Methods of treatment with a myosin inhibitor using protein levels

Abstract

Methods of treatment with a myosin inhibitor using protein levels; methods of monitoring response to treatment based on such protein levels; and methods of determining whether to treat a patient with a myosin inhibitor based on such protein levels are described herein. Such methods may include obtaining a biological sample of a subject, measuring one or more protein levels in the sample, analyzing the one or more protein levels, and determining the treatment response based on the analysis, and may further include treating the subject based on the determination of treatment response.

Description

Attorney Docket No.267224-557784 Methods of Treatment with a Myosin Inhibitor Using Protein Levels CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of United States provisional patent application nos.63 / 546,878, filed November 1, 2023, and 63 / 562,333, filed March 7, 2024, the entire contents of each of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present invention relates to methods of treatment with a myosin inhibitor using protein levels, as well as methods of monitoring response to treatment based on such protein levels and methods of determining whether to treat a patient with a myosin inhibitor based on such protein levels. BACKGROUND

[0003] Clinical investigation of myosin inhibitors for treating heart disease has accelerated over the past decade. The myosin inhibitor, mavacamten (MYK-461), was investigated in clinical trials for obstructive and non-obstructive hypertrophic cardiomyopathy (HCM), including the PIONEER-HCM (NCT02842242), MAVERICK-HCM (NCT03442764), EXPLORER-HCM (NCT03470545), and VALOR-HCM (NCT04349072) trials, as well as long term extension studies thereof (MAVA-LTE (NCT03723655), PIONEER-OLE (NCT03496168)). Mavacamten was approved by the FDA for treatment of adults with symptomatic New York Heart Association (NYHA) class II–III obstructive hypertrophic cardiomyopathy to improve functional capacity and symptoms. The myosin inhibitor aficamten has also been investigated in clinical trials for HCM, including ACACIA-HCM (NCT06081894), MAPLE-HCM (NCT05767346), and SEQUOIA-HCM (NCT05186818). Other myosin inhibitors have also been studied in clinical and pre-clinical settings.

[0004] Despite the advances in the clinic in the use of myosin inhibitors for treating HCM and other heart diseases, there remains a need for new and improved methods of treatment with myosin inhibitors, as well as methods of monitoring response to treatment and methods of determining whether to treat a patient with a myosin inhibitor. 1 57961429.1Attorney Docket No.267224-557784 SUMMARY

[0005] Methods of treatment with a myosin inhibitor using protein levels, methods of monitoring response to treatment based on such protein levels, and methods of determining whether to treat a patient with a myosin inhibitor based on such protein levels, are described herein. Such methods may include obtaining a biological sample of a subject, measuring one or more protein levels in the sample, analyzing the one or more protein levels, and determining the treatment response based on the analysis, and may further include treating the subject or modifying treatment based on the determination of treatment response. DRAWINGS

[0006] Figure 1 is (A) a diagram of hemoglobin (Hb), haptoglobin (Hp), and LV septum at baseline and at Week 30 of mavacamten treatment and (B) a plot of Log2RFU (relative fluorescence units) at baseline and Week 30 of mavacamten treatment. The RFU for hemoglobin is shown on the plot on the left and the RFU for haptoglobin (mixed type) is shown on the right.

[0007] Figure 2 shows the cell-free plasma hemolysis biomarkers at baseline and Week 30.

[0008] Figure 3 shows the correlation between hemoglobin and haptoglobin levels.

[0009] Figure 4 shows the resting and Valsalva LVOT (left ventricular outflow tract) gradients by hemolysis biomarker profile change.

[0010] Figure 5 shows plots of Log2RFU at baseline and Week 30 of mavacamten treatment for Myosin light chain 1, ACTN2, MYOM2, and MYPC1.

[0011] Figure 6 shows plots of Log2RFU at baseline and Week 30 of mavacamten treatment for N-terminal pro-BNP, SP-D, and Heparin cofactor II.

[0012] Figure 7 shows a study scheme for VALOR-HCM described in Example 7.

[0013] Figure 8 shows a study scheme for EXPLORER-HCM described in Example 8.

[0014] Figure 9 shows a study scheme for MAVERICK-HCM described in Example 9.

[0015] Figure 10 shows selected scatter plots of the change in LA volume index or change in LVOT gradient (resting or Valsalva) versus Log2 fold change in a specified protein level based on data from EXPLORER-HCM.

[0016] Figure 11 shows selected scatter plots of the change in LA volume index or change in LVOT gradient (resting or Valsalva) versus Log2 fold change in a specified protein level based on data from VALOR-HCM. 2 57961429.1Attorney Docket No.267224-557784

[0017] Figure 12 shows selected scatter plots of the change in LA volume index or change in LVOT gradient (resting or Valsalva) versus Log2 fold change in a specified protein level based on data from LTE-EXPLORER.

[0018] Figure 13 shows selected scatter plots of the change in LA volume index or change in LVOT gradient (resting or Valsalva) versus Log2 fold change in a specified protein level based on data from LTE-MAVERICK. DETAILED DESCRIPTION

[0019] Definitions

[0020] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0021] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention.

[0022] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0023] The terms “a” or “an,” as used in herein means one or more.

[0024] The terms “comprise,” “include,” and “have,” and the derivatives thereof, are used herein interchangeably as comprehensive, open-ended terms. For example, use of “comprising,” “including,” or “having” means that whatever element is comprised, had, or included, is not the only element encompassed by the subject of the clause that contains the verb.

[0025] As used herein, “treatment” or “treating,” or “ameliorating” are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but 3 57961429.1Attorney Docket No.267224-557784 not limited to a therapeutic benefit. Therapeutic benefit means eradication or amelioration of the underlying disorder being treated and / or eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. Treatment includes causing the clinical symptoms of the disease to slow in development by administration of a composition; suppressing the disease, that is, causing a reduction in the clinical symptoms of the disease; inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a composition after the initial appearance of symptoms; and / or relieving the disease, that is, causing the regression of clinical symptoms by administration of a composition after their initial appearance.

[0026] “Patient” or “subject” refers to a living organism suffering from or prone to a disease or condition that can be treated by using the methods provided herein. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, cats, monkeys, goat, sheep, cows, deer, and other non- mammalian animals. In some embodiments, a patient or subject is a human.

[0027] Embodiments

[0028] As described above, there is a need for new and improved methods of treatment with myosin inhibitors, as well as methods of monitoring response to treatment and methods of determining whether to treat a patient with a myosin inhibitor. Research regarding proteomics in myosin inhibitor therapy has been limited, with some data available regarding NT-proBNP and cardiac troponin (hs-cTnI) levels previously reported (Olivotto et al., The Lancet, 396(10253):P759-769;2020).

[0029] It has presently been discovered that certain proteins, including sarcomeric and hemolytic proteins, as well as muscle / contractility related proteins, cardiac biomarker proteins, and cardiopulmonary biomarker proteins, are useful as biomarkers in myosin inhibitor therapy. For example, certain protein levels are upregulated or downregulated as a result of administration of a myosin inhibitor (e.g., mavacamten). Moreover, changes in certain protein levels during myosin inhibitor therapy are indicative of treatment response.

[0030] Determining treatment response based on protein levels can be applied in methods of treating a patient with a myosin inhibitor. Protein levels may be determined prior to treatment, 4 57961429.1Attorney Docket No.267224-557784 following treatment, and / or periodically during treatment, and the treatment response may be used to determine the course of treatment. These and other methods of treatment and various embodiments thereof are described in further detail below.

[0031] The present disclosure provides a method of treating a patient in need thereof with a myosin inhibitor comprising administering a myosin inhibitor to the patient; and determining, or having determined, the treatment response of the patient based on one or more protein levels obtained from a biological sample from the patient following administration.

[0032] The present disclosure also provides a method of treating a patient in need thereof with a myosin inhibitor comprising administering a myosin inhibitor to the patient; measuring, or having measured, one or more protein levels in a biological sample from the patient following administration of the myosin inhibitor to the patient; analyzing, or having analyzed, the one or more protein levels; and determining, or having determined, the treatment response of the patient based on the one or more protein levels.

[0033] The present methods may further comprise modifying the treatment of the patient based on the treatment response of the patient. The present methods may further comprise modifying the treatment of the patient based on the protein level(s). In such cases, the protein level(s) may be used to determine the treatment response of the patient. For example, in some embodiments, modifying the treatment comprises administering a higher dose of the myosin inhibitor. As another example, the modification may comprise administering the myosin inhibitor at a greater frequency. In some embodiments, modifying the treatment comprises administering a lower dose of the myosin inhibitor. In some instances, the modification may comprise administering the myosin inhibitor at a lesser frequency. In some embodiments, modifying the treatment comprises administering a different myosin inhibitor. And, in some embodiments, modifying the treatment comprises temporarily discontinuing administration of the myosin inhibitor. Alternatively, the present methods may comprise maintaining the treatment of the patient based on the treatment response of the patient, e.g., by maintaining the dose and / or frequency of dosing of the myosin inhibitor, and maintaining treatment with the same myosin inhibitor.

[0034] The protein level(s) may be analyzed to determine a treatment response of the patient. A treatment response may be related to efficacy, safety or pharmacokinetics. For example, a certain change from baseline beyond a threshold may indicate that the myosin inhibitor is providing an efficacious response to treatment. Alternatively, a change in protein level from a 5 57961429.1Attorney Docket No.267224-557784 first protein level measured prior to a myosin inhibitor treatment period, to a second protein level measured after the myosin inhibitor treatment period, may be analyzed to determine the treatment response during the treatment period. In some embodiments, the treatment response indicates whether the patient is benefitting from the treatment. In some embodiments, the treatment response indicates the level of exposure to the patient of the myosin inhibitor. In some embodiments, the treatment response indicates the reduction in left ventricular outflow tract (LVOT) gradient in the patient, e.g., resting LVOT gradient or Valsalva LVOT gradient. In some embodiments, the treatment response indicates the reduction in left atrial volume index (LAVI) in the patient. In some embodiments, the treatment response indicates the increase in peak oxygen consumption (pVO2) of the patient. In some embodiments, the treatment response indicates the improvement in New York Heart Association (NYHA) classification of the patient. In some embodiments, the treatment response indicates the Kansas City Cardiomyopathy Questionnaire (KCCQ) score of the patient or the Hypertrophic Cardiomyopathy Symptom Questionnaire (HCMSQ) score of the patient. In some embodiments, the patient is identified as a non-responder to treatment. In some embodiments, the patient is identified as a responder to treatment.

[0035] In some embodiments, the treatment of the patient with the myosin inhibitor is performed in the absence of monitoring LVOT gradient. In some embodiments, the treatment of the patient is performed in the absence of monitoring for left ventricular ejection fraction (LVEF). In some embodiments, the treatment of the patient is performed in the absence of monitoring blood plasma concentration of the myosin inhibitor.

[0036] Various mathematical and statistical methods may be used as part of the present methods to analyze the protein levels that are measured. In some embodiments, analyzing the one or more protein levels comprises comparing a protein level to a threshold. The threshold may be a percent threshold or other statistical threshold, which may be calculated in comparison to a reference protein level, such as a baseline protein level, or by comparison of a first and second protein levels measured before and after a treatment period, respectively.. In some embodiments, the method further comprises measuring, or having measured, one or more baseline protein levels in a biological sample from the patient prior to administration of the myosin inhibitor to the patient. In some embodiments, analyzing the one or more protein levels comprises comparing a protein level to a baseline protein level, or comparing a first protein level 6 57961429.1Attorney Docket No.267224-557784 to a second protein level. In some embodiments, analyzing the one or more protein levels comprises determining a percent change from baseline in a protein level and comparing the percent change from baseline to a threshold percentage. In some embodiments, analyzing the one or more protein levels comprises determining whether a change from baseline in a protein level is a statistically significant change. First and second protein levels (with intervening treatment period) may also be used to determine percent change and statistically significant change). Various statistical methods can be employed to determine whether a change is statistically significant. Analyzing protein levels may include determining a fold change, determining a percentage change, determining false discovery rate (FDR), and / or determining a p value. Various statistical methods are known in the art for these statistical calculations.

[0037] In some embodiments, the methods comprise modifying treatment when a protein is downregulated by myosin inhibitor treatment. In such instances, the level of the protein(s) decreases compared to baseline following a period of myosin inhibitor treatment. Such downregulated proteins may include IZUM4, Heparin cofactor II, Haptoglobin mixed type, and / or Haptoglobin. Different modifications to treatment may be employed in response to a determination of downregulation in order to provide improved therapy to the patient. In some embodiments, the change from baseline in the one or more protein levels is below a threshold, and the method further comprises modifying treatment by administering a lower dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more protein levels is below a threshold, and the method further comprises modifying treatment by temporarily discontinuing administration of the myosin inhibitor. In some embodiments, the change from baseline in the one or more protein levels is below a threshold, and the method further comprises modifying treatment by administering a different myosin inhibitor. In some embodiments, the change from baseline in the one or more protein levels is above a threshold, and the method further comprises modifying treatment by administering a higher dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more protein levels is above a threshold, and the method further comprises modifying treatment by administering a different myosin inhibitor. In various embodiments, instead of a baseline protein level being used as comparison, a first protein level measured during treatment is used as a comparison against a second protein level measured after the first protein level and following a treatment period. In some embodiments, the present methods may comprise maintaining the 7 57961429.1Attorney Docket No.267224-557784 treatment of the patient based on the downregulation of the protein level(s), e.g., by maintaining the dose and / or frequency of dosing of the myosin inhibitor, and maintaining treatment with the same myosin inhibitor.

[0038] In some embodiments, treatment is modified in order to achieve level(s) of the one or more downregulated proteins at target level(s) (e.g., below a target threshold or within a target range). Further, treatment may be modified to maintain the level(s) of the one or more downregulated proteins at target level(s), e.g., a steady state or normal level. In such embodiments, protein level(s) may be periodically monitored and analyzed over a course of treatment.

[0039] In some embodiments, the methods comprise modifying treatment when a protein is upregulated by mavacamten treatment. In such instances, the level of the protein(s) increases compared to baseline following a period of myosin inhibitor treatment. Such upregulated proteins may include N-terminal pro-BNP, BNP, myosin light chain 1, Laminin02, BNP-32, MYOM2, SP-D, ACTN2, C1QR1, MYPC1, VAP-1, CK-MM, Hemoglobin, SYWC, and / or VEGF-D. Different modifications to treatment may be employed in response to a determination of upregulation in order to provide improved therapy to the patient. In some embodiments, the change from baseline in the one or more protein levels is above a threshold, and the method further comprises modifying treatment by administering a lower dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more protein levels is above a threshold, and the method further comprises modifying treatment by temporarily discontinuing administration of the myosin inhibitor. In some embodiments, the change from baseline in the one or more protein levels is above a threshold, and the method further comprises modifying treatment by administering a different myosin inhibitor. In some embodiments, the change from baseline in the one or more protein levels is below a threshold, and the method further comprises modifying treatment by administering a higher dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more protein levels is below a threshold, and the method further comprises modifying treatment by administering a different myosin inhibitor. In various embodiments, instead of a baseline protein level being used as comparison, a first protein level measured during treatment is used as a comparison against a second protein level measured after the first protein level and following a treatment period. In some embodiments, the present methods may comprise maintaining the treatment of the patient based 8 57961429.1Attorney Docket No.267224-557784 on the upregulation of the protein level(s), e.g., by maintaining the dose and / or frequency of dosing of the myosin inhibitor, and maintaining treatment with the same myosin inhibitor.

[0040] In some embodiments, treatment is modified in order to achieve level(s) of the one or more upregulated proteins at target level(s) (e.g., above a target threshold or within a target range). Further, treatment may be modified to maintain the level(s) of the one or more upregulated proteins at target level(s), e.g., a steady state or normal level. In such embodiments, protein level(s) may be periodically monitored and analyzed over a course of treatment.

[0041] As noted above, it has presently been discovered that hemolytic proteins are useful as biomarkers in myosin inhibitor therapy. Example 1 describes a study of hemolytic protein levels in HCM patients receiving mavacamten. Reduced cell-free plasma hemoglobin (Hb) and increased cell-free plasma haptoglobin (Hp) levels were observed following mavacamten administration, which suggests a reduction in intravascular hemolysis by mavacamten in obstructive HCM. The results are consistent with improved hemodynamic effects (less outflow obstruction) and indicate that treatment with mavacamten may reduce shear forces on red blood cells in the heart. Accordingly, in some embodiments, the one or more protein levels comprises one or more hemolysis protein levels.

[0042] In some embodiments, the change from baseline in the one or more hemolysis protein levels is below a threshold change, and the method further comprises modifying treatment by administering a different dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more hemolysis protein levels is below a threshold change, and the method further comprises modifying treatment by temporarily discontinuing administration of the myosin inhibitor. In some embodiments, the change from baseline in the one or more hemolysis protein levels is below a threshold change, and the method further comprises modifying treatment by administering a different myosin inhibitor. In some embodiments, the change from baseline in the one or more hemolysis protein levels is above a threshold change, and the method further comprises modifying treatment by administering a different dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more hemolysis protein levels is above a threshold change, and the method further comprises modifying treatment by temporarily discontinuing administration of the myosin inhibitor. In some embodiments, the change from baseline in the one or more hemolysis protein levels is above a threshold change, and the method further comprises modifying treatment by administering a different myosin inhibitor. In some 9 57961429.1Attorney Docket No.267224-557784 embodiments, the threshold change is a percent change or a fold change. In various embodiments, instead of a baseline protein level used as comparison, a first protein level measured during treatment is used as a comparison against a second protein level measured after the first protein level and following a treatment period.

[0043] In some embodiments, treatment is modified in order to achieve level(s) of the one or more hemolysis proteins at target level(s) (e.g., above or below a target threshold or within a target range). Further, treatment may be modified to maintain the level(s) of the one or more hemolysis proteins at target level(s), e.g., a steady state or normal level. In such embodiments, hemolysis protein level(s) may be periodically monitored and analyzed over a course of treatment.

[0044] In some embodiments, the one or more hemolysis protein levels are levels of one or more proteins selected from the group consisting of hemoglobin, hemoglobin subunit beta (also known as beta globin), HBG2 (hemoglobin subunit gamma-2), haptoglobin, and haptoglobin mixed type, or any combination thereof. In some embodiments, the one or more hemolysis protein levels are levels of hemoglobin, haptoglobin, or any combination thereof. In some embodiments, the one or more hemolysis protein levels comprises a hemoglobin level. In some embodiments, the one or more hemolysis protein levels comprises a haptoglobin level.

[0045] As noted above, it has presently been discovered that sarcomere proteins and proteins related to muscle / contractility are useful as biomarkers in myosin inhibitor therapy. Example 2 describes a study and data showing that mavacamten reduced sarcomeric biomarkers and biomarkers related to muscle / contractility. Accordingly, in some embodiments, the one or more protein levels comprises one or more sarcomere protein levels. And, in some embodiments, the one or more protein levels comprises one or more muscle / contractility-related protein levels.

[0046] In some embodiments, the change from baseline in the one or more sarcomere protein levels and / or the muscle / contractility-related protein levels is below a threshold change, and the method further comprises modifying treatment by administering a different dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more sarcomere protein levels and / or muscle / contractility-related protein levels is below a threshold change, and the method further comprises modifying treatment by temporarily discontinuing administration of the myosin inhibitor. In some embodiments, the change from baseline in the one or more sarcomere protein levels and / or muscle / contractility-related protein levels is below a threshold 10 57961429.1Attorney Docket No.267224-557784 change, and the method further comprises modifying treatment by administering a different myosin inhibitor. In some embodiments, the change from baseline in the one or more sarcomere protein levels and / or muscle / contractility-related protein levels is above a threshold change, and the method further comprises modifying treatment by administering a different dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more sarcomere protein levels and / or muscle / contractility-related protein levels is above a threshold change, and the method further comprises modifying treatment by temporarily discontinuing administration of the myosin inhibitor. In some embodiments, the change from baseline in the one or more sarcomere protein levels and / or muscle / contractility-related protein levels is above a threshold change, and the method further comprises modifying treatment by administering a different myosin inhibitor. In some embodiments, the threshold change is a percent change or a fold change. In various embodiments, instead of a baseline protein level used as comparison, a first protein level measured during treatment is used as a comparison against a second protein level measured after the first protein level and following a treatment period.

[0047] In some embodiments, treatment is modified in order to achieve level(s) of the one or more sarcomere proteins and / or muscle / contractility-related proteins at target level(s) (e.g., above or below a target threshold or within a target range). Further, treatment may be modified to maintain the level(s) of the one or more sarcomere proteins and / or muscle / contractility-related proteins at target level(s), e.g., a steady state or normal level. In such embodiments, sarcomere protein level(s) and / or muscle / contractility-related protein level(s) may be periodically monitored and analyzed over a course of treatment.

[0048] In some embodiments, the one or more sarcomere protein levels are levels of one or more proteins selected from the group consisting of myosin light chain 1, alpha actinin 2 (ACTN2), myosin-binding pro-C1 (MYPC1), myomesin 2 (MYOM2), or any combination thereof. In some embodiments, the one or more sarcomere protein levels are levels of myosin light chain 1, alpha actinin 2 (ACTN2), or any combination thereof. In some embodiments, the one or more muscle / contractility-related protein levels are levels of one or more proteins selected from the group consisting of myosin light chain 1, ACTN2, MYPC1, Laminin-2, MYOM2, and CK-MM, or any combination thereof.

[0049] Other proteins (non-hemolytic, non-sarcomeric) have also been presently discovered to be useful as biomarkers in myosin inhibitor therapy. Table 1 provides a list of proteins found to 11 57961429.1Attorney Docket No.267224-557784 be differentially expressed during myosin inhibitor therapy, specifically in clinical trials of mavacamten. In some embodiments, the one or more protein levels comprises one or more proteins selected from the group consisting of the proteins in Table 1, or any combination thereof. Useful proteins include cardiac biomarker proteins and cardiopulmonary biomarker proteins, as well as IZUM4. In some embodiments, the one or more protein levels are cardiac biomarkers selected from the group consisting of NT-proBNP, BNP, BNP-32 and heparin cofactor II or any combination thereof. In some embodiments, the one or more protein levels are cardiac biomarkers selected from the group consisting of BNP, BNP-32 and heparin cofactor II or any combination thereof. In some embodiments, the one or more protein levels are cardiopulmonary biomarkers selected from the group consisting of NT-proBNP, BNP, BNP-32, heparin cofactor II, SP-D, C1QR1, and VEGF-D or any combination thereof. In some embodiments, the one or more protein levels are cardiopulmonary biomarkers selected from the group consisting of BNP, BNP-32, heparin cofactor II, SP-D, C1QR1, and VEGF-D or any combination thereof. In some embodiments, the one or more protein levels are cardiopulmonary biomarkers selected from the group consisting of heparin cofactor II, SP-D, C1QR1, and VEGF- D or any combination thereof. In some embodiments, the one or more protein levels are cardiopulmonary biomarkers selected from the group consisting of NT-proBNP, BNP, BNP-32, heparin cofactor II, SP-D, C1QR1, VEGF-D, and SYWC or any combination thereof. In some embodiments, the one or more protein levels are cardiopulmonary biomarkers selected from the group consisting of BNP, BNP-32, heparin cofactor II, SP-D, C1QR1, VEGF-D, and SYWC or any combination thereof. In some embodiments, the one or more protein levels are cardiopulmonary biomarkers selected from the group consisting of heparin cofactor II, SP-D, C1QR1, VEGF-D, and SYWC or any combination thereof. In some embodiments, the one or more protein levels include the level of IZUM4. The mechanistic role of IZUM4 in HCM needs further exploration.

[0050] In some embodiments, the change from baseline in the one or more cardiac biomarker and / or cardiopulmonary biomarker and / or IZUM4 protein levels is below a threshold change, and the method further comprises modifying treatment by administering a different dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more cardiac biomarker and / or cardiopulmonary biomarker and / or IZUM4 protein levels is below a threshold change, and the method further comprises modifying treatment by temporarily discontinuing 12 57961429.1Attorney Docket No.267224-557784 administration of the myosin inhibitor. In some embodiments, the change from baseline in the one or more cardiac biomarker and / or cardiopulmonary biomarker and / or IZUM4 protein levels is below a threshold change, and the method further comprises modifying treatment by administering a different myosin inhibitor. In some embodiments, the change from baseline in the one or more cardiac biomarker and / or cardiopulmonary biomarker and / or IZUM4 protein levels is above a threshold change, and the method further comprises modifying treatment by administering a higher dose of the myosin inhibitor. In some embodiments, the change from baseline in the one or more cardiac biomarker and / or cardiopulmonary biomarker and / or IZUM4 protein levels is above a threshold change, and the method further comprises modifying treatment by temporarily discontinuing administration of the myosin inhibitor. In some embodiments, the change from baseline in the one or more cardiac biomarker and / or cardiopulmonary biomarker and / or IZUM4 protein levels is above a threshold change, and the method further comprises modifying treatment by administering a different myosin inhibitor. In some embodiments, the threshold change is a percent change or a fold change. In various embodiments, instead of a baseline protein level used as comparison, a first protein level measured during treatment is used as a comparison against a second protein level measured after the first protein level and following a treatment period.

[0051] In some embodiments, the one or more protein levels measured comprises the level of a protein selected from the group consisting of N-terminal pro-BNP, BNP, Myosin light chain 1, Laminin-2, BNP-32, MYOM2, IZUM4, Heparin cofactor II, SP-D, ACTN2, C1QR1, MYPC1, Haptoglobin Mixed Type, VAP-1, HPT, CK-MM, Hemoglobin, SYWC, VEGF-D, HBG2 and beta-globin or any combination thereof.

[0052] In some embodiments, the one or more protein levels analyzed does not comprise the level of NT-proBNP nor cardiac troponin. In some embodiments, the one or more protein levels analyzed comprises multiple protein levels wherein the multiple protein levels include NT- proBNP and / or cardiac troponin.

[0053] In some embodiments, a single protein level is measured and analyzed to determine treatment response. In some embodiments, multiple protein levels are measured and analyzed to determine treatment response. In some such embodiments, the multiple protein levels (e.g., the change in multiple protein levels from respective baseline levels) are analyzed as a composite and / or to calculate a score, which may be compared to a threshold score. In some embodiments, 13 57961429.1Attorney Docket No.267224-557784 an algorithm may be used to analyze the multiple protein levels and determine a treatment response.

[0054] The methods described herein are useful in the field of myosin inhibitor therapy, either as part of a therapeutic method or separately as a diagnostic method. Myosin inhibitor therapy is understood to have utility in treating cardiovascular diseases. Thus, the present methods may be utilized in treating patients suffering from cardiovascular diseases. In some embodiments, the patient is suffering from obstructive hypertrophic cardiomyopathy (oHCM), nonobstructive hypertrophic cardiomyopathy (nHCM), heart failure with preserved ejection fraction (HFpEF), diastolic dysfunction, left ventricular hypertrophy (LVH), malignant LVH, ischemia, or angina, or any combination thereof. In some embodiments, the patient is suffering from diastolic dysfunction, left ventricular hypertrophy (LVH), angina, ischemia, hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM), or HFpEF, mixed left ventricular (LV) systolic and diastolic dysfunction, or idiopathic right ventricular (RV) hypertrophy. In some embodiments, angina is microvascular angina. In some embodiments, the LVH is malignant LVH. In some embodiments, HCM is obstructive HCM. In some embodiments, the HCM is non-obstructive HCM.

[0055] In some embodiments, the patient is suffering from HCM. In some embodiments, the patient is suffering from oHCM. In some embodiments, the patient is suffering from nHCM. In some embodiments, the patient is suffering from HFpEF. In some embodiments, the patient is suffering from LVH. In some embodiments, the patient is suffering from diastolic dysfunction.

[0056] The protein levels in the patient are measured from a biological sample from the patient. In some embodiments, the method further comprises obtaining, or having obtained, a biological sample from the patient. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a blood serum sample. In some embodiments, the biological sample is a blood plasma sample.

[0057] The analysis of protein level(s) described herein may also be employed in a manner to improve the safety of myosin inhibitor therapy. In some embodiments, the method further comprises measuring, or having measured, the LVEF of the patient when one or more protein levels are above a threshold. In some embodiments, the method further comprises measuring, or having measured, the LVEF of the patient when the change from baseline in one or more protein levels is above a threshold change. In some embodiments, the method further comprises 14 57961429.1Attorney Docket No.267224-557784 measuring, or having measured, the LVEF of the patient when one or more protein levels is below a threshold. In some embodiments, the method further comprises measuring, or having measured, the LVEF of the patient when the change from baseline in one or more protein levels is below a threshold change.

[0058] As noted above, the determination of treatment response may be utilized to modify the dose of the myosin inhibitor. In some embodiments, the dose is modified based on the analysis of the protein level(s) and / or the determination of treatment response. In some embodiments, the method comprises administering an initial dose of a myosin inhibitor for a first time period prior to measuring the one or more protein levels. In some embodiments, the method comprises modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of the myosin inhibitor that is higher than the initial dose. In some embodiments, the method comprises modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of the myosin inhibitor that is lower than the initial dose. In some embodiments, the method comprises continuing administration of the initial dose of the myosin inhibitor due to a determination that the treatment response is a desired treatment response In some embodiments, the method comprises temporarily discontinuing administration of the myosin inhibitor based on the treatment response or based on the analysis of the one or more protein level(s).

[0059] In some embodiments, the myosin inhibitor is mavacamten and the dose of mavacamten is modified based upon the analysis of the protein level(s) and / or the determination of treatment response. In some embodiments, the method comprises administering an initial dose of 5 mg per day of mavacamten for a first time period prior to measuring the one or more protein levels. In some embodiments, the method comprises modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of 10 mg per day of mavacamten for a second time period subsequent to measuring the one or more protein levels. In some embodiments, the method comprises modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of 2.5 mg per day of mavacamten for a second time period subsequent to measuring the one or more protein levels. In some embodiments, the 15 57961429.1Attorney Docket No.267224-557784 method comprises continuing administration of 5 mg per day of mavacamten based on determination that the treatment response is a desired treatment response.

[0060] In some embodiments, the method comprises administering an initial dose of 2.5 mg per day of mavacamten for a first time period prior to measuring the one or more protein levels. In some embodiments, the method comprises modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of 5 mg per day of mavacamten for a second time period subsequent to measuring the one or more protein levels. In some embodiments, the method comprises modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of 1 mg per day of mavacamten for a second time period subsequent to measuring the one or more protein levels, or temporarily discontinuing administration of mavacamten during the second time period. In some embodiments, the method comprises continuing administration of 2.5 mg per day of mavacamten based on determination that the treatment response is a desired treatment response.

[0061] In embodiments comprising increasing or decreasing a dose, the dose may be increased or decreased among dosing levels. In some embodiments the dosing levels comprise 2.5 mg, 5 mg, and 10 mg. In some embodiments, the dosing levels further comprise 15 mg. In some embodiments, the dosing levels further comprise 1 mg. In some embodiments, the dosing levels further comprise 0 mg. In some such embodiments, the myosin inhibitor is mavacamten.

[0062] Also disclosed herein is a method of determining response to myosin inhibitor therapy in a patient comprising analyzing one or more protein levels in a biological sample from the patient.

[0063] Also disclosed herein is a method of determining whether to treat a patient with a myosin inhibitor and treating the patient with a myosin inhibitor, the method comprising: determining, or having determined, whether to treat the patient with a myosin inhibitor based on one or more protein levels in a biological sample from the patient; and treating the patient by administering a myosin inhibitor to the patient.

[0064] Also disclosed herein is a method of determining whether to treat a patient with a myosin inhibitor and treating the patient with a myosin inhibitor, the method comprising: measuring, or having measured, one or more protein levels in a biological sample from the 16 57961429.1Attorney Docket No.267224-557784 patient; analyzing, or having analyzed, the one or more protein levels; determining, or having determined, based on the one or more protein levels in the biological sample, whether to treat the patient with a myosin inhibitor; and treating the patient by administering a myosin inhibitor to the patient.

[0065] In some embodiments, analyzing the one or more protein levels comprises comparing a protein level to a threshold. In some embodiments, analyzing the one or more protein levels results in finding that one or more protein levels are above a threshold. In some embodiments, when one or more protein levels are above a threshold, the method comprises determining, or having determined, that the patient is indicated for treatment with a myosin inhibitor.

[0066] In some embodiments, analyzing the one or more protein levels results in finding that one or more protein levels are below a threshold. In some embodiments, when one or more protein levels are below a threshold, the method comprises determining, or having determined, that the patient is indicated for treatment with a myosin inhibitor.

[0067] In some embodiments, the patient is a NYHA class II oHCM patient. In some embodiments it is determined to treat the NYHA class II oHCM patient based on the analysis of the one or more protein levels. In some embodiments, it is determined to treat the NYHA class II oHCM patient based on the analysis of one or more hemolysis protein levels. In some embodiments, it is determined to treat the NYHA class II oHCM patient based on the analysis of one or more sarcomere protein levels. In some embodiments, it is determined to treat the NYHA class II oHCM patient based on the analysis of one or more protein levels selected from the proteins in Table 1.

[0068] In some embodiments, the method further comprises monitoring the one or more protein levels following treatment. In some embodiments, the method further comprises modifying treatment when a protein is downregulated by myosin inhibitor treatment. In such instances, the level of the protein(s) decreases compared to baseline following a period of myosin inhibitor treatment. Such downregulated proteins may include IZUM4, Heparin cofactor II, Haptoglobin mixed type, and / or Haptoglobin. Different modifications to treatment may be employed in response to a determination of downregulation in order to provide improved therapy to the patient. For example, treatment may be modified in order to achieve target protein level(s) in the patient, such as maintaining steady state protein level(s). 17 57961429.1Attorney Docket No.267224-557784

[0069] In some embodiments, the methods comprise modifying treatment when a protein is upregulated by mavacamten treatment. In such instances, the level of the protein(s) increases compared to baseline following a period of myosin inhibitor treatment. Such upregulated proteins may include N-terminal pro-BNP, BNP, myosin light chain 1, Laminin02, BNP-32, MYOM2, SP-D, ACTN2, C1QR1, MYPC1, VAP-1, CK-MM, Hemoglobin, SYWC, and / or VEGF-D. Different modifications to treatment may be employed in response to a determination of upregulation in order to provide improved therapy to the patient. For example, treatment may be modified in order to achieve target protein level(s) in the patient, such as maintaining steady state protein level(s).

[0070] In some embodiments, the present methods may comprise maintaining the treatment of the patient, e.g., by maintaining the dose and / or frequency of dosing of the myosin inhibitor, and maintaining treatment with the same myosin inhibitor.

[0071] Also disclosed herein is a method of selecting a myosin inhibitor therapy and treating a patient in need thereof with the myosin inhibitor therapy, comprising: measuring, or having measured, one or more protein levels in a biological sample from the patient; analyzing, or having analyzed, the one or more protein levels; selecting, or having selected, based on the one or more protein levels in the biological sample, a myosin inhibitor therapy for the patient; and treating the patient by administering a myosin inhibitor to the patient.

[0072] In some embodiments, the method further comprises monitoring the one or more protein levels during the treatment.

[0073] In some embodiments of the present methods, the one or more protein levels measured comprises the level of a protein selected from Table 1: Table 1 SeqId Protein UniProt Full Protein Name18 57961429.1Attorney Docket No.267224-557784 13534-20 MYOM2 P54296 Myomesin-2 20549-1 IZUM4 Q1ZYL8 Izumo sperm-egg fusion t , e a a , a

[0074] Table 2 presents statistical data on differential expression of the proteins in Table 1. Proteins to be measured and analyzed according to the present methods may be selected based upon such statistical data. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 0.1. In some embodiments, the one or more protein levels measured comprises the 19 57961429.1Attorney Docket No.267224-557784 level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 0.2. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 0.3. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 0.4. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 0.5. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 0.6. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 0.7. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 0.8. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 0.9. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Table 2 with a logFC.max or –logFC.max is greater than 1.

[0075] Tables 4A–D presents statistical data on correlation of proteins with clinical variables. Proteins to be measured and analyzed according to the present methods may be selected based upon such statistical data. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Tables 4A–D with a p value less than 0.01. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Tables 4A-D with a p value less than 0.005. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Tables 4A–D with a p value less than 0.001. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Tables 4A–D with a p value less than 0.0005. In some embodiments, the one or more protein levels measured comprises the level of a protein selected from Tables 4A–D with a p value less than 0.0001.

[0076] In some embodiments, the present methods include administration of a myosin inhibitor. In some embodiments, a myosin inhibitor is a compound of formula (I): 20 57961429.1Attorney Docket No.267224-557784 or pharmaceutically acceptable 1R is C1-8alkyl, C3-8cycloalkyl, substituted with one or two halo; R2is phenyl optionally substituted with one or two halo; R3is C1-8alkyl or C3-8cycloalkyl, wherein each R3is optionally substituted with halo, hydroxyl or C1-2alkoxy; R4is H; and X is H.

[0077] In some embodiments, a myosin inhibitor of formula (I) or a pharmaceutically acceptable salt thereof is selected from group (I) consisting of: , , ,Attorney Docket No.267224-557784 , ,mavacamten or a pharmaceutically acceptable salt thereof having the following structure:

[0079] Mavacamten is also known as MYK-461. Its chemical name is (S)-3-Isopropyl-6-((1- phenylethyl)amino)pyrimidine-2, 4(1H,3H)-dione or 6-[[(1S)-1-phenylethyl]amino]-3-propan-2- yl-1H-pyrimidine-2,4-dione. In some embodiments, mavacamten is in free base form.

[0080] In some embodiments, a myosin inhibitor of formula (I) is MYK-581 or a pharmaceutically acceptable salt thereof having the following structure. 22 57961429.1Attorney Docket No.267224-557784

[0081] MYK-581’s chemical name is (S)-6-((1-(3-fluorophenyl)ethyl)amino)-3- isopropylpyrimidine-2,4(1H,3H)-dione.

[0082] Myosin inhibitors of formula (I), including the compounds of group (I), mavacamten, or MYK-581, or a pharmaceutically acceptable salt thereof, can be obtained according to the production methods described in U.S. Patent No.9,181,200, which is incorporated herein by reference in its entirety and for all purposes.

[0083] In some embodiments, a myosin inhibitor is a compound of formula (II): or pharmaceuticallyn is 1 or 2; R1is fluoro, chloro, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy, C1-4 haloalkoxy, or C2-4 alkynyl, wherein at least one R1is fluoro; and one of R2aand R2bis fluoro and the other of R2aand R2bis H.

[0084] In some embodiments, a myosin inhibitor of formula (II) or a pharmaceutically acceptable salt thereof is selected from group (II) consisting of: 23 57961429.1Attorney Docket No.267224-557784 .pharmaceutically acceptable salt thereof, can be obtained according to the production methods described in International Application Number PCT / US2019 / 058297, filed on October 29, 2019, which is incorporated herein by reference in its entirety and for all purposes.

[0086] In some embodiments, a myosin inhibitor is a compound of formula (III): or pharmaceuticallyG1is -CR4R5- or -O-; G2 is a bond or -CR6R7-; G3 is -CR8- or -N-; 24 57961429.1Attorney Docket No.267224-557784 R1, R3, R4, R5, R6, R7, and R8are each independently H, C1-C6alkyl, halo, or hydroxyl; R2is H, C2-C6 alkyl, halo, or hydroxyl; Z is a bond, C1-C6 alkyl, -O-, -N(R9)-, -RXO-, -ORY, or –RZS-; R9is H, C1-C6alkyl, or cycloalkyl; A is selected from the group consisting of substituted C2 alkynyl, unsubstituted C2 alkynyl, substituted phenyl, unsubstituted phenyl, and 5- or 6-membered heteroaryl comprising at least one annular N atom, wherein the 5- or 6-membered heteroaryl is unsubstituted or substituted with one or more R10substituents: each R10is independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or –C(O)ORa; B is selected from the group consisting of H, C1-C6 alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl, wherein the C1-C6 alkyl, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl of B is unsubstituted or substituted with one or more R11substituents; each R11is independently selected from the group consisting of substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, unsubstituted C1-C6alkyl, C1-C6alkyl substituted with one or more R12substituents, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2- C6 alkynyl, halo, —ORb, —C(O)Rc, — C(O)ORd, oxo, and —NReRf; each R12is independently selected from the group consisting of halo, -ORb, —C(O)Rg, — C(O)ORh, and —C(O)NRiRj; each Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh, Ri, and Rjis independently H or C1-C6 alkyl; and RX, RY, and RZare each C1-C6 alkyl.

[0087] In some embodiments, a myosin inhibitor of formula (III) or a pharmaceutically acceptable salt thereof is selected from group (III) consisting of: 25 57961429.1Attorney Docket No.267224-557784 , .

[0088] In some embodiments, the myosin inhibitor is aficamten. In some embodiments, the myosin inhibitor is aficamten or a pharmaceutically acceptable salt thereof. The structure of aficamten is shown below. 57961429.1Attorney Docket No.267224-557784

[0089] Myosin inhibitors of formula (III), including the compounds of group (III), or a pharmaceutically acceptable salt thereof, can be obtained according to the production methods described in International Publication Number WO 2019 / 144041, published on July 25, 2019, which is incorporated herein by reference in its entirety and for all purposes.

[0090] In some embodiments, myosin inhibitors include the compounds disclosed in PCT patent applications, published as WO2020 / 005887, WO2020 / 005888, WO2020 / 047447, each of which is incorporated herein by reference in its entirety and for all purposes.

[0091] Also disclosed herein is a myosin inhibitor for use in a method as described herein. In some embodiments, the myosin inhibitor is mavacamten. EXAMPLES

[0092] SomaScan Platform: The Somascan Platform was used as indicated in the Examples below. The SomaScan® platform (SomaLogic Operating Co., Inc., Boulder, CO) is a highly multiplexed, sensitive, quantitative, and reproducible proteomic tool for discovering biomarkers for drug discovery, pre-clinical and clinical drug development, and clinical diagnostics. The SomaScan Assay v4.1 measures approximately 7,000 unique human protein analytes in small volumes of biological samples. The assay was developed for performance in human serum and plasma and has a large dynamic range that can quantify the relative levels of proteins in plasma that span 10 logs in abundance. The SomaScan platform is enabled by the generation of protein- capture reagents called SOMAmer® (Slow Off-rate Modified Aptamer) reagents. SOMAmer reagents consist of short single-stranded DNA sequences that incorporate hydrophobic modifications. The SomaScan platform measures native proteins in complex matrices by transforming available binding sites on individual proteins into a corresponding SOMAmer reagent concentration, which is then quantified by hybridization to microarrays. The systematic evolution of ligands by exponential enrichment (SELEX) procedure was used for SOMAmer library optimization. The assay entails combining SOMAmers with analyte, standard chemistry for isolating target-bound SOMAmers, and microarray quantification of SOMAmer DNA. The ~7000 SOMAmer reagents include 6401 unique Uniprot IDs and 4241 (66%) of unique Uniprot IDs are specific to SomaScan.

[0093] Differential Expression Analyses: Differential expression analyses were performed as indicated in the Examples below. The differential expression analyses were completed using the 27 57961429.1Attorney Docket No.267224-557784 open-source software R. Linear models were implemented using the Limma package for linear model analysis of microarray and RNA-seq data. Limma fits linear models to each protein. For the EXPLORER and VALOR data sets, the models were specified to include main effects for treatment and time, treatment-by-time interactions, along with covariates to be adjusted for. For the long-term extension (LTE) data sets (LTE-EXPLORER and LTE-MAVERICK), the models were specified to include the effect of time on expression, along with covariate adjustment. For the EXPLORER data set, the models were adjusted for covariates including ergometer type, baseline beta blocker status, baseline NYHA classification, and the Somalogic plate identifier. The EXPLORER analysis was performed both with and without adjusting for the top three surrogate variables (SVs) identified by surrogate variable analysis (SVA). SVs identified through SVA represent potentially unknown confounding factors identified directly from the data. For the LTE data sets, the models were adjusted for the plate identifier and the top three SVs. For the VALOR data set, the models were adjusted for surgery type, baseline NYHA class, plate identifier, and the top three SVs. For EXPLORER and VALOR, contrasts were applied to evaluate treatment-by-time interactions to determine whether the effect of time on protein expression differed between the placebo and Mavacamten treatment groups. Fold changes were estimated for the applied contrasts to estimate differences between changes in log2-scaled expression over time for Mavacamten versus placebo, while accounting for covariates of interest, and are expression on the log2 scale. For the LTE studies, contrasts were preformed to evaluate expression changes from baseline at sequential time points, quantified by log2-scale fold changes at each time point relative to baseline. Statistical testing was implemented with moderated t-tests, with adjustment for multiple testing using the Benjamini-Hochberg procedure to estimate false discovery rates (FDRs). In the moderated t-tests, the standard errors are compressed towards a common value across proteins using a Bayesian approach. Differential expression was defined for interaction contrasts with FDR < 0.05. When this threshold is applied across all proteins from a given data set, it is expected that approximately 5% of the hits are not differentially expressed, consistent with the null hypothesis. Log2 fold changes quantify the effect sizes for the treatment- by-time interactions (EXPLORER and VALOR) or effects of Mavacamten over time (LTE). 28 57961429.1Attorney Docket No.267224-557784

[0094] Example 1 – The effect of mavacamten treatment on hemolysis biomarkers

[0095] Introduction: In patients with obstructive hypertrophic cardiomyopathy (HCM), blood flow changes from left ventricular outflow tract (LVOT) obstruction can lead to hemolysis and release of red blood cell proteins into circulation. Increased cell-free plasma hemoglobin (Hb) and reduced haptoglobin (Hp) are biomarkers of intravascular hemolysis. Mavacamten, a cardiac myosin inhibitor, reduced LVOT obstruction in the phase 3 EXPLORER-HCM study; however, its effect on hemolysis biomarkers is unknown.

[0096] Aim: To assess the effect of 30 weeks of mavacamten treatment versus placebo on levels of intravascular hemolysis biomarkers in patients with obstructive HCM.

[0097] Methods: Hemolysis biomarkers were measured as part of exploratory proteomic analyses from randomized EXPLORER-HCM (NCT03470545) patients at baseline and Week 30 (mavacamten, n = 115; placebo, n = 120) using a proteomic platform (SomaScan v4.1). The EXPLORER-HCM study is described herein in Example 8.

[0098] Results: At Week 30, mavacamten treatment showed decreases in cell-free plasma Hb (Log2 fold change = -1.57, FDR = 1.7×10-4) and increases in cell-free plasma Hp (Log2 fold change = 1.83, FDR = 1.2×10-4) vs. placebo (Figure 1). Hb and Hp levels were negatively correlated at baseline and at Week 30. In general, patients can be grouped into high- or low- hemolysis biomarker profile (high Hb with low Hp or low Hb with high Hp, respectively) at baseline and Week 30. Patients who received mavacamten and changed from high- to low hemolysis biomarker profile at Week 30 trended towards greater LVOT reductions under resting and Valsalva conditions. No changes were detected for either group in whole blood Hb or hematocrit. Figure 1 shows the cell-free plasma Hb and Hp levels at baseline and at Week 30. Figure 2 shows the cell-free plasma hemolysis biomarkers at baseline and Week 30. Figure 3 shows the correlation between hemoglobin and haptoglobin levels. Figure 4 shows the resting and Valsalva LVOT gradients by hemolysis biomarker profile change.

[0099] Conclusions: Reduced cell-free plasma Hb and increased cell-free plasma Hp levels, suggest a reduction in intravascular hemolysis by mavacamten in obstructive HCM. These results are consistent with improved hemodynamic effects (less outflow obstruction) and indicate that mavacamten may reduce shear forces on red blood cells in the heart. Whole-blood Hb and hematocrit levels did not change after treatment with mavacamten or placebo 29 57961429.1Attorney Docket No.267224-557784

[0100] Example 2 – The effect of mavacamten on sarcomeric biomarkers

[0101] Methods: Sarcomeric biomarkers were measured as part of exploratory proteomic analyses from randomized EXPLORER-HCM (NCT03470545) patients at baseline and Week 30 using a proteomic platform (SomaScan). The EXPLORER-HCM study is described herein in Example 8.

[0102] Results: Mavacamten reduced sarcomeric biomarkers related to muscle / contractility, including myosin light chain 1, ACTN2, MYOM2, and MYPC1. Figure 5 shows the box plots of Log2RFU change from baseline to Week 30 for the placebo and mavacamten (MYK-461) treatment populations. RFU are relative fluorescence units, indicating the presence of the protein in the sample on a Log2 scale. Figure 5 also shows the Log FC and FDR values for myosin light chain 1, ACTN2, MYOM2, and MYPC1. FDR refers to false discovery rate and FC refers to fold change. The differential expression at Week 30 compared to baseline of myosin light chain 1, ACTN2, MYOM2, and MYPC1 was statistically significant.

[0103] Example 3 – The effect of mavacamten on cardiac / pulmonary biomarkers

[0104] Methods: Biomarkers were measured as part of exploratory proteomic analyses from randomized EXPLORER-HCM (NCT03470545) patients at baseline and Week 30 using a proteomic platform (SomaScan). The EXPLORER-HCM study is described herein in Example 8.

[0105] Results: Mavacamten treatment resulted in differential expression of cardiac / pulmonary biomarkers, including NTproBNP, SP-D, and Heparin cofactor II. Figure 6 shows the box plots of Log2RFU change from baseline to Week 30 for the placebo and mavacamten (MYK-461) treatment populations. RFU are relative fluorescence units, indicating the presence of the protein in the sample on a Log2 scale. Figure 6 also shows the Log FC and FDR values for NTproBNP, SP-D, and Heparin cofactor II. FDR refers to false discovery rate and FC refers to fold change. The differential expression at Week 30 compared to baseline of NTproBNP, SP-D, and Heparin cofactor II was statistically significant. 30 57961429.1Attorney Docket No.267224-557784

[0106] Example 4 – Differential expression analysis reveals a peripheral signature of the mavacamten response in EXPLORER-HCM

[0107] Methods: Biomarkers were measured as part of exploratory proteomic analyses from randomized EXPLORER-HCM (NCT03470545) patients at baseline and Week 30 using a proteomic platform (SomaScan). The EXPLORER-HCM study is described herein in Example 8. The model used to determine differential expression was as follows: Protein expression ~ time + treatment + time * treatment + covariates + (1|USUBJID). A significant interaction was defined as mava wk30 – mava baseline ≠ placebo wk30 – placebo baseline. Results were filtered for FDR < 0.05.

[0108] Results:

[0109] The results showed that mavacamten treatment reduced BNP protein levels. Mavacamten treatment also reduced ACTN2 protein levels. The Z-line is a highly organized multiprotein complex at the boundary between sarcomeres in which actin and titin filaments from adjacent sarcomeres are anchored and crosslinked by α-actinin. α-Actinin plays a central role in connecting proteins at the Z-line and also interacts with a wealth of other Z-line proteins. Mavacamten treatment also reduced MYPC1 and MYOM2 protein levels. Myomesin-2, M- protein is expressed in adult cardiac muscle and fast skeletal muscle, and functions to stabilize the three-dimensional arrangement of proteins comprising M-band structures in a sarcomere.

[0110] Example 5 – Differential expression analysis following mavacamten at week 16 in VALOR-HCM

[0111] Methods: Biomarkers were measured as part of exploratory proteomic analyses from randomized VALOR-HCM (NCT04349072) patients at baseline and Weeks 16 and 32 using a proteomic platform (SomaScan). The VALOR-HCM study is described herein in Example 7.

[0112] Results: There were no detected changes between week 32 and week 16 for the mavacamten treated group, generally consistent with sustained changes. Also, all proteins showed concordant change directionality for both 8 and 16 week timepoints. Most changes at week 8 were also observed at week 16 (80%), generally consistent with progressive rather than transient changes.

[0113] Table 3 shows proteins with significant differential expression in both VALOR (Week 16) and EXPLORER (Week 30) studies. 31 57961429.1Attorney Docket No.267224-557784 Table 3 Target logFC.exp FDR.exp logFC.val FDR.val Haptoglobin 1.74 3.5E-04 1.86 6.1E-03

[0114] Comparing differential expression in VALOR and EXPLORER, only 12% of proteins had un-matched change directionality. There was consistent directionality for 88% of the proteins (r = 0.96, p = 8.4e-43). Overall, changes were highly concordant in both magnitude and directionality.

[0115] Conclusions: Key results from EXPLORER were validated in VALOR, i.e., decreased sarcomere protein expression and improved hemolysis-related protein expression. Changes were generally non-transient and sustained after 16 weeks. The VALOR design facilitated the combination of placebo / treatment groups for enhanced power / sensitivity of differential expression detection (novel pd markers).

[0116] Example 6 – Multi-study analysis of differentially expressed biomarkers

[0117] Data sets from clinical trials were analyzed for proteins that were differentially expressed after mavacamten treatment as compared to baseline. Data sets from the following 32 57961429.1Attorney Docket No.267224-557784 clinical trials were used: EXPLORER-HCM, VALOR-HCM, and MAVA-LTE (LTE- EXPLORER and LTE-MAVERICK).

[0118] A significance threshold of FDR < 0.05 was applied for all analyses. The EXPLORER and VALOR results are based on comparing the effects of 30 or 16 weeks, respectively, for Mavacamten versus placebo treatment. The LTE-MAVERICK results are based on differential expression compared to baseline at weeks 24, 48, 72, 96, 120, and 144. The LTE-EXPLORER results are based on differential expression compared to baseline at weeks 24, 48, 72, and 96. LTE proteins were deemed significant if identified as significant for two or more time points with identical fold change directionality.

[0119] Significant aptamers were identified, with exemplary species listed in Table 2. There was consistent fold change directionality across all studies that showed significance.

[0120] Table 2 shows aptamer / protein identification information (SeqId, Protein, UniProt), the minimum FDR from all studies that yielded significance (fdr.min), the maximum log2 fold change from all studies that yielded significance (logFC.max), the number of studies for which significance was identified (n hits), and the studies for which significance was identified (hits). Table 2 SeqId Protein UniProt fdr. logFC. n hits33 57961429.1Attorney Docket No.267224-557784 20549 IZUM4 Q1ZYL8 1.59 0.78 3 LTE-EXP, EXPLORER, -1 E-35 VALOR34 57961429.1Attorney Docket No.267224-557784 Example 7. VALOR TRIAL: A Randomized, Double-blind, Placebo-Controlled Study to Evaluate Mavacamten in Adults with Symptomatic Obstructive Hypertrophic Cardiomyopathy Who Are Eligible For Septal Reduction Therapy

[0121] VALOR-HCM is a Phase 3 study to evaluate the effect of mavacamten treatment on reducing the number of septal reduction therapy (SRT) procedures performed in subjects with symptomatic obstructive hypertrophic cardiomyopathy (oHCM [also known as HOCM]) who are eligible for SRT based on American College of Cardiology Foundation (ACCF) / American Heart Association (AHA) and / or European Society of Cardiology (ESC) guidelines (ie, guidelines). The VALOR-HCM study (NCT04349072) reached primary completion on February 7, 2022. The VALOR-HCM study was performed according to the following protocol. Overall Design:

[0122] This is a Phase 3, randomized, double-blind, placebo-controlled, multicenter study of males and females ≥ 18 years with oHCM who meet ACCF / AHA and / or ESC guideline criteria for SRT (e.g., LVOT gradient of ≥50mmHg and NYHA Class III-IV) and have been referred for an invasive procedure. After completing screening assessments, eligible subjects will be randomized 1:1 to the mavacamten or placebo treatment groups. Randomization will be stratified by the type of SRT procedure recommended (myectomy or alcohol septal ablation [ASA]) and NYHA functional class.

[0123] The study duration will be up to 138 weeks, including a 2-week screening period (Week –2), 128 weeks of treatment, and an 8-week posttreatment follow-up visit (Week 136).

[0124] There will be 3 dosing periods as follows: ^ Placebo-controlled dosing period (Day 1 to Week 16): Subjects will receive double-blind mavacamten or placebo once daily for 16 weeks. ^ Active-controlled dosing period (Week 16 to Week 32): All subjects will receive mavacamten once daily for 16 weeks. Dose will be blinded. ^ Long-term extension (LTE) dosing period (Week 32 to Week 128): All subjects will receive mavacamten once daily for 96 weeks. Dose will remain blinded unless the sponsor chooses to unblind once the primary analysis is complete.

[0125] Study Procedures and Treatment: ^ Study visits will occur at screening, Day 1, every 4 weeks through Week 32, every 12 weeks thereafter until Week 128 (end of treatment, EOT), and Week 136 (end of study). 35 57961429.1Attorney Docket No.267224-557784 Visits must take place at the study center at Day 1 and Weeks 8, 16, 24, and 32, every 12 weeks thereafter through Week 128, and Week 136. For selected sites, study visits may take place at a subject’s home with a qualified home health care professional who is contracted by the sponsor at Weeks 4, 12, 20, and 28. Subjects who prematurely discontinue study drug at any time (except for SRT) will attend a treatment discontinuation visit within 14 days of study drug discontinuation and will be followed every 24 weeks thereafter until Week 128. ^ On Day 1, eligible subjects will be randomized in a double-blind manner via an interactive response system (IXRS) to the mavacamten or placebo groups. Randomization will be stratified by the type of septal reduction therapy (SRT) procedure recommended (myectomy or alcohol septal ablation (ASA)) and New York Heart Association (NYHA) functional class. Subjects will begin mavacamten 5 mg or matching placebo once daily by mouth for 16 weeks with subsequent assessments for dose adjustments. ^ At Weeks 16, 32, 80, and 128, subjects will be reevaluated for SRT eligibility. The investigator will confirm that the subject remains on maximal medical therapy, determine NYHA class, and enter the information in the electronic case report form (eCRF). Every effort should be made to have the same investigator who evaluates NYHA at screening also evaluate NYHA at Weeks 16, 32, 80, and 128. Independently, and blinded to the investigator, a transthoracic echocardiography (TTE) will be performed to assess LVOT gradients at rest, provocation, and post exercise. At Weeks 16 and 32, TTE will be read at the core echocardiography laboratory, and a categorical LVOT gradient result (< 50 mmHg or ≥ 50 mmHg) will be reported to the study site by the core laboratory. At Weeks 80 and 128, LVOT < 50 mmHg or ≥ 50 mmHg will be determined by site-read echocardiography. The investigator will remain blinded to the LVOT gradient result until after NYHA results have been entered in the eCRF. Results of medical therapy, NYHA functional class, and LVOT will be reviewed by the investigator, who will determine whether the subject meets ACCF / AHA and / or ESC eligibility criteria for SRT (yes or no). The investigator will discuss the recommendation with the subject. If the recommendation is to proceed with SRT, the subject may schedule the SRT at a recommended HCM center to occur after a recommended study drug washout period ≥ 6 weeks, or the subject may decline the recommendation and remain on study drug. ^ After Week 16 assessments, subjects in the mavacamten treatment group who elect to continue treatment (i.e., do not make a decision to have SRT) will continue once-daily dosing 36 57961429.1Attorney Docket No.267224-557784 with mavacamten at the dose they had been receiving at Week 16 for an additional 16 weeks; subjects in the placebo group who elect to continue treatment (i.e., do not make a decision to have SRT) will begin dosing with mavacamten 5 mg once daily for 16 weeks with subsequent assessments for dose adjustments (placebo-to-active group). During the active-controlled dosing period, mavacamten dose will remain blinded. ^ After Week 32 assessments, all subjects (mavacamten group and placebo-to-active group) who elect to continue treatment (ie, do not make a decision to have SRT) will continue daily dosing with mavacamten at the dose they had been receiving at Week 32 for an additional 96 weeks to Week 128 (EOT). During the LTE dosing period, mavacamten dose will remain blinded unless the sponsor chooses to unblind once the primary analysis is complete. Subjects will be reevaluated for SRT eligibility at Weeks 80 and 128. ^ During the study, dose may be titrated based on LVEF and LVOT by TTE read at the core echocardiography laboratory and according to dose titration guidelines. Throughout the study, all dose adjustments will occur in a blinded manner via the IXRS. ^ During the placebo-controlled dosing period (Day 1 to Week 16), all subjects will be evaluated for possible down-titration at Week 4 and up-titration at Weeks 8 and 12. Although subjects in the placebo group will be evaluated for dose titration, they will remain on placebo. ^ During the active-controlled dosing period (Weeks 16 to 32), subjects in the placebo-to- active group, who begin dosing with mavacamten at Week 16, will be evaluated for possible down-titration at Week 20 and up-titration at Weeks 24 and 28. ^ During the LTE dosing period (Weeks 32 to 128), mavacamten dose may be up-titrated at any scheduled visit after Week 32 if the site-read LVOT gradient with Valsalva maneuver is ≥ 30 mmHg and LVEF is ≥ 50%. All dose increases during LTE dosing must be approved by the medical monitor before they are implemented. Subjects who have their mavacamten dose increased during the LTE period will attend an unscheduled study visit 4 weeks after the dose increase and then resume the regular study visit schedule. ^ Dose may be down-titrated for safety at any time. Safety will be monitored throughout the study. ^ Table 6.0 provides dose titration guidelines for the study 37 57961429.1Attorney Docket No.267224-557784 Table 6.0 Dose Titration Guidelines LVEF ≥ 50% Mavacamten Group Placebo-to-Active Group g, 0 d p gStudy Scheme:

[0126] The study scheme is shown in Figure 7. Study Scheme Notes:

[0127] a. During the placebo-controlled dosing period (Day 1 to Week 16) subjects will be evaluated for possible down-titration at Week 4 and up-titration at Weeks 8 and 12 by 38 57961429.1Attorney Docket No.267224-557784 independent assessment of TTE by the echocardiography core laboratory and according to dose- titration guidelines. Dose may be down-titrated for safety at any time. b. Subjects in the placebo-to-active group, who begin dosing with mavacamten at Week 16, will be evaluated for possible down-titration at Week 20 and up-titration at Weeks 24 and 28. Dose may be down-titrated for safety at any time. c. During the long-term extension (LTE) dosing period (Weeks 32 to 128), mavacamten dose may be up-titrated at any scheduled visit after Week 32 if the site-read LVOT gradient with Valsalva maneuver is ≥ 30 mmHg and LVEF is ≥ 50%. All dose increases during LTE dosing must be approved by the MyoKardia medical monitor before they are implemented. Subjects who have their mavacamten dose increased during the LTE period will attend an unscheduled study visit 4 weeks after the dose increase and then resume the regular study visit schedule. Dose may be down-titrated for safety at any time. d. At any time during the study, subjects may withdraw from study drug and proceed with SRT at a recognized HCM center after a recommended study drug washout period ≥ 6 weeks. Subjects who discontinue study drug to undergo SRT will undergo EOT assessments within 14 days and will have a telephone follow-up with the study site to assess adverse events 8 weeks after treatment discontinuation (or prior to SRT, whichever is earlier). Subjects will be followed every 24 weeks from the date of SRT to Week 128. Study Drug Schedule:

[0128] On Day 1, subjects will begin blinded dosing with mavacamten or matching placebo once daily for 16 weeks (placebo-controlled period). After the Week 16 study assessments, subjects in the mavacamten group will continue mavacamten, and subjects in the placebo group will begin dosing with mavacamten, once daily from Weeks 16 to 32 (active- controlled period). During the active-controlled period, mavacamten dose will be blinded. Beginning at Week 16 and throughout the remainder of the study, the placebo group will be referred to as the placebo-to-active group. After the Week 32 assessments, all subjects will continue once-daily mavacamten until Week 128 (LTE period). During the LTE period, mavacamten dose will remain blinded unless the sponsor chooses to unblind once the primary analysis is complete. 39 57961429.1Attorney Docket No.267224-557784 Example 8. EXPLORER-HCM TRIAL: A Phase 3, double blind, randomized, placebo controlled, multicenter, international, parallel group study to evaluate the safety, tolerability, and efficacy of mavacamten compared with placebo (1:1) in participants with symptomatic oHCM

[0129] A Phase 3, double blind, randomized, placebo controlled, multicenter, international, parallel group study to evaluate the safety, tolerability, and efficacy of mavacamten compared with placebo (1:1) in participants with symptomatic oHCM was conducted. The study, referred to as EXPLORER-HCM (NCT03470545), was completed on May 6, 2020. 251 participants were enrolled (123 on mavacamten, 128 on placebo). A subset of participants consented to participate in a CMR substudy at selected sites. Randomization was stratified according to NYHA functional classification (II or III), current treatment with β-blocker (yes or no), planned type of ergometer used during the study (treadmill or exercise bicycle), and consent for the CMR substudy (yes or no). Study Design:

[0130] The study included 3 periods carried out according to the following design:

[0131] 1) Screening period (Day -35 to Day -1): Participants will undergo a variety of general, cardiopulmonary, laboratory, symptom, and PRO assessments over 1 to 2 days in order to assess eligibility. Key Screening tests include electrocardiogram (ECG); transthoracic echocardiography (TTE) conducted at rest, with Valsalva maneuver, and post-exercise; as well as cardiopulmonary exercise testing (CPET). The following screening assessments may be repeated, as long as within the 35-days screening window: blood tests, ECG, and / or TTE . Repeat assessments are allowed if central core labs require a repeat submission due to quality and in order to better assess inclusion / exclusion values. Participants who screen fail may be considered for rescreening based on the investigator’s discretion, taking into consideration the reason(s) for screen fail. One attempt at rescreening will be allowed, and all procedures must be repeated.

[0132] 2) Double-blind treatment period (Day 1 [randomization] to Week 30 / end of treatment [EOT]): The double-blind treatment period will include a two-step dose titration scheme designed to achieve safe and effective dosing for each participant based on their own response parameters. Participants who meet all eligibility criteria at Screening will first be randomized via an interactive response system in a 1:1 ratio to receive treatment with 40 57961429.1Attorney Docket No.267224-557784 mavacamten 5 mg starting dose or matching placebo once daily (QD). Subsequently, assessments including ECG, PK (trough plasma concentrations), and TTE will be performed at each of 7 study visits, beginning at Week 4, and read by core laboratories. At Week 8 and Week 14, the dose may be increased, decreased, or remain unchanged based upon results of Week 6 and Week 12 assessments, respectively, and based primarily on measurements of provoked left ventricular outflow tract (LVOT) gradient and bounded by a target plasma concentration (PK) range and clinical tolerability (LVEF). At Week 8, the dose may be increased to a maximum daily dose of 10 mg (i.e., increase from 5 mg QD to 10 mg QD), and at Week 14 to a maximum daily dose of 15 mg (i.e., increase from 10 mg QD to 15 mg QD). Dose increases are designed to be step wise and are not allowed to skip doses (eg, from 5 mg to 15 mg).

[0133] At Week 30 / EOT, participants will complete CPET and post-exercise TTE. For any participants permanently discontinuing treatment prior to Week 30, an early termination (ET) visit should be conducted as soon as possible, including CPET and post-exercise TTE. Participants with ET will also be encouraged to complete all remaining study visits and assessments, including the Week 30 visit.

[0134] 3) Posttreatment follow-up period (Week 30 / EOT to Week 38 / end of study [EOS]): When double-blind treatment ends at Week 30, participants will be contacted by phone at Week 34 and return to the site at Week 38 for an EOS visit. At the EOS visit, specified assessments will be repeated. This posttreatment follow-up period applies only to participants who are receiving study drug after Week 22. Study design is shown in Figure 8. Safety Monitoring:

[0135] Safety monitoring was carried out as follows:

[0136] To maintain safety throughout the double-blind treatment period, a clinic visit will occur every 2 to 4 weeks, beginning at Week 4 for an initial evaluation of clinical tolerability and safety. Clinic visits will include but are not limited to clinical evaluation (symptoms, PRO evaluations, adverse event [AE] / serious adverse event [SAE] assessment), ECGs, PK sample, TTEs, and laboratory assessments. Results of TTE performed by study site sonographers at each scheduled visit following randomization should be kept blinded to the investigator and other study site personnel. An exception may occur if left ventricular ejection fraction (LVEF) ≤30% is measured at the site, then the investigator will be immediately notified and study drug will be permanently discontinued as described within the protocol. 41 57961429.1Attorney Docket No.267224-557784

[0137] Assessments at Weeks 4, 6, 8, 12, 18, 22, and 26 will be used to guide dose reduction or temporary discontinuation if indicated, based on predefined criteria detailed within the protocol. If at any time during the double blind treatment period the mavacamten dose is decreased from the previous dose, the participant will continue on the reduced dose to the EOT (Week 30) unless further safety concerns or intolerability arise.

[0138] At selected sites, participants will have the option to participate in the CMR substudy. Approximately 80 participants will be enrolled (~40 per treatment group). In addition to the main study schedule of procedures, participants will undergo CMR at Day 1 and Week 30 (or up to 5 days before each visit). Study Treatment:

[0139] Participants received mavacamten immediate release capsules 5 mg or matching placebo QD for the first 8 weeks of the dosing period with trough PK samples drawn at Week 4, Week 6, and Week 8. If at Week 4 the trough PK was between 700 ng / mL and 1000 ng / mL, the dose was decreased to 2.5 mg at Week 6.

[0140] Otherwise, the dose was adjusted (increase, decrease, or remain unchanged) at Week 8 based on Week 6 assessments and Week 14 based on Week 12 assessments. The permissible doses after dose adjustment at Week 8 was 2.5 mg, 5 mg, 10 mg, or placebo. The permissible doses after dose adjustment at Week 14 was 2.5 mg, 5 mg, 10 mg, 15 mg, or placebo.

[0141] For added safety, if 700 ng / mL < Week 8 PK < 1000 ng / mL then an unscheduled visit was arranged 2 weeks later (Week 10) to reduce dose. After Week 14, assessments continued every 4 weeks to Week 30 / EOT for safety monitoring.

[0142] At any time if PK plasma concentration ≥ 1000 ng / mL, then study drug was temporarily discontinued.

[0143] Each participant was in the study for up to 43 weeks: for Screening, up to 5 weeks; for study conduct, 38 weeks (±7 days). Example 9. MAVERICK-HCM TRIAL: A Randomized, Double-blind, Placebo- controlled, Concentration-Guided Study, Exploratory Study of Mavacamten in Subjects with Symptomatic Non-obstructive Hypertrophic Cardiomyopathy (nHCM) and Preserved Left Ventricular Ejection Fraction 42 57961429.1Attorney Docket No.267224-557784

[0144] This is a Phase 2 trial designed to assess the safety and tolerability of a range of exposures over 16 weeks of treatment in subjects with symptomatic, non-obstructive HCM. All study subjects were required to be diagnosed with non-obstructive HCM, with left ventricular wall thickness either ≥15mm or ≥13mm with a family history of HCM, LVEF ≥ 55%, NYHA classifications of Class II or III, and NT-proBNP levels of greater than 300 pg / mL at rest. Baseline characteristics, such as age, weight, gender, pathogenic mutation status, background beta blocker use, NYHA classification and exercise capacity were approximately evenly distributed between active and placebo arms. The MAVERICK-HCM (NCT03442764) study was completed on January 7, 2020. The MAVERICK-HCM study was performed according to the following protocol. Methods:

[0145] This double-blind study enrolled 59 individuals with nHCM (Left ventricular outflow tract gradient <30 mmHg; resting or provoked), NYHA Class II or III, and LVEF ≥55%. Subjects were randomized 1:1:1 to one of two target plasma drug concentrations (Group 1: ~200 ng / mL and Group 2: ~500 ng / mL) or placebo for 16 weeks, followed by an 8-week washout. The starting dose of mavacamten was 5 mg daily, with one-step dose titration at Week 6 based on plasma drug concentration. Predefined criteria, including LVEF (LVEF ≤ 45%), guided study drug discontinuation if indicated. Cardiopulmonary exercise testing was performed at baseline and Week 16 to assess the impact on exercise capacity. Study Design and Plan:

[0146] This study is to evaluate the safety, tolerability, preliminary efficacy, PD, and PK of 2 target drug concentrations of Mavacamten compared with placebo in subjects with symptomatic nHCM. Study Scheme is shown in Figure 9.

[0147] Approximately 60 subjects with symptomatic nHCM are randomized and receive a 16-week course of Mavacamten doses titrated to achieve 1 of 2 target drug concentrations (Group 1: ~200 ng / mL; Group 2: ~500 ng / mL) or placebo once daily (QD). Dose adjustments will be based on PK parameters. Assessments include safety, standardized cardiopulmonary exercise testing (CPET) with measurement of peak oxygen consumption, echocardiography to evaluate left ventricular ejection fraction (LVEF) and parameters of diastolic function, symptoms, quality of life, daily step counts, and NT-proBNP at rest and after exercise. In 43 57961429.1Attorney Docket No.267224-557784 addition, subjects may consent to hypertrophic cardiomyopathy genotyping and pharmacogenetic sampling.

[0148] For subjects who consented and had prior HCM genotype test results demonstrating a pathogenic mutation known to be associated with HCM, no further genotype assessment were performed if the data could be provided from a clinical laboratory source document and the subject consents to share this information. Subjects who had not been tested and subjects who did not have an HCM genotype test results demonstrating a pathogenic mutation known to be associated with HCM might consent separately to have blood drawn prior to dosing on Day 1 for assessment of HCM genotype. For subjects who consented to pharmacogenetic assessment, blood samples were collected prior to dosing for analysis of genetic biomarkers of efficacy, safety, PD, or PK parameters as determined by future studies, using clinically meaningful endpoints, through additional DNA sequencing or other genetic testing.

[0149] Cardiac Troponin I levels were evaluated on plasma and serum samples of subjects at baseline and at various time points in the trial (Abbott Architect Stat Troponin-I assay (Ref.2K41)). Cardiac Troponin T levels were evaluated on plasma and serum samples of subjects at baseline and at various time points in the trial (Roche Elecsys Troponin T hs assay) (Ref.08469873190) performed on a cobas e 801 analyzer). NT-proBNP levels were evaluated on plasma samples using the Roche Elecsys proBNPII assay (Ref.07027664190) on a cobas e 801 analyzer. Study Treatment:

[0150] A concentration guided approach was used to evaluate what doses of mavacamten resulted in improvement of diastolic function in nHCM subjects. Subjects were randomized via an interactive response system to 3 groups in a 1:1:1 ratio: 2 active treatment groups and 1 matching placebo.

[0151] 5 mg QD was used as the starting dose for the study. All subjects in the active treatment groups started on 5 mg QD. Subjects were assessed for plasma concentration of mavacamten in blood samples taken at Week 4 visit. Pharmacokinetic (PK) modeling was used to guide blinded dose adjustment at the Week 6 visit, based on the plasma concentrations collected at Week 4. Subjects in the placebo group underwent the same assessments in order to preserve the blind. The study drug was provided in mavacamten capsules in available strengths 44 57961429.1Attorney Docket No.267224-557784 of 2.5 mg, 5 mg, 10 mg and 15 mg. Subjects were instructed to take the drug under fasting conditions, at approximately the same time each day, and with 8 ounces of water.

[0152] A target mavacamten blood plasma concentration of 200 ng / mL was the goal in Group 1 subjects. To achieve the target concentration, if a subject’s Week 4 concentration was >450 ng / mL, the subject’s dose was decreased to 2.5 mg QD; if Week 4 concentration was 110- 450 ng / mL, the dose was maintained at 5 mg QD; and if Week 4 concentration was <110 ng / mL, the dose was increased to 10 mg QD.

[0153] A target Mavacamten blood plasma concentration of 500 ng / mL was the goal in Group 2 subjects. To achieve the target concentration, if a subject’s Week 4 concentration was >450 ng / mL, the subject’s dose was decreased to 2.5 mg QD; if Week 4 concentration was 300- 450 ng / mL, the dose was maintained at 5 mg QD; if Week 4 concentration was greater than or equal to 175 and less than 300 ng / mL, the dose was increased to 10 mg QD; and if Week 4 concentration was <175 ng / mL, the dose was increased to 15 mg QD.

[0154] Subjects were monitored for adverse events (AE), including high blood plasma concentration, systolic dysfunction, QT prolongation, and LVEF decrease. If any of the following thresholds were hit PK 1000 or more, QTcF 500, or LVEF 45%, the subjects were discontinued on drug. Specifically, high blood plasma concentration was defined as blood plasma concentration greater than or equal to 1000 ng / mL; QT prolongation was defined as QTcF greater than or equal to 500 ms; and LVEF shortening was defined as LVEF less than or equal to 45% (including serious adverse event (SAE) for LVEF less than or equal to 30%).

[0155] Efficacy and pharmacodynamics assessments were also made. Resting transthoracic echocardiography measurements were taken at Weeks 4, 8, 12 and 16. Ejection fraction (2-D) and LV frantional shortening were analyzed along with other echocardiographic at baseline measures including measure of diastolic function. Post-exercise stress echocardiography was also performed following a standard symptom-limited exercise test performed by the subjects. Instantaneous peak LVOT gradient was assessed immediately post- exercise. Cardiopulmonary exercise testing (CPET) was also performed. CPET was conducted using a standardized treadmill or upright bicycle ergometer on Day 1 and at Week 16. Subjects were encouraged to perform maximally to achieve expected heart rate. Oxygen uptake (VO2), carbon dioxide production (VCO2), volume expired (VE), VE / VO2, ventilatory efficiency 45 57961429.1Attorney Docket No.267224-557784 (VE / VCO2), respiratory exchange ratio, circulatory power, and metabolic equivalent of the task were assessed.

[0156] Pharmacokinetic assessments were also made during the study. Blood samples were collected for mavacamten plasma concentration assessments at Weeks 4, 8, 12 and 16. At Week 16, a predose and postdose PK blood sample was taken. Example 10 – Correlations Between Clinical Variables and Proteins Identified by Differential Expression

[0157] Statistical correlations were determined between (A) change from baseline in protein levels (for proteins identified by differential expression, e.g., as described in Examples 1- 6) and (B) improvement in clinical variables relevant to HCM from four clinical studies: EXPLORER-HCM, VALOR-HCM, LTE-EXPLORER and LTE-MAVERICK. Clinical variables included LVOT gradient (pd oHCM), LAVI (diastolic function), and NT-ProBNP (positive control). Tables 4A-D (below) and Figures 10-13 show correlations with an absolute correlation coefficient > 0.2 and p < 0.05 for proteins that were identified in three or four of the four clinical studies analyzed.

[0158] Table 4A lists the correlation data for proteins in the EXPLORER-HCM study. Table 4B lists the correlation data for proteins in the VALOR-HCM study. Table 4C lists the correlation data for proteins in the LTE-EXPLORER study. Table 4D lists the correlation data for the LTE-MAVERICK study. Rho.pcbo refers to the correlation coefficient for the placebo data. Pval.pcbo refers to the p value for the placebo data. Rho.mava refers to the correlation coefficient for the patients receiving mavacamten. Pval.mava refers to the p value for the patients receiving mavacamten. N.pcbo refers to the number of the patients in the study who received placbo. N.mava refers to the number of patients in the study who received mavacamten treatment.

[0159] Figures 10-13 show selected scatter plots of the change in LA volume index or change in LVOT gradient (resting or Valsalva) versus Log2 fold change in the protein level. Figure 10 is based on data from EXPLORER-HCM. Figure 11 is based on data from VALOR- HCM. Figure 12 is based on data from LTE-EXPLORER. Figure 13 is based on data from LTE-MAVERICK. As seen by the plots and corresponding calculations of correlation coefficient (Rho) and p value (P), the correlations were significant. 46 57961429.1487755-422762.oNtekcoDAy 4e enrlbottaTAe PP I I IpyN NIB-B- rIrIrTd2i-ototot eneororn 2t323 4 4 4cacaca1 1 xioplaplP P iaN N ni-P-PM M MfUofcofcocD-D-D- R RQ QM,rPnini B Bm mrm a N NU U er L B BZIZIZIniPrSPSPS1 1 nianir nirC Cbot-et-paepaepelgN N H H HotpaH 115 5 51 1 1 8 8 86 6 64 4 dIq1-1- 1-1 11-1- 1-1- -9-9-95-5-5- 4-4-4- 323-2- 3- .1e 555755757433232454545616160 0 01959595 63643592S67676161817373020202333333919191 141034114169759830180000.0604.0-hrm3.50. . . .3.3.3.00 0 0 0 0 0 027629 9 5 2 4 2 8 7 0 3 2 ..6 4 0 579616672760.0.0.0.0.0.0.0.0.0laob1 3 4 972 3 7 5 6 2 1618481380385940.0-)gH mm,gnitser(TOVLepeypTyd Tedxeixn P P P1iin nbi iN N NniM M1-b bPT T T ololol C nieB-B-B-ah, , P P Pg gWorororcninAo ogotop p p tbi hobV H H Hmlo ememeYSrPlalalagil2-glH H Hnininiogto mnpt rnmirm isni23apaert-et-etP Poym -PH H - N N NNBNBMaLNB3-374- -0 0 0 4 4 4 7 d 1 1 1515191515405 139-3-3-6-6-6-1-303 4510505051515107Iq1-1-1- -1-1-6-71- .192297979794949489eS5556555575773433241767676161818173697584840 2000.04877 25515-4.022576328..0oNt13ek0.co0-Dyenr)ogttH A m m,gnitser(TO VLe p e p e yypT TyIIITded drI xexexoroi i itctacfaM,M,M,2 2 of ninini nininicocbobobb b bM M4ni 2 2 1 1l lol olololO O MranirN N1RC Cg g gMgogogo C CY Y UpaepeD-T TQP PotoptotT T M- W W M MZIH HPS C CA A1Y Y Capapa P PM M H H H H HK mCememeH H HYSYS00 64 2-24-134-8 8 483954-56- -383326019-14- -0 0 7 4 4 4 7 7469 9 3 3 3 3 3 6 6 6 6 1 1.13- 8 - 8 - 4 - 4- - - - - - - - -92535 5 1 1 5 4 4 1 4 4 5 54550500 5 5 5 0 0 411310233339189894167670303039797761291491 7 749489896975CL L L L L L L L L L L L L L L L L L LP P PN N N1ni III IB-B-B-arIrIroro ohcotototprlaprt clacacaapla hgil2-2-2-fofofonniininietomrm mnir er sninniinniini2323 4 4 4cic cnr nir nirt P-ert-et- P P PN N NNBNBN oym m m -P-PM M Ma a aU U UpBepepeD- MaLaLaLNBNBZIZIZIH H HPS1 1 151515191515 5 6d1I-15-15- -51-51-51-56-177-147-14714-13- -139-149-8 8 8 4495 5 5 -.14- 6- - 092qe5 5 5 7 7 7 3 3 3 3 2 2 5 5 5 1616 9 41S67676761616181818181737302020233313353916975611486 07-7E535- 24 32 32 2.7162.oNt 3ek34co.0Dyenr)gg gH)gH)g / L)g g)g g / L)g g / L)g gottm H m H H mmm(H H H H H m H mm(H mm(H m A m m ,amxm mxmxm vl ,m,am,m,aedm,m,am,m,ae m,m,ae m,m,aagnvlaa gnvlanIgnvlgnvl dnIgnvl dnIgnvlsl its sl its sleitsasl itsasleitsas eitsasv(era(v(era(v(mueraveravmue lravmue lrav TTlo ( ( ( (l( (l( (T T T o oVT T T TVT TVT TO O O O O O O O O O O O O VLVLVLVLVLALVLVLVLVLALVLVLALVLVLe p e p e ypTyTyTded dxexei ixiM,Mn,Min, n n nbinioboboibiobiob11 lglglg 1 1 lgloglgC CDR R-Q Qotoptot- apapa P-PTo o oPTPTPmem mW W PS1C1CA Ae eH H H V V H H H H H HYSYS6 4 4 43-232 7 7 0 00-96-3633-34-34- -41-9139-30 4 4 4 7 75 -35-65-6 6 1 1.15- 5- - -92591 1 5 5 5 4 4 0 0 0 1 1510 0 4114141030303121297979794949748798969754a8v7a75m5.-n61526161422a76va2 m.. 3 6 3 4olaNtv 00303000p.0.0.0.0ekcaoDDva42 5 4y 4e em.o9243 7.5.6.2nrlbh6. 0 0 05ottar 0 - - -TA))2gm / )g)gHLH H mm(m m elbm,aa xie m mvd,a,arlaasnIvlvleasasVllaav(mulalalv(v(ci T o T TnilOVO O CVLALVLVLIIrotcafo nincboleniD i1g -tFora-P orp GPeAme EH V H V 87 439d5.1I- - q6169- -589924e1 1S34 1 031294316975

Claims

Attorney Docket No.267224-557784 CLAIMS We Claim:

1. A method of treating a patient in need thereof with a myosin inhibitor, comprising: administering a myosin inhibitor to the patient; and determining, or having determined, a treatment response of the patient based on one or more protein levels obtained from a biological sample from the patient following administration 2. A method of treating a patient in need thereof with a myosin inhibitor, comprising: administering a myosin inhibitor to the patient; measuring, or having measured, one or more protein levels in a biological sample from the patient following administration of the myosin inhibitor to the patient; analyzing, or having analyzed, the one or more protein levels; and determining, or having determined, a treatment response of the patient based on the one or more protein levels.

3. The method of any preceding claim, further comprising modifying the treatment of the patient based on the treatment response of the patient.

4. The method of any preceding claim, wherein modifying the treatment comprises administering a higher dose of the myosin inhibitor or administering the myosin inhibitor at a greater frequency.

5. The method of any preceding claim, wherein modifying the treatment comprises administering a lower dose of the myosin inhibitor or administering the myosin inhibitor at a lesser frequency.

6. The method of any preceding claim, wherein modifying the treatment comprises administering a different myosin inhibitor or wherein modifying the treatment comprises temporarily discontinuing administration of the myosin inhibitor. 53 57961429.1Attorney Docket No.267224-557784 7. The method of claim 1 or 2, comprising maintaining the treatment of the patient based on the treatment response of the patient and / or based on the analysis of the one or more protein level(s), optionally wherein maintaining the treatment comprises administering the same myosin inhibitor at the same dose and with the same frequency of dosing..

8. The method of any preceding claim, wherein the treatment response indicates whether the patient is benefitting from the treatment.

9. The method of any preceding claim, wherein the treatment response indicates the level of exposure to the patient of the myosin inhibitor.

10. The method of any preceding claim, wherein the treatment response indicates the reduction in LVOT gradient or LA volume index in the patient.

11. The method of any preceding claim, wherein analyzing the one or more protein levels comprises comparing a protein level to a threshold.

12. The method of any preceding claim, further comprising measuring, or having measured, one or more baseline protein levels in a biological sample from the patient prior to administration of the myosin inhibitor to the patient.

13. The method of any preceding claim, wherein analyzing the one or more protein levels comprises comparing a protein level to a baseline protein level, or comparing a first protein level to a second protein level, wherein the first protein level is measured prior to a myosin inhibitor treatment period and the second protein level is measured after the myosin inhibitor treatment period .

14. The method of claim 13, wherein analyzing the one or more protein levels comprises determining a percent change from baseline in a protein level and comparing the percent change from baseline to a threshold percentage, or determining a percent change from the 54 57961429.1Attorney Docket No.267224-557784 first protein level to the second protein level and comparing the percent change to a threshold percentage.

15. The method of claim 13, wherein analyzing the one or more protein levels comprises determining whether a change from baseline in a protein level is a statistically significant change, or determining whether a change from the first protein level to the second protein level is a statistically significant change.

16. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is below a threshold, the method further comprising modifying treatment by administering a lower dose of the myosin inhibitor.

17. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is below a threshold, the method further comprising modifying treatment by temporarily discontinuing administration of the myosin inhibitor 18. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is below a threshold, the method further comprising modifying treatment by administering a different myosin inhibitor.

19. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is above a threshold, the method further comprising modifying treatment by administering a higher dose of the myosin inhibitor.

20. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is above a threshold, the method further comprising modifying treatment by administering a different myosin inhibitor.

21. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is above a threshold, the method further comprising modifying treatment by administering a lower dose of the myosin inhibitor. 55 57961429.1Attorney Docket No.267224-557784 22. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is above a threshold, the method further comprising modifying treatment by temporarily discontinuing administration of the myosin inhibitor 23. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is above a threshold, the method further comprising modifying treatment by administering a different myosin inhibitor.

24. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is below a threshold, the method further comprising modifying treatment by administering a higher dose of the myosin inhibitor.

25. The method of any preceding claim, wherein the change from baseline in the one or more protein levels is below a threshold, the method further comprising modifying treatment by administering a different myosin inhibitor.

26. The method of any preceding claim, wherein the one or more protein levels comprises one or more hemolysis protein levels.

27. The method of claim 26, wherein the change from baseline in the one or more hemolysis protein levels is below a threshold change, the method further comprising modifying treatment by administering a different dose of the myosin inhibitor.

28. The method of claim 26, wherein the change from baseline in the one or more hemolysis protein levels is below a threshold change, the method further comprising modifying treatment by temporarily discontinuing administration of the myosin inhibitor.

29. The method of claim 26, wherein the change from baseline in the one or more hemolysis protein levels is below a threshold change, the method further comprising modifying treatment by administering a different myosin inhibitor. 56 57961429.1Attorney Docket No.267224-557784 30. The method of claim 26, wherein the change from baseline in the one or more hemolysis protein levels is above a threshold change, the method further comprising modifying treatment by administering a different dose of the myosin inhibitor.

31. The method of claim 26, wherein the change from baseline in the one or more hemolysis protein levels is above a threshold change, the method further comprising modifying treatment by temporarily discontinuing administration of the myosin inhibitor.

32. The method of claim 26, wherein the change from baseline in the one or more hemolysis protein levels is above a threshold change, the method further comprising modifying treatment by administering a different myosin inhibitor.

33. The method of any one of claims 27-32, wherein the threshold change is a percent change or a fold change.

34. The method of any one of claims 26-33, wherein the one or more hemolysis protein levels are levels of one or more proteins selected from the group consisting of hemoglobin, hemoglobin subunit beta, HBG2, haptoglobin, and haptoglobin mixed type, or any combination thereof.

35. The method of claim 34, wherein the one or more hemolysis protein levels are levels of hemoglobin, haptoglobin, or a combination thereof.

36. The method of any preceding claim, wherein the one or more protein levels comprises one or more sarcomere protein levels and / or one or more muscle / contractility-related protein levels.

37. The method of claim 36, wherein the change from baseline in the one or more sarcomere protein levels and / or one or more muscle / contractility-related protein levels is below a 57 57961429.1Attorney Docket No.267224-557784 threshold change, the method further comprising modifying treatment by administering a different dose of the myosin inhibitor.

38. The method of claim 36, wherein the change from baseline in the one or more sarcomere protein levels and / or one or more muscle / contractility-related protein levels is below a threshold change, the method further comprising modifying treatment by temporarily discontinuing administration of the myosin inhibitor.

39. The method of claim 36, wherein the change from baseline in the one or more sarcomere protein levels and / or one or more muscle / contractility-related protein levels is below a threshold change, the method further comprising modifying treatment by administering a different myosin inhibitor.

40. The method of claim 36, wherein the change from baseline in the one or more sarcomere protein levels and / or one or more muscle / contractility-related protein levels is above a threshold change, the method further comprising modifying treatment by administering a different dose of the myosin inhibitor.

41. The method of claim 36, wherein the change from baseline in the one or more sarcomere protein levels and / or one or more muscle / contractility-related protein levels is above a threshold change, the method further comprising modifying treatment by temporarily discontinuing administration of the myosin inhibitor.

42. The method of claim 36, wherein the change from baseline in the one or more sarcomere protein levels and / or one or more muscle / contractility-related protein levels is above a threshold change, the method further comprising modifying treatment by administering a different myosin inhibitor.

43. The method of any one of claims 37-42, wherein the threshold change is a percent change or a fold change. 58 57961429.1Attorney Docket No.267224-557784 44. The method of any one of claims 36-43, wherein the one or more sarcomere protein levels are levels of one or more proteins selected from the group consisting of myosin light chain 1, alpha actinin 2 (ACTN2), myosin-binding pro-C1 (MYPC1), and myomesin 2 (MYOM2), or any combination thereof.

45. The method of claim 44, wherein the one or more sarcomere protein levels are levels of myosin light chain 1, alpha actinin 2, or any combination thereof.

46. The method of any one of claims 36-43, wherein the one or more muscle / contractility- related protein levels are levels of one or more proteins selected from the group consisting of myosin light chain 1, ACTN2, MYPC1, Laminin-2, MYOM2, and CK- MM, or any combination thereof.

47. The method of any preceding claim, wherein the one or more protein levels comprises the level of one or more cardiac biomarker proteins and / or one or more cardiopulmonary biomarker proteins.

48. The method of claim 47, wherein the change from baseline in the one or more cardiac biomarker protein levels and / or cardiopulmonary biomarker protein levels is below a threshold change, the method further comprising modifying treatment by administering a different dose of the myosin inhibitor.

49. The method of claim 47, wherein the change from baseline in the one or more cardiac biomarker protein levels and / or cardiopulmonary biomarker protein levels is below a threshold change, the method further comprising modifying treatment by temporarily discontinuing administration of the myosin inhibitor.

50. The method of claim 47, wherein the change from baseline in the one or more cardiac biomarker protein levels and / or cardiopulmonary biomarker protein levels is below a threshold change, the method further comprising modifying treatment by administering a different myosin inhibitor. 59 57961429.1Attorney Docket No.267224-557784 51. The method of claim 47, wherein the change from baseline in the one or more cardiac biomarker protein levels and / or cardiopulmonary biomarker protein levels is above a threshold change, the method further comprising modifying treatment by administering a higher dose of the myosin inhibitor.

52. The method of claim 47, wherein the change from baseline in the one or more cardiac biomarker protein levels and / or cardiopulmonary biomarker protein levels is above a threshold change, the method further comprising modifying treatment by temporarily discontinuing administration of the myosin inhibitor 53. The method of claim 47, wherein the change from baseline in the one or more cardiac biomarker protein levels and / or cardiopulmonary biomarker protein levels is above a threshold change, the method further comprising modifying treatment by administering a different myosin inhibitor.

54. The method of any one of claims 47-53, wherein the threshold change is a percent change or a fold change.

55. The method of any one of claims 47-54, wherein the one or more cardiac biomarker protein levels are levels of proteins selected from the group consisting of BNP, BNP-32, and heparin cofactor II, or any combination thereof.

56. The method of any one of claims 47-54, wherein the one or more cardiopulmonary biomarker protein levels are levels of proteins selected from the group consisting of BNP, BNP-32, heparin cofactor II, SP-D, C1QR1, VEGF-D, and SYWC, or any combination thereof.

57. The method of any preceding claim, wherein the one or more protein levels comprises the level of IZUM4. 60 57961429.1Attorney Docket No.267224-557784 58. The method of any preceding claim, wherein the one or more protein levels measured comprises the level of a protein selected from the group consisting of N-terminal pro- BNP, BNP, Laminin-2, BNP-32, IZUM4, Heparin cofactor II, SP-D, C1QR1, Haptoglobin Mixed Type, VAP-1, HPT, Hemoglobin, SYWC, Myosin light chain 1, MYOM2, ACTN2, MYPC1, CK-MM, VEGF-D, HBG2, and beta-globin, or any combination thereof.

59. The method of claim 58, wherein the one or more protein levels measured comprises the level of a protein selected from the group consisting of N-terminal pro-BNP, BNP, Laminin-2, BNP-32, IZUM4, Heparin cofactor II, SP-D, C1QR1, Haptoglobin Mixed Type, VAP-1, HPT, Hemoglobin, SYWC, Myosin light chain 1, MYOM2, ACTN2, MYPC1, CK-MM, and VEGF-D, or any combination thereof.

60. The method of any preceding claim, wherein the one or more protein levels measured does not comprise the level of NT-proBNP nor cardiac troponin.

61. The method of any preceding claim, wherein the one or more protein levels measured comprises multiple protein levels wherein the multiple protein levels include NT-proBNP and / or cardiac troponin.

62. The method of any preceding claim, further comprising modifying treatment to achieve level(s) of the one or more proteins at target level(s).

63. The method of any preceding claim, further comprising modifying treatment to maintain the level(s) of the one or more proteins at target level(s).

64. The method of any preceding claim, wherein the myosin inhibitor is mavacamten or a pharmaceutically acceptable salt thereof.

65. The method of any preceding claim, wherein the myosin inhibitor is mavacamten. 61 57961429.1Attorney Docket No.267224-557784 66. The method of any preceding claim, wherein the myosin inhibitor is or a pharmaceutically67. The method of any preceding claim, wherein the myosin inhibitor is .

68. The method of any preceding claim, wherein the myosin inhibitor is aficamten or a pharmaceutically acceptable salt thereof.

69. The method of any preceding claim, wherein the myosin inhibitor is aficamten.

70. The method of any preceding claim, wherein the myosin inhibitor is or a pharmaceutically71. The method of any preceding claim, wherein the myosin inhibitor is 62 57961429.1Attorney Docket No.267224-557784 .

72. The method of any preceding claim, wherein the patient is suffering from HCM.

73. The method of any preceding claim, wherein the patient is suffering from oHCM.

74. The method of any preceding claim, wherein the patient is suffering from nHCM.

75. The method of any preceding claim, wherein the patient is suffering from HFpEF.

76. The method of any preceding claim, wherein the patient is suffering from LVH.

77. The method of any preceding claim, wherein the patient is suffering from diastolic dysfunction.

78. The method of any preceding claim, further comprising obtaining, or having obtained, a biological sample from the patient.

79. The method of any preceding claim, wherein the biological sample is a blood sample.

80. The method of any preceding claim, wherein the biological sample is a blood serum sample.

81. The method of any preceding claim, wherein the biological sample is a blood plasma sample.

82. The method of any preceding claim, further comprising measuring, or having measured, an LVEF of the patient when one or more protein levels are above a threshold. 63 57961429.1Attorney Docket No.267224-557784 83. The method of any preceding claim, further comprising measuring, or having measured, an LVEF of the patient when the change from baseline in one or more protein levels is above a threshold change.

84. The method of any preceding claim, further comprising measuring, or having measured, an LVEF of the patient when one or more protein levels is below a threshold.

85. The method of any preceding claim, further comprising measuring, or having measured, an LVEF of the patient when the change from baseline in one or more protein levels is below a threshold change.

86. The method of any preceding claim, comprising administering an initial dose of 5 mg per day of mavacamten for a first time period prior to measuring the one or more protein levels.

87. The method of claim 86, comprising modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of 10 mg per day of mavacamten for a second time period subsequent to measuring the one or more protein levels.

88. The method of claim 86, comprising modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of 2.5 mg per day of mavacamten for a second time period subsequent to measuring the one or more protein levels.

89. The method of any preceding claim, comprising administering an initial dose of 2.5 mg per day of mavacamten for a first time period prior to measuring the one or more protein levels. 64 57961429.1Attorney Docket No.267224-557784 90. The method of claim 89, comprising modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of 5 mg per day of mavacamten for a second time period subsequent to measuring the one or more protein levels.

91. The method of claim 89, comprising modifying the treatment of the patient with the myosin inhibitor based on the treatment response of the patient, wherein modifying the treatment comprises administering a second dose of 1 mg per day of mavacamten for a second time period subsequent to measuring the one or more protein levels, or temporarily discontinuing administration of mavacamten during the second time period.

92. A method of determining response to myosin inhibitor therapy in a patient comprising analyzing one or more protein levels in a biological sample from the patient.

93. A method of determining whether to treat a patient with a myosin inhibitor and treating the patient with a myosin inhibitor, the method comprising: determining, or having determined, whether to treat the patient with a myosin inhibitor based on one or more protein levels in a biological sample from the patient; and treating the patient by administering a myosin inhibitor to the patient 94. A method of determining whether to treat a patient with a myosin inhibitor and treating the patient with a myosin inhibitor, the method comprising: measuring, or having measured, one or more protein levels in a biological sample from the patient; analyzing, or having analyzed, the one or more protein levels; determining, or having determined, based on the one or more protein levels in the biological sample, whether to treat the patient with a myosin inhibitor; and treating the patient by administering a myosin inhibitor to the patient.

95. The method of any preceding claim, wherein analyzing the one or more protein levels comprises comparing a protein level to a threshold. 65 57961429.1Attorney Docket No.267224-557784 96. The method of any preceding claim, wherein analyzing the one or more protein levels results in finding that one or more protein levels are above a threshold.

97. The method of any preceding claim, wherein, when one or more protein levels are above a threshold, the method comprises determining, or having determined, that the patient is indicated for treatment with a myosin inhibitor.

98. The method of any preceding claim, wherein analyzing the one or more protein levels results in finding that one or more protein levels are below a threshold.

99. The method of any preceding claim, wherein, when one or more protein levels are below a threshold, the method comprises determining, or having determined, that the patient is indicated for treatment with a myosin inhibitor.

100. The method of any preceding claim, wherein the patient has been identified as a NYHA class II oHCM patient.

101. The method of claim 100, wherein it is determined to treat the NYHA class II oHCM patient based on the analysis of the one or more protein levels.

102. The method of claim 100, wherein it is determined to treat the NYHA class II oHCM patient based on the analysis of one or more hemolysis protein levels.

103. The method of claim 100, wherein it is determined to treat the NYHA class II oHCM patient based on the analysis of one or more sarcomere protein levels and / or one or more muscle / contractility protein levels.

104. The method of claim 100, wherein it is determined to treat the NYHA class II oHCM patient based on the analysis of one or more cardiac biomarker protein levels and / or one or more cardiopulmonary biomarker protein levels. 66 57961429.1Attorney Docket No.267224-557784 105. The method of claim 100, wherein it is determined to treat the NYHA class II oHCM patient based on the analysis of one or more protein levels in Table 1 and / or Tables 4A, 4B, 4C, and / or 4D.

106. A method of selecting a myosin inhibitor therapy and treating a patient in need thereof with the myosin inhibitor therapy, comprising: measuring, or having measured, one or more protein levels in a biological sample from the patient; analyzing, or having analyzed, the one or more protein levels; selecting, or having selected, based on the one or more protein levels in the biological sample, a myosin inhibitor therapy for the patient; and treating the patient by administering a myosin inhibitor to the patient.

107. The method of claim 106, further comprising monitoring the one or more protein levels during the treatment. 67 57961429.1

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