tumor evaluations are repeated every 8 weeks or more frequently if clinically necessary. Subjects with bone metastases being followed by bone scan are scanned every 8 weeks (±1 week) for the first 6 months and then every 16 weeks (±2 weeks) thereafter. During long-term follow-up evaluations, evaluations of subjects who have not yet progressed and have not started alternative anticancer therapy are performed every 16 weeks unless more frequently requested by the study sponsor or the study physician in charge. All lesions measured at baseline are measured at each subsequent disease evaluation and clearly recorded in the scan report. Any non-measurable lesions noted at baseline are noted in the scan report as present or absent. All subjects excluded from the study treatment for reasons other than progressive disease (PD) should be re-evaluated at the time of treatment discontinuation, except if a tumor evaluation has been performed within the past 4 weeks. Subjects are followed for PD until disease progression or study discontinuation.

[0359] There is a measurable disease, and all subjects who have been administered at least one cycle of SRA737 and have a baseline evaluation of the disease, plus at least one post-baseline evaluation, are evaluable for response. Subjects who produce clear evidence of PD without a formal disease evaluation, and subjects without a formal disease evaluation prior to study discontinuation, are considered non-responders. Complete remission and PR need to be confirmed by subsequent evaluations at least 4 weeks later. For the determination of stable disease (SD), relevant criteria need to be met at least once, at least 6 weeks after the first dose of SRA737 has been administered.

[0360] If rapid tumor progression occurs before completion of 4 weeks of treatment, the subject is classified as having early progression.

[0361] Tumor response should be classified as "not evaluable" (NE) only when it cannot be classified under another response category, for example, when baseline and / or follow-up evaluations are not performed or are not performed appropriately.

[0362] Define the response criteria as follows. a. Complete remission (CR): Disappearance of all target lesions. All pathological lymph nodes generally have a short axis reduction to <10 mm (regardless of whether they are target or non-target). b. Partial remission (PR): Take the baseline diameter sum as a criterion, with at least a 30% decrease in the sum of the diameters of the target lesions. c. Progressive disease (PD): Take the shortest sum in the study as a criterion (if this is the minimum in the study, this includes the baseline sum), with at least a 20% increase in the sum of the diameters of the target lesions. In addition to the 20% relative increase, the sum generally also indicates an absolute increase of at least 5 mm. (Note: The appearance of one or more new lesions is also considered progression.) d. Stable disease (SD): Take the shortest diameter sum during the study as a criterion, with neither a reduction sufficient to qualify for PR nor an increase sufficient to qualify for PD.

[0363] Results The dose escalation phase utilized an accelerated titration design starting with 20 mg of SRA737, orally administered QD on a 28-day cycle. When SRA737-related ≥ grade 2 toxicity was observed during Cycle 1, a rolling-6 design was continued for incremental dose escalations in a single-subject cohort.

[0364] The cohort expansion phase was initiated simultaneously when the circulating plasma concentration of SRA737 exceeded the minimum effective concentration of SRA737 modeled from the mouse efficacy study. Subsequently, information on dose selection for the expanded cohort was provided from the experience obtained during the ongoing dose escalation phase. The cohort expansion phase enrolled subjects with genetically defined tumors that were prospectively selected by next-generation sequencing (FoundationOne) and harbored genomic alterations hypothesized to confer sensitivity to Chk1 inhibition. Subjects with the following tumors were eligible for enrollment: i) high-grade serous ovarian cancer (HGSOC), ib) HGSOC with presumed elevated CCNE1 gene network amplification, ii) colorectal cancer (CRC), iii) metastatic castration-resistant prostate cancer (mCRPC), iv) non-small cell lung cancer (NSCLC), and v) squamous cell carcinoma (head and neck (SCCHN), anal (SCCA)).

[0365] This Phase 1 / 2 signal-seeking study (NCT02797964) was designed to investigate the safety and tolerability of continuous daily dosing of SRA737 and to evaluate preliminary antitumor activity in tumors with genetic alterations that may confer increased endogenous RS and Chk1i sensitivity. Genetic screening was performed to identify and select subjects harboring two or more of these genetic alterations. The study was designed as an extensive investigation to evaluate the association between various causes of endogenous RS and the antitumor activity of SRA737 monotherapy in various cancer indications.

[0366] The dose-limiting toxicity (DLT) was evaluated following the pharmacokinetic (PK) starting dose and throughout cycle 1 of treatment and was defined as an adverse event that was very highly or highly associated with SRA737, i.e., grade ≥ 4 neutropenia or thrombocytopenia lasting > 7 days, grade ≥ 3 febrile neutropenia or thrombocytopenia with bleeding, grade ≥ 3 non-hematological toxicity, or the inability to administer ≥ 75% of the SRA737 dose planned in cycle 1 due to drug-related toxicity.

[0367] Preliminary antitumor activity was evaluated by target tumor response and overall response according to RECIST v1.1.

[0368] Dose and Pharmacokinetics (PK) In the dose-escalation phase, 18 subjects were administered SRA737 across 9 dose levels (i.e., 20, 40, 80, 160, 300, 600, 1000, and 1300 mg) ranging from 20 to 1300 mg QD. Of these subjects, 3 experienced DLT (failure to administer 75% of the planned dose). Two experienced DLT at 1300 mg QD due to gastrointestinal intolerance and one experienced DLT at 500 mg BID due to thrombocytopenia. The maximum tolerated dose (MTD) was established at 1000 mg QD or 500 mg BID.

[0369] The plasma pharmacokinetics of SRA737 were reproducible and generally dose-proportional. Cmax and AUC0-24 at the PK dose of 1000 mg were 2098 ng / mL and 20270 ng·h / mL, respectively, and Cmin (289 ng / mL) exceeded that determined to have antitumor activity (100 nM, approximately 37.9 ng / mL) in the preclinical efficacy model. Also, all doses of ≥ 300 mg QD exceeded this threshold level.

[0370]

Table 11

[0371] The registration of the cohort expansion phase was initiated with 600 mg of SRA737. Based on the overall tolerability, including the particularly common gastrointestinal events (nausea, vomiting, diarrhea), the recommended dose used in the expanded cohort was later determined to be 800 mg QD (RP2D).

[0372] In the cohort expansion phase, out of 512 prospectively identified subjects, 355 were tested for genetic changes associated with Chk1 sensitivity. Among these subjects, 237 (67%) met the genetic eligibility criteria and 94 were registered in the expanded cohort across six tumor types.

[0373] Safety Adverse reactions (TEAE) occurring during treatment were reported in 106 (99%) of the subjects, and 97 subjects (91%) experienced events related to at least one SRA737. Most of the TEAEs were of mild to moderate severity (90% grade 1 / grade 2). The most common TEAEs were diarrhea (68%), nausea (66%), vomiting (51%), and fatigue (47%).

[0374] The most common ≥ grade 3 TEAEs were neutropenia and disease progression (each 8%), lymphopenia (5%), infections and hyponatremia (each 4%), and nausea, abdominal pain, upper abdominal pain, fatigue, elevated AST, dyspnea, pleural effusion, and maculopapular rash (each 3%). The most common ≥ grade 3 SRA737-related TEAEs were neutropenia (8%) and maculopapular rash (3%).

[0375] Six grade 5 TEAEs were reported up to a maximum of 30 days after the previous treatment. None were considered related to SRA737. The median exposure period was 2.3 cycles (range <1 - 9 cycles). No evidence of toxicity or cumulative toxicity and / or reduced tolerability was observed up to a maximum of 9 cycles of SRA737 administered.

[0376]

Table 12

[0377] Reaction Over both the incremental and expansion cohorts, 107 subjects have received treatment with SRA737. In the cohort expansion phase, the largest number of subjects were enrolled in the HGSOC cohort (n = 38), followed by the CRC cohort (n = 27).

[0378] The mean number of previous treatment plans across all tumor types was 4.2, consistent with a highly pretreated study population. The HGSOC cohort had approximately five lines of treatment history.

[0379] The median treatment delay from commitment to enable gene profiling until Day 1 of Cycle 1 was approximately 2 months (61 days), highlighting the challenges of prospective gene screening using tumor tissue.

[0380] Of the treated subjects, 64 (60%) were considered evaluable for RECIST target tumor response and had an available gene profile.

[0381] The best treatment effect of SD was seen in 34 (32%) subjects. At the data cut-off point (May 3, 2019), long stable disease (SD) lasting ≥ 4 months was recorded in 22 (21%) subjects, observed in all cohort expansion tumor types (excluding SCCHN. 4 subjects).

[0382] No subjects had a confirmed RECIST partial or complete response, although a subset of subjects demonstrated a marked target tumor reduction. Antitumor activity was observed in subjects with tumors of HGSOC, CRC, mCRPC, and NSCLC.

[0383] In this study, HGSOC appeared to be the most sensitive tumor to SRA737 monotherapy (maximum target tumor reduction of 27% and 29%, disease control rate (DCR) = 54%). Along with the signal exploration objectives of this study, tumor response was further investigated with respect to the gene profiles determined for the registered and treated tumor types.

[0384] RS driver genes, including functional categories (G1 / S, tumor genes, DNA repair genes), were investigated to identify individual genes with enhanced sensitivity to gene networks and / or treatment with SRA737.

[0385] The number of subjects with tumor changes in the selected gene networks varied based on i) the occurrence of specific gene changes within the range of explored indications and ii) the registered measurement criteria.

[0386] The subject cohort was specifically enrolled to presumptively enhance mutations in the CCNE group, but genetic analysis revealed that only 4 / 24 HGSOC subjects had tumors with clearly positive CCNE1 amplification, 3 were stable disease (SD), and 1 was progressive disease (PD). Although a very limited dataset, no clear correlation with sensitivity was observed in relation to CCNE1 amplification.

[0387] Among the gene networks of >10 subjects, activating mutations in the RAS network trended towards a low DCR (25%) in 15 / 20 PD, and a +20% change in the overall mean tumor %, as well as a short study duration (DOS) of 2 cycles. Given a strong negative correlation with activity, subsequent pathway / signal exploration analysis was performed excluding RAS-mutated tumors.

[0388] In contrast to RAS mutations, changes in the P13K gene network (PIK3CA, AKT, PTEN) were associated with a DCR of 77%.

[0389] Similarly, genetic alterations in multiple components of the FA / BRCA gene network were associated with a 71% DCR and a DOS of 3.8 cycles.

[0390] The majority of subjects with a significant response (tumor shrinkage and / or SD > 4 months) harbored alterations in either or both the PI3K gene network and the FA / BRCA gene network. Alterations in the CCNE gene network (DCR = 67%) partially overlapped with FA / BRCA subjects but were mutually exclusive with alterations in the PI3K network. Many subjects with demonstrable tumor reduction harbored two genetic alterations in the FA / BRCA gene network, frequently involving secondary mutations in DDR kinases genes (ATR, PRKDC).

[0391] Summary In this first-in-human trial of SRA737 monotherapy, the maximum tolerated dose (MTD) was 1000 mg / day, and based on overall tolerability and PK, the recommended monotherapy dose is 800 mg / day. These results highlight the safety and tolerability of SRA737.

[0392] The median treatment delay to C1D1 (day 1 of treatment cycle 1) exceeded 2 months due to the commitment to enable gene profiling, highlighting the challenges of prospective gene screening using tumor tissue. In the population of subjects with progressive disease (4+ lines of treatment history), this delay in treatment initiation may, although debatable, exacerbate the underlying disease progression.

[0393] Signal-seeking studies were widely investigated across tumor indications and tumor RS driver genetic characteristics to identify potentially SRA737-sensitive settings. Preliminary evidence suggests that some endogenous causes of RS may contribute to or enhance Chk1-dependence across some solid tumor types.

[0394] The highly pre-treated HGSOC cohort (with approximately five types of treatment history) demonstrated directionally favorable disease control (DCR = 54%), and notable maximum tumor reductions were 29% and 27%. No clear trend towards improved sensitivity was observed in CCNE1 gene amplification. The small subset of subjects enrolled with clear CCNE1 amplification made it difficult to draw the most reliable conclusions.

[0395] From i) the evaluation of significant reduction in tumor volume and ii) the longest DOS, it became clear that subjects with tumors harboring FA / BRCA network mutations showed the most favorable outcomes (DCR = 71%, DOS = 3.8 cycles). The genes of this network encode factors that directly or indirectly regulate replication fork metabolism in response to RS. Importantly, these trends in sensitivity were observed across multiple indications, suggesting the possibility of histology-independent sensitization.

[0396] Also, the same fundamental tumor genetic characteristics associated with improved sensitivity in this study were also observed in subjects treated in a clinical study of SRA737 + low-dose gemcitabine (LDG) (NCT02797977).

[0397] Within the FA / BRCA pathway, some significant tumor reductions occurred in subjects harboring two genetic changes, including secondary mutations in frequently DDR checkpoint kinase genes (ATR, PRKDC). This phenomenon, also observed in the SRA737 + LDG clinical study, suggests that overlapping complex mutations may lead to and / or result from increased endogenous RS and genomic instability.

[0398] By identifying both positive and negative selection gene markers determined in this clinical study, a ctDNA-enriched strategy focused for future clinical study development is provided, which could potentially accelerate prospective enrollment.

[0399] Collectively, these data demonstrate promising evidence of the anti-tumor activity of SRA737 and identify several gene networks associated with improved SRA737 sensitivity.

[0400] These findings suggest that additional RS, such as through exogenous sources, may be desirable to produce a long objective response with highly selective Chk1i, as demonstrated by the anti-tumor activity demonstrated in the SRA737-02 clinical study in which enhanced LDG was combined with SRA737.

[0401] Example 7: A Phase 1 / 2 clinical study to confirm the efficacy of combination therapy with SRA737 in selected tumors with genetic changes conferring Chk1 sensitivity A clinical trial is conducted in humans (the "cohort expansion phase") to confirm the effectiveness of the methods of combination therapy with SRA737 and patient selection strategies for the treatments disclosed herein, in prospectively selected genetically defined subjects having tumor types known to have a high prevalence of genomic changes predicted to sensitize tumors to Chk1 inhibition. In the cohort expansion phase, approximately 20 prospectively selected genetically defined subjects are enrolled in each of four indication-specific cohorts, namely high-grade serous ovarian cancer (HGSOC), small cell lung cancer (SCLC), soft tissue sarcoma (STS), and cervical / anal genital cancer. Based on PK data establishing a dosing regimen that produces effective concentrations of SRA737 (see Example 5), 500 mg of SRA737 and 100 mg / m 2 of gemcitabine are used as the starting dose levels. The dosing schedule may vary in some cases during the course of the trial. SRA737 capsules are taken on an empty stomach (subjects fast for at least 2 hours before and 1 hour after dosing) unless otherwise instructed.

[0402] Subjects have a. Histologically or cytologically proven progressive malignancies of the following types for which no other conventional therapy is considered appropriate. i. High-grade serous ovarian cancer (HGSOC) 1. Histologically confirmed high-grade serous ovarian cancer, fallopian tube cancer, or primary peritoneal cancer 2. Platinum-resistant or refractory disease, or if the subject is intolerant to platinum therapy ii. Small cell lung cancer 1. Unless otherwise approved by the sponsor of the clinical trial, generally, the subject has received at least 1 but no more than 3 prior treatments for the progressive disease. iii. Soft tissue sarcoma 1. Includes undifferentiated pleomorphic sarcoma / malignant fibrous histiocytoma (MFH) (including high-grade spindle cell sarcoma / polymorphic liposarcoma), leiomyosarcoma, and dedifferentiated liposarcoma. Some other types of STS may be eligible with approval from the sponsor of the clinical trial. 2. Unless otherwise approved by the sponsor of the clinical trial, generally, the subject has received at least 1 but no more than 3 prior treatments for the progressive disease. iv. Cervical cancer / anal genital cancer 1. Includes all cervical cancers and progressive / metastatic squamous cell carcinomas of the anus, penis, vagina, and vulva 2. Unless otherwise approved by the sponsor of the clinical trial, generally, the subject has received at least 1 but no more than 3 prior treatments for the progressive disease. v. Urothelial cancer 1. Histologically confirmed locally advanced and unresectable or metastatic urothelial cancer of the bladder, upper ureter, or urethra 2. Generally, the subject has received at least 1 but no more than 3 prior treatments for the progressive disease. b. Measurable disease according to RECIST v1.1 (see below) c. The subject has predicted sensitivity to Chk1 inhibition based on factors including gene profiling of tumor tissue or ctDNA, HPV status, and germline BRCA1 and BRCA2 gene status. All subjects have gene profiling from tumor tissue or ctDNA, and the profiling is performed prospectively if required to evaluate Chk1 sensitivity or retrospectively otherwise. i. For subjects with HGSOC, a description of the wild-type status of somatic or germline BRCA1 and BRCA2 confers eligibility without the need for prospective gene profiling. If a description of the BRCA status is not available, in some cases, gene profiling is prospectively performed to determine eligibility. ii. Subjects with SCLC are eligible without the need for prospective gene profiling based on the very high prevalence of cancer-related changes in tumor suppressor genes (e.g., TP53 and RB1) in this population. iii. For subjects with STS and all other subjects for whom gene profiling is prospectively performed, eligibility is determined by review by the trial sponsor of genetic abnormalities detected in genes in the following categories. Key tumor suppressor genes that regulate G1 cell cycle progression / arrest, such as RB1 and TP53. For relevant cancers, the status of positive human papillomavirus (HPV) is also considered for eligibility. a. DNA damage response pathways, including ATM, CDK12, BRCA1, BRCA2, mismatch repair gene changes, and / or high microsatellite instability b. Genetic indicators of replication stress, such as gain of function / amplification of Chk1 or ATR or other related genes c. Oncogenic drivers such as MYC, CCNE1 iv. For subjects with anal-genital cancer, a known HPV-positive status confers eligibility in some cases without the need for prospective gene profiling. If the HPV status is unknown or negative, gene profiling (or, where appropriate, a human papillomavirus test) is prospectively performed in some cases to determine eligibility. Subjects with cervical or anal squamous cell carcinoma are eligible without the need for prospective gene profiling based on the very high prevalence of HPV positivity in these populations.

[0403] Instead, the subject has one of the progressive malignancies histologically or cytologically proven as described above, and it is demonstrated that the tumor tissue or ctDNA harbors one or more mutations expected to confer sensitivity of the tumor to Chk1 inhibition. Eligibility is determined by review by the sponsor of genetic abnormalities detected in genes in the following categories. a. Key tumor suppressor genes that regulate the progression / arrest of the G1 cell cycle, such as RB1, TP53. In the case of related cancers, the positive human papillomavirus (HPV) status is also considered for eligibility. b. DNA damage response pathways, including ATM, CDK12, BRCA1, BRCA2, mismatch repair gene changes, and / or high microsatellite instability c. Genetic indicators of replication stress, such as gain of function / amplification of Chk1 or ATR or other related genes d. Oncogenic drivers, such as MYC, KRAS

[0404] The subject is excluded based on the following criteria. a. Received the following previous or current anticancer therapies within the noted time frame and recovered from toxicity prior to administration of SRA737. i. Radiation therapy, chemotherapy, PARP inhibitor, other targeted therapy, or other IMP within 2 weeks ii. Nitrosourea or mitomycin C within 6 weeks iii. Any previous treatment with a Chk1 inhibitor at any time or previous treatment with an ATR inhibitor within 6 months b. Three or fewer previous treatment plans for progressive disease (not applicable to the HGSOC expanded cohort) c. Other malignancies within the past 2 years, excluding appropriately treated tumors d. In the opinion of the trial responsible physician, if the subject is very likely to experience clinically significant myelosuppression e. Occurrence of toxic symptoms during continuation of previous treatment with an NCI-CTCAE grade greater than 1 f. History of allergy to gemcitabine g. New or ongoing brain metastases. Subjects with brain metastases that are asymptomatic over an 8-week period, stable on X-ray, and not treated with steroids during that period may, in some cases, be included with the approval of the sponsor. h. High medical risk due to non-malignant systemic diseases i. Serologically positive for hepatitis B, hepatitis C, or HIV j. Severe heart disease, baseline left ventricular ejection fraction < 45%, history of myocardial ischemia within the past 6 months, or history of arrhythmia requiring treatment, unless approved by the sponsor k. Previous bone marrow transplantation or extensive radiotherapy to the bone marrow exceeding 25% within the past 8 weeks l. Peanut allergy m. QTcF > 450 msec in adult males and > 470 msec in adult females n. In some cases, gastrointestinal (GI) dysfunction or GI diseases that significantly modify the absorption of SRA737 o. Inability to swallow the capsule without chewing or crushing p. A participant in or planning to participate in another interventional clinical trial q. Any other condition that, in the opinion of the principal investigator of the trial, would render the subject an inappropriate candidate

[0405] All enrolled subjects with measurable disease who are administered at least 75% (stage 1) or 83% (stage 2) of SRA737 (or equivalent if the sponsor chooses to evaluate an alternative dosing schedule) in one cycle and have a baseline evaluation of the disease and at least one post-baseline evaluation are evaluated for response. All subjects enrolled in the cohort expansion phase have measurable disease, have had at least one cycle of study drug administered as defined above, have a baseline evaluation of the disease, plus at least one post-baseline disease evaluation, and are confirmed to meet the gene selection requirements, and are evaluated for response.

[0406] In addition, the subject has a measurable disease and has received at least 83% of SRA737 in one cycle (if the sponsor elects to evaluate an alternative dosing schedule). Subjects who progress to PD, intolerable toxicity, or death prior to evaluation after baseline are evaluable and are classified as non-responders.

[0407] Analysis of all efficacy endpoints is based on the response-evaluable population and is evaluated using the RECIST v1.1 criteria as described below.

[0408] Other endpoints include duration of response (DOR), disease control rate (DCR), time to response (TTR), PFS, time to progression (TTP), and OS. Other exploratory objectives are described in Table 11.

[0409]

Table 13

[0410] Additional trials are conducted with SRA737 in combination with other therapies, including administering a chemotherapeutic agent, administering an antibody or antibody fragment, administering radiation therapy, administering an exogenous inducer of replication stress, or combinations thereof. Other trials are conducted with SRA737 in combination with other therapies, including administering olaparib, niraparib, rucaparib, talazoparib, cisplatin, a ribonucleotide reductase inhibitor, etoposide, SN-38 / CPT-11, mitomycin C, or combinations thereof.

[0411] The results of this study confirm the efficacy of SRA737 combination therapy for the treatment of tumors with known genetic changes that are expected to confer sensitivity to Chk1 inhibition (Figure 17). Proof-of-concept clinical activity was seen in tumor types such as anogenital (e.g., anus), neck, and rectum. The combination of LDG and SRA737 has good tolerability. This first-in-human clinical study provides proof-of-concept that sub-therapeutic doses of LDG effectively enhance SRA737 anti-tumor activity.

[0412] RECIST criteria The evaluation of disease response in this study is performed according to the Revised RECIST Criteria V1.1. The RECIST criteria are described in detail by Eisenhauer, et al. (New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1) Eur J Cancer [Internet] 2009), which is incorporated herein by reference for all teachings.

[0413] At baseline, tumor lesions / lymph nodes are typically classified as measurable or non-measurable as follows.

[0414] Measurable Tumor lesion: Typically, at least one dimension with a minimum size of the following sizes (the longest diameter in the measurement plane should be recorded) is accurately measured. - 10 mm on CT scan (CT scan slice thickness of 5 mm or less. For imaging guidance, see Appendix II of Eisenhauer et al. [Eisenhauer, 2009]). - 10 mm caliper measurement by clinical examination (lesions that cannot be accurately measured with calipers should be recorded as non-measurable) - 20 mm on chest X-ray

[0415] Malignant lymph nodes: Pathologically enlarged, to be considered measurable, lymph nodes are usually 15 mm in short axis when evaluated by CT scan (CT scan slice thickness is recommended to be 5 mm or less). At baseline and during follow-up evaluations, usually only the short axis is measured and followed up.

[0416] Non-measurable All other lesions, including small lesions (pathological lymph nodes with a longest diameter < 10 mm or a short axis of ≧ 10 to < 15 mm) and truly non-measurable lesions. Lesions considered truly non-measurable are generally identified by physical examination that cannot be measured by reproducible imaging techniques, including leptomeningeal disease, ascites, pleural effusion or pericardial effusion, inflammatory breast disease, lymphatic invasion of the skin or lung, abdominal tumors / visceral hypertrophy.

[0417] Bone lesions, cystic lesions, and lesions previously treated with local therapy require specific comments.

[0418] Bone lesions: - Bone scans, PET scans, or plain films are generally not considered appropriate imaging techniques for measuring bone lesions. However, these techniques can be used to confirm the presence or absence of bone lesions. - Lytic bone lesions or mixed lytic-osteoblastic lesions with identifiable soft tissue components that can be evaluated by tomographic imaging techniques such as CT or MRI are usually considered measurable lesions if the soft tissue component meets the above definition of measurability. - Osteoblastic bone lesions are usually non-measurable.

[0419] Cystic lesions: - Lesions that meet the criteria of radiologically defined simple cysts are usually not considered malignant lesions (neither measurable nor non-measurable) because they are simple cysts by definition. - "Cystic lesions" considered to represent cystic metastases are usually considered measurable lesions if they meet the above definition of measurability. However, if non-cystic lesions are present in the same subject, these are preferred for selection as target lesions.

[0420] Lesions previously treated with local therapy: - Tumor lesions located in areas previously irradiated or treated with other local area therapies are not usually considered measurable unless there is evidence of progression at the lesion. Study protocols usually specify the conditions under which such lesions are considered usually measurable.

[0421] Methods of evaluation All measurements, when clinically evaluated, are usually made using calipers and recorded in metric units. All baseline evaluations are generally performed as close as possible to the start of treatment and are never performed more than 4 weeks prior to the start of treatment.

[0422] The same evaluation methods and techniques are usually used to characterize each lesion identified and reported at baseline and during follow-up evaluations. Lesions being followed that cannot be imaged are usually evaluated by imaging-based evaluation rather than clinical examination, unless they are evaluable by clinical examination.

[0423] Clinical lesions are usually considered measurable when they are superficial and have a diameter ≥ 10 mm (e.g., skin nodules) when evaluated using calipers. For skin lesions, documentation by color photography including a ruler to estimate the size of the lesion is recommended. As described above, when lesions can be evaluated by both clinical examination and imaging, imaging evaluation is generally performed because it is more objective and in some cases is also reviewed at the end of the study.

[0424] When identifying particularly new lesions, CT is generally preferred over chest X-ray, especially when progression is an important evaluation item because CT is more sensitive than X-ray. However, in some cases, lesions on chest X-ray are considered measurable if they are clearly demarcated and surrounded by aerated lung.

[0425] CT is generally the best available and reproducible method currently available for measuring lesions selected for response assessment. This guideline defines the measurability of lesions on CT scans based on the assumption that the CT slice thickness is 5 mm or less. When the CT scan has a slice thickness greater than 5 mm, the minimum size of a measurable lesion is twice the slice thickness. Also, in certain situations (e.g., in the case of body scans), MRI is also acceptable. Details regarding the use of both CT and MRI for the assessment of objective tumor response evaluation are provided from the publication by Eisenhauer et al.

[0426] Ultrasound is generally not useful for the assessment of lesion size and is generally not used as a measurement method. Ultrasonography generally cannot be reproduced in its entirety for later independent review, and since ultrasonography is operator-dependent, it generally cannot be guaranteed that the same technique and measurements will be taken from one assessment to the next (as described in more detail by Eisenhauer, et al. (2009)). When new lesions are identified by ultrasound during the course of a study, confirmation by CT or MRI is usually recommended. If there are concerns about radiation exposure with CT, in some cases, MRI is used in selected cases instead of CT.

[0427] The use of endoscopic and laparoscopic techniques for objective tumor evaluation is generally not recommended. However, they are generally useful for confirming pathologic complete remission when a biopsy is obtained or for determining recurrence in clinical trials where recurrence following complete remission or surgical resection is an evaluation item.

[0428] Generally, tumor markers alone are not used to assess objective tumor response. However, when the marker is initially above the normal upper limit, the tumor marker is usually normalized so that the subject can be considered to be in complete remission.

[0429] Cytology and histology are generally used to distinguish PR from CR when required by protocol, rarely (e.g., in tumor types such as germ cell tumors where known residual benign tumors may remain). When it is known that an effusion is a potential side effect of treatment (e.g., using certain taxane compounds or angiogenesis inhibitors), if the measurable tumor meets the criteria for a responding or stable disease to distinguish between a response (or stable disease) and progressive disease, a cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment is considered.

[0430] Tumor Response Assessment To assess objective response or future progression, the total baseline body tumor burden is generally estimated and used as a comparator for subsequent measurements. Measurable disease is generally defined by the presence of at least one measurable lesion.

[0431] When multiple measurable lesions are present at baseline, up to a maximum of five lesions in total (and a maximum of two lesions per organ) representing all involved organs are generally identified as target lesions, recorded at baseline, and measured (i.e., if the subject has only one or two organ sites involved, a maximum of two and four lesions respectively are recorded). Target lesions are generally selected based on their size (the longest diameter of the lesion), generally represent all involved organs, but more generally are lesions suitable for reproducible repeated measurements. In some cases, the largest lesion is not suitable for reproducible measurement, and in this situation, as illustrated in Figure 3 of Eisenhauer, et al. (2009), the next largest lesion that can be reproducibly measured is usually selected.

[0432] Lymph nodes are noteworthy because they are normal anatomical structures that are visible by imaging in some cases, even if not invaded by a tumor. Pathological nodules that are defined as measurable and in some cases identified as target lesions generally meet the criterion of a short axis of ≥15 mm by CT scan. Only the short axis of these nodules typically contributes to the baseline sum. The short axis of a nodule is the diameter that is typically used by a radiologist to determine whether the nodule is invaded by a solid tumor. The size of a nodule is usually reported as two-dimensional in the plane in which the image is obtained (in the case of a CT scan, this is almost always the axial plane; in the case of an MRI, the acquisition plane is in some cases axial, sagittal, or coronal). The smaller of these measurements is the short axis. For example, an abdominal lymph node reported as 20 mm × 30 mm has a short axis of 20 mm and is eligible as a measurable malignant nodule. In this example, 20 mm needs to be recorded as the nodule measurement. All other pathological nodules (with a short axis of ≥10 mm but <15 mm) are usually considered non-target lesions. Nodules with a short axis <10 mm are generally considered non-pathological and are generally not recorded or followed up.

[0433] The sum of the diameters of all target lesions (the longest in the case of non-nodular lesions and the short axis in the case of nodular lesions) is usually calculated and reported as the baseline diameter sum. When including lymph nodes in the sum, only the short axis is then added to the sum as described above. The baseline diameter sum is generally used as a criterion to further characterize any objective tumor regression in the measurable dimension of the disease.

[0434] All other lesions (or sites of the disease) that include pathological lymph nodes are generally identified as non-target lesions and are generally recorded at baseline. Measurements are usually not required, and these lesions are usually followed up as "present," "absent," or rarely "obvious worsening" (details follow below). Additionally, it is possible to record multiple non-target lesions that invade the same organ as a single entry in the case record form (e.g., "multiple enlarged pelvic lymph nodes" or "multiple liver metastases").

[0435] Response Criteria Complete Remission (CR): Disappearance of all target lesions. Any pathological lymph nodes are reduced to < 10 mm in short axis (regardless of whether they are target or non-target).

[0436] Partial Remission (PR): Taking the baseline sum of diameters as a reference, at least a 30% decrease in the sum of the diameters of the target lesions.

[0437] Progressive Disease (PD): Taking the shortest sum (if this is the minimum in the study, this includes the baseline sum) as a reference, at least a 20% increase in the sum of the diameters of the target lesions. In addition to a 20% relative increase, the sum generally demonstrates an absolute increase of at least 5 mm. (Note: The appearance of one or more new lesions is also generally considered progression.)

[0438] Stable Disease (SD): Taking the shortest sum of diameters as a reference, there is neither a reduction sufficient to qualify for PR nor an increase sufficient to qualify for PD.

[0439] Lymph nodes identified as target lesions generally record the actual short-axis measurements (measured in the same anatomical plane as the baseline examination), even if the nodules regress below 10 mm. That is, since normal lymph nodes are generally defined as having a short axis of < 10 mm, when a lymph node is included as a target lesion, even if the criteria for complete remission are met, the "sum" of the lesions is not zero in some cases. Therefore, case report forms or other data collection methods are designed to record target nodular lesions in a separate section in some cases, and each nodule generally achieves a short axis of < 10 mm to qualify for CR. In the case of PR, SD, and PD, the actual short-axis measurements of the nodules are preferably included in the sum of the target lesions.

[0440] During the study, all lesions (nodular and non-nodular) recorded at baseline are usually recorded at their actual measurements in each subsequent evaluation, even if very small (e.g., 2 mm). However, sometimes, lesions or lymph nodes recorded as target lesions at baseline become very thin on CT scans, so the radiologist may feel resistance to assigning accurate measurements in some cases and report them as "too small to measure" in some cases. When this occurs, it is generally important to record the value in the case report. In the radiologist's opinion, if the lesion has probably disappeared, the measurement is generally recorded as 0 mm. If the lesion is thought to be present and appears thin but is too small to measure, a default value of 5 mm is usually assigned. (Note: Generally, lymph nodes usually have a size that can be defined when normal and are frequently surrounded by fat, as in the retroperitoneum, so this rule is less likely to be used for lymph nodes. However, if a lymph node is thought to be present and appears thin but is too small to measure, a default value of 5 mm is usually assigned in this situation as well.) This default value is derived from a 5-mm CT slice thickness (but usually does not change with varying CT slice thicknesses). Since the measurements of these lesions are potentially non-reproducible, providing this default value usually prevents false responses or progression based on measurement errors. However, if repeated, if the radiologist can provide the actual measurement, it is usually recorded even if it is less than 5 mm.

[0441] When "fragmenting" non-nodular lesions, the longest diameters of the fragmented parts are usually added together to calculate the target lesion sum. Similarly, when lesions adhere, the plane between them, which would be useful for obtaining the maximum diameter measurements of each individual lesion, is generally maintained. If the lesions are truly adherent such that they are no longer separable, the vector of the longest diameter in this case is generally the maximum longest diameter of the "adherent lesions".

[0442] In some cases, some non-target lesions are actually measurable, but they are not usually measured. Instead, they are generally only qualitatively evaluated at the time points specified in the protocol.

[0443] Complete remission (CR): Disappearance of all non-target lesions and normalization of tumor marker levels. The size of all lymph nodes is not pathological (<10 mm short axis).

[0444] Non-CR / non-PD: Persistence of one or more non-target lesions (s) and / or maintenance of tumor marker levels beyond normal limits

[0445] Progressive disease (PD): Obvious worsening of existing non-target lesions (see comments below) (Note: The appearance of one or more new lesions is also considered progression.)

[0446] When the subject also has a measurable disease, to achieve "obvious worsening" based on non-target disease, even if there is SD or PR in the target disease, the overall level of the target disease substantially deteriorates as if the total systemic progressive tumor tissue volume has increased sufficiently to warrant discontinuation of therapy. A mild "expansion" in the size of one or more non-target lesions is usually not sufficient to qualify for obvious worsening. Therefore, it is generally very rare to designate overall progression based only on changes in non-target disease despite SD or PR in the target disease.

[0447] Subjects having only unmeasurable diseases occur in some Phase III trials when having a measurable disease is not a criterion for study participation. The same general concept applies here as above, but in this example, there is no assessment of measurable diseases that takes into account the interpretation of an increase in the burden of unmeasurable diseases. Worsening in non-target diseases is usually not easily quantifiable (by definition: all lesions are truly unmeasurable), so a useful test that is usually applicable when evaluating subjects for obvious worsening is whether an increase in the overall disease burden based on changes in unmeasurable diseases is of a magnitude comparable to the increase required to declare PD for measurable diseases, i.e., an increase in the amount of systemic tumor tissue representing an additional 73% increase in "volume" equivalent to a 20% increase in diameter in measurable lesions. Examples include an increase in pleural effusion from "trace" to "large", an expansion of lymphatic disease from localized to extensive, or in some cases, described in the protocol as "sufficient to require a change in therapy". If "obvious worsening" is seen, the subject is usually considered to have overall PD at that time. It would be ideal to have objective criteria applicable to unmeasurable diseases, but due to the nature of the disease itself, it is generally very difficult to do so. Therefore, the increase is usually substantial.

[0448] The appearance of new malignant lesions usually indicates disease progression, so some comments regarding the detection of new lesions are usually important. Generally, there are no specific criteria for identifying new radiographic lesions. However, the discovery of new lesions is usually obvious. That is, it usually does not result from differences in scan technology, changes in imaging methods, or findings that are thought to represent something other than a tumor (e.g., some "new" bone lesions are, in some cases, only the healing or inflammation of existing lesions). This is particularly important when the subject's baseline lesions show partial or complete remission. For example, necrosis of liver lesions is frequently reported as a "new" cystic lesion in CT scan reports, which it usually is not.

[0449] Lesions identified in follow-up studies in anatomical locations not scanned at baseline are generally considered new lesions and generally indicate disease progression. An example of this is a subject who has an internal disease at baseline and during the study a brain CT or MRI is indicated, which reveals metastases. Brain metastases in the subject are generally considered evidence of PD even if the patient did not have a brain imaging at baseline.

[0450] If a new lesion is not clear, for example due to its small size, continuous therapy and follow-up generally clarify whether it represents a truly new disease. If repeated scans confirm the presence of a new lesion beyond doubt, progression is then declared using the date of the initial scan.

[0451] Although FDG-PET response assessment requires additional studies, it may be appropriate to incorporate the use of FDG-PET scans to complement CT scans in the assessment of progression (especially of a potentially "new" disease). New lesions based on FDG-PET imaging are usually identified according to the following algorithm. a. FDG-PET negative at baseline and FDG-PET positive at follow-up * generally indicates signs of PD based on a new lesion (* a lesion that is "positive" on an FDG-PET scan generally means one that avidly desires FDG with uptake more than twice that of the surrounding tissue on the attenuation-corrected image). b. FDG-PET not performed at baseline and FDG-PET positive at follow-up - FDG-PET positivity at follow-up corresponds to a new site of disease confirmed by CT, which is generally PD. - If FDG-PET positivity at follow-up evaluation is not confirmed as a new site of disease on CT, an additional follow-up evaluation CT scan is usually performed to determine whether there is truly progression occurring at that site (if performed, the date of PD will be the date of the initial abnormal FDG-PET scan). A "positive" FDG-PET scan lesion generally means one that avidly takes up FDG with uptake more than twice that of the surrounding tissue on the attenuation-corrected image. - If FDG-PET positivity at follow-up evaluation corresponds to an existing site of disease on CT that is not progressing based on the anatomical images, this is generally not PD.

[0452] Evaluation of Best Overall Response The best overall response is generally the best response recorded from the start of the investigational treatment to the end of treatment. In the event that the response is not documented after the end of the therapy of this trial, the post-treatment evaluation is generally considered in the determination of the best overall response, unless alternative anticancer therapy is given. Assignment of the best overall response for a subject depends on the findings for both the target and non-target diseases and generally also takes into account the appearance of new lesions.

[0453] In general, it is assumed that response evaluations are performed at each protocol-specified time point. Table 12 provides a summary of the calculation of the overall response status at each time point for subjects with measurable disease at baseline.

[0454] When a subject has only non-measurable (and thus non-target) disease, Table 13 is generally used.

[0455] When imaging / measurement is not performed at all at a particular time point, the subject is generally not evaluable (NE) at that time point. If only a subset of lesion measurements is performed at the time of evaluation, generally, the case is considered NE at that time point as well, unless there is a persuasive claim that the response at the time point assigned by the contribution of the individual missing lesion(s) does not change. This is most likely to occur in the case of PD. For example, if a subject has a baseline sum of 50 mm with three measured lesions and at follow-up evaluation only two lesions are evaluated but they show a sum of 80 mm, the subject is generally considered to have achieved a PD state regardless of the contribution of the missing lesion.

[0456] The best overall response is generally determined when all data for the subject are known.

[0457] Determination of the best response in a clinical trial where complete or partial remission confirmation is not generally required. The best effect in these trials is generally defined as the best effect over all time points (e.g., a subject with SD at the first evaluation, PR at the second evaluation, and PD at the last evaluation has a best overall response of PR). When SD is considered the best response, SD generally also meets the minimum time from the protocol-specified baseline. If the minimum time is not met when SD is the best time point effect otherwise, the best response of the subject generally depends on subsequent evaluations. For example, a subject with SD at the first evaluation and PD at the second evaluation and not meeting the minimum duration of SD shows a best effect of PD. The same subject who becomes non-assessable after the first SD evaluation is generally considered non-evaluable.

[0458] Even if nodular diseases are included in the sum of target lesions and the nodules shrink to a "normal" size (<10 mm), in some cases, they have the measured values reported on the scan. This measured value is generally recorded even if the nodules are normal so as not to exaggerate progression if progression is based on an increase in the size of the nodules. As described above, this means that in some cases, subjects with CR do not have a "zero" total in the case report form (CRF).

[0459] Subjects with a wide range of deterioration in health that require discontinuation of treatment without objective evidence of disease progression at that time are generally reported as "threatening deterioration." Generally, every effort is made to document objective progression even after treatment discontinuation. Threatening deterioration is generally not a descriptor of objective response. It is a reason to stop the investigational therapy. The objective response status of such subjects is generally determined by the evaluation of target and non-target diseases shown in Tables 12 and 13.

[0460] The conditions defining "EP, early death, and inevaluability" are specific to the study and are generally clearly described in each protocol (depending on the treatment period, treatment cycle).

[0461] In some situations, it is difficult to distinguish residual disease from normal tissue. When the assessment of complete remission depends on this determination, it is generally recommended to investigate the residual lesion (fine needle aspirate / biopsy) before assigning a state of complete remission. In some cases, when residual radiographic abnormalities are considered to represent fibrosis or scarring, FDG-PET is used to upgrade the response to CR in a manner similar to biopsy.

[0462] In cases of unclear findings of progression (e.g., very small, uncertain new lesions, cystic changes or necrosis in existing lesions), treatment generally continues until the next scheduled evaluation in some cases. If progression is confirmed at the next scheduled evaluation, the date of progression is generally the earlier date when progression was suspected.

[0463]

Table 14

[0464]

Table 15

[0465] Duration of response The duration of the overall effect is generally measured from the first time the measurement criteria are met for CR / PR (the first recorded one) until the first day on which a recurrent or progressive disease is recorded in the study.

[0466] The duration of overall complete remission is generally measured from the first time the measurement criteria are met for CR until the first day on which a recurrent disease is objectively documented.

[0467] Stable diseases are generally measured from the start of treatment (in a randomized trial, from the date of randomization) until the criteria for progression are met, taking the minimum sum as the reference in the study (if the baseline sum is the minimum, this is the basis for the calculation of PD).

[0468] Results This phase 1 / 2 signal-seeking study (NCT02797977) was designed to investigate the safety and tolerability of SRA737 in combination with sub-therapeutic (low-dose) gemcitabine (LDG), and to evaluate the preliminary antitumor activity in tumors with genetic changes that can confer increased endogenous RS and Chk1i sensitivity. A prospective genetic screening was performed to identify and select subjects who harbored one or more of these genetic changes. The study was designed as an extensive investigation to explore the relationship between various causes of endogenous replication stress and the SRA737+LDG antitumor activity in the expansion phase, in order to elaborate on potential gene signatures and / or tumor phenotypes that may warrant additional therapeutic investigation.

[0469] Inducers of replication stress enhance Chk1i Replication stress (RS) is revealed by the deceleration and stalling of replication forks that generate vulnerable and exposed single-stranded DNA that is prone to damage. Chk1 plays an essential role in maintaining the stability of replication forks and the cellular response to RS in order to maintain genomic stability. RS driver genes can be divided into several functional categories, including G1 / S tumor suppressors, oncogenes, and DNA repair genes. An inclusive list of candidate "RS driver" genes was generated based on the compilation of available preclinical and clinical data, mapped to these functional categories, and used for prospective subject selection to enable the direct clinical exploration of these potential Chk1i-sensitizing genetic features.

[0470] To determine whether additional exogenous RS could reinforce the endogenous RS conferred by tumor genetics, subjects were treated with the potent RS inducer LDG with the intent of enhancing the antitumor activity of SRA737 to further increase tumor dependence on Chk1.

[0471] Methods In the dose-escalation phase, subjects with solid tumors in cohorts of 3-6 subjects were administered escalating doses of SRA737 in combination with gemcitabine at doses below various therapeutic doses. SRA737 was administered for 2 days on days 1, 8, and 15 of a 28-day cycle following LDG administration. The lead-in dose for pharmacokinetic (PK) analysis was performed 4-7 days prior to cycle 1 (C1).

[0472] The expansion cohort was initiated simultaneously when the circulating plasma concentration of SRA737 exceeded the minimum effective concentration of SRA737 modeled from the mouse efficacy study.

[0473] Subsequently, information on dosing in the expanded cohort was provided based on the experience obtained during the ongoing dose escalation phase. The cohort expansion phase enrolled subjects with genomically defined tumors harboring genomic alterations hypothesized to confer sensitivity to Chk1 inhibition, prospectively selected by next-generation sequencing (FoundationOne). Subjects with the following tumors, namely i) soft tissue sarcoma, ii) high-grade serous ovarian cancer (HGSOC), iii) small cell lung cancer, and iv) ano-genital cancer / cervical cancer were eligible for enrollment. Subjects with ano-genital or cervical cancer were eligible for enrollment without prospective genetic profiling based on the near-ubiquitous prevalence of HPV positivity in this population.

[0474] This Phase 1 / 2 signal-seeking study was generally conducted at Phase 1 cancer units in specialty areas. Subjects aged ≥18 years with an ECOG performance status of 0–1, measurable disease (by RECIST 1.1), and archival tumor tissue (or willing to consent to a biopsy) were eligible to participate in the study. Subjects in the expansion phase had received 1–3 prior treatments for progressive disease, with no restrictions on prior treatment in the case of HGSOC.

[0475] Dose-limiting toxicity (DLT) was evaluated following the PK lead-in dose and throughout cycle 1 of treatment and was defined as the inability to administer 5 out of 6 doses (83%) of SRA737 or all doses of gemcitabine planned in cycle 1 due to an adverse event (AE) highly likely or likely related to the investigational agents (SRA737 and gemcitabine), namely grade 4 neutropenia or thrombocytopenia (>7 days duration), febrile neutropenia, thrombocytopenia with bleeding (≥grade 3), non-hematologic toxicity (≥grade 3), or toxicity related to the investigational agents (SRA737 and gemcitabine).

[0476] Preliminary antitumor activity was evaluated by target tumor response and overall response according to RECIST v1.1.

[0477] Genomics: Genomic information was required to participate in the clinical trial, but several subjects had registered with a failed FMI report (Foundation Medicine), and thus their genomics were unknown. Some patient reports have not yet been received and are currently unknown. Most of the patients sequenced were sequenced using only the FMI assay. However, several patients were sequenced using both FMI and the Guardant 360 panel (one patient was sequenced only by G360). In most cases, the assays were highly concordant. However, the Guardant 360 panel has fewer genes than FMI, and thus some genes will be "missing".

[0478] HPV Status: When possible, the HPV status was determined using standard IHC-based methods. Foundation Medicine provided data on the HPV status of the sequenced tumors. Only HPV strains 6, 11, 16, and 18 were sequenced. 16 and 18 are the most common strains in cervical cancer (75%) and anal cancer (79%), but several other subtypes have been suggested as high-risk / cancer-related (HPV-33 is approximately 5% of anal cancers).

[0479] Characteristics and Dose Evaluation of Subjects A total of 55 subjects were administered SRA737 at doses of 40 - 600 mg of SRA737, variously combined with LDG doses of 50 - 300 mg / m2 in 13 dose-escalating cohorts. Dose-limiting toxicity (DLT) as defined by the protocol was not observed, but tolerability was particularly evident at the highest dose tested. The pharmacokinetic profile of SRA737 revealed an AUC0-24 of 3550 ng·h / mL and a Cmax of 548 ng / mL at 150 mg of SRA737. At this dose, the Cmin (52 ng / mL) exceeded that determined to be effective in preclinical models.

[0480] Enrollment into the expansion cohort was initiated at 500 mg of SRA737 + 100 mg / m2 of LDG.

[0481] Based on the overall tolerability, the recommended dose for use in the expanded cohort was determined to be 500 mg of SRA737 + 250 mg / m2 of LDG (RP2D).

[0482] Of the 335 prospectively identified subjects, 204 were tested for genetic changes associated with Chk1 sensitivity. Of these subjects, 176 (86%) met the genetic eligibility criteria and 86 were enrolled in the four expanded cohorts.

[0483] Safety Treatment-emergent adverse events (TEAEs) were reported in 137 (99%) of the subjects, and 131 subjects (94%) experienced at least one investigational drug (SRA737 and / or gemcitabine)-related event. The majority of TEAEs were of mild to moderate severity (91% grade 1 / grade 2). The most common TEAEs were nausea (60%), vomiting (50%), diarrhea (45%), fatigue (43%), anemia (33%), and fever (31%). The most common ≥ grade 3 TEAEs were neutropenia (9%), anemia and increased ALT (6% each), increased AST (5%), thrombocytopenia (4%), hyponatremia and lymphopenia (3% each). The most common ≥ grade 3 investigational drug (SRA737 and / or gemcitabine)-related TEAEs were neutropenia (9%), increased ALT (5%), thrombocytopenia and increased AST (4%), and anemia (3%). Five grade 5 TEAEs were reported up to 30 days after the previous treatment. None were considered related to SRA737. Median exposure duration: 2 cycles (range <1 to 13 cycles). There was no evidence of acute or cumulative toxicity and / or decreased tolerability up to 13 cycles.

[0484]

Table 16

[0485] Results of SRA737-02 A total of 141 subjects received treatment with SRA737(+LDG) across both the incremental and expansion cohorts. The largest number of subjects (n = 35) were enrolled in the ano-genital cancer / cervical cancer cohort, followed by the HGSOC (n = 28) and SCLC (n = 23) cohorts.

[0486] The mean number of prior treatment regimens across all tumor types was 2.8, and subjects in the HGSOC cohort had been treated an average of 4.2 times previously, consistent with a highly pre-treated cohort.

[0487] The median treatment duration for all subjects was 2 cycles, with a maximum treatment duration of 13 cycles. Twenty-two subjects were still receiving investigational treatment at the time of the data cut-off (May 3, 2019).

[0488] Forty-one subjects had a best response of stable disease (SD). Long SD lasting ≥4 months was recorded in 32 subjects and was observed in all expansion cohorts.

[0489] Of the treated subjects, 81 / 141 (57%) were considered evaluable for RECIST target tumor response, and of these, 54 had an available gene profile.

[0490] Partial response (PR) was observed in 6 subjects (Figure 17). These included 3 subjects with ano-genital cancer and 1 subject each with rectal, cervical, and ovarian cancer. In general, tumor responses were first recorded at the end of cycle 2 (the first scan during the study).

[0491] The highly pre-treated HGSOC cohort (with approximately 4 lines of treatment history) demonstrated a directionally favorable disease control rate (DCR = 67%), with a maximum tumor reduction of 38% observed (38%).

[0492] The ORR for subjects with squamous ano-genital cancer / cervical cancer was 22% (4 / 18).

[0493] Anogenital cancer was identified as the most sensitive indication to SRA737+LDG in this clinical study (ORR=30%, DCR=60%).

[0494] The magnitude of target tumor reduction in anogenital tumors was significant. At the data cutoff, two subjects achieved ongoing reductions of -66% and -51% respectively, and the third subject achieved a -41% reduction.

[0495] Furthermore, several subjects with anogenital cancer had notable response durations. 5 / 10 (50%) of the subjects received the investigational treatment for ≥4 months, with a maximum duration of approximately 11 months. At the data cutoff, the investigational treatment was ongoing in 5 subjects (5 / 10, 50%).

[0496] In line with the signal exploration objective of this study, tumor response was further investigated with respect to the gene profiles determined for the registered and treated tumor types.

[0497] RS driver genes, including functional categories (G1 / S, cancer cells, DNA repair genes), were investigated across multiple indications to identify individual genes with enhanced sensitivity to gene network and / or treatment with SRA737+LDG.

[0498] The number of subjects with tumor changes in the selected gene networks varied based on i) the occurrence of specific gene changes within the range of explored indications, and ii) the registered measurement criteria.

[0499] Mutations in the RAS gene network were associated with relatively poor responses.

[0500] In contrast, changes in the PI3K gene network (PIK3CA, AKT, or PTEN mutations) resulted in a strong 75% DCR and PR in two subjects.

[0501] Similarly, gene mutations in multiple components of the FA / BRICA gene network resulted in an 81% DCR and a 25% response rate (RR).

[0502] The defined CCNE network observations were based on limited data (n = 6), but changes in the CCNE network were associated with a favorable DCR (67%), and tumor responses were observed in subjects with HGSOC.

[0503] Some of the strong responses observed in this study were associated with gene changes in the FA / BRCA network and were frequently accompanied by secondary changes in one of two DDR checkpoint kinase genes (ATR, PRKDC).

[0504] The study results suggest that mutations associated with multiple replication forks may exacerbate endogenous RS and genomic instability and / or their consequences. Consistent with this hypothesis, subjects with available genetic features that achieved PR were generally determined to have changes in multiple gene networks.

[0505] In particular, an increase in tumor mutational burden (TMB) was associated with specific tumor responses, especially in subjects with anorectal and rectal cancers. Specifically, 3 out of 4 subjects with anorectal cancer presenting an increase in TMB showed strong responses that included some of the most profound tumor reductions observed in the SRA737-02 study.

[0506] Summary In this first-in-human trial of SRA737+LDG, the RP2D (recommended Phase II dose) was determined to be 500 mg of SRA737 plus 250 mg / m 2 of gemcitabine. Consistent with the RS-inducing properties of LDG, this combination utilized gemcitabine doses substantially below the standard of therapeutic dose levels (10 - 25%), such as doses below the therapeutic dose of gemcitabine. The combination of SRA737+LDG was safe and well-tolerated.

[0507] Overall, the safety and efficacy data determined in this study support that SRA737+LDG appears to readily lead to development as a single therapy and potentially be combinable with other therapeutic substances.

[0508] This signal discovery study comprehensively investigated the entire spectrum of tumor phenotypes and tumor RS driver genetic features to identify potential settings sensitive to SRA737 in the context of the enhancing effect of exogenous RS inducers. Preliminary evidence suggests that some endogenous causes of RS combined with LDG significantly enhance Chk1 activity. For example, mutations in the PI3K gene network were correlated with both tumor response and strong DDR (75%).

[0509] FA / BRCA network mutations were associated with the most favorable outcomes (ORR = 25%, DCR = 81%) in this study. The FA / BRCA gene network encodes a series of Fanconi anemia and other proteins that are directly or indirectly involved in replication fork metabolism and the management of RS.

[0510] The sensitivity of SRA737+LDG associated with mutations in the PI3K and FA / BRCA gene networks observed in this study is consistent with similar findings from the SRA737 monotherapy clinical study (NCT02797964), strengthening the notion that these changes function as Chk1i-sensitizing genetic contexts. Furthermore, these network changes occur across several tumor phenotypes, suggesting the potential for histology-independent sensitization.

[0511] Particularly, subjects whose tumors harbored multiple gene network changes tended to have more favorable tumor reduction and longer DOS (study duration). This phenomenon, also observed in the clinical study of SRA737 monotherapy, suggests that overlapping complex mutations may lead to and / or result from an increase in endogenous RS and genomic instability.

[0512] In particular, in the anal-genital cohort, a preliminary correlation was also observed with an increase in the tumor mutational burden (TMB), a notable tumor response (3 out of 4 patients achieved significant tumor reduction). The increase in TMB (e.g., above the intermediate TMB level) is consistent with increased genomic instability and represents a possible intensification strategy.

[0513] Overall, these data provide clear evidence of the anti-tumor activity of SRA737+LDG. Multiple partial responses were observed, generally first recorded at the first study scan (end of cycle 2).

[0514] Efficacy was determined across several tumor symptoms, and the most robust efficacy signal was observed in the squamous anal-genital / neck cohort (ORR = 22%, DCR = 50%).

[0515] Specifically, significant and clear anti-tumor activity was observed in subjects with advanced anal-genital cancer (ORR = 30%, DCR = 60%), including notable tumor reduction (e.g., -66% tumor reduction, resolution of pleural effusion) and promising treatment duration (e.g., approximately 11 months).

[0516] Second metastatic anal-genital cancer represents an unmet major medical need with no approved therapies and a significant inhibition of life expectancy. These promising data indicate that SRA737+LDG will be an effective treatment option for these patients.

[0517] Example 10: Evaluation of Genetic Abnormalities and Tumor Mutational Burden Genetic abnormalities and TMB were evaluated in tumor samples from individual patients using the FoundationOne CDx™ assay.

[0518] FoundationOne CDx (trademark) (F1CDx) is a next-generation sequencing-based in vitro diagnostic device for detecting substitutions, insertions and deletions (indels) of up to 324 or more genes, copy number alterations (CNA), and selected gene rearrangements, and for selecting genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed and paraffin-embedded (FFPE) tumor tissue specimens. The test can be used as a companion diagnostic to identify patients who may benefit from treatment with targeted therapies. Additionally, the F1CDx assay can provide tumor mutation profiling for use in oncology for patients with solid malignancies.

[0519] Genes with full coding exon regions evaluated in the F1CDx assay for the detection of substitutions, insertions-deletions (indels), and copy number alterations (CNA) are shown in Table 1. Further details of the FoundationOne CDx assay can be found at www.accessdata.fda.gov / cdrh_docs / pdf17 / P170019B.pdf, and some of those details are described below.

[0520] FoundationOne CDx (F1CDx) is a single-site assay performed by Foundation Medicine, Inc. The assay includes reagents, software, instrumentation, and procedures for testing DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumor samples. The assay employs a single DNA extraction method from routine FFPE biopsies or surgical resection specimens, and 50–1000 ng of the specimen is subjected to the construction of a whole-genome shotgun library and hybridization-based capture of all coding exons from more than 300 cancer-related genes, one promoter region, one non-coding RNA (ncRNA), and selected intron regions from 34 commonly rearranged genes (21 of which also have coding exons) (see FoundationOne CDx for the complete list of genes included in F1CDx). Thus, in total, the assay detects changes in up to 324 or more genes. Using the Illumina® HiSeq 4000 platform, libraries selected by hybridization capture are sequenced to a uniform and high depth (median targeted coverage >500×, >99% of exons with >100× coverage). Sequence data are processed using a customized analysis pipeline designed to detect all classes of genomic changes, including base substitutions, indels, copy number changes (amplifications and homozygous deletions), and selected genomic rearrangements (e.g., gene fusions). Additionally, genomic signatures, including microsatellite instability (MSI) and tumor mutational burden (TMB), are reported.

[0521] Specimen collection and preparation. Formalin-fixed, paraffin-embedded (FFPE) tumor specimens are collected and prepared following standard pathology practices. FFPE specimens can be received either as unstained slides or as FFPE blocks. Prior to starting the assay, slides stained with hematoxylin and eosin (H&E) are prepared and then used to confirm the tumorology of the disease and to identify appropriate tissue (0.6 mm 3) The tumor content (≥20% tumor) and the presence of sufficient nucleated cells are reviewed by a qualified pathologist to confirm that the assay can proceed.

[0522] Tumor mutational burden (TMB). TMB is measured by counting all synonymous and non-synonymous variants with an allele frequency of 5% or greater and excluding potential germline variants according to publicly available databases of known germline polymorphisms, including the Single Nucleotide Polymorphism Database (dbSNP) and the Exome Aggregation Consortium (ExAC). Additional germline changes that remain after database querying are evaluated for potential germline status and excluded using the somatic-germline / zygosity (SGZ) algorithm. Furthermore, known likely driver mutations are excluded to eliminate bias in the dataset. The resulting number of mutations is then divided by the coding region corresponding to the total number of variants counted, i.e., 793 kb. The resulting number is reported as mutations per megabase (mutations / Mb).

[0523] High TMB (TBM-H) corresponds to approximately 20 or more somatic mutations per megabase (mutations / Mb). TMB-I corresponds to between approximately 6 and 19 mutations / Mb. Low TMB corresponds to approximately 5 mutations / Mb or less.

[0524] Identified VUS (variants of unknown significance) The VARsome evaluation algorithm was used to evaluate genetic mutations within replication fork genes identified using the FoundationOne CDx assay that were not characterized as being pathogenic. Some of the genetic abnormalities identified as VUS were associated with responsiveness to Chk1i therapy.

[0525] Results Intermediate TMB level (TMB-I) can serve as a biomarker for SR737 therapy in combination with LDG for various cancers, such as anorectal cancers like anal cancer. The classification of TMB and the impact of TMB can be tumor-specific, and there are new but strong clinical correlations with TMB and immunotherapy activity.

[0526] Figures 27A - 27B show waterfall plots demonstrating the response to treatment with SRA737 in combination with low-dose gemcitabine in subjects with specific tumors, namely anorectal (e.g., anal), rectal, high-grade serous ovarian cancer (HGSOC), and cervical. Subjects with at least intermediate TMB are shown.

[0527] The above invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding. However, it will be readily apparent to those skilled in the art that specific changes and modifications can be made to them without departing from the spirit or scope of the appended claims, in view of the teachings of the present invention.

[0528] Therefore, the above are merely illustrative of the principles of the present invention. Those skilled in the art will understand that although not explicitly described or illustrated herein, they can embody the principles of the present invention and devise various configurations within the spirit and scope thereof. Furthermore, all examples and conditional language recited herein are principally intended to assist the reader's understanding of the principles of the present invention and the concepts contributed by the inventors to promote this technology, and should not be construed as being limited to such specifically recited examples and conditions. Moreover, all descriptions in this specification listing the principles, aspects, and embodiments of the present invention, as well as its specific examples, are intended to encompass both their structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function regardless of structure. Additionally, nothing disclosed herein is intended to be dedicated to the public, whether or not such disclosure is explicitly recited in the claims.

[0529] Therefore, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, Title 35, United States Code, Section 112(f) or Title 35, United States Code, Section 112(6) is explicitly defined as being exercised for a limitation in a claim only when the exact phrase "means for" or the exact phrase "step for" begins the limitation in the claim. If such exact phrases are not used for a limitation in the claim, then Title 35, United States Code, Section 112(f) or Title 35, United States Code, Section 112(6) is not exercised.

Claims

[Claim 1] 1. A method of treating cancer in a subject, comprising: administering a therapeutically effective amount of a checkpoint kinase 1 (Chk1) inhibitor to a subject identified as having cancer cells with a genetic abnormality in one or more genes selected from cell cycle regulatory genes, replication stress genes, DNA damage response genes and repair network genes, and oncogenic driver genes; A method comprising:

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