Methods of treatment with myosin inhibitors based on protein levels

Abstract

Described herein are methods of treatment with myosin inhibitors based on 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. 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 therapeutic response based on the analysis, and may also include treating the subject based on the determination of a therapeutic response.

Description

Cross-references to related applications

[0001] This application claims priority and benefit to U.S. Provisional Patent Applications Nos. 63 / 546,878 and 63 / 562,333, filed November 1, 2023 and March 7, 2024, respectively, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to a method for treating patients with myosin inhibitors based on protein levels, a method for monitoring the response to treatment based on such protein levels, and a method for determining whether to treat a patient with a myosin inhibitor based on such protein levels. Background Technology

[0003] Over the past decade, clinical research on myosin inhibitors for the treatment of heart disease has accelerated. The myosin inhibitor mavacamten (MYK-461) has been investigated in clinical trials for the treatment of 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 their long-term extension studies (MAVA-LTE (NCT03723655), PIONEER-OLE (NCT03496168)). Mavacamten has received FDA approval for the treatment of adults with symptomatic New York Heart Association (NYHA) class II-III obstructive hypertrophic cardiomyopathy to improve function and symptoms. The myosin inhibitor aficamten has also been investigated in clinical trials for the treatment of HCM, including ACACIA-HCM (NCT06081894), MAPLE-HCM (NCT05767346), and SEQUOIA-HCM (NCT05186818). Other myosin inhibitors have also been studied in clinical and preclinical settings.

[0004] Despite clinical advances in the use of myosin inhibitors to treat HCM and other heart diseases, there is still a need for new and improved myosin inhibitor treatments, as well as methods to monitor responses to treatment and determine whether patients should be treated with myosin inhibitors. Summary of the Invention

[0005] This article describes methods for treating patients with myosin inhibitors based on protein levels, methods for monitoring responses to treatment based on such protein levels, and methods for determining whether to treat patients with myosin inhibitors based on such protein levels. Such methods may include obtaining biological samples from subjects, measuring the levels of one or more proteins in the samples, analyzing said one or more protein levels, determining a treatment response based on the analysis, and may also include treating or modifying treatment in subjects based on the determination of a treatment response. Attached Figure Description

[0006] Figure 1 shows (A) diagrams of hemoglobin (Hb), haptoglobin (Hp), and left ventricular (LV) septum at baseline and after 30 weeks of macaverate treatment, and (B) diagrams of Log2RFU (relative fluorescence units) at baseline and after 30 weeks of macaverate treatment. The left-hand graph shows the RFU of hemoglobin, and the right-hand graph shows the RFU of haptoglobin (mixed type).

[0007] Figure 2 Biomarkers of cell-free plasma hemolysis at baseline and week 30 are shown.

[0008] Figure 3 The correlation between hemoglobin and haptoglobin levels was shown.

[0009] Figure 4 The resting and Valsalva LVOT (left ventricular outflow tract) gradients caused by changes in the hemolytic biomarker spectrum are shown.

[0010] Figure 5 The Log2RFU plots of myosin light chain 1, ACTN2, MYOM2, and MYPC1 at baseline and after 30 weeks of treatment with macvaciate are shown.

[0011] Figure 6 Log2RFU plots of N-terminal pro-BNP, SP-D, and heparin cofactor II are shown at baseline and at 30 weeks of treatment with macvaciate.

[0012] Figure 7 The research scheme of VALOR-HCM described in Example 7 is shown.

[0013] Figure 8 The research scheme of EXPLORER-HCM described in Example 8 is shown.

[0014] Figure 9 The research scheme of MAVERICK-HCM described in Example 9 is shown.

[0015] Figure 10Selected scatter plots show changes in left atrial (LA) volume index or LVOT gradient (resting or Valsalva) compared to fold changes in specific protein levels in Log2, based on EXPLORER-HCM data.

[0016] Figure 11 Selected scatter plots are shown, comparing changes in the LA volume index or LVOT gradient (resting or Valsalva) with fold changes in specific protein levels in Log2, based on VALOR-HCM data.

[0017] Figure 12 Selected scatter plots show changes in left atrial (LA) volume index or LVOT gradient (resting or Valsalva) compared to fold changes in specific protein levels in Log2, based on LTE-EXPLORER data.

[0018] Figure 13 Selected scatter plots are shown, based on LTE-MAVERICK data, comparing changes in the LA volume index or LVOT gradient (resting or Valsalva) with fold changes in Log2 at specific protein levels. Detailed Implementation

[0019] definition While various embodiments and aspects of the invention have been shown and described herein, such embodiments and aspects are provided by way of example only and will be apparent to those skilled in the art. Various modifications, variations, and substitutions will now be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the inventive embodiments described herein can be used to practice the invention.

[0020] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice of this invention.

[0021] The section headings used herein are for organizational purposes only and are not intended to limit the subject matter. All documents or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are hereby expressly incorporated in their entirety by reference for any purpose.

[0022] As used herein, the terms “a” or “an” refer to one or more.

[0023] The terms “contains,” “includes,” and “has,” and their derivatives, are used interchangeably in this text as comprehensive, open-ended terms. For example, the use of “contains,” “includes,” or “has” implies that whatever elements are contained, have, or include, they are not the only elements covered by the subject of the clause containing the verb.

[0024] As used herein, the terms “treatment”, “treating”, or “relief” are used interchangeably. These terms refer to methods for achieving beneficial or desired outcomes, including but not limited to therapeutic benefits. A therapeutic benefit means the eradication or relief of an underlying condition being treated and / or the eradication or relief of one or more physiological symptoms associated with the underlying condition, resulting in an observed improvement in the subject, although the subject may still have the underlying condition. Treatment includes slowing the development of clinical symptoms of the disease by administration of the composition; suppressing the disease, i.e., reducing the clinical symptoms that cause the disease; inhibiting the disease, i.e., preventing the development of clinical symptoms by administration of the composition after the first appearance of symptoms; and / or alleviating the disease, i.e., causing the remission of clinical symptoms by administration of the composition after the first appearance of clinical symptoms.

[0025] "Patient" or "subject" means a living organism that suffers from or is susceptible to a disease or ailment that can be treated using the methods provided herein. The term does not necessarily indicate that a subject has been diagnosed with a specific disease, but generally refers to an individual under medical supervision. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, cats, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the patient or subject is a human.

[0026] Implementation Plan As mentioned above, there is a need for new and improved myosin inhibitor treatments, as well as methods for monitoring responses to treatment and determining whether patients should be treated with myosin inhibitors. Proteomic studies on myosin inhibitor therapy are limited, with some available data previously reported on NT-proBNP and cardiac troponin (hs-cTnI) levels (Olivotto et al., The Lancet, 396(10253): P759-769; 2020).

[0027] Certain proteins, including sarcoma proteins and hemolytic proteins, as well as muscle / contraction-related proteins, cardiac biomarker proteins, and cardiopulmonary biomarker proteins, have been identified as potential biomarkers for myosin inhibitor therapy. For example, administration of myosin inhibitors (e.g., macvacitazone) can upregulate or downregulate the levels of certain proteins. Furthermore, changes in the levels of certain proteins during myosin inhibitor therapy can indicate a treatment response.

[0028] Determining treatment response based on protein levels can be applied to methods of treating patients with myosin inhibitors. Protein levels can be determined before, after, and / or periodically during treatment, and the treatment response can be used to determine the course of treatment. These and other treatment methods and their various implementation schemes will be described in further detail below.

[0029] This disclosure provides a method for treating a patient in need with a myosin inhibitor, comprising: administering a myosin inhibitor to the patient; and determining, or having determined, the patient's treatment response based on the levels of one or more proteins obtained from a biological sample from the patient after administration.

[0030] This disclosure also provides a method for treating a patient in need with a myosin inhibitor, comprising: administering a myosin inhibitor to the patient; measuring or having measured the level of one or more proteins in a biological sample from the patient after administering the myosin inhibitor to the patient; analyzing or having analyzed the one or more protein levels; and determining or having determined the patient's treatment response based on the one or more protein levels.

[0031] Current methods may also include modifying a patient's treatment based on their treatment response. Current methods may also include modifying a patient's treatment based on protein levels. In such cases, one or more protein levels can be used to determine a patient's treatment response. For example, in some embodiments, modifying treatment includes administering a higher dose of a myosin inhibitor. As another example, modification may include administering a myosin inhibitor at a greater frequency. In some embodiments, modifying treatment includes administering a lower dose of a myosin inhibitor. In some cases, modification may include administering a myosin inhibitor at a lower frequency. In some embodiments, modifying treatment includes administering a different myosin inhibitor. And, in some embodiments, modifying treatment includes temporarily discontinuing the administration of a myosin inhibitor. Alternatively, current methods may include maintaining a patient's treatment based on their treatment response, for example, by maintaining the dose and / or frequency of administration of the myosin inhibitor, and by maintaining treatment with the same myosin inhibitor.

[0032] One or more protein levels can be analyzed to determine a patient's treatment response. Treatment response may be related to efficacy, safety, or pharmacokinetics. For example, a change relative to baseline exceeding a threshold may indicate an effective response to treatment with a myosin inhibitor. Alternatively, treatment response during treatment can be determined by analyzing changes in protein levels between a first protein level measured before the start of myosin inhibitor treatment and a second protein level measured after the start of myosin inhibitor treatment. In some embodiments, treatment response indicates whether the patient benefits from treatment. In some embodiments, treatment response indicates the level of myosin inhibitor exposure in the patient. In some embodiments, treatment response indicates a decrease in the patient's left ventricular outflow tract (LVOT) gradient, such as the resting LVOT gradient or the Valsalva LVOT gradient. In some embodiments, treatment response indicates a decrease in the patient's left atrial volume index (LAVI). In some embodiments, treatment response indicates an increase in the patient's peak oxygen consumption (pVO2). In some embodiments, treatment response indicates an improvement in the patient's New York Heart Association (NYHA) classification. In some embodiments, treatment response indicates the patient's Kansas City Cardiomyopathy Questionnaire (KCCQ) score or the patient's Hypertrophic Cardiomyopathy Symptom Questionnaire (HCMSQ) score. In some implementations, the patient is identified as unresponsive to treatment. In other implementations, the patient is identified as responsive to treatment.

[0033] In some implementations, patients are treated with myosin inhibitors without monitoring the LVOT gradient. In some implementations, patients are treated without monitoring the left ventricular ejection fraction (LVEF). In some implementations, patients are treated without monitoring blood plasma myosin inhibitor concentrations.

[0034] Various mathematical and statistical methods can be used as part of the current method to analyze the measured protein levels. In some embodiments, analyzing the one or more protein levels includes comparing the protein levels to a threshold. This threshold may be a percentage threshold or other statistical threshold, which can be calculated by comparing with a reference protein level (such as a baseline protein level), or by comparing first and second protein levels measured respectively before and after a treatment period. In some embodiments, the method further includes measuring or having measured one or more baseline protein levels in a biological sample from the patient before administering a myosin inhibitor to the patient. In some embodiments, analyzing one or more protein levels includes comparing the protein levels to a baseline protein level, or comparing a first protein level to a second protein level. In some embodiments, analyzing one or more protein levels includes determining the percentage change in the protein level relative to a baseline and comparing the percentage change relative to the baseline to a threshold percentage. In some embodiments, analyzing one or more protein levels includes determining whether the change in the protein level relative to the baseline is a statistically significant change. First and second protein levels (with a treatment period in between) can also be used to determine percentage changes and statistically significant changes. Various statistical methods can be used to determine whether a change is statistically significant. Analyzing protein levels may include determining fold changes, percentage changes, false discovery rates (FDR), and / or p-values. Various statistical methods are known in the art for these calculations.

[0035] In some embodiments, the method includes modifying treatment when myosin inhibitor treatment downregulates a protein. In such cases, after a period of myosin inhibitor treatment, the levels of one or more proteins decrease compared to baseline. Such downregulated proteins may include IZUM4, heparin cofactor II, mixed haptoglobin, and / or haptoglobin. Depending on the determination of downregulation, different adjustments may be made to the treatment to provide improved care for the patient. In some embodiments, the change in the levels of the one or more proteins relative to baseline is below a threshold, and the method further includes modifying treatment by administering a lower dose of the myosin inhibitor. In some embodiments, the change in the levels of the one or more proteins relative to baseline is below a threshold, and the method further includes modifying treatment by temporarily discontinuing the administration of the myosin inhibitor. In some embodiments, the change in the levels of the one or more proteins relative to baseline is below a threshold, and the method further includes modifying treatment by administering a different myosin inhibitor. In some embodiments, the change in the levels of the one or more proteins relative to baseline is above a threshold, and the method further includes modifying treatment by administering a higher dose of the myosin inhibitor. In some embodiments, the change in the levels of the one or more proteins relative to baseline is above a threshold, and the method further includes modifying treatment by administering a different myosin inhibitor. In various implementations, instead of using baseline protein levels for comparison, a first protein level measured during treatment is compared to a second protein level measured after the first protein level has been reached at the end of the treatment period. In some implementations, current methods may include maintaining patient treatment based on downregulation of one or more protein levels, for example, by maintaining the dose and / or frequency of administration of a myosin inhibitor, and by maintaining treatment with the same myosin inhibitor.

[0036] In some implementations, the treatment is modified to bring the levels of one or more downregulated proteins to one or more target levels (e.g., below a target threshold or within a target range). Furthermore, the treatment can be modified to maintain the levels of one or more downregulated proteins at one or more target levels, such as a stable state or normal levels. In such implementations, the levels of one or more proteins can be monitored and analyzed periodically during treatment.

[0037] In some embodiments, the method includes modifying treatment when a protein is upregulated by treatment with macvacitabine. In such cases, after a period of treatment with a myosin inhibitor, the levels of one or more proteins are elevated compared to baseline. Such upregulated proteins may include N-terminal pro-BNP, BNP, myosin light chain 1, laminin O2, BNP-32, MYOM2, SP-D, ACTN2, C1QR1, MYPC1, VAP-1, CK-MM, hemoglobin, SYWC, and / or VEGF-D. In response to the identified upregulation, different modifications to treatment may be made to provide improved treatment for the patient. In some embodiments, the change in the level of the one or more proteins relative to baseline is above a threshold, and the method further includes modifying treatment by administering a lower dose of the myosin inhibitor. In some embodiments, the change in the level of the one or more proteins relative to baseline is above a threshold, and the method further includes modifying treatment by temporarily discontinuing the administration of the myosin inhibitor. In some embodiments, the change in the level of the one or more proteins relative to baseline is above a threshold, and the method further includes modifying treatment by administering a different myosin inhibitor. In some embodiments, the change in the level of the one or more proteins relative to baseline is below a threshold, and the method further includes modifying treatment by administering a higher dose of a myosin inhibitor. In some embodiments, the change in the level of the one or more proteins relative to baseline is below a threshold, and the method further includes modifying treatment by administering a different myosin inhibitor. In various embodiments, instead of using baseline protein levels as a comparison, a first protein level measured during treatment is compared to a second protein level measured after the first protein level has been reached after the treatment period. In some embodiments, the current method may include maintaining patient treatment based on upregulation of one or more protein levels, for example, by maintaining the dose and / or frequency of administration of the myosin inhibitor, and by maintaining treatment with the same myosin inhibitor.

[0038] In some embodiments, the treatment is modified to bring the levels of one or more of the upregulated proteins to one or more target levels (e.g., above a target threshold or within a target range). Furthermore, the treatment is modified to maintain the levels of one or more of the upregulated proteins at one or more target levels, such as a stable state or normal levels. In such embodiments, the levels of one or more proteins can be monitored and analyzed periodically during treatment.

[0039] As described above, hemolytic proteins have been found to serve as biomarkers for myosin inhibitor therapy. Example 1 describes a study of hemolytic protein levels in HCM patients receiving macvacitabine. Following administration of macvacitabine, a decrease in cell-free plasma hemoglobin (Hb) levels and an increase in cell-free plasma haptoglobin (Hp) levels were observed, indicating that macvacitabine can reduce intravascular hemolysis in obstructive HCM patients. The results were consistent with improved hemodynamic effects (less outflow tract obstruction) and suggested that treatment with macvacitabine may reduce shear forces on red blood cells in the heart. Therefore, in some embodiments, the levels of one or more proteins include levels of one or more hemolytic proteins.

[0040] In some embodiments, the change in the level of the one or more hemolysin proteins relative to baseline is less than a threshold change, and the method further includes modifying the treatment by administering different doses of myosin inhibitors. In some embodiments, the change in the level of the one or more hemolysin proteins relative to baseline is less than a threshold change, and the method further includes modifying the treatment by temporarily discontinuing the administration of myosin inhibitors. In some embodiments, the change in the level of the one or more hemolysin proteins relative to baseline is less than a threshold change, and the method further includes modifying the treatment by administering different myosin inhibitors. In some embodiments, the change in the level of the one or more hemolysin proteins relative to baseline is greater than a threshold change, and the method further includes modifying the treatment by administering different doses of myosin inhibitors. In some embodiments, the change in the level of the one or more hemolysin proteins relative to baseline is greater than a threshold change, and the method further includes modifying the treatment by temporarily discontinuing the administration of myosin inhibitors. In some embodiments, the change in the level of the one or more hemolysin proteins relative to baseline is greater than a threshold change, and the method further includes modifying the treatment by administering different myosin inhibitors. In some embodiments, the threshold change is a percentage change or a multiple change. In various embodiments, instead of using baseline protein levels as a comparison, a first protein level measured during treatment is compared with a second protein level measured after the first protein level following the treatment period.

[0041] In some embodiments, the treatment is modified to bring the levels of one or more of the one or more hemolysin proteins to one or more target levels (e.g., above or below a target threshold or within a target range). Furthermore, the treatment is modified to maintain the levels of one or more of the one or more hemolysin proteins at one or more target levels, such as a stable state or normal levels. In such embodiments, the levels of one or more hemolysin proteins can be monitored and analyzed periodically during treatment.

[0042] In some embodiments, the level of the one or more hemolysin proteins is the level of one or more proteins selected from the group consisting of: hemoglobin, hemoglobin β subunit (also known as β-globin), HBG2 (hemoglobin γ-2 subunit), haptoglobin, and mixed haptoglobin, or any combination thereof. In some embodiments, the level of the one or more hemolysin proteins is the level of hemoglobin, haptoglobin, or any combination thereof. In some embodiments, the level of the one or more hemolysin proteins includes the level of hemoglobin. In some embodiments, the level of the one or more hemolysin proteins includes the level of haptoglobin.

[0043] As described above, sarcomere proteins and muscle / contractility-related proteins have been found to serve as biomarkers for myosin inhibitor therapy. Example 2 describes a study and data showing that macvaketide reduces sarcomere biomarkers and muscle / contractility-related biomarkers. Therefore, in some embodiments, the one or more protein levels include one or more sarcomere protein levels. Moreover, in some embodiments, the one or more protein levels include one or more muscle / contractility-related protein levels.

[0044] In some embodiments, the changes in the levels of the one or more sarcomere proteins and / or muscle / contraction-related proteins relative to baseline are less than a threshold change, and the method further includes modifying the treatment by administering different doses of myosin inhibitors. In some embodiments, the changes in the levels of the one or more sarcomere proteins and / or muscle / contraction-related proteins relative to baseline are less than a threshold change, and the method further includes modifying the treatment by temporarily discontinuing the administration of myosin inhibitors. In some embodiments, the changes in the levels of the one or more sarcomere proteins and / or muscle / contraction-related proteins relative to baseline are less than a threshold change, and the method further includes modifying the treatment by administering different myosin inhibitors. In some embodiments, the changes in the levels of the one or more sarcomere proteins and / or muscle / contraction-related proteins relative to baseline are greater than a threshold change, and the method further includes modifying the treatment by administering different doses of myosin inhibitors. In some embodiments, the changes in the levels of the one or more sarcomere proteins and / or muscle / contraction-related proteins relative to baseline are greater than a threshold change, and the method further includes modifying the treatment by temporarily discontinuing the administration of myosin inhibitors. In some embodiments, the change in the levels of one or more sarcomere proteins and / or muscle / contraction-related proteins relative to baseline exceeds a threshold change, and the method further includes modifying the treatment by administering different myosin inhibitors. In some embodiments, the threshold change is a percentage change or a fold change. In various embodiments, instead of using baseline protein levels as a comparison, a first protein level measured during treatment is compared with a second protein level measured after the first protein level has been reached during the treatment period.

[0045] In some embodiments, the treatment is modified to bring the levels of one or more sarcomere proteins and / or muscle / contraction-related proteins to one or more target levels (e.g., above or below a target threshold or within a target range). Furthermore, the treatment can be modified to maintain the levels of one or more sarcomere proteins and / or muscle / contraction-related proteins at one or more target levels, such as a stable state or normal levels. In such embodiments, the levels of one or more sarcomere proteins and / or one or more muscle / contraction-related proteins can be monitored and analyzed periodically during treatment.

[0046] In some embodiments, the levels of the one or more sarcomere proteins are levels of one or more proteins selected from the group consisting of: myosin light chain 1, α-actin-conjugated protein 2 (ACTN2), myosin-binding protein C1 (MYPC1), titin 2 (MYOM2), or any combination thereof. In some embodiments, the levels of the one or more sarcomere proteins are levels of myosin light chain 1, α-actin-conjugated protein 2 (ACTN2), or any combination thereof. In some embodiments, the levels of the one or more muscle / contraction-related proteins 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.

[0047] Other proteins (non-hemolytic, non-sarcomeretic) have also been identified as potential biomarkers for myosin inhibitor therapy. Table 1 provides a list of differentially expressed proteins observed during myosin inhibitor therapy, particularly in the Mavacare clinical trials. In some embodiments, the levels of one or more proteins comprise 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 levels of one or more proteins 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 levels of one or more proteins are cardiac biomarkers selected from the group consisting of BNP, BNP-32, and heparin cofactor II or any combination thereof. In some embodiments, the levels of one or more proteins 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 levels of the one or more proteins 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 levels of the one or more proteins include the level of IZUM4. The mechanistic role of IZUM4 in HCM requires further investigation.

[0048] In some embodiments, the changes in the levels of one or more cardiac biomarkers and / or cardiopulmonary biomarkers and / or IZUM4 protein relative to baseline are below a threshold change, and the method further includes modifying treatment by administering a different dose of a myosin inhibitor. In some embodiments, the changes in the levels of one or more cardiac biomarkers and / or cardiopulmonary biomarkers and / or IZUM4 protein relative to baseline are below a threshold change, and the method further includes modifying treatment by temporarily discontinuing the administration of a myosin inhibitor. In some embodiments, the changes in the levels of one or more cardiac biomarkers and / or cardiopulmonary biomarkers and / or IZUM4 protein relative to baseline are below a threshold change, and the method further includes modifying treatment by administering a different myosin inhibitor. In some embodiments, the changes in the levels of one or more cardiac biomarkers and / or cardiopulmonary biomarkers and / or IZUM4 protein relative to baseline are above a threshold change, and the method further includes modifying treatment by administering a higher dose of a myosin inhibitor. In some embodiments, the changes in the levels of one or more cardiac biomarkers and / or cardiopulmonary biomarkers and / or IZUM4 protein relative to baseline are above a threshold change, and the method further includes modifying treatment by temporarily discontinuing the administration of a myosin inhibitor. In some embodiments, the change in the levels of one or more cardiac biomarkers and / or cardiopulmonary biomarkers and / or IZUM4 protein relative to baseline is greater than a threshold change, and the method further includes modifying the treatment by administering different myosin inhibitors. In some embodiments, the threshold change is a percentage change or a fold change. In various embodiments, instead of using baseline protein levels as a comparison, a first protein level measured during treatment is compared with a second protein level measured after the first protein level has been reached at the end of the treatment period.

[0049] In some embodiments, the measured levels of one or more proteins include the levels of proteins 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, mixed haptoglobin, VAP-1, HPT, CK-MM, hemoglobin, SYWC, VEGF-D, HBG2, and β-globin, or any combination thereof.

[0050] In some embodiments, the levels of one or more proteins analyzed do not include levels of NT-proBNP or cardiac troponin. In some embodiments, the levels of one or more proteins analyzed include multiple protein levels, wherein said multiple protein levels include NT-proBNP and / or cardiac troponin.

[0051] In some embodiments, individual protein levels are measured and analyzed to determine a treatment response. In some embodiments, multiple protein levels are measured and analyzed to determine a treatment response. In some such embodiments, the multiple protein levels (e.g., changes in multiple protein levels relative to their respective baseline levels) are analyzed as a whole and / or a score is calculated, which can be compared to a threshold score. In some embodiments, algorithms may be used to analyze multiple protein levels and determine a treatment response.

[0052] The methods described herein can be used in the field of myosin inhibitor therapy, either as part of a treatment approach or as a standalone diagnostic method. Myosin inhibitor therapy is considered a potential treatment for cardiovascular disease. Therefore, the current methods can be used to treat patients with cardiovascular disease. In some implementations, the patient has obstructive hypertrophic cardiomyopathy (oHCM), non-obstructive hypertrophic cardiomyopathy (nHCM), heart failure with preserved ejection fraction (HFpEF), diastolic dysfunction, left ventricular hypertrophy (LVH), malignant LVH, ischemia, or angina, or any combination of the above conditions. In some implementations, the patient has diastolic dysfunction, left ventricular hypertrophy (LVH), angina, ischemia, hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM), or heart failure with preserved ejection fraction (HFpEF), mixed left ventricular (LV) systolic and diastolic dysfunction, or idiopathic right ventricular (RV) hypertrophy. In some implementations, the angina is microvascular angina. In some implementations, the LVH is malignant LVH. In some implementations, the HCM is an obstructive HCM. In some implementations, the HCM is a non-obstructive HCM.

[0053] In some implementations, the patient has hemodialysis (HCM). In some implementations, the patient has oHCM. In some implementations, the patient has nHCM. In some implementations, the patient has HFpEF. In some implementations, the patient has LVH. In some implementations, the patient has diastolic dysfunction.

[0054] The patient's protein levels are measured from a biological sample taken from the patient. In some embodiments, the method further includes 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.

[0055] The analysis of one or more protein levels described herein can also be used in some way to improve the safety of myosin inhibitor therapy. In some embodiments, the method further includes measuring or having measured the patient's LVEF when one or more protein levels are above a threshold. In some embodiments, the method further includes measuring or having measured the patient's LVEF when the change in one or more protein levels relative to baseline is greater than a threshold change. In some embodiments, the method further includes measuring or having measured the patient's LVEF when one or more protein levels are below a threshold. In some embodiments, the method further includes measuring or having measured the patient's LVEF when the change in one or more protein levels relative to baseline is less than a threshold change.

[0056] As described above, the determination of a treatment response can be used to modify the dosage of a myosin inhibitor. In some embodiments, the dosage is modified based on the analysis of one or more protein levels and / or the determination of a treatment response. In some embodiments, the method includes administering an initial dose of the myosin inhibitor within a first time period, followed by the measurement of one or more protein levels. In some embodiments, the method includes modifying a patient's myosin inhibitor treatment based on the patient's treatment response, wherein the modified treatment includes administering a second dose of the myosin inhibitor higher than the initial dose. In some embodiments, the method includes modifying a patient's myosin inhibitor treatment based on the patient's treatment response, wherein the modified treatment includes administering a second dose of the myosin inhibitor lower than the initial dose. In some embodiments, the method includes continuing to administer the initial dose of the myosin inhibitor because the treatment response is determined to be a desired treatment response. In some embodiments, the method includes temporarily discontinuing the administration of the myosin inhibitor based on the treatment response or based on the analysis of said one or more protein levels.

[0057] In some embodiments, the myosin inhibitor is macvacalecine, and the dose of macvacalecine is modified based on analysis of one or more protein levels and / or determination of treatment response. In some embodiments, the method includes administering an initial dose of macvacalecine at 5 mg daily for a first time period, followed by measurement of the one or more protein levels. In some embodiments, the method includes modifying a patient's myosin inhibitor treatment based on the patient's treatment response, wherein modifying the treatment includes administering a second dose of macvacalecine at 10 mg daily for a second time period after measurement of the one or more protein levels. In some embodiments, the method includes modifying a patient's myosin inhibitor treatment based on the patient's treatment response, wherein modifying the treatment includes administering a second dose of macvacalecine at 2.5 mg daily for a second time period after measurement of the one or more protein levels. In some embodiments, the method includes continuing administration of macvacalecine at 5 mg daily based on determination that the treatment response is the desired treatment response.

[0058] In some embodiments, the method includes administering an initial dose of 2.5 mg macvacitabine daily for a first time period, followed by measuring the levels of the one or more proteins. In some embodiments, the method includes modifying a patient's myosin inhibitor treatment based on the patient's treatment response, wherein modifying the treatment includes administering a second dose of 5 mg macvacitabine daily for a second time period after measuring the levels of the one or more proteins. In some embodiments, the method includes modifying a patient's myosin inhibitor treatment based on the patient's treatment response, wherein modifying the treatment includes administering a second dose of 1 mg macvacitabine daily for a second time period after measuring the levels of the one or more proteins, or temporarily discontinuing macvacitabine administration during the second time period. In some embodiments, the method includes continuing administration of 2.5 mg macvacitabine daily based on determining that the treatment response is the desired treatment response.

[0059] In embodiments that include increasing or decreasing the dose, the dose may be increased or decreased at various dose levels. In some embodiments, the dose levels include 2.5 mg, 5 mg, and 10 mg. In some embodiments, the dose level also includes 15 mg. In some embodiments, the dose level also includes 1 mg. In some embodiments, the dose level also includes 0 mg. In some such embodiments, the myosin inhibitor is macvaketide.

[0060] This article also discloses a method for determining a patient’s response to myosin inhibitor therapy, which includes analyzing the levels of one or more proteins in a biological sample from the patient.

[0061] This article also discloses a method for determining whether a patient should be treated with a myosin inhibitor and for treating a patient with a myosin inhibitor, the method comprising: determining, or having determined, whether a patient should be treated with a myosin inhibitor based on the levels of one or more proteins in a biological sample from the patient; and treating the patient by administering a myosin inhibitor to the patient.

[0062] This article also discloses a method for determining whether a patient should be treated with a myosin inhibitor and for treating a patient with a myosin inhibitor, the method comprising: measuring or having measured the level of one or more proteins in a biological sample from the patient; analyzing or having analyzed the level of said one or more proteins; determining or having determined whether the patient should be treated with a myosin inhibitor based on the level of said one or more proteins in the biological sample; and treating the patient by administering a myosin inhibitor to the patient.

[0063] In some embodiments, analyzing the levels of the one or more proteins includes comparing the protein levels to a threshold. In some embodiments, analyzing the levels of the one or more proteins results in the discovery that the levels of one or more proteins are above a threshold. In some embodiments, when the levels of one or more proteins are above a threshold, the method includes determining, or having determined, that the patient is suitable for treatment with a myosin inhibitor.

[0064] In some embodiments, analysis of the levels of one or more proteins results in the discovery that the levels of one or more proteins are below a threshold. In some embodiments, when the levels of one or more proteins are below a threshold, the method includes determining, or having determined, that the patient is suitable for treatment with a myosin inhibitor.

[0065] In some embodiments, the patient is an NYHA class II oHCM patient. In some embodiments, treatment for NYHA class II oHCM patients is determined based on the analysis of levels of one or more of the aforementioned proteins. In some embodiments, treatment for NYHA class II oHCM patients is determined based on the analysis of levels of one or more hemolytic proteins. In some embodiments, treatment for NYHA class II oHCM patients is determined based on the analysis of levels of one or more sarcomere proteins. In some embodiments, treatment for NYHA class II oHCM patients is determined based on the analysis of levels of one or more proteins selected from the proteins in Table 1.

[0066] In some embodiments, the method further includes monitoring the levels of the one or more proteins after treatment. In some embodiments, the method further includes modifying the treatment when myosin inhibitor treatment downregulates a protein. In such cases, after a period of myosin inhibitor treatment, the levels of one or more proteins decrease compared to baseline. Such downregulated proteins may include IZUM4, heparin cofactor II, mixed haptoglobin, and / or haptoglobin. Depending on the determination of downregulation, different adjustments can be made to the treatment to provide improved care for the patient. For example, the treatment can be modified to achieve levels of one or more target proteins in the patient, such as maintaining levels of one or more stable proteins.

[0067] In some implementations, the method includes modifying treatment when proteins are upregulated by macvaciate. In such cases, after a period of treatment with a myosin inhibitor, the levels of one or more proteins are elevated compared to baseline. Such upregulated proteins may include N-terminal pro-BNP, BNP, myosin light chain 1, laminin O2, BNP-32, MYOM2, SP-D, ACTN2, C1QR1, MYPC1, VAP-1, CK-MM, hemoglobin, SYWC, and / or VEGF-D. In response to the identified upregulation, various modifications can be made to the treatment to provide improved care for the patient. For example, treatment can be modified to achieve levels of one or more target proteins in the patient, such as maintaining levels of one or more stable proteins.

[0068] In some implementations, current methods may include maintaining the patient’s treatment, for example by maintaining the dose and / or frequency of administration of the myosin inhibitor, and maintaining treatment with the same myosin inhibitor.

[0069] This article also discloses a method for selecting myosin inhibitor therapy and treating a patient in need with myosin inhibitor therapy, comprising: measuring or having measured the level of one or more proteins in a biological sample from the patient; analyzing or having analyzed the level of said one or more proteins; selecting or having selected myosin inhibitor therapy for the patient based on said one or more protein levels in the biological sample; and treating the patient by administering a myosin inhibitor to the patient.

[0070] In some implementations, the method also includes monitoring the levels of the one or more proteins during treatment.

[0071] In some embodiments of this method, the measured levels of one or more proteins include levels of proteins selected from Table 1: Table 1

[0072] Table 2 lists the statistical data on differential protein expression in Table 1. Based on these statistics, proteins to be measured and analyzed according to this method can be selected. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 0.1. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 0.2. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 0.3. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 0.4. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 0.5. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 0.6. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 0.7. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 0.8. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 0.9. In some embodiments, the measured levels of one or more proteins include the levels of proteins selected from Table 2 whose logFC.max or –logFC.max is greater than 1.

[0073] Table 4A to Figure 4Table D lists statistical data on the correlation between proteins and clinical variables. Proteins that can be measured and analyzed according to current methods can be selected based on such statistical data. In some embodiments, the measured levels of one or more proteins include levels of proteins selected from Tables 4A to 4D with p-values ​​less than 0.01. In some embodiments, the measured levels of one or more proteins include levels of proteins selected from Tables 4A to 4D with p-values ​​less than 0.005. In some embodiments, the measured levels of one or more proteins include levels of proteins selected from Tables 4A to 4D with p-values ​​less than 0.001. In some embodiments, the measured levels of one or more proteins include levels of proteins selected from Tables 4A to 4D with p-values ​​less than 0.0005. In some embodiments, the measured levels of one or more proteins include levels of proteins selected from Tables 4A to 4D with p-values ​​less than 0.0001.

[0074] In some embodiments, the current method includes administering a myosin inhibitor. In some embodiments, the myosin inhibitor is a compound of formula (I): (I) Or its pharmaceutically acceptable salt, wherein R 1 It is C 1-8 Alkyl, C 3-8 cycloalkyl or phenyl, wherein R 1 It may be optionally replaced by one or two halogenated groups; R 2 It is a phenyl group that is optionally substituted with one or two halogroups; R 3 It is C 1-8 Alkyl or C 3-8 cycloalkyl, wherein each R 3 Optionally coated with a halogenated group, hydroxyl group, or C 1-2 Alkyl substitution; R 4 It is H; and X is H.

[0075] In some embodiments, the myosin of formula (I) or a pharmaceutically acceptable salt thereof is selected from group (I) consisting of: , , , , , , , , , , .

[0076] In some embodiments, the myosin inhibitor of formula (I) is magnaciate or a pharmaceutically acceptable salt thereof having the following structure: Mavakatai.

[0077] Mavaketene is also known as MYK-461. Its chemical name is ( S )-3-Isopropyl-6-((1-Phenylacetyl)amino)pyrimidine-2,4-(1 H ,3 H )-dione or 6-[[(1S)-1-phenylethyl]amino]-3-propane-2-yl-1H-pyrimidin-2,4-dione. In some embodiments, mavaketene is in the form of a free base.

[0078] In some implementations, the myosin inhibitor of formula (I) is MYK-581 having the following structure or a pharmaceutically acceptable salt thereof.

[0079] MYK-581.

[0080] The chemical name of MYK-581 is (S)-6-((1-(3-fluorophenyl)ethyl)amino)-3-isopropylpyrimidine-2,4(1H,3H)-dione.

[0081] Myosin inhibitors of formula (I), including compounds of group (I) such as macvaketene or MYK-581 or pharmaceutically acceptable salts thereof, are available according to the manufacturing method described in U.S. Patent No. 9,181,200, which is incorporated herein by reference in its entirety and for all purposes.

[0082] In some implementations, the myosin inhibitor is a compound of formula (II): (II) Or its pharmaceutically acceptable salt, wherein: n is 1 or 2; R 1 It is fluorine, chlorine, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy or C 2-4 Alkyne group, wherein at least one R 1 It is fluorine; and R 2a and R 2b At least one of them is fluorine, and R 2aand R 2b The other one is H.

[0083] In some embodiments, the myosin of formula (II) or a pharmaceutically acceptable salt thereof is selected from group (II) consisting of: , , , , , , , , , , , .

[0084] Myosin inhibitors of formula (II), including compounds of group (II) or pharmaceutically acceptable salts thereof, are available according to the method of manufacture described in International Application No. PCT / US2019 / 058297 filed on October 29, 2019, which is incorporated herein by reference in its entirety and for all purposes.

[0085] In some implementations, the myosin inhibitor is a compound of formula (III): (III) Or its pharmaceutically acceptable salt, wherein G1 is -CR 4 R 5 -or -O-; G2 is a key or -CR 6 R 7 -; G3 is -CR 8 -or -N-; R 1 R 3 R 4 R 5 R 6 R 7 and R 8 Each is independently H, C1-C6 alkyl, halogroup or hydroxyl group; R 2 It is H, C2-C6 alkyl, halogroup or hydroxyl; Z represents a bond, C1-C6 alkyl group, -O-, or -N(R). 9 )-、-R X O-、-OR Y or -R Z S-; R 9 It is H, C1-C6 alkyl or cycloalkyl; A is selected from the group consisting of substituted C2-alkynyl groups, unsubstituted C2-alkynyl groups, substituted phenyl groups, unsubstituted phenyl groups, and 5- or 6-membered heteroaryl groups containing at least one cyclic N atom, wherein the 5- or 6-membered heteroaryl group is unsubstituted or is surrounded by one or more R atoms. 10 Substituent substitution: Each R 10 All are independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 ynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or –C(O)OR a ; 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 group of B is unsubstituted or substituted by one or more R groups. 11 Substituent substitution; Each R 11 Each is independently selected from the group consisting of: substituted or unsubstituted heterocyclic alkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, unsubstituted C1-C6 alkyl, and alkyl groups containing one or more R groups. 12 Substituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, halogroup, -OR b —C(O)R c — C(O)OR d , oxo group and -NR e R f ; Each R 12 Each group is independently selected from the following groups: halogenated group, -OR b —C(O)R g —C(O)OR h and —C(O)NR i R j ; Each R a R b R c R d R e R f R g R h R i and R j All are independently H or C1-C6 alkyl; and R X R Y and R Z All are C1-C6 alkyl groups.

[0086] In some embodiments, the myosin of formula (III) or a pharmaceutically acceptable salt thereof is selected from group (III) consisting of: , , , , , , , , , .

[0087] In some embodiments, the myosin inhibitor is avketene. In some embodiments, the myosin inhibitor is avketene or a pharmaceutically acceptable salt thereof. The structure of avketene is shown below.

[0088] Avkatai Myosin inhibitors of formula (III), including compounds of group (III) or pharmaceutically acceptable salts thereof, are available according to the method of manufacture described in International Application No. WO 2019 / 144041, published on July 25, 2019, which is incorporated herein by reference in its entirety and for all purposes.

[0089] In some embodiments, the myosin inhibitors include compounds disclosed in PCT patent applications (publication numbers WO2020 / 005887, WO2020 / 005888, WO2020 / 047447), each of which is incorporated herein by reference in its entirety and for all purposes.

[0090] This document also discloses a myosin inhibitor that can be used in the methods described herein. In some embodiments, the myosin inhibitor is mavaketene.

[0091] Example SomaScan platform:The Somascan platform is used as illustrated in the following examples. The SomaScan® platform (SomaLogic Operating Co., Inc., Boulder, CO) is a highly multiplexed, sensitive, quantitative, and reproducible proteomics tool for discovering biomarkers for drug discovery, preclinical and clinical drug development, and clinical diagnostics. The SomaScan Assay v4.1 detects approximately 7,000 unique human protein analytes in small biological samples. This assay was developed for performance in human serum and plasma and has a large dynamic range, quantifying the relative levels of proteins in plasma with abundances spanning 10 log₂. The SomaScan platform is made possible by the development of a protein-capturing reagent called SOMAmer® (Slow Dissociation Rate Modified Aptamer). SOMAmer reagents consist of short, single-stranded DNA sequences incorporating hydrophobic modifications. The SomaScan platform measures native proteins in complex matrices by converting available binding sites on individual proteins into corresponding SOMAmer reagent concentrations, followed by quantification via hybridization with a microarray. The SOMAmer library was optimized using the exponentially enriched ligand phylogenetic evolution (SELEX) procedure. The assay involves binding SOMAmers to analytes, separating target-bound SOMAmers using standard chemical methods, and microarray quantification of SOMAmer DNA. Approximately 7000 SOMAmer reagents were included, comprising 6401 unique Uniprot IDs, of which 4241 (66%) unique Uniprot IDs were specific to SomaScan.

[0092] Differential expression analysis:Differential expression analysis was performed as shown in the following examples. The differential expression analysis was performed using the open-source software R. Linear models were implemented using the Limma package for linear model analysis of microarray and RNA-seq data. Limma fitted a linear model for each protein. For the EXPLORER and VALOR datasets, the models were specified to include the main effects of treatment and time, the interaction between treatment and time, and the covariates to be adjusted. For the Long Term Extended (LTE) datasets (LTE-EXPLORER and LTE-MAVERICK), the models were specified to include the effect of time on expression and covariate adjustments. For the EXPLORER dataset, the model covariates were adjusted, including ergometer type, baseline beta-blocker status, baseline NYHA classification, and Somalogic plate identifier. EXPLORER analysis was performed with and without adjustment for the top three substitute variables (SVs) identified by analysis of substitute variables (SVA). The SVs identified by SVA represent potential unknown confounding factors directly identified from the data. For the LTE dataset, the model plate identifier and the top three SVs were adjusted. For the VALOR dataset, the model was adjusted for surgery type, baseline NYHA grade, plate identifier, and the first three SVs. For EXPLORER and VALOR, we applied contrastives to assess the interaction between treatment and time to determine whether the effect of time on protein expression differed between the placebo and macvacitabine treatment groups. The fold change of the contrastives applied was estimated to estimate the difference in log2-scale expression over time between macvacitabine and placebo, taking into account the covariates of interest and being expression on a log2 scale. For the LTE study, contrastives were performed to assess expression changes relative to baseline at consecutive time points and quantified by the log2-scale fold change relative to baseline at each time point. Statistical tests were performed using adjusted t-tests, and multiple tests were adjusted using the Benjamini-Hochberg procedure to estimate the false discovery rate (FDR). In the adjusted t-tests, the standard error was compressed to the common value of the proteins using a Bayesian method. Differential expression was defined as an interaction contrast with an FDR < 0.05. When this threshold is applied to all proteins in a given dataset, approximately 5% of the hits are expected to have no differential expression, consistent with the null hypothesis. Log2 fold change quantifies the effect size of treatment and time interaction (EXPLORER and VALOR) or the effect of malvacalite over time (LTE).

[0093] Example 1 - Effect of malvacalite treatment on hemolytic biomarkers Abstract: In patients with obstructive hypertrophic cardiomyopathy (HCM), changes in blood flow caused by left ventricular outflow tract (LVOT) obstruction can lead to hemolysis and release erythrocyte proteins into the circulation. Increased cell-free plasma hemoglobin (Hb) and decreased haptoglobin (Hp) are biomarkers of intravascular hemolysis. Mavacarita (a cardiac myosin inhibitor) reduced LVOT obstruction in the phase 3 EXPLORER-HCM study; however, its effect on hemolysis biomarkers remains unclear.

[0094] Objective: To evaluate the effect of 30 weeks of malvacalide treatment versus placebo treatment on the levels of intravascular hemolytic biomarkers in patients with obstructive hemorrhage (HCM).

[0095] Methods: At baseline and week 30 (Mavacaitai, n=115; placebo, n=120), hemolytic biomarkers were measured using the proteomics platform (SomaScan v4.1) as part of an exploratory proteomics analysis in randomized EXPLORER-HCM (NCT03470545) patients. Example 8 of this paper describes the EXPLORER-HCM study.

[0096] Results: At week 30, compared with placebo, malvacalite treatment showed a reduction in cell-free plasma Hb (Log2 fold change = -1.57, FDR = 1.7 × 10⁻⁶). -4 Cell-free plasma Hp increased (Log2 fold change = 1.83, FDR = 1.2 × 10⁻⁶). -4 (Figure 1). Hb and Hp levels were negatively correlated at baseline and week 30. Generally, patients could be grouped into high or low hemolytic biomarker profiles (high Hb / low Hp or low Hb / high Hp, respectively) at baseline and week 30. Patients receiving macvafinemab and transitioning from a high to a low hemolytic biomarker profile at week 30 showed a greater tendency for LVOT reduction at rest and in the Valsalva state. No changes were detected in whole blood Hb or hematocrit in either group of subjects. Figure 1 shows cell-free plasma Hb and Hp levels at baseline and week 30. Figure 2 Biomarkers of cell-free plasma hemolysis at baseline and week 30 are shown. Figure 3 The correlation between hemoglobin and bead protein levels was shown. Figure 4 The resting and Valsalva LVOT gradients caused by changes in the hemolytic biomarker spectrum are shown.

[0097] Conclusion: Decreased and increased cell-free plasma hemoglobin (Hb) and hematocrit levels suggest that macvacitabine can reduce intravascular hemolysis in obstructive hemolytic disease (HCM). These results are consistent with improved hemodynamic effects (less outflow tract obstruction) and indicate that macvacitabine can reduce shear stress on erythrocytes in the heart. Whole blood hemoglobin and hematocrit levels did not change after treatment with macvacitabine or placebo. Example 2 - Effects of malvacalite on sarcomere biomarkers Methods: At baseline and week 30, sarcomere biomarkers were measured using the SomaScan proteomics platform as part of an exploratory proteomics analysis of randomized EXPLORER-HCM (NCT03470545) patients. Example 8 of this paper describes the EXPLORER-HCM study.

[0098] Results: Mavakaite reduced sarcomere biomarkers associated with muscle / contractility, including myosin light chain 1, ACTN2, MYOM2, and MYPC1. Figure 5 Box plots of Log2RFU changes from baseline to week 30 are shown for the placebo and MYK-461 (mavacaitai) treatment groups. RFU is a relative fluorescence unit indicating the presence of protein in the sample, expressed on a Log2 scale. Figure 5 Log FC and FDR values ​​for myosin light chain 1, ACTN2, MYOM2, and MYPC1 are also shown. FDR refers to the false discovery rate, and FC refers to the fold change. The differential expression of myosin light chain 1, ACTN2, MYOM2, and MYPC1 at week 30 compared with baseline was statistically significant.

[0099] Example 3 - Effects of malvacalite on cardiac / lung biomarkers Methods: Biomarkers were measured at baseline and at week 30 using the SomaScan proteomics platform as part of an exploratory proteomics analysis of randomized EXPLORER-HCM (NCT03470545) patients. Example 8 of this paper describes the EXPLORER-HCM study.

[0100] Results: Mavacait treatment led to differential expression of cardiac / lung biomarkers (including NTproBNP, SP-D and heparin cofactor II). Figure 6 Box plots of Log2RFU changes from baseline to week 30 are shown for the placebo and MYK-461 (mavacaitai) treatment groups. RFU is a relative fluorescence unit indicating the presence of protein in the sample, expressed on a Log2 scale. Figure 6Log FC and FDR values ​​for NTproBNP, SP-D, and heparin cofactor II are also shown. FDR refers to the false discovery rate, and FC refers to the fold change. The differential expression of NTproBNP, SP-D, and heparin cofactor II at week 30 compared with baseline was statistically significant.

[0101] Example 4 - Differential expression analysis reveals the peripheral tagging of the magnacate reaction in EXPLORER-HCM Methods: Biomarkers were measured at baseline and week 30 using the SomaScan proteomics platform as part of an exploratory proteomics analysis in randomized EXPLORER-HCM (NCT03470545) patients. The EXPLORER-HCM study is described in Example 8 of this paper. The model used to determine differential expression was as follows: Protein expression ~ time + treatment + time * treatment + covariate + (1|USUBJID). A significant interaction was defined as: macaverine at week 30 – macaverine at baseline ≠ placebo at week 30 – placebo at baseline. Results were screened for FDR < 0.05.

[0102] result: Results showed that macvaciate treatment reduced BNP protein levels. Macvaciate treatment also reduced ACTN2 protein levels. Z-lines are highly ordered multi-protein complexes located at the boundaries between sarcomeres, where actin and titin filaments from adjacent sarcomeres are anchored and cross-linked via α-actin. α-actin plays a central role in connecting proteins at Z-lines and also interacts with various other Z-line proteins. Macvaciate treatment also reduced MYPC1 and MYOM2 protein levels. Tipin-2 (M protein) is expressed in adult cardiac and fast-moving skeletal muscle and functions to stabilize the three-dimensional arrangement of proteins containing M-band structures within sarcomeres.

[0103] Example 5 - Differential expression analysis after malvacalite administration at week 16 in the VALOR-HCM study Methods: Biomarkers were measured at baseline and at weeks 16 and 32 using the SomaScan proteomics platform as part of an exploratory proteomics analysis of randomized VALOR-HCM (NCT04349072) patients. Example 7 of this article describes the VALOR-HCM study.

[0104] Results: No changes were detected in the malvacalite treatment group between weeks 32 and 16, which was consistent with the sustained changes observed. Furthermore, all proteins showed a consistent direction of change at both the 8-week and 16-week time points.

[0105] Most of the changes observed in week 8 were also observed in week 16 (80%), which is generally consistent with gradual rather than transient changes.

[0106] Table 3 shows the proteins that showed significant differential expression in both the VALOR (week 16) and EXPLORER (week 30) studies.

[0107] Table 3

[0108] Comparing differential expression in VALOR and EXPLORER, only 12% of proteins showed inconsistent direction of change. 88% of proteins exhibited consistent directionality (r = 0.96, p = 8.4e-43). Overall, the changes were highly consistent in both magnitude and direction.

[0109] Conclusion: The key results of EXPLORER were validated in VALOR, namely decreased sarcomere protein expression and improved hemolysis-related protein expression. These changes were generally non-transient and persisted after 16 weeks. The VALOR design facilitated the combination of placebo / treatment groups, thereby enhancing the power / sensitivity of differential expression detection (a novel pd biomarker).

[0110] Example 6 - Multi-study analysis of differentially expressed biomarkers Data sets from clinical trials were analyzed to identify differentially expressed proteins compared to baseline after treatment with magnatecide. Data sets from the following clinical trials were used: EXPLORER-HCM, VALOR-HCM, and MAVA-LTE (LTE-EXPLORER and LTE-MAVERICK).

[0111] All analyses used a significance threshold of FDR < 0.05. EXPLORER and VALOR results were based on comparisons of the effects of macavericine treatment versus placebo at 30 or 16 weeks, respectively. LTE-MAVERICK results were based on differential expression compared to baseline at weeks 24, 48, 72, 96, 120, and 144. LTE-EXPLORER results were based on differential expression compared to baseline at weeks 24, 48, 72, and 96. LTE protein was considered significant if it was identified as significant at two or more time points with the same fold change direction.

[0112] Important aptamers were identified, and exemplary species are listed in Table 2. In all studies showing significance, the direction of fold change was consistent.

[0113] Table 2 shows the aptamer / protein identification information (sequence number, protein, UniProt), the minimum FDR (fdr.min) among all studies that yielded significant results, the maximum log2 fold change (logFC.max) among all studies that yielded significant results, the number of studies that yielded significant results (n hits), and the number of studies that yielded significant results (hits).

[0114] Table 2

[0115] Example 7. VALOR test: An evaluation of malvacate in symptomatic infarction patients meeting the criteria for ventricular septal reduction therapy. Randomized, double-blind, placebo-controlled study of efficacy in adult patients with obstructive hypertrophic cardiomyopathy VALOR-HCM was a phase 3 study designed to evaluate the effect of macvaciate treatment on reducing the number of septal reduction therapy (SRT) procedures performed in patients with symptomatic obstructive hypertrophic cardiomyopathy (oHCM [also known as HOCM]) who meet the guidelines of the American College of Cardiology Foundation (ACCF) / American Heart Association (AHA) and / or European Society of Cardiology (ESC) (i.e., the guidelines). The VALOR-HCM study (NCT04349072) reached primary completion on February 7, 2022. The VALOR-HCM study was conducted according to the following protocol.

[0116] Overall Design: This is a phase 3 randomized, double-blind, placebo-controlled, multicenter study of men and women ≥18 years of age with oHCM who meet the SRT criteria in the ACCF / AHA and / or ESC guidelines (e.g., LVOT gradient ≥50 mmHg and NYHA class III-IV) and have been referred for invasive surgery. Upon completion of screening assessment, eligible participants will be randomly assigned in a 1:1 ratio to either the macvaciate treatment group or the placebo group. Randomization will be stratified according to the recommended SRT procedure type (myocardiectomy or alcohol septal ablation [ASA]) and NYHA functional classification.

[0117] The study will last for 138 weeks, including a 2-week screening period (week -2), a 128-week treatment period, and an 8-week post-treatment follow-up period (week 136).

[0118] There will be 3 dosing periods, as shown below: • Placebo-controlled dosing period (day 1 to week 16): Subjects will receive either malvacalite or placebo once daily for 16 weeks in a double-blind manner.

[0119] • Active control dosing period (weeks 16 to 32): All subjects will receive macvacitabine once daily for 16 weeks. Dosage will be blinded.

[0120] • Long-Term Extended Dosing Period (Week 32 to Week 128): All subjects will receive macvacitabine once daily for 96 weeks. Dosing will remain blinded unless the sponsor chooses to unblind after the primary analysis is completed.

[0121] Research procedures and treatments: • Study visits will be conducted at screening, on day 1, every 4 weeks thereafter until week 32, every 12 weeks thereafter until week 128 (end of treatment, EOT), and week 136 (end of study). Visits to the research center are mandatory on day 1 and at weeks 8, 16, 24, and 32, and every 12 weeks thereafter until week 128 and week 136. For selected research centers, study visits at weeks 4, 12, 20, and 28 may be conducted at the participant's home by a qualified home healthcare professional contracted by the sponsor. Participants who prematurely discontinue the study drug (except SRT) at any time will have a treatment cessation visit within 14 days of discontinuation, followed by visits every 24 weeks thereafter until week 128.

[0122] • On day 1, eligible participants will be randomly assigned in a double-blind manner to either the macvaciate group or the placebo group using an interactive response system (IXRS). Randomization will be stratified based on the recommended type of septal reduction therapy (SRT) procedure (myocardectomy or alcohol septal ablation (ASA)) and the New York Heart Association (NYHA) functional classification. Participants will begin macvaciate 5 mg or a matched placebo orally once daily for 16 weeks, followed by evaluation to adjust the dose.

[0123] • At weeks 16, 32, 80, and 128, participants' eligibility for SRT will be reassessed. Investigators will confirm whether participants are still receiving maximum medical therapy, determine their NYHA classification, and enter the information into the electronic case report form (eCRF). Every effort should be made to ensure that the NYHA classification assessment at weeks 16, 32, 80, and 128 is performed by the same investigator who assessed the NYHA classification at screening. Transthoracic echocardiography (TTE) will be performed independently and in investigator-blinded mode to assess LVOT gradients at rest, after stimulation, and after exercise. At weeks 16 and 32, TTE will be read at the core echocardiography laboratory, which will report the LVOT gradient classification results (<50 mmHg or ≥ 50 mmHg) to the research center. At weeks 80 and 128, LVOT <50 mmHg or ≥ 50 mmHg will be determined by echocardiography read at the research center. Researchers will remain blinded on LVOT gradient results until NYHA results are entered into the electronic case report form (eCRF). Researchers will review drug treatment, NYHA functional classification, and LVOT results to determine if the subject meets the ACCF / AHA and / or ESC SRT eligibility criteria (yes or no). Researchers will discuss this recommendation with the subject. If SRT is recommended, the subject can schedule it at the recommended HCM center after a recommended study drug washout period of ≥ 6 weeks, or the subject can decline the recommendation and continue using the study drug.

[0124] • Following the assessment at week 16, subjects in the macvacaimet treatment group who chose to continue treatment (i.e., decided not to receive SRT) will continue to receive macvacaimet once daily at the dose they received at week 16 for another 16 weeks; subjects in the placebo group who chose to continue treatment (i.e., decided not to receive SRT) will begin receiving macvacaimet 5 mg once daily for 16 weeks, followed by dose adjustment assessment (placebo to active group). During the active control dosing period, the dosage of macvacaimet will remain blinded.

[0125] • Following the assessment at week 32, all subjects who opted to continue treatment (i.e., decided against SRT) (in the macvacaletate group and the placebo-to-active group) will continue to receive macvacaletate once daily at the dose they received at week 32 for an additional 96 weeks through week 128 (end of treatment (EOT)). During LTE dosing, macvacaletate dosing will remain blinded unless the sponsor chooses to unblind after the primary analysis is completed. Subject eligibility for SRT will be reassessed at weeks 80 and 128.

[0126] • During the study, doses will be titrated based on LVEF and LVOT reads via TTE in the core echocardiography laboratory and according to dose titration guidelines. All dose adjustments will be performed blinded via IXRS throughout the study.

[0127] • During the placebo-controlled dosing period (day 1 through week 16), all subjects will be assessed for possible down titration at week 4 and possible up titration at weeks 8 and 12. While subjects in the placebo group will undergo dose titration assessments, they will continue to receive placebo.

[0128] • During the active control dosing period (weeks 16 to 32), subjects in the placebo-to-active group who started receiving macvacate from week 16 will be assessed for possible down titration at week 20 and possible up titration at weeks 24 and 28.

[0129] • During the LTE dosing period (weeks 32 to 128), if the LVOT gradient of Valsalva maneuver read by the research center is ≥ 30 mmHg and LVEF ≥ 50% at any scheduled visit after week 32, the mavacaitai dose may be titrated up. All dose increases during the LTE dosing period must be approved by the medical monitor before implementation. Subjects who increase their mavacaitai dose during the LTE period will have an unscheduled study visit 4 weeks after the dose increase, followed by a resumption of regular study visit schedules.

[0130] • For safety reasons, the dose may be titrated down at any time. Safety will be monitored throughout the study.

[0131] Table 6.0 provides the dosing titration guidelines for this study. Table 6.0 Dosage Titration Guidelines

[0132] Research timeline: Research timeline as follows Figure 7 As shown.

[0133] Research timeline description: a. During the placebo-controlled dosing period (day 1 to week 16), independent TTE assessments will be conducted by the echocardiography core laboratory, and potential down-tipping will be assessed at week 4, and potential up-tipping will be assessed at weeks 8 and 12, in accordance with dose titration guidelines. Down-tipping may be performed at any time for safety reasons.

[0134] b. In the placebo-to-active group, subjects who started receiving magnaciate from week 16 will be assessed for possible down-titration at week 20 and possible up-titration at weeks 24 and 28. Down-titration may be performed at any time for safety reasons.

[0135] c. During the Long-Term Extended Dosing (LTE) period (weeks 32 to 128), if the LVOT gradient of Valsalva action read at any planned visit after week 32 is ≥ 30 mmHg and LVEF ≥ 50%, the mavacaitai dose may be titrated up. All dose increases during LTE dosing must be approved by the MyoKardia medical monitor prior to implementation. Subjects who increase their mavacaitai dose during LTE will have an unscheduled study visit 4 weeks after the dose increase, followed by a resumption of regular study visit schedules. For safety reasons, the dose may be titrated down at any time.

[0136] d. At any time during the study period, subjects may discontinue the study drug and undergo SRT at an accredited HCM center after the recommended ≥ 6-week study drug washout period. Subjects who discontinue the study drug to receive SRT will be assessed for EOT within 14 days and will be followed up by telephone with the research center 8 weeks after treatment discontinuation (or before SRT, whichever is earlier) to assess for adverse events. Subjects will be followed up every 24 weeks from the date of SRT until week 128.

[0137] Drug research timeline: On day 1, participants will begin blinded dosing of either macaverite or a matched placebo (placebo-controlled period) once daily for 16 weeks. Following study evaluation at week 16, participants in the macaverite group will continue receiving macaverite, while participants in the placebo group will begin dosing macaverite once daily from week 16 to week 32 (active control period). During the active control period, the macaverite dosage will remain blinded. From week 16 onwards and for the remainder of the study, the placebo group will be referred to as the placebo-to-active control group. Following evaluation at week 32, all participants will continue dosing macaverite once daily until week 128 (LTE period). During the LTE period, the macaverite dosage will remain blinded unless the sponsor chooses to unblind after the primary analysis is completed.

[0138] Example 8. EXPLORER-HCM test: An evaluation of malvacalide versus placebo (1:1) in symptomatic obesity A phase 3, double-blind, randomized, placebo-controlled, multicenter study on the safety, tolerability, and efficacy in patients with oHCM. International and parallel group studies A phase 3, double-blind, randomized, placebo-controlled, multicenter, international, parallel-group study was conducted to evaluate the safety, tolerability, and efficacy of macvacitabine versus placebo (1:1) in patients with symptomatic hypertrophic cardiomyopathy (oHCM). The study, named EXPLORER-HCM (NCT03470545), was completed on May 6, 2020. A total of 251 participants were enrolled (123 received macvacitabine and 128 received placebo). Some participants consented to participate in a CMR substudy at a selected research center. Randomization was stratified by NYHA functional class (II or III), current beta-blocker therapy (yes or no), type of ergometer planned for use during the study (treadmill or exercise bike), and consent to participate in the CMR substudy (yes or no).

[0139] Research Design: The study consisted of three phases and was conducted according to the following design: 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 to evaluate eligibility. Key screening tests include electrocardiogram (ECG); transthoracic echocardiography (TTE) at rest, after Valsalva maneuver, and after exercise; and cardiopulmonary exercise test (CPET). The following screening assessments can be repeated as long as they are within the 35-day screening window: blood tests, ECG, and / or TTE. Repeat assessments are permitted if the central core laboratory requires a resubmission due to quality issues and for better evaluation of inclusion / exclusion criteria. Based on the investigator's judgment, rescreening of unqualified participants may be considered, taking into account one or more reasons for disqualification. One rescreening attempt is permitted, and all procedures must be repeated.

[0140] 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 schedule designed to ensure safe and effective dosing based on each participant's individual response parameters. Participants who meet all eligibility criteria at screening will first be randomized 1:1 via an interactive response system to receive either a starting dose of 5 mg magnaciate or a matched placebo once daily (QD). Subsequently, starting from Week 4, assessments including ECG, PK (glutamic-plasmic concentration), and TTE will be performed at each of the seven study visits and read by the core laboratory. At Weeks 8 and 14, the dose may be increased, decreased, or remained unchanged based on the assessment results at Weeks 6 and 12, respectively, and is primarily based on induced left ventricular outflow tract (LVOT) gradient measurements and is limited by the 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., from 5 mg QD to 10 mg QD), and at week 14, it may be increased to a maximum daily dose of 15 mg (i.e., from 10 mg QD to 15 mg QD). The dose increases are designed to be done in stages, and skipping doses is not permitted (e.g., from 5 mg to 15 mg).

[0141] At week 30 / EOT, participants will complete CPET and post-exercise TTE. For any participant who permanently discontinues treatment before week 30, an early termination (ET) visit, including CPET and post-exercise TTE, should be scheduled as soon as possible. Participants with ET will also be encouraged to complete all remaining study visits and assessments, including the week 30 visit.

[0142] 3) Post-treatment follow-up period (Week 30 / EOT to Week 38 / End of Study [EOS]): Participants will be contacted by phone at week 34 when the double-blind treatment ends at week 30, and will return to the research center for an EOS visit at week 38. During the EOS visit, the designated assessments will be repeated. This post-treatment follow-up period applies only to participants who received the study drug after week 22. The study design is in... Figure 8 As shown in the image.

[0143] Security monitoring: Safety monitoring is conducted in the following manner: To ensure safety throughout the double-blind treatment period, outpatient visits will be conducted every 2 to 4 weeks, beginning in week 4, to initially assess clinical tolerability and safety. Outpatient visits will include, but are not limited to, clinical assessments (symptoms, PRO assessment, adverse event [AE] / serious adverse event [SAE] assessment), ECG, PK sample, TTE, and laboratory assessments. TTE results performed by the research center sonographer at each planned visit following randomization should be blinded to the investigator and other research center personnel. If a left ventricular ejection fraction (LVEF) measured at the research center is ≤30%, an exception may occur, and the investigator will be immediately notified and the investigation drug will be permanently discontinued as described in the protocol.

[0144] Dose reduction or temporary discontinuation (if necessary) will be guided by assessments at weeks 4, 6, 8, 12, 18, 22, and 26, based on the predefined criteria detailed in the protocol. If the dose of macvaciate is reduced from the previous dose at any time during double-blind treatment, the subject will continue to the reduced dose until the end of the treatment period (week 30) unless further safety concerns or intolerance arise.

[0145] At selected research centers, participants will have the option to participate in a CMR sub-study. Approximately 80 participants will be recruited (about 40 per treatment group). In addition to the main study's procedural schedule, participants will undergo CMR examinations on Day 1 and at Week 30 (or up to 5 days prior to each visit).

[0146] Research on treatment: Participants received either 5 mg of macaverite immediate-release capsules or a matched placebo (QD) for the first 8 weeks of the dosing period, and glutathione PK samples were collected at weeks 4, 6, and 8. If glutathione PK was between 700 ng / mL and 1000 ng / mL at week 4, the dose was reduced to 2.5 mg at week 6.

[0147] Otherwise, adjust the dose at week 8 based on the assessment results at week 6 (increase, decrease, or remain unchanged), and at week 14 based on the assessment results at week 12. Permissible doses after dose adjustment at week 8 are 2.5 mg, 5 mg, 10 mg, or placebo. Permissible doses after dose adjustment at week 14 are 2.5 mg, 5 mg, 10 mg, 15 mg, or placebo.

[0148] To enhance safety, if 700 ng / mL < week 8 PK < 1000 ng / mL, an unplanned visit will be scheduled 2 weeks later (week 10) to reduce the dose. After week 14, assessments will continue every 4 weeks until week 30 / EOT for safety monitoring.

[0149] If the PK plasma concentration is ≥ 1000 ng / mL at any time, the investigational drug should be temporarily discontinued.

[0150] Each participant may participate in the study for a maximum of 43 weeks: up to 5 weeks for screening and 38 weeks (±7 days) for study implementation.

[0151] Example 9. MAVERICK-HCM test: A study on malvacalide for symptomatic non-obstructive hypertrophic cardiomyopathy. A randomized, double-blind, placebo-controlled, concentration-guided exploratory study in subjects with (nHCM) and preserved left ventricular ejection fraction. Inquiry This was a phase 2 trial designed to evaluate the safety and tolerability of a range of subjects exposed to symptomatic non-obstructive hemorrhage (HCM) over a 16-week treatment period. All study participants were diagnosed with non-obstructive HCM, with a left ventricular wall thickness ≥15 mm or ≥13 mm and a family history of HCM, LVEF ≥55%, NYHA class II or III, and a resting NT-proBNP level greater than 300 pg / mL. Baseline characteristics, such as age, weight, sex, pathogenic mutation status, background beta-blocker use, NYHA class, and exercise capacity, were substantially homogeneous between the active and placebo groups. The MAVERICK-HCM (NCT03442764) study was completed on January 7, 2020. The MAVERICK-HCM study was conducted according to the following protocol.

[0152] method: This double-blind study enrolled 59 patients with nHCM (left ventricular outflow tract gradient <30 mmHg; at rest or induced), NYHA class II or III, and LVEF ≥55%. Subjects were randomly assigned 1:1:1 to either of two target plasma drug concentration groups (Group 1: ~200 ng / mL and Group 2: ~500 ng / mL) or a placebo group for 16 weeks, followed by an 8-week washout period. Mavacate was started at a dose of 5 mg daily, with single-step dose titration based on plasma drug concentration at week 6. Predefined criteria, including LVEF (LVEF ≤ 45%), guided study drug discontinuation (if necessary). Cardiopulmonary exercise testing was performed at baseline and at week 16 to assess the impact on exercise capacity.

[0153] Research design and planning: This study aims to evaluate the safety, tolerability, preliminary efficacy, drug progression (PD), and pharmacokinetics (PK) of magvacalite at two target concentrations compared to placebo in patients with symptomatic nHCM. The study timeline is as follows: Figure 9 As shown.

[0154] Approximately 60 patients with symptomatic nHCM were randomized to receive magnaciate for 16 weeks, with titration to achieve one of two target drug concentrations (Group 1: ~200 ng / mL; Group 2: ~500 ng / mL) or placebo once daily (QD). Dose adjustments were based on pharmacokinetic parameters. Assessments included safety, standardized cardiopulmonary exercise testing (CPET) (measuring peak oxygen consumption), echocardiography (assessing left ventricular ejection fraction (LVEF) and diastolic function parameters), symptoms, quality of life, daily steps, and NT-proBNP at rest and after exercise. Furthermore, participants could consent to hypertrophic cardiomyopathy genotyping and pharmacogenetic sampling.

[0155] For subjects who consent and whose previous HCM genotyping results show the presence of a known pathogenic mutation associated with HCM, further genotyping evaluation is not required if data is available from clinical laboratory source documentation and the subject consents to share this information. Subjects who have not been tested and whose HCM genotyping results do not show a known pathogenic mutation associated with HCM may individually consent to blood collection for HCM genotyping prior to day 1 dosing. For subjects who consent to pharmacogenetic evaluation, blood samples will be collected prior to dosing to analyze genetic biomarkers of efficacy, safety, and PD or PK parameters, which will be determined by additional DNA sequencing or other genetic testing if clinically significant endpoints are to be used in future studies.

[0156] Cardiac troponin I levels were assessed in plasma and serum samples from subjects at baseline and at various time points during the trial (Abbott Architect Stat Troponin-I kit (Ref. 2K41)). Cardiac troponin T levels were assessed in plasma and serum samples from subjects at baseline and at various time points during the trial (Roche Elecsys Troponin T hs kit (reference number 08469873190), performed on a cobas e 801 analyzer). NT-proBNP levels in plasma samples were assessed on the cobas e 801 analyzer using the Roche Elecsys proBNPII kit (reference number 07027664190).

[0157] Research on treatment: The effect of macvacitabine dosage on improving diastolic function in patients with nHCM was assessed using a concentration-guided approach. Subjects were randomly assigned in a 1:1:1 ratio to three groups using an interactive response system: two active treatment groups and one matched placebo group.

[0158] This study used a 5 mg QD as the starting dose. All subjects in the active treatment group started with a 5 mg QD. Plasma concentrations of macvacarbide in blood samples collected at the week 4 visit were assessed. Based on the plasma concentrations collected at week 4, pharmacokinetic (PK) modeling was used to guide blinded dose adjustments at the week 6 visit. Subjects in the placebo group underwent the same assessment to maintain blinding. The study drug was provided in macvacarbide capsules in strengths of 2.5 mg, 5 mg, 10 mg, and 15 mg. Subjects were instructed to take the drug on an empty stomach at approximately the same time each day with 8 ounces of water.

[0159] The target plasma concentration of 200 ng / mL for macaverate was set for Group 1 subjects. To achieve the target concentration, if the concentration in a subject was >450 ng / mL at week 4, the dose was reduced to 2.5 mg QD; if the concentration was 110–450 ng / mL at week 4, the dose was maintained at 5 mg QD; and if the concentration was <110 ng / mL at week 4, the dose was increased to 10 mg QD.

[0160] The target plasma concentration of 500 ng / mL for macaverate was set for Group 2 subjects. To achieve the target concentration, if the concentration at week 4 was >450 ng / mL, the dose was reduced to 2.5 mg QD; if the concentration at week 4 was 300-450 ng / mL, the dose was maintained at 5 mg QD; if the concentration at week 4 was greater than or equal to 175 ng / mL but less than 300 ng / mL, the dose was increased to 10 mg QD; and if the concentration at week 4 was <175 ng / mL, the dose was increased to 15 mg QD.

[0161] Monitor subjects for adverse events (AEs), including high plasma concentrations, systolic dysfunction, QT interval prolongation, and decreased LVEF. Discontinue medication if any of the following thresholds are met: PK 1000 or higher, QTcF 500, or LVEF 45%. Specifically, high plasma concentrations are defined as plasma concentrations greater than or equal to 1000 ng / mL; QT interval prolongation is defined as QTcF greater than or equal to 500 ms; and LVEF shortening is defined as LVEF less than or equal to 45% (including serious adverse events (SAEs) with LVEF less than or equal to 30%).

[0162] Efficacy and pharmacodynamic assessments were also conducted. Resting transthoracic echocardiography measurements were performed at weeks 4, 8, 12, and 16. Ejection fraction (2-D) and LV fraction shortening, as well as other baseline echocardiographic measurements (including diastolic function measurements), were analyzed. Post-exercise stress echocardiography was performed after subjects underwent a standard symptom-limiting exercise test. Instantaneous peak LVOT gradient was assessed immediately after exercise. Cardiopulmonary exercise testing (CPET) was also performed. CPET was conducted on day 1 and at week 16 using a standardized treadmill or upright bicycle ergometer. Subjects were encouraged to exert maximum effort to achieve the expected heart rate. Oxygen intake (VO2), carbon dioxide production (VCO2), expiratory volume (VE), VE / VO2, ventilatory efficiency (VE / VCO2), respiratory exchange rate, circulatory power, and metabolic equivalents were assessed during this task.

[0163] Pharmacokinetic assessments were also performed during the study. Blood samples were collected at weeks 4, 8, 12, and 16 to assess macralat plasma concentrations. At week 16, PK blood samples were collected before and after administration.

[0164] Example 10 - Correlation between clinical variables and proteins identified through differential expression Statistical relevance was determined by: (A) changes in protein levels relative to baseline (for proteins identified by differential expression, e.g., as described in Examples 1-6) and (B) improvements in HCM-related clinical variables 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 to 4D (below) and Figures 10 to 13 The correlations of proteins identified in three or four of the four clinical studies analyzed are shown, where the absolute correlation coefficients are >0.2 and p<0.05.

[0165] Table 4A lists the protein correlation data from the EXPLORER-HCM study. Table 4B lists the protein correlation data from the VALOR-HCM study. Table 4C lists the protein correlation data from the LTE-EXPLORER study. Table 4D lists the correlation data from the LTE-MAVERICK study. Rho.pcbo refers to the correlation coefficient for placebo data. Pval.pcbo refers to the p-value for placebo data. Rho.mava refers to the correlation coefficient for patients receiving macaverite. Pval.mava refers to the p-value for patients receiving macaverite. N.pcbo refers to the number of patients receiving placebo in the study. N.mava refers to the number of patients receiving macaverite treatment in the study.

[0166] Figures 10 to 13 Selected scatter plots show changes in LA volume index or LVOT gradient (resting or Valsalva) versus changes in protein levels by a Log2-fold. Figure 10 Data based on EXPLORER-HCM. Figure 11 Data based on VALOR-HCM. Figure 12 Data based on LTE-EXPLORER. Figure 13 Based on LTE-MAVERICK data, the correlation is significant, as can be seen from the graph and the corresponding correlation coefficient (Rho) and p-value (P).

[0167] Table 4A

[0168] Table 4B

[0169] Table 4C

[0170] Table 4D

Claims

1. A method of treating a patient in need with a myosin inhibitor, comprising: The patient was given a myosin inhibitor; and The patient's treatment response has been determined or has been determined based on the levels of one or more proteins obtained from biological samples from the patient after administration.

2. A method of treating a patient in need with a myosin inhibitor, comprising: Administer myosin inhibitors to the patients; The levels of one or more proteins in a biological sample from the patient were measured after the patient was given the myosin inhibitor; The levels of one or more of the aforementioned proteins have been analyzed or analyzed; and The patient's treatment response has been determined or has been determined based on the levels of one or more of the aforementioned proteins.

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

4. The method of any of the preceding claims, 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 of the preceding claims, wherein the modification of the treatment comprises administering a lower dose of the myosin inhibitor or administering the myosin inhibitor at a less frequent frequency.

6. The method of any of the preceding claims, wherein the modification of the treatment comprises administering a different myosin inhibitor, or wherein the modification of the treatment comprises temporarily discontinuing the administration of the myosin inhibitor.

7. The method of claim 1 or 2, comprising maintaining treatment of the patient based on the patient's treatment response and / or based on analysis of the levels of the one or more proteins, optionally wherein maintaining treatment comprises administering the same myosin inhibitor at the same dose and at the same dosing frequency.

8. The method of any of the preceding claims, wherein the treatment response indicates whether the patient benefits from the treatment.

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

10. The method of any of the preceding claims, wherein the treatment response indicates a decrease in the patient's LVOT gradient or VA volume index.

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

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

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

14. The method of claim 13, wherein analyzing the one or more protein levels comprises determining a percentage change in the protein level relative to a baseline and comparing the percentage change relative to the baseline with a threshold percentage, or determining a percentage change from the first protein level to the second protein level and comparing the percentage change with a threshold percentage.

15. The method of claim 13, wherein analyzing the one or more protein levels comprises determining whether a change in the protein level relative to a baseline 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 of the preceding claims, wherein the change in the level of the one or more proteins relative to a baseline is below a threshold, the method further comprising modifying the treatment by administering a lower dose of the myosin inhibitor.

17. The method of any of the preceding claims, wherein the change in the level of the one or more proteins relative to a baseline is below a threshold, the method further comprising modifying treatment by temporarily discontinuing the administration of the myosin inhibitor.

18. The method of any of the preceding claims, wherein the change in the level of one or more proteins relative to a baseline is below a threshold, the method further comprising modifying the treatment by administering different myosin inhibitors.

19. The method of any of the preceding claims, wherein the change in the level of one or more proteins relative to a baseline is above a threshold, the method further comprising modifying the treatment by administering a higher dose of the myosin inhibitor.

20. The method of any of the preceding claims, wherein the change in the level of one or more proteins relative to a baseline is above a threshold, the method further comprising modifying the treatment by administering different myosin inhibitors.

21. The method of any of the preceding claims, wherein the change in the level of one or more proteins relative to a baseline is above a threshold, the method further comprising modifying the treatment by administering a lower dose of the myosin inhibitor.

22. The method of any of the preceding claims, wherein the change in the level of one or more proteins relative to a baseline is above a threshold, the method further comprising modifying treatment by temporarily discontinuing the administration of the myosin inhibitor.

23. The method of any of the preceding claims, wherein the change in the level of one or more proteins relative to a baseline is above a threshold, the method further comprising modifying the treatment by administering different myosin inhibitors.

24. The method of any of the preceding claims, wherein the change in the level of the one or more proteins relative to a baseline is below a threshold, the method further comprising modifying the treatment by administering a higher dose of the myosin inhibitor.

25. The method of any of the preceding claims, wherein the change in the level of one or more proteins relative to a baseline is below a threshold, the method further comprising modifying the treatment by administering different myosin inhibitors.

26. The method of any of the preceding claims, wherein the one or more protein levels include one or more hemolytic protein levels.

27. The method of claim 26, wherein the change in the level of one or more hemolytic proteins relative to baseline is less than a threshold change, the method further comprising modifying treatment by administering different doses of the myosin inhibitor.

28. The method of claim 26, wherein the change in the level of one or more hemolytic proteins relative to baseline is less than 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 in the level of one or more hemolytic proteins relative to baseline is less than a threshold change, the method further comprising modifying the treatment by administering different myosin inhibitors.

30. The method of claim 26, wherein the change in the level of one or more hemolytic proteins relative to baseline is greater than a threshold change, the method further comprising modifying the treatment by administering different doses of the myosin inhibitor.

31. The method of claim 26, wherein the change in the level of one or more hemolytic proteins relative to baseline is greater than 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 in the level of one or more hemolytic proteins relative to baseline is greater than a threshold change, the method further comprising modifying the treatment by administering different myosin inhibitors.

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

34. The method of any one of claims 26-33, wherein the level of the one or more hemolytic proteins is the level of one or more proteins selected from the group consisting of hemoglobin, hemoglobin β subunit, HBG2, haptoglobin and mixed haptoglobin or any combination thereof.

35. The method of claim 34, wherein the level of one or more hemolytic proteins is the level of hemoglobin, haptoglobin, or a combination thereof.

36. The method of any of the preceding claims, wherein the one or more protein levels comprise one or more sarcomere protein levels and / or one or more muscle / contraction-related protein levels.

37. The method of claim 36, wherein the change in the level of one or more sarcomere proteins and / or the level of one or more muscle / contraction-related proteins relative to baseline is less than a threshold change, the method further comprising modifying treatment by administering different doses of the myosin inhibitor.

38. The method of claim 36, wherein the change in the level of one or more sarcomere proteins and / or the level of one or more muscle / contraction-related proteins relative to baseline is less than a threshold change, the method further comprising modifying treatment by temporarily discontinuing the administration of the myosin inhibitor.

39. The method of claim 36, wherein the change in the level of one or more sarcomere proteins and / or the level of one or more muscle / contraction-related proteins relative to baseline is less than a threshold change, the method further comprising modifying the treatment by administering different myosin inhibitors.

40. The method of claim 36, wherein the change in the level of one or more sarcomere proteins and / or the level of one or more muscle / contraction-related proteins relative to baseline is greater than a threshold change, the method further comprising modifying the treatment by administering different doses of the myosin inhibitor.

41. The method of claim 36, wherein the change in the level of one or more sarcomere proteins and / or the level of one or more muscle / contraction-related proteins relative to baseline is greater than a threshold change, the method further comprising modifying treatment by temporarily discontinuing the administration of the myosin inhibitor.

42. The method of claim 36, wherein the change in the level of one or more sarcomere proteins and / or the level of one or more muscle / contraction-related proteins relative to baseline is above a threshold change, the method further comprising modifying the treatment by administering different myosin inhibitors.

43. The method of any one of claims 37-42, wherein the threshold change is a percentage change or a multiple change.

44. The method of any one of claims 36-43, wherein the level of the one or more sarcomere proteins is the level of one or more proteins selected from the group consisting of myosin light chain 1, α-actin 2 (ACTN2), myosin-binding protein C1 (MYPC1), and titin 2 (MYOM2) or any combination thereof.

45. The method of claim 44, wherein the level of one or more sarcomere proteins is the level of myosin light chain 1, α-actin 2, or any combination thereof.

46. ​​The method of any one of claims 36-43, wherein the level of one or more muscle / contraction-related proteins is the level 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 of the preceding claims, wherein the one or more protein levels include the levels of one or more cardiac biomarker proteins and / or one or more cardiopulmonary biomarker proteins.

48. The method of claim 47, wherein the change in the level of one or more cardiac biomarker proteins and / or the level of cardiopulmonary biomarker proteins relative to baseline is less than a threshold change, the method further comprising modifying treatment by administering different doses of the myosin inhibitor.

49. The method of claim 47, wherein the change in the level of one or more cardiac biomarker proteins and / or the level of cardiopulmonary biomarker proteins relative to baseline is less than 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 in the level of one or more cardiac biomarker proteins and / or the level of cardiopulmonary biomarker proteins relative to baseline is less than a threshold change, the method further comprising modifying the treatment by administering different myosin inhibitors.

51. The method of claim 47, wherein the change in the level of one or more cardiac biomarker proteins and / or the level of cardiopulmonary biomarker proteins relative to baseline is greater than a threshold change, the method further comprising modifying the treatment by administering a higher dose of the myosin inhibitor.

52. The method of claim 47, wherein the change in the level of one or more cardiac biomarker proteins and / or the level of cardiopulmonary biomarker proteins relative to baseline 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 in the level of one or more cardiac biomarker proteins and / or the level of cardiopulmonary biomarker proteins relative to baseline is above a threshold change, the method further comprising modifying the treatment by administering different myosin inhibitors.

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

55. The method of any one of claims 47-54, wherein the level of one or more cardiac biomarker proteins is the level 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 levels of one or more cardiopulmonary biomarker proteins 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 of the preceding claims, wherein the one or more protein levels include the level of IZUM4.

58. The method of any preceding claim, wherein the measured level of said one or more proteins comprises the level of said proteins selected from the group consisting of N-terminal pro-BNP, BNP, laminin-2, BNP-32, IZUM4, heparin cofactor II, SP-D, C1QR1, mixed haptoglobin, VAP-1, HPT, hemoglobin, SYWC, myosin light chain 1, MYOM2, ACTN2, MYPC1, CK-MM, VEGF-D, HBG2 and β-globin or any combination thereof.

59. The method of claim 58, wherein the measured levels of the one or more proteins comprise the levels of proteins selected from the group consisting of N-terminal pro-BNP, BNP, laminin-2, BNP-32, IZUM4, heparin cofactor II, SP-D, C1QR1, mixed haptoglobin, VAP-1, HPT, hemoglobin, SYWC, myosin light chain 1, MYOM2, ACTN2, MYPC1, CK-HM, and VEGF-D, or any combination thereof.

60. The method of any of the preceding claims, wherein the measured levels of the one or more proteins do not include levels of NT-proBNP or cardiac troponin.

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

62. The method of any of the preceding claims, further comprising modifying the treatment to bring one or more levels of the one or more proteins to one or more target levels.

63. The method of any of the preceding claims, further comprising modifying the treatment to maintain one or more levels of the one or more proteins at one or more target levels.

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

65. The method of any of the preceding claims, wherein the myosin inhibitor is mavaketene.

66. The method of any of the preceding claims, wherein the myosin inhibitor is Or its pharmaceutically acceptable salt.

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

69. The method of any of the preceding claims, wherein the myosin inhibitor is Avkaite.

70. The method of any preceding claim, wherein the myosin inhibitor is Or its pharmaceutically acceptable salt.

71. The method of any preceding claim, wherein the myosin inhibitor is 。 72. The method of any of the preceding claims, wherein the patient suffers from HCM.

73. The method of any of the preceding claims, wherein the patient suffers from oHCM.

74. The method of any of the preceding claims, wherein the patient suffers from nHCM.

75. The method of any of the preceding claims, wherein the patient suffers from HFpEF.

76. The method as claimed in any of the preceding claims, wherein the patient has LVH.

77. The method of any of the preceding claims, wherein the patient suffers from diastolic dysfunction.

78. The method of any of the preceding claims, further comprising a biological sample obtained or already obtained from the patient.

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

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

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

82. The method of any of the preceding claims, further comprising measuring or having measured the LVEF of the patient when one or more protein levels are above a threshold.

83. The method of any of the preceding claims, further comprising measuring or having measured the patient's LVEF when the change in one or more protein levels relative to baseline is greater than a threshold change.

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

85. The method of any of the preceding claims, further comprising measuring or having measured the patient's LVEF when the change in one or more protein levels relative to baseline is less than a threshold change.

86. The method of any of the preceding claims, comprising administering an initial dose of 5 mg of malvacate daily for a first time period prior to measuring the levels of the one or more proteins.

87. The method of claim 86, further comprising modifying the myosin inhibitor treatment of the patient based on the patient's treatment response, wherein modifying the treatment comprises administering a second dose of 10 mg of malvacalite daily for a second duration after measuring the levels of the one or more proteins.

88. The method of claim 86, further comprising modifying the myosin inhibitor treatment of the patient based on the patient's treatment response, wherein modifying the treatment comprises administering a second dose of 2.5 mg of malvacate daily for a second duration after measuring the levels of the one or more proteins.

89. The method of any of the preceding claims, comprising administering an initial dose of 2.5 mg of malvacate daily for a first time period prior to measuring the levels of the one or more proteins.

90. The method of claim 89, further comprising modifying the myosin inhibitor treatment of the patient based on the patient's treatment response, wherein modifying the treatment comprises administering a second dose of 5 mg malvacate daily for a second duration after measuring the levels of the one or more proteins.

91. The method of claim 89, comprising modifying the myosin inhibitor treatment of the patient based on the patient's treatment response, wherein modifying the treatment comprises administering a second dose of 1 mg macvastatin daily for a second time period after measuring the levels of the one or more proteins, or temporarily discontinuing the administration of macvastatin for the second time period.

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

93. A method for determining whether to treat a patient with a myosin inhibitor and for treating said patient with a myosin inhibitor, the method comprising: Whether to treat the patient with a myosin inhibitor is determined or has been determined based on the levels of one or more proteins in a biological sample from the patient. and The patient was treated by administering a myosin inhibitor.

94. A method for determining whether to treat a patient with a myosin inhibitor and for treating said patient with a myosin inhibitor, the method comprising: The levels of one or more proteins in a biological sample from the patient have been measured or have been measured. The levels of one or more of the aforementioned proteins have been analyzed or analyzed. Based on the levels of one or more proteins in the biological sample, it has been determined, or is already determined, whether the patient should be treated with a myosin inhibitor; and The patient was treated by administering a myosin inhibitor.

95. The method of any of the preceding claims, wherein analyzing the one or more protein levels comprises comparing the protein levels with a threshold.

96. The method of any of the preceding claims, wherein analyzing the levels of the one or more proteins results in the discovery that the levels of one or more proteins are above a threshold.

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

98. The method of any of the preceding claims, wherein analysis of the one or more protein levels results in the discovery that the levels of one or more proteins are below a threshold.

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

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

101. The method of claim 100, wherein treatment for the NYHA grade II oHCM patient is determined based on analysis of the levels of the one or more proteins.

102. The method of claim 100, wherein treatment of the NYHA grade II oHCM patient is determined based on analysis of the levels of one or more hemolytic proteins.

103. The method of claim 100, wherein treatment of the NYHA grade II oHCM patient is determined based on analysis of the levels of one or more sarcomere proteins and / or one or more muscle / contractile proteins.

104. The method of claim 100, wherein treatment for the NYHA grade II oHCM patient is determined based on analysis of one or more cardiac biomarker protein levels and / or one or more cardiopulmonary biomarker protein levels.

105. The method of claim 100, wherein the treatment of the NYHA grade II oHCM patient is determined based on the analysis of one or more protein levels in Table 1 and / or Table 4A, Table 4B, Table 4C and / or Table 4D.

106. A method of selecting and treating a patient in need with a myosin inhibitor therapy, comprising: The levels of one or more proteins in a biological sample from the patient have been measured or have been measured. The levels of one or more of the aforementioned proteins have been analyzed or analyzed. Based on the levels of one or more proteins in the biological sample, a myosin inhibitor therapy has been selected or is selected for the patient; and The patient was treated by administering a myosin inhibitor.

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

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