CN118382439A - Compounds for the treatment of anemia and other disorders associated with MDS - Google Patents

Abstract

Provided herein are uses of certain pyruvate kinase activators, or pharmaceutically acceptable salts or compositions thereof, for treating anemia and other disorders associated with MDS.

Description

Compounds for the treatment of anemia and other disorders associated with MDS

RELATED APPLICATIONS

The present application claims priority from U.S. provisional application number 63/280,069 filed on day 11, month 16 of 2021 and U.S. provisional application number 63/357,240 filed on day 6, month 30 of 2022, each of which is incorporated herein by reference in its entirety.

Background

Myelodysplastic syndrome (MDS) is a rare group of heterogeneous hematological malignancies characterized by hematopoietic dysfunction, progressive cytopenia, and an increased risk of progression to Acute Myelogenous Leukemia (AML). MDS occurs when hematopoietic cells in the bone marrow become abnormal (dysplasia) and fail to produce new blood cells. Many blood cells formed from bone marrow cells often die or are destroyed by the body, resulting in an individual not having sufficient normal blood cells. Although different cell types are affected by this phenomenon, the most common finding in MDS is erythrocyte shortage (anemia). However, hemolytic anemia is also found to occur in patients with MDS, although this is rare. See, for example, leukemia Research Reports,2016, volume 5: pages 23-26.

The average diagnostic age of a patient with MDS is 71 years, and long-term low hemoglobin levels can have a severe impact on it. This can lead to fatigue, deterioration of cardiopulmonary function, increased number of falls and severe decline in cognitive ability. Thus, the treatment of anemia is critical to overall health and quality of life. Currently, the infusion of concentrated erythrocytes (PRBC) is the standard method of treating anemia associated with MDS. However, a problem with patients with MDS-related anemia that rely on infusion is that these patients are at higher risk of developing iron overload and infusion reactions, and report reduced quality of life.

There is therefore a need for alternative methods of treating anemia associated with MDS.

Disclosure of Invention

It has now been found that certain pyruvate kinase activators may also be effective in treating anemia associated with MDS, particularly anemia associated with very low risk, and medium risk MDS. These include activators having the following structural formula:

And pharmaceutically acceptable salts thereof. In one aspect, the pyruvate kinase activators of the present disclosure have been found to increase hemoglobin levels, decrease reticulocyte count, and/or improve Red Blood Cell (RBC) function in an animal model of MDS. See, for example, fig. 1 and 2.

Accordingly, provided herein are methods of treating anemia and other disorders associated with MDS in a subject, comprising administering to the subject one or more of the pyruvate kinase activators of the present disclosure, or a pharmaceutically acceptable salt or composition thereof.

Also provided are methods of increasing hemoglobin levels in a subject having MDS comprising administering to the subject one or more of the pyruvate kinase activators of the present disclosure, or a pharmaceutically acceptable salt or composition thereof.

Also provided are methods of treating acquired PK deficiency (PKD) in a subject having MDS, comprising administering to the subject one or more of the pyruvate kinase activators of the present disclosure, or a pharmaceutically acceptable salt or composition thereof.

Also provided are methods of treating acquired PKD-related anemia in a subject with MDS, the methods comprising administering to the subject one or more of the pyruvate kinase activators of the present disclosure, or a pharmaceutically acceptable salt or composition thereof.

Also provided are methods of treating cytopenia in a subject with MDS, the methods comprising administering to the subject one or more of the pyruvate kinase activators of the present disclosure, or a pharmaceutically acceptable salt or composition thereof.

Further provided are methods of treating hemolytic anemia in a subject having MDS, the methods comprising administering one or more of the pyruvate kinase activators of the present disclosure, or a pharmaceutically acceptable salt or composition thereof, to the subject.

Further provided are methods of treating ineffective erythropoiesis in a subject with MDS, the methods comprising administering to the subject one or more of the pyruvate kinase activators of the present disclosure, or a pharmaceutically acceptable salt or composition thereof.

Further provided are methods of slowing disease progression in a subject with MDS. In some aspects, administration of one or more of the pyruvate kinase activators of the present disclosure, or a pharmaceutically acceptable salt or composition thereof, to a subject can reduce inflammation in the bone marrow of the subject, thereby slowing disease progression in the subject.

The invention also provides a method of treating a disease associated with mitochondrial dysfunction in a subject in need thereof, the method comprising administering to the subject one or more of the pyruvate kinase activators of the present disclosure or a pharmaceutically acceptable salt or composition thereof.

Drawings

Figures 1A-1D show the effect of hemoglobin levels after 6 weeks (figure 1A), 8 weeks (figure 1B), 12 weeks (figure 1C) and 18 weeks (figure 1D) of administration of mitepin in food in a mouse model of anemia associated with MDS.

Fig. 2A-2D show the results of hematological parameters of red blood cell count (RBC) (fig. 2A), reticulocyte percentage (fig. 2B), reticulocyte concentration (fig. 2C), and hematocrit percentage (fig. 2D) of a mouse model of MDS-related anemia after administration of mitsubpitaled.

Figure 3 shows a design of a multiple phase clinical trial for the treatment of anemia associated with MDS.

Fig. 4 shows an overview of a phase 2 study design using compound 1 in subjects with anemia arising from low risk myelodysplastic syndrome (LR-MDS).

Fig. 5 shows an overview of a phase 2 study design using compound 1 in subjects with anemia arising from low risk myelodysplastic syndrome (LR-MDS).

Fig. 6A-6E show flow cytometry data of Polg ^D257A mouse bone marrow aspirate after treatment with compound 1.

Fig. 7A to 7D show flow cytometry data of bone marrow aspirate of NHD13 mice after treatment with mitsubpitaled or compound 1.

Fig. 8A-8C show the results of hematological parameters of hemoglobin (fig. 8A), RBC (fig. 8B) and reticulocyte concentration (fig. 8C) of NHD13 mice after treatment with mitepiva or compound 1.

Detailed Description

In a first embodiment, provided herein is a method of treating myelodysplastic syndrome (MDS) -related anemia in a subject suffering from MDS, comprising administering to the subject a therapeutically effective amount of a compound having a structural formula selected from the group consisting of:

Or a pharmaceutically acceptable salt of any of the foregoing compounds.

In a second embodiment, provided herein is a method of treating myelodysplastic syndrome (MDS) -associated hemolytic anemia in a subject having MDS, the method comprising administering to the subject a therapeutically effective amount of a compound having a structural formula selected from the group consisting of:

Or a pharmaceutically acceptable salt of any of the foregoing compounds.

In a third embodiment, provided herein is a method of increasing hemoglobin levels in a subject having myelodysplastic syndrome (MDS), comprising administering to the subject a therapeutically effective amount of a compound having a structural formula selected from the group consisting of:

Or a pharmaceutically acceptable salt of any of the foregoing compounds.

In a fourth embodiment, provided herein is a method of treating acquired PK deficiency (PKD) in a subject having myelodysplastic syndrome (MDS), comprising administering to the subject a therapeutically effective amount of a compound having a structural formula selected from the group consisting of:

Or a pharmaceutically acceptable salt of any of the foregoing compounds.

In a fifth embodiment, provided herein is a method of treating acquired PK deficiency (PKD) -related anemia in a subject having myelodysplastic syndrome (MDS), comprising administering to the subject a therapeutically effective amount of a compound having a structural formula selected from the group consisting of:

Or a pharmaceutically acceptable salt of any of the foregoing compounds.

In a sixth embodiment, provided herein is a method of treating cytopenia in a subject having myelodysplastic syndrome (MDS), the method comprising administering to the subject a therapeutically effective amount of a compound having a structural formula selected from the group consisting of:

Or a pharmaceutically acceptable salt of any of the foregoing compounds.

In a seventh embodiment, provided herein is a method of treating a mitochondrial dysfunction-related disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having a structural formula selected from the group consisting of:

Or a pharmaceutically acceptable salt of any of the foregoing compounds.

In an eighth embodiment, provided herein is a method of treating ineffective erythropoiesis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having a structural formula selected from the group consisting of:

Or a pharmaceutically acceptable salt of any of the foregoing compounds.

As used herein, the term "hemolytic anemia" refers to a subtype of anemia in which a subject's low red blood cell count is due to destruction of red blood cells (rather than insufficient production of red blood cells). As used herein, unless otherwise indicated, the term "anemia" refers to low red blood cell count due to insufficient erythropoiesis (including ineffective erythropoiesis).

The terms "anemia associated with MDS", "anemia associated with MDS" and "anemia due to MDS" are synonymous and refer to anemia that develops or is obtained in a subject due to or suffering from MDS.

The terms "anemia associated with acquired PK deficiency (PKD)" and "PKD-associated anemia" are synonymous and refer to anemia that occurs in a subject due to acquired PKD having or suffering from MDS. In some embodiments, the acquired PK deficiency-related anemia of a subject with MDS is hemolytic anemia.

The terms "subject" and "patient" are synonymous and refer to mammals in need of treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). Unless stated to the contrary, a subject is a human in need of treatment. In some embodiments, the subject is an adult (e.g., age. Gtoreq.18 years). In other embodiments, the subject is a human child (e.g., < 18 years old). In other embodiments, the subject is a human female (adult or child). In other embodiments, the subject is a human male (adult or child).

The term "administering" refers to providing, implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a pharmaceutically acceptable salt or composition thereof, in or out of a subject.

The terms "treat (TREATMENT, TREAT and treating)" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or one or more symptoms of a disease described herein. In some embodiments, the treatment (i.e., therapeutic treatment) may be administered after one or more signs or symptoms of the disease have occurred or have been observed. In other embodiments, treatment may be performed in the absence of signs or symptoms of the disease. For example, treatment (i.e., prophylactic treatment) may be administered to a susceptible subject prior to the onset of symptoms (e.g., based on a history of symptoms and/or based on exposure to pathogens). Treatment may also continue after the symptoms subside, for example, to delay or prevent recurrence. In certain embodiments, treating comprises delaying the appearance of at least one symptom of the disorder over a period of time.

The term "effective amount" or "therapeutically effective amount" of a compound or pharmaceutically acceptable salt thereof described herein refers to an amount of a compound or pharmaceutically acceptable salt thereof sufficient to provide a therapeutic benefit in the treatment of a disorder described herein. In one aspect, an effective amount is about 0.01mg/kg body weight/day to about 100mg/kg body weight/day of the provided compound or pharmaceutically acceptable salt, such as, for example, about 0.1mg/kg body weight/day to about 100mg/kg body weight/day. In another aspect, an effective amount is about 0.01mg to about 2000mg of the provided compound, or a pharmaceutically acceptable salt thereof, which can be administered once or twice daily.

As used herein, recitation of ranges of values is intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value falling within the range is incorporated into the specification as if it were individually recited herein, unless explicitly recited otherwise. For example, the range of values from X to Y includes both X and Y and all values between X and Y.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

By "pharmaceutically acceptable salt" is meant a salt which, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art, for example, as described in detail in Berge et al, J.pharmaceutical Sciences,1977, volume 66, pages 1-19. Pharmaceutically acceptable salts of the compounds disclosed herein include salts derived from suitable inorganic and organic acids. Examples of pharmaceutically acceptable acid addition salts are salts of amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, benzenesulfonates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfate, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphate, gluconate, gentisates, hemisulfates, heptanates, caprates, hydroiodinates, 2-hydroxyethanesulfonates, lactonates, lactates, laurates, laurylsulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmates, pamonates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, tosylate, undecanoates, valerates, and the like.

In one aspect, as part of the ninth embodiment, a compound administered to a subject (e.g., as set forth in any one of the first to eighth embodiments) has the following structural formula:

Or a pharmaceutically acceptable salt thereof. This compound is also referred to herein as its chemical name 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one, or simply compound 1.

In one aspect, as part of the tenth embodiment, a compound administered to a subject (e.g., as set forth in any one of the first to eighth embodiments) has the following structural formula:

Or a pharmaceutically acceptable salt thereof. This compound is also referred to herein as its chemical name 2- ((1H-pyrazol-3-yl) methyl) -4-methyl-6- ((1-methyl-1H-pyrazol-3-yl) methyl) -4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one, or simply compound 2.

In one aspect, as part of the eleventh embodiment, a compound administered to a subject (e.g., as set forth in any one of the first to eighth embodiments) has the following structural formula:

Or a pharmaceutically acceptable salt thereof. This compound is also known as "mitsunobu", "AG-348", or its chemical name "N- (4- (4- (cyclopropylmethyl) piperazine-1-carbonyl) phenyl) quinoline-8-sulfonamide". Alternatively, as part of the eleventh embodiment, the mitsui or a pharmaceutically acceptable salt thereof (e.g., as described in any one of the first to eighth embodiments) is administered to the individual in crystalline form. In another alternative, as part of the eleventh embodiment, the mitsubvalvular or a pharmaceutically acceptable salt thereof (e.g., as described in any of the first to eighth embodiments) is administered to the subject in an amorphous form. In a further alternative embodiment, as part of the eleventh embodiment, the mitsubvalvular or a pharmaceutically acceptable salt thereof (e.g., as described in any of the first to eighth embodiments) is administered to the subject as a mixture of solid forms (e.g., a mixture of one or more crystalline forms or a mixture of one or more crystalline forms and an amorphous form).

In one aspect, as part of the twelfth embodiment, the compound administered to the subject (e.g., as described in any one of the first to eighth embodiments) is a pharmaceutically acceptable salt of mitsubvalvular. Alternatively, as part of the twelfth embodiment, the compound administered to the subject (e.g., as described in any one of the first to eighth embodiments) is the sulfate salt of mitsubpitaled. In another alternative, as part of the twelfth embodiment, the compound administered to the subject (e.g., as described in any one of the first to eighth embodiments) is a hemisulfate salt of mitsubvalvular. In another alternative, as part of the twelfth embodiment, the compound administered to the subject (e.g., as described in any one of the first to eighth embodiments) is hydrated hemisulfate of mitsubvalvular. In another alternative, as part of the twelfth embodiment, the compound administered to the subject (e.g., as described in any one of the first to eighth embodiments) is a hemisulfate sesquihydrate of mitsubpitava, also known as mitsubpitava sulfate or 1- (cyclopropylmethyl) -4- (4- (quinoline-8-sulfonylamino) benzoyl) piperazin-1-ium hemisulfate sesquihydrate, having formula a:

Formula A. In another alternative, as part of the twelfth embodiment, the compound administered to the subject (e.g., as described in any one of the first to eighth embodiments) is a sulfate trihydrate salt, also known (and equivalent to) as mirtazapiva trihydrate or 1- (cyclopropylmethyl) -4- (4- (quinoline-8-sulfonylamino) benzoyl) piperazin-1-ium sulfate trihydrate, having formula B:

The hemi-sulfate salt of mitsubpitava (i.e., mitsubpitava sulfate) can be crystalline, for example, as disclosed in form a of U.S. publication No. 20200277279. Form a is characterized by using one or more of the following x-ray powder diffraction patterns of Cu ka radiation at 2Θ angles (±0.2°): 9.9 °, 15.8 °, and 22.6 °;15.0 °, 17.1 °, 21.3 ° and 21.9 °;9.9 °, 15.0 °, 15.8 °, 17.1 °, 21.3 °, 21.9 °, and 22.6 °;9.9 °, 11.4 °, 15.0 °, 15.3 °, 15.8 °, 17.1 °, 17.7 °, 21.3 °, 21.9 °, 22.6 ° and 23.5 °; Or 4.9°、9.9°、11.0°、11.4°、11.7°、12.3°、12.8°、13.6°、13.9°、14.2°、15.0°、15.3°、15.8°、17.1°、17.4°、17.7°、18.8°、19.1°、19.8°、21.3°、21.9°、22.6°、23.0°、23.2°、23.5°、23.8°、24.1°、24.5°、25.3°、25.6°、26.1°、27.1°、28.1° and 29.8 °. In some embodiments, form a is characterized by x-ray powder diffraction peaks at 9.9 °, 15.8 °, and 22.6 ° at 2Θ angles (±0.2°). In certain embodiments, form a is characterized by an x-ray powder diffraction peak at 9.9 °, 15.8 °, and 22.6 ° at 2Θ angle (±0.2°) and at least one additional x-ray powder diffraction peak selected from 15.0 °, 17.1 °, 21.3 °, and 21.9 ° at 2Θ angle (±0.2°). in certain embodiments, form a is characterized by an x-ray powder diffraction peak at 9.9 °, 15.8 °, and 22.6 ° at 2Θ angle (±0.2°) and at least two additional x-ray powder diffraction peaks selected from 15.0 °, 17.1 °, 21.3 °, and 21.9 ° at 2Θ angle (±0.2°). In yet another alternative, form a is characterized by x-ray powder diffraction peaks at 9.9 °, 15.8 °, and 22.6 ° at 2Θ angles (±0.2°) and at least three additional x-ray powder diffraction peaks selected from 15.0 °, 17.1 °, 21.3 °, and 21.9 ° at 2Θ angles (±0.2°). in certain embodiments, form a is characterized by x-ray powder diffraction peaks at 9.9 °, 15.0 °, 15.8 °, 17.1 °, 21.3 °, 21.9 °, and 22.6 ° at 2Θ angles (±0.2°). In certain embodiments, form a is characterized by x-ray powder diffraction peaks at 9.9 °, 11.4 °, 15.0 °, 15.3 °, 15.8 °, 17.1 °, 17.7 °, 21.3 °, 21.9 °, 22.6 °, and 23.5 ° at 2Θ angles (±0.2°). In certain embodiments, form a is characterized by X-ray powder diffraction peaks at 4.9°、9.9°、11.0°、11.4°、11.7°、12.3°、12.8°、13.6°、13.9°、14.2°、15.0°、15.3°、15.8°、17.1°、17.4°、17.7°、18.8°、19.1°、19.8°、21.3°、21.9°、22.6°、23.0°、23.2°、23.5°、23.8°、24.1°、24.5°、25.3°、25.6°、26.1°、27.1°、28.1° and 29.8 ° at 2Θ angles (±0.2°). In yet another alternative, form a is characterized by a Differential Scanning Calorimetry (DSC) thermogram comprising endothermic peaks at about 159 ℃ ± 5 ℃ and 199 ℃ ± 5 ℃. In yet another alternative, crystalline form a is characterized by a thermogram of thermogravimetric analysis (TGA) comprising a weight loss of about 4.5% ± 0.5% at up to 180 ℃ ± 2 ℃. In some embodiments, the sesquisulfate salt of mitsuiv is 1- (cyclopropylmethyl) -4- (4- (quinoline-8-sulfonylamino) benzoyl) piperazin-1-ium sesquisulfate salt form a.

The term "amorphous" means a solid that exists in an amorphous state or form. Amorphous solids are molecularly unordered and therefore have no distinguishable lattices or unit cells, and no definable long range order. The solid state ordering of the solids can be determined by standard techniques known in the art, for example, by X-ray powder diffraction (XRPD) or Differential Scanning Calorimetry (DSC). Amorphous solids can also be distinguished from crystalline solids, for example, by using birefringence of a polarizing microscope.

The 2-theta values of the X-ray powder diffraction patterns of the crystalline forms described herein may vary somewhat from instrument to instrument and also depend on variations in sample preparation and batch-to-batch variations due to factors such as temperature variations, sample displacement, and the presence or absence of internal standards. Thus, unless otherwise defined, the XRPD patterns/distributions described herein should not be considered absolute and may vary by ±0.2 degrees. It is well known in the art that such variations will explain the factors described above without hampering the clear identification of the crystal form. Unless otherwise indicated, the 2-theta values provided herein are all obtained using Cu kα1 radiation.

For example, a compound or pharmaceutically acceptable salt of any one of the first to twelfth embodiments may be formulated as a pharmaceutical composition and administered. The pharmaceutical compositions may be prepared by methods known in the pharmacological arts. In one aspect, the pharmaceutical composition is administered orally in an orally acceptable dosage form including, but not limited to, granules or minitablets, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.

In one aspect, as part of the thirteenth embodiment, subjects treated by the compounds, pharmaceutically acceptable salts, or compositions of the present disclosure (including those subjects and compounds described in any of the first, second, and fourth to twelfth embodiments) experience a hemoglobin response.

As used herein, "hemoglobin response" refers to an increase in the baseline Hb level (i.e., hb concentration) from a subject, wherein the subject's hemoglobin response is measured during treatment with a compound disclosed herein or a pharmaceutically acceptable salt or composition thereof or over a period of time following administration of the compound disclosed herein or a pharmaceutically acceptable salt or composition thereof. Unless specified to the contrary, the term "during treatment" or "after administration" when used in connection with a compound, pharmaceutically acceptable salt, or composition of the present disclosure refers to ongoing treatment or administration (i.e., the subject will continue to receive treatment or administration of the compound, pharmaceutically acceptable salt, or composition of the present disclosure). The terms hemoglobin (Hb) level and hemoglobin concentration are used synonymously herein. As used herein, the term "baseline" refers to a level or concentration measured or determined prior to or during treatment with a compound disclosed herein or a pharmaceutically acceptable salt or composition thereof. For example, as used herein, the term "baseline hemoglobin level" refers to the hemoglobin (Hb) level of a subject measured or determined prior to or during treatment with a compound disclosed herein or a pharmaceutically acceptable salt or composition thereof. In one aspect, a "hemoglobin response" refers to an increase in the baseline Hb level (i.e., hb concentration) from a subject, wherein the hemoglobin response of the subject is measured over a period of time during treatment. In another aspect, a "hemoglobin response" refers to an increase in baseline Hb level (i.e., hb concentration) from a subject, wherein the subject's hemoglobin response is measured over a period of time after administration, e.g., 1 week of treatment, 2 weeks of treatment, 3 weeks of treatment, 4 weeks of treatment, 3 months of treatment, 6 months of treatment, or 1 year or more of treatment.

In one aspect, as part of the fourteenth embodiment, during treatment with a compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof, the hemoglobin level of the subject receiving treatment (including any one of the first to thirteenth embodiments) increases from baseline over a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks. In one aspect, as part of the fourteenth embodiment, during treatment with a compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof, the hemoglobin level of the subject receiving treatment (including any one of the first to thirteenth embodiments) increases from baseline over a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks. Alternatively, as part of the fourteenth embodiment, during treatment with a compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof, the hemoglobin level of the subject receiving treatment (including any one of the first to thirteenth embodiments) increases from baseline over a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, or at least 20 weeks. In another alternative, as part of the fourteenth embodiment, the hemoglobin level (including any one of the first to thirteenth embodiments) of the subject being treated is between 1 week and 20 weeks, between 1 week and 18 weeks, between 1 week and 16 weeks, between 4 weeks and 20 weeks, between 4 weeks and 18 weeks, between 4 weeks and 16 weeks, between 6 weeks and 20 weeks, between 6 weeks and 18 weeks, between 6 weeks and 16 weeks, between 8 weeks and 20 weeks, between 8 weeks and 18 weeks, between 8 weeks and 16 weeks, between 10 weeks and 20 weeks during treatment with a compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof, week 10 to week 18 or week 10 to week 16 increases from baseline. In another alternative, as part of the fourteenth embodiment, the hemoglobin level (including any one of the first to thirteenth embodiments) of the subject receiving treatment increases from baseline from week 8 to week 16 during treatment with the compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof. In another alternative, as part of the fourteenth embodiment, the hemoglobin level (including any one of the first to thirteenth embodiments) of the subject being treated is between 1 week and 20 weeks, between 1 week and 18 weeks, between 1 week and 16 weeks, between 4 weeks and 20 weeks, between 4 weeks and 18 weeks, between 4 weeks and 16 weeks, between 6 weeks and 20 weeks, between 6 weeks and 18 weeks, between 6 weeks and 16 weeks, between 8 weeks and 20 weeks, between 8 weeks and 18 weeks, between 8 weeks and 16 weeks, between 10 weeks and 20 weeks during treatment with a compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof, The continuous time points from week 10 to week 18 or from week 10 to week 16 are increased from baseline by ≡2, ≡3, ≡4, ≡5 or ≡6. In another alternative, as part of the fourteenth embodiment, the hemoglobin level (including any one of the first to thirteenth embodiments) of the subject receiving treatment increases from baseline during treatment with the compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof from ≡2 consecutive time points from week 8 to week 16. In another alternative, as part of the fourteenth embodiment, during treatment with a compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof, the subject receiving treatment increases in hemoglobin levels (including any one of the first to thirteenth embodiments) from baseline for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In another alternative, as part of the fourteenth embodiment, the hemoglobin level of the subject receiving treatment (including any one of the first to thirteenth embodiments) increases from baseline for ≡8 weeks or longer during treatment with the compound of the disclosure or a pharmaceutically acceptable salt or composition thereof.

In one aspect, as part of the fifteenth embodiment, during treatment with a compound of the present disclosure, or a pharmaceutically acceptable salt or composition thereof, the hemoglobin level (including any one of the first to fourteenth embodiments) of the subject receiving the treatment is improved by at least 1.0g/dL (e.g., an increase from baseline of ≡1.0 g/dL). Alternatively, as part of the fifteenth embodiment, during treatment with a compound of the present disclosure, or a pharmaceutically acceptable salt or composition thereof, the hemoglobin level (including any one of the first to fourteenth embodiments) of the subject receiving the treatment is improved by at least 1.5g/dL (e.g., an increase from baseline of ≡1.5 g/dL). In another alternative, as part of the fifteenth embodiment, the hemoglobin level (including any one of the first to fourteenth embodiments) of a subject receiving treatment is improved by at least 2.0g/dL (e.g., an increase from baseline of ≡2.0 g/dL) during treatment with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof. In another alternative, as part of the fifteenth embodiment, the hemoglobin level (including any one of the first to fourteenth embodiments) of the subject receiving treatment is improved by more than 2.0g/dL (e.g., increased by > 2.0g/dL from baseline) during treatment with a compound of the disclosure or a pharmaceutically acceptable salt or composition thereof.

In one aspect, as part of the sixteenth embodiment, a subject (including any of the first to fifteenth embodiments) treated by a compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof is categorized as an infusion-independent (NTD) subject prior to treatment.

As used herein, the terms non-infusion-dependent (NTD) and "no infusion" refer to those subjects who do not require periodic blood transfusion prior to treatment with a compound of the present disclosure or a pharmaceutically acceptable salt or composition thereof. In one aspect, NTD refers to those subjects having < 3 Red Blood Cell (RBC) units within 16 weeks prior to administration of the first dose of the disclosed compound or pharmaceutically acceptable salt or composition thereof and no infusion within 8 weeks prior to administration of the first dose of the disclosed compound or pharmaceutically acceptable salt or composition thereof.

As used herein, the term infusion-dependent (TD) refers to those subjects that require regular blood transfusion.

In one aspect, as part of the seventeenth embodiment, a subject (including any of the first to fifteenth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof becomes infusion-independent during treatment. In some aspects, as part of the seventeenth embodiment, a subject (including any of the first to fifteenth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof becomes infusion-independent for a period of time during treatment.

As used herein, the terms "infusion-independent" or "infusion-independent" are used interchangeably herein and refer to those subjects that have not (i.e., have not undergone) an infusion of red blood cells for a period of time (also referred to herein as "no infusion"). In some aspects, the term "infusion-independent" or "infusion-independent" refers to a subject that has not undergone an infusion of erythrocytes for 16 consecutive weeks. In some aspects, subjects not undergoing erythrocyte infusion for 16 consecutive weeks are referred to as no infusion. In other aspects, the term "infusion-independent" or "infusion-independent" refers to a subject that has not undergone an infusion of erythrocytes for ≡8 weeks in succession. In some aspects, subjects not undergoing erythrocyte infusion for 8 consecutive weeks or more are referred to as no infusion.

In one aspect, as part of the eighteenth embodiment, a subject (including any of the first to fifteenth or seventeenth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof is classified as having a high infusion burden (HTB). Alternatively, as part of the eighteenth embodiment, a subject (including any of the first to fifteenth or seventeenth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof is classified as having a high infusion burden (HTB) prior to treatment. In another alternative, as part of the eighteenth embodiment, a subject treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof, including any one of the first to fifteenth or seventeenth embodiments, is categorized as having a high infusion burden (HTB), characterized in that the subject receives at least 8 Red Blood Cell (RBC) units over a16 week period and has greater than or equal to 4 infusion events over an 8 week period. In another alternative, as part of the eighteenth embodiment, a subject treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof (including any of the first to fifteenth or seventeenth embodiments) is categorized as having a high infusion burden (HTB), characterized in that the subject receives at least 8 RBC units within a16 week period prior to treatment and has greater than or equal to 4 infusion events within an 8 week period. In another alternative, as part of the eighteenth embodiment, a subject treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof (including any of the first to fifteenth or seventeenth embodiments) is categorized as having a high infusion burden (HTB), characterized in that the subject receives at least 8 RBC units within a16 week period and at least 4 RBC units within an 8 week period over a16 week period prior to treatment.

In one aspect, as part of the nineteenth embodiment, a subject (including any of the first to fifteenth or seventeenth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof is classified as having a low infusion burden (LTB). Alternatively, as part of the nineteenth embodiment, a subject (including any of the first to fifteenth or seventeenth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof is categorized as having a low infusion burden (LTB) prior to treatment. In another alternative, as part of the nineteenth embodiment, a subject (including any of the first to fifteenth or seventeenth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof is categorized as having a low infusion burden (LTB), characterized in that the subject receives 3 to 7 RBC units for at least 2 infusion events over a 16 week period prior to treatment, up to 3 infusion events over an 8 week period. In another alternative, as part of the nineteenth embodiment, a subject (including any of the first to fifteenth or seventeenth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof is categorized as having a low infusion burden (LTB), characterized in that the subject receives 3 to 7 RBC units for at least 2 infusion events over a 16 week period, up to 3 infusion events over an 8 week period at a time point during the treatment.

In one aspect, as part of the twentieth embodiment, a subject (including any one of the first to twenty-first embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof becomes infusion-independent during treatment for 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or 10 weeks or more, consecutive. Alternatively, as part of the twentieth embodiment, a subject (including any of the first to twenty-first embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof becomes independent of infusion for ≡8 weeks continuously during treatment. In another alternative, as part of the twentieth embodiment, a subject (including any of the first to twenty-first embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof becomes infusion-independent for ≡8 weeks of treatment, wherein the subject is classified as having a low infusion burden (LTB) prior to treatment.

In one aspect, as part of the twenty-first embodiment, a subject (including any of the first to twentieth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof undergoes a change in total number of infused Red Blood Cell (RBC) units from baseline during treatment. In one aspect, as part of the twenty-first embodiment, a subject (including any of the first to twentieth embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences a reduction in total infused Red Blood Cell (RBC) units during treatment as compared to the subject's baseline total infused Red Blood Cell (RBC) units.

In one aspect, as part of the twenty-second embodiment, a subject (including any of the first to twenty-first embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences a change in total infused Red Blood Cell (RBC) units from baseline, characterized by a decrease in total infused RBC units of ∈10% ∈20% ∈30% ∈40% ∈50% ∈60% ∈70% or ∈80% during treatment. Alternatively, as part of the twenty-second embodiment, a subject (including any of the first to twenty-first embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences a change in total infused Red Blood Cell (RBC) units from baseline, characterized by a decrease in total infused RBC units of ∈10% >, > 20% >, > 30% >, > 40% >, > 50% >, > 60% >, > 70% or ∈80% for ∈1 week, ∈2 weeks, ∈3 weeks, ∈4 weeks, ∈5 weeks, +.6 weeks, +.7 weeks, +.8 weeks, +.9 weeks or +.. In another alternative, as part of the twenty-second embodiment, the total infused Red Blood Cell (RBC) units of a subject (including any of the first through twenty-first embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof are changed from baseline, characterized by a decrease of ≡50% in total infused RBC units during treatment. In another alternative, as part of the twenty-second embodiment, a subject (including any of the first to twenty-first embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences a change in total infused Red Blood Cell (RBC) units from baseline, characterized by a decrease of ∈50% in total infused RBC units for ∈1 week ∈2 weeks ∈3 weeks ∈4 weeks ∈5 weeks ∈6 weeks ∈7 weeks ∈8 weeks ∈9 weeks or ∈10 weeks during treatment. In another alternative, as part of the twenty-second embodiment, the total infused Red Blood Cell (RBC) units of a subject (including any of the first to twenty-first embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof undergo a change from baseline, characterized in that the total infused RBC units decrease by ≡50% over a period of ≡8 weeks consecutive during treatment. In another alternative, as part of the twenty-second embodiment, a subject (including any of the first to twenty-first embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences a change in total infused Red Blood Cell (RBC) units from baseline, characterized by a decrease in total infused RBC units of greater than or equal to 50% over a period of greater than or equal to 8 consecutive weeks during treatment, wherein the subject is classified as having a high infusion burden (HTB) prior to treatment.

In one aspect, as part of the twenty-third embodiment, a subject (including any of the first to twenty-second embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences a decrease in 2, 3-phosphoglycerate (2, 3-DPG) concentration during treatment as compared to the subject's baseline 2,3-DPG concentration.

In one aspect, as part of the twenty-fourth embodiment, a subject (including any of the first to twenty-third embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences an increase in Adenosine Triphosphate (ATP) concentration (as compared to the subject's baseline ATP concentration) during treatment. Alternatively, as part of the twenty-fourth embodiment, a subject (including any of the first to twenty-third embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences an increase in Adenosine Triphosphate (ATP) concentration of more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, and more than 75% during treatment (as compared to the subject's baseline ATP concentration). In another alternative, as part of the twenty-fourth embodiment, a subject (including any of the first to twenty-third embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences an increase in Adenosine Triphosphate (ATP) concentration of 68%, 71% or 74% during treatment (as compared to the subject's baseline ATP concentration). In another alternative, as part of the twenty-fourth embodiment, a subject (including any of the first to twenty-third embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences an increase in Adenosine Triphosphate (ATP) concentration of greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, and greater than 75% after daily dosing for 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, or 65 days (as compared to the subject's baseline ATP concentration). In another alternative, as part of the twenty-fourth embodiment, a subject (including any of the first to twenty-third embodiments) treated with the disclosed compounds or pharmaceutically acceptable salts or compositions thereof experiences an increase in Adenosine Triphosphate (ATP) concentration of 68% after 56 days of daily administration, 71% after 56 days of daily administration, or 74% after 56 days of daily administration (as compared to the subject's baseline ATP concentration).

In one aspect, as part of the twenty-fifth embodiment, the MDS described herein (including any one of the first through sixth and tenth through twenty-fourth embodiments) is low risk MDS, as characterized by the MDS's revised version international prognosis scoring system (IPSS-R). See, e.g., greenberg PL, tuechlerH, schanz J et al, revised International Prognostic Scoring System formyelodysplastic syndromes, blood,2012, volume 120: pages 2454-2465. Low risk MDS includes, for example, IPSS-R scores greater than 1.5 to 3. Alternatively, as part of the twenty-fifth embodiment, the MDS described herein (including any one of the first through sixth and tenth through twenty-fourth embodiments) is an extremely low risk MDS, as characterized by the MDS's revised version international prognosis scoring system (IPSS-R). Very low risk MDS includes, for example, an IPSS-R score of less than or equal to 1.5. In another alternative, as part of the twenty-fifth embodiment, the MDS described herein (including any one of the first through sixth and tenth through twenty-fourth embodiments) is medium risk MDS, as characterized by the MDS's revised version international prognosis scoring system (IPSS-R). Medium risk MDS includes, for example, IPSS-R scores greater than 3 to 4.5. In yet another alternative, the term "low risk MDS" encompasses extremely low risk MDS and low risk MDS as described above as part of the twenty-fifth embodiment, as described herein (including any one of the first to sixth and tenth to twenty-fourth embodiments).

In one aspect, as part of the twenty-sixth embodiment, the subject described herein (including any one of the first to twenty-fifth embodiments) is a male. Alternatively, as part of the twenty-sixth embodiment, the subject described herein (including any one of the first to twenty-fifth embodiments) is a female. Alternatively, as part of the twenty-sixth embodiment, the subject described herein (including any of the first to twenty-fifth embodiments) is an adult male. Alternatively, as part of the twenty-sixth embodiment, the subject described herein (including any of the first to twenty-fifth embodiments) is an adult female. In another alternative, the subject described herein (including any of the first to twenty-fifth embodiments) is a boy or girl as part of the twenty-sixth embodiment. Alternatively, as part of the twenty-sixth embodiment, the subject described herein (including any of the first to twenty-fifth embodiments) is an adult male (i.e., age ≡18 years). In yet another alternative, the subject described herein (including any of the first to twenty-fifth embodiments) is a child (i.e., age < 18 years) as part of the twenty-sixth embodiment.

In one aspect, as part of the twenty-seventh embodiment, a therapeutically effective amount of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one, or a pharmaceutically acceptable salt thereof, is administered to a subject described herein, including any of the first to twenty-sixth embodiments. Alternatively, as part of the twenty-seventh embodiment, a composition comprising a therapeutically effective amount of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier is administered to a subject described herein, including any of the first to twenty-sixth embodiments.

In one aspect, as part of the twenty-eighth embodiment, a therapeutically effective amount of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one disclosed herein (including any of the first to twenty-seventh embodiments) is 2mg daily, 3mg daily, or 5mg daily. Alternatively, as part of the twenty-eighth embodiment, a therapeutically effective amount of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one disclosed herein (including any of the first to twenty-seventh embodiments) is 2mg administered once daily (QD), 3mg administered QD, or 5mg administered QD. In another alternative, as part of the twenty-eighth embodiment, a therapeutically effective amount (including any of the first through twenty-seventh embodiments) of a pharmaceutically acceptable salt of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one disclosed herein is an amount equivalent to 2mg daily, 3mg daily, or 5mg daily of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one as the free base. In another alternative, as part of the twenty-eighth embodiment, a therapeutically effective amount of a pharmaceutically acceptable salt of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one disclosed herein (including any of the first to twenty-seventh embodiments) is an amount equivalent to 2mg administered once daily (QD), 3mg administered QD, or 5mg administered as the free base 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one. In another alternative, as part of the twenty-eighth embodiment, the therapeutically effective amount of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one disclosed herein (including any of the first to twenty-seventh embodiments) ranges from 0.5mg to 10mg. In another alternative, as part of the twenty-eighth embodiment, A therapeutically effective amount of a pharmaceutically acceptable salt of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one disclosed herein (including any of the first to twenty-seventh embodiments) is equivalent to 0.5mg to 10mg of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one as the free base. in another alternative, as part of the twenty-eighth embodiment, the therapeutically effective amount of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one disclosed herein (including any of the first to twenty-seventh embodiments) ranges from 0.5mg to 10mg twice daily (BID). In another alternative, as part of the twenty-eighth embodiment, a therapeutically effective amount of a pharmaceutically acceptable salt of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one disclosed herein (including any of the first to twenty-seventh embodiments) is equivalent to from 0.5mg to 10mg of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5', 4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one. In another alternative, as part of the twenty-eighth embodiment, a therapeutically effective amount of compound 1 disclosed herein (including any one of the first to twenty-seventh embodiments) ranges from 0.25mg to 15mg per day. In another alternative, as part of the twenty-eighth embodiment, a therapeutically effective amount of a pharmaceutically acceptable salt of compound 1 disclosed herein (including any of the first to twenty-seventh embodiments) is an amount equivalent to an amount ranging from 0.25mg to 15mg per day as the free base. In another alternative, as part of the twenty-eighth embodiment, a therapeutically effective amount of compound 1 disclosed herein (including any of the first to twenty-seventh embodiments) ranges from 0.25mg to 2mg QD or BID or 1.5mg to 5.5mg QD or BID or 4mg to 6mg QD or BID. In another alternative, as part of the twenty-eighth embodiment, a therapeutically effective amount of a pharmaceutically acceptable salt of compound 1 disclosed herein (including any of the first to twenty-seventh embodiments) is an amount equivalent to an amount ranging from QD or BID0.25mg to 2mg or QD or BID 1.5mg to 5.5mg or QD or BID 4mg to 6mg as the free base. In another alternative, as part of the twenty-eighth embodiment, a therapeutically effective amount of compound 1 disclosed herein (including any one of the first to twenty-seventh embodiments) is QD or BID 1mg or 5mg. In another alternative, as part of the twenty-eighth embodiment, a therapeutically effective amount of a pharmaceutically acceptable salt of compound 1 disclosed herein (including any of the first to twenty-seventh embodiments) is an amount equivalent to QD or BID 1mg or 5mg as the free base.

In one aspect, as part of the twenty-ninth embodiment, a subject described herein (including any one of the first to twenty-eighth embodiments) is treated (i.e., administered a compound, pharmaceutically acceptable salt, or composition described herein) for a period of at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 24 weeks, at least 30 weeks, or at least 6 weeks. Alternatively, as part of the twenty-ninth embodiment, the subject described herein (including any of the first to twenty-eighth embodiments) is treated (i.e., administered a compound, pharmaceutically acceptable salt, or composition described herein) for a period of up to 12 weeks, up to 14 weeks, up to 16 weeks, up to 24 weeks, up to 30 weeks, up to 36 weeks, up to 50 weeks, up to 100 weeks, or up to 160 weeks. In another alternative, as part of the twenty-ninth embodiment, the subject described herein (including any of the first to twenty-eighth embodiments) is treated (i.e., administered a compound, pharmaceutically acceptable salt, or composition described herein) for a period of 16 weeks, 24 weeks, or 156 weeks. In another alternative, as part of the twenty-ninth embodiment, the subject described herein (including any one of the first to twenty-eighth embodiments) is treated (i.e., administered a compound, pharmaceutically acceptable salt, or composition described herein) indefinitely or for the remainder of the subject's life.

In one aspect, there is provided the use of one or more of the disclosed pyruvate kinase activators, or a pharmaceutically acceptable salt or composition thereof, in the manufacture of a medicament for treating a disclosed disorder (e.g., as described in any of the embodiments disclosed above). In another aspect, there is also provided the use of one or more of the disclosed pyruvate kinase activators, or a pharmaceutically acceptable salt or composition thereof, for treating a disclosed disorder (e.g., as described in any of the embodiments disclosed above).

Further details are described in the examples section below and are part of the present invention.

Example(s)

Preparation of the Compounds

Example 1: preparation of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one (Compound 1).

2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one can be prepared following the procedure outlined for compound E8-4 of example 8A in U.S. Pat. No. 11,040,036, the entire contents of which are incorporated herein by reference. Pharmaceutically acceptable salts of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one can also be prepared according to the disclosure of U.S. Pat. No. 11,040,036.

2- ((1H-pyrazol-3-yl) methyl) -4-methyl-6- ((1-methyl-1H-pyrazol-3-yl) methyl) -4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one can be prepared following the procedure outlined in U.S. Pat. No. 11,040,036 for compound E8-41, the entire contents of which are incorporated herein by reference. Pharmaceutically acceptable salts of 2- ((1H-pyrazol-3-yl) methyl) -4-methyl-6- ((1-methyl-1H-pyrazol-3-yl) methyl) -4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one can also be prepared according to the disclosure of U.S. Pat. No. 11,040,036.

Mitapravastatin is prepared according to the procedure outlined in WO 2011/002817 for compound VIII-8, the disclosure of which is incorporated herein by reference in its entirety. Additional methods, salts, and crystalline forms are also found in WO 2016/201227, and salts and crystalline forms are found in WO 2019/104134 and WO 2020/237047, each of these disclosures being incorporated herein by reference in their entirety.

Mouse model for MDS-related anemia

An in vivo mouse study was performed to determine the effect of mitsubvalvular and compound 1 on erythropoiesis maturation under ineffective erythropoiesis conditions comparable to those of MDS patients. To achieve this goal, two mouse models that have proven ineffective in erythropoiesis were used. To determine the effect of Mitapitava and Compound 1 under these conditions, bone marrow aspirates of diseased mice were collected and erythroblasts populations at various stages of maturation were evaluated using flow cytometry. Hemoglobin (Hb), red Blood Cell (RBC) count and reticulocyte fraction in whole blood were also monitored every 4 weeks on a Sysmex XN-1000 ^TM hematology analyzer to determine whether disease burden and effects on erythroblasts maturation could be detected peripherally. Pharmacodynamic and pharmacokinetic analysis was also performed to confirm proper exposure and target participation of the compounds (data not shown).

Example 2: study of Mitapilar in Polg ^D257A mouse model

Mitochondrial destruction is associated with the occurrence of anemia in patients with MDS (see, e.g., chen et al, blood,2009, volume 114, 19: pages 4045-4053). Mitochondria play a key role in regulating apoptosis, a mechanism highlighted in the loss of hematopoietic function in MDS (Green et al, science,1998, volume 281, 5381: pages 1309-1312; kerbauy et al, exp Hematol, 2007, volume 35, 11: pages 1739-1746; ahlqvist et al, nature Communications,2015, volume 6: page 6494). B6.129S7 (Cg) -Polg ^tm1Prol/J or Polg ^D257A is a mouse model in which the N-terminus of the DNA polymerase gamma gene is mutated, resulting in impaired mitochondrial proofreading activity and thus increased mitochondrial DNA mutagenesis (Jackson Laboratory # 017341). See Chen et al Blood,2009, volume 114, 19: pages 4045-4053; and Ahlqvist et al, natureCommunications,2015, volume 6: page 6494. Accumulation of these mutations results in hematopoietic disorders and increased apoptosis, comparable to the disease mechanisms in MDS patients (Chen et al Blood,2009, volume 114, 19: pages 4045-4053). Furthermore, there is evidence that MDS has acquired pyruvate deficiency, which means that PKR may be a potential therapeutic target for this condition (Bovin P et al, britishJournal of hematology,1975, volume 18, 3: pages 175-187; VALENTINE WN et al, blood,1973, volume 41, 6: pages 857-875; arnold H et al, clinica Chimica acta, 1974, volume 57: pages 187-189; lin G et al, chin J Hematol, 1997, volume 18, 7: pages 350-353). Here we used Polg ^D257A mouse models to evaluate the therapeutic benefit of the PK activator, mitsubvalvular, in this anemia mechanism.

Once the anemic phenotype was determined by hematological analysis, male and female Polg ^D257A mice were fed 200 mg/kg/day of mitsui ad libitum starting from 7.5 months of age for 18 weeks. Whole blood was collected into EDTA tubes via the tail vein and analyzed using a Sysmex XN-2000 ^TM hematology system according to the manufacturer's protocol. Hemoglobin, red Blood Cells (RBCs), and reticulocytes were monitored every 2 to 4 weeks to track disease progression and severity. Compared to untreated mutants, RBC counts were increased by 45% and hemoglobin levels were increased by 2.4g/dL in the mice receiving the mitepival treatment after 18 weeks of mitepival treatment. To supplement this, the frequency of reticulocytes was reduced by 30% in treated mice, indicating improved erythropoiesis compared to untreated mice.

See FIGS. 1A-1D and 2A-2D (C57 BL/6J mice as controls). Interestingly, male mutagens began to develop signs of therapeutic benefit 6 weeks after treatment, with hemoglobin increased by 1g/dL to 2g/dL, whereas female mutagens responded after 18 weeks. The net reticulocyte count was significantly reduced (about 30%) in response to the hemoglobin increase. See fig. 2C.

Example 3: study of Mitapravastatin and Compound 1 in Polg ^D257A mouse model

A second experiment was performed using the Polg ^D257A model in example 2 (RRID: IMSR_JAX: 017341). In this experiment, 20 mice (10 males and 10 females per group) were fed to Mitapitava or compound 1, as described in example 2. For the Mitapide group, the dose of compound was 200 mg/kg/day, and for the compound 1 group, the dose of compound 1 was 10 mg/kg/day. Treatment was started when mice were 4 months old and continued for 8 months (rolling into groups). After 8 months of treatment (at 12 months of age), bone marrow aspirates were evaluated by flow cytometry using putative erythropoiesis markers following the modified procedure outlined by Suragani et al (Nat Med, month 4, 2014, volume 20, 4: pages 408-414). The bone marrow aspirate was treated with ACK lysis buffer for 3 minutes to remove red blood cells from the sample. Throughout the remainder, the remaining cells were washed and stored in pbs+5% fbs. Flow cytometry panels include live/dead (Indo 1), B220 (BV 421), CD5 (BV 421), CD71 (AF 700), and Ter119 (PE) antibodies. The gating strategy for analysis is as follows: live/B220-CD 5-/Ter119+. Ter119+ cells were then gated into the population using CD71 and FSC as outlined in the literature (Suragani et al, nat Med, month 2014, vol.20, 4: pages 408-414; FIG. 6E). As shown by the bone marrow flow cytometry data shown in fig. 6A-6D, treatment with compound 1 resulted in a decrease in basophilic erythroblasts (EryA) while resulting in an increase in late basophilic and multi-stained erythrocytes (EryB) and orthochromatic and reticulocytes (EryC), indicating that treatment with compound 1 improved erythroblast maturation in Polg ^D257A mouse bone marrow. this evidence suggests that treatment with compound 1 improves erythropoiesis in the Polg ^D257A model. An increase in the number of ProE (pre-erythrocytes) was also observed in Polg ^D257A mice, however, compound 1 treatment had no additional effect on the ProE population (compared to vehicle control Polg ^D257A mice). fig. 6D. Since the mitsui treatment group was not completed, flow cytometry data from Polg ^D257A mice receiving mitsui treatment were not available.

Analysis of Hb, RBC and reticulocytes in whole blood showed that a significant disease burden was established in this model, with the mean Hb concentration of vehicle control Polg ^D257A mice being 7.9g/dL, and the wild type control mice being 13.9g/dL. The data is not shown. After 24 weeks (6 months so far) treatment, the mitepin increased Hb and RBC counts by 7% in Polg ^D257A mice, with no change in reticulocytes. The data is not shown. No change in Hb, RBC count or reticulocyte fraction was observed after 32 weeks of treatment with compound 1. The data is not shown.

The Polg ^D257A model also supports the use of the disclosed PK activators to treat diseases or disorders associated with mitochondrial dysfunction. Erythrocytes, despite their lack of mitochondria, remain later in the hematopoietic process. Thus, the effect of PK activators on erythropoiesis and increased hemoglobin production is the result of PK activation in the blood cell progenitor stem cells. The Polg ^D257A model has proofreading defective mitochondrial DNA polymerase and has a delayed effect on Hematopoietic Stem Cell (HSC) differentiation (see CELL STEM CELL, volume 8, phase 5, 2011, month 5, 6, pages 499-510). The consequence of rapid accumulation of mitochondrial DNA mutations is the differentiation of progenitor stem cells and the loss of downstream progenitor cells. These defects may be due to reduced ATP production caused by mitochondrial dysfunction. Since HSCs express both PKR and PKM2 during differentiation, and since the disclosed PK activators enhance the function of both isoenzymes, the increase in ATP production in the glycolytic pathway compensates for the loss of ATP production in mitochondrial dysfunction cells. PK activators exhibit improved differentiation of HSC multilineage stem cells, suggesting that PK activators have an opportunity to support adult stem cell differentiation in patients with potentially impaired mitochondrial function.

Example 4: NHD13 mouse model

NUP98-HOXD (NHD 13) transgenic MDS-related mouse models will also be used to evaluate the therapeutic benefit of the disclosed compounds. See, e.g., lin et al, blood, 7.1.2005, volume 106, phase 1: pages 287-295; and Suragani et al, nat Med, month 4, 2014, volume 20, phase 4: pages 408-414. Male and female NHD13 mice, 4 or 10 month old, are fed daily with the disclosed compounds (e.g., mitepin or compound 1). Whole blood cell analysis and erythrocyte precursor analysis will be performed 2 months after treatment and the results analyzed.

Evidence of the effect of the disclosed PKR activators on ineffective erythropoiesis that may be converted to MDS (e.g., very low risk, and medium risk MDS) includes: the characteristics of ineffective erythropoiesis between thalassemia and MDS are similar; PKR activators improve the survival and differentiation of erythrocytes in bone marrow; and PKR activators improve RBC function via activation of glycolysis to increase energy ATP, nucleotide biosynthesis, and antioxidant stress. A proposed human clinical study is illustrated in fig. 3.

Example 5: study of Mitapitavan or Compound 1 in NHD13 mouse model (4 months of age)

Using example 4, 20 NHD13 mice (10 males and 10 females per group; RRID: IMSR_JAX: 010505) were randomly divided into treatment groups using a hemoglobin level matching profile. Mice were given Mitaprava or Compound 1 in the same manner (ad libitum) as in example 3 for 20 weeks. After 20 weeks of treatment with mitsubpitaled or compound 1, bone marrow aspirates of NHD13 mice were collected as described above and analyzed by flow cytometry. The results of the evaluation of erythroblasts in bone marrow showed little difference between the erythroblasts of wild type and NHD13 vehicle control mice. In addition, no significant drop in survival was observed in vehicle control NHD13 mice previously reported (Lin et al Neoplasia, 7 month 2005, volume 106, phase 1). Taken together, these data indicate that the NHD13 model produces a milder disease pattern than described in the literature, which complicates the assessment of therapeutic efficacy. Thus, it is difficult to detect any therapeutic benefit from the administration of mitsubvalvular or compound 1 from flow cytometry data (fig. 7A-7D). Whole blood analysis also supports this mild disease state. At nine months of age, NHD13 mice fed standard diet (vehicle control) had an average hemoglobin level of 10.4g/dL, which was only 4g/dL lower than the wild type control. Evaluation of the therapeutic effect on NHD13 mice, both mitsubishi and compound 1 showed a significant decrease in reticulocyte fraction (20%). (fig. 8C) treatment with mitsubnatal or compound 1 also demonstrated a significant decrease in reticulocyte count (15% decrease in mitsubnatal and 25% decrease in compound 1). The data is not shown. These data indicate that erythropoiesis was improved in NHD13 mice after treatment, whereas the response to Hb or RBC counts was minimal compared to progressive anemia of vehicle control (fig. 8A-8B).

Example 6: study of Mitapitavan or Compound 1 in NHD13 mouse model (10 months of age)

A second experiment can also be performed on 10 month old NHD13 mice, with the expectation that the disease burden will be more severe and likely to be detected in bone marrow by flow cytometry, as described in example 5. In this study, NHD13 mice can be randomized into treatment groups of 20 mice (10 males and 10 females) each using a matching profile of hemoglobin levels at 10 months of age. The mitsubvalvular and/or compound 1 can be administered in ad libitum fed foods for 8 weeks as described in example 5. Hemoglobin, red cell count and reticulocyte fraction were monitored every 4 weeks. Terminal harvesting, including bone marrow flow cytometry analysis, may be performed at 12 months of age.

Study of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one on patients with anemia due to low risk myelodysplastic syndrome at stage 2a/2b

A 2a/2b phase multicentric study will be conducted to evaluate the efficacy and safety of 2- ((1H-pyrazol-3-yl) methyl) -6- ((6-aminopyridin-2-yl) methyl) -4-methyl-4H-thiazolo [5',4':4,5] pyrrolo [2,3-d ] pyridazin-5 (6H) -one (referred to herein as compound 1) in subjects suffering from anemia due to low risk myelodysplastic syndrome (LR-MDS).

Study design

The 2a part of the study was a single arm evaluation of 1 dose level of compound 1 (QD 5mg once daily) to establish a proof of concept of compound 1 in LR-MDS. A eligible subject will orally administer compound 1 during the core period of 16 weeks. Subjects who completed the 16 week core period will be eligible to continue to receive the same dose of compound 1 for an extended period of time, up to 156 weeks. An overview of the study design at stage 2a is illustrated in figure 4.

The 2b phase part of the study was a double blind, randomized, placebo controlled evaluation aimed at evaluating the efficacy and safety of compound 1 (QD 2mg, QD 3mg and QD 5 mg) relative to placebo. The initiation of the phase 2b portion of the study will be based on pre-specified pass/fail criteria. Eligible subjects will receive QD 2mg compound 1 (dose level 1), 3mg compound 1 (dose level 2), QD 5mg compound 1 (dose level 3) or matched placebo for QD oral administration at a 1:1:1:1 ratio. Subjects who completed the 24-week double-blind period will be eligible to receive compound 1 for up to 156 weeks in an extended period. Randomization will be graded according to baseline infusion burden (low infusion burden [ NTD subjects and LTB subjects ], high infusion burden (HTB)). An overview of the phase 2 study design is illustrated in fig. 5.

During the extended period of phase 2b, all subjects will receive compound 1. Subjects receiving placebo during the double blind period will be randomized to receive QD 2mg compound 1 (dose level 1), QD 3mg compound 1 (dose level 2) or QD 5mg compound 1 (dose level 3) in a 1:1:1 ratio in order to assess the long term safety and efficacy of these multiple doses of compound 1. Subjects receiving compound 1 during the double blind period will be eligible to continue to receive the same dose of compound 1.

Inclusion criteria

The subject was eligible to incorporate the phase 2a portion of the study if all of the following conditions were met:

1. the age at which informed consent is provided is at least 18 years.

2. Diagnosis was recorded according to the World Health Organization (WHO) classification, conforming to the IPSS-R low risk disease classification (risk score:.ltoreq.3.5) and based on primary cells < 5% determined from bone marrow biopsy/puncture results of participants during the screening period.

3. Based on the history of infusions in the participant medical records, no infusions or with LTB according to the revised IWG 2018 standard: ntd: < 3 RBC units 16 weeks prior to administration of the first dose of study drug, and no infusion 8 weeks prior to administration of the first dose of study drug; or b.ltb: 3 to 7 RBC units within 16 weeks prior to administration of the first dose of study drug, and < 4 RBC units within 8 weeks prior to administration of the first dose of study drug.

Hb concentration < 11.0g/dL during stage 4.4 Zhou Shaixuan.

5. Eastern tumor cooperative group (ECOG) physical stamina score 0,1 or 2.

6. If iron chelation therapy is received, the iron chelation therapy dose must be stable and begin at > 56 days prior to administration of the first study drug.

7. For women with fertility (WOCBP) and men with WOCBP partners, it is necessary to use 2 contraceptive regimens as part of their daily lifestyle or agree from the time of informed consent, throughout the study period and within 28 days after the last dose of study medication by the woman, and within 90 days after the last dose of study medication by the men, of which 1 must be considered as efficient. The second contraceptive mode may be an acceptable barrier method.

8. Prior to any study-related procedures, participants were informed in written consent and willing to follow all study procedures during the study.

The subject was eligible to incorporate the phase 2b portion of the study if all of the following conditions were met:

1. the age at which informed consent is provided is at least 18 years.

2. According to the WHO classification, diagnosis was recorded of < 5% of primary cells determined from bone marrow biopsies/punctures of participants during the screening period, in accordance with the IPSS-R low risk disease classification (risk score:.ltoreq.3.5).

3. Based on the history of infusions in the participant medical records, no infusions either have LTB or have HTB according to the revised IWG 2018 standard: ntd: 16 weeks prior to randomization < 3 RBC units, and 8 weeks prior to randomization without infusion, or b.ltb: 3 to 7 RBC units 16 weeks prior to randomization, and <4 RBC units 8 weeks prior to randomization, or c.htb: no less than 8 RBC units 16 weeks prior to randomization, and no less than 4 RBC units 8 weeks prior to randomization.

Hb concentration < 11.0g/dL during stage 4.4 Zhou Shaixuan.

5. Up to 2 previous therapies, including Erythropoiesis Stimulating Agents (ESAs) (e.g., erythropoietin [ EPO ], epo+granulocyte colony stimulating factor [ G-CSF ]), and/or Luo Texi prions.

Ecog physical state score 0,1 or 2.

7. If iron chelation therapy is received, the iron chelation therapy dose must be stable and begin at > 56 days prior to randomization.

8. For WOCBP and men with WOCBP partners, it is necessary to

The contraindications of sexual activity that could lead to pregnancy are taken as part of its daily lifestyle or agree to use 2 contraceptive regimens within 28 days of the last study medication taken by the woman, and within 90 days of the last study medication taken by the man, from the time of providing informed consent, of which 1 must be considered as efficient. The second contraceptive mode may be an acceptable barrier method.

9. The participants were informed written consent prior to any study-related procedures and were willing to follow all studies during the study

Exclusion criteria

Subjects will be excluded from phase 2a of the study if any of the following conditions are met:

1. A history of Acute Myeloid Leukemia (AML) is known.

2. Secondary MDS, defined as MDS that is known to occur as a result of chemical injury or as a result of chemotherapy and/or radiation therapy to treat other diseases.

3. Prior exposure to pyruvate kinase activators, treatment for high risk MDS (hypomethylating agents [ HMA ], isocitrate dehydrogenase [ IDH ] inhibitors, or allogeneic or autologous stem cell transplantation), and/or disease modifying agents (e.g., immunomodulatory drugs such as lenalidomide). If the participants received disease modifying agent for less than or equal to 1 week more than or equal to 8 weeks prior to administration of the first dose of study medication, the investigator may decide not to exclude them.

4. Currently, luo Texi common, EPO or G-CSF are being used. Treatment with EPO or G-CSF must be discontinued for > 28 days prior to administration of the first dose of study drug; the Luo Texi treatment must be discontinued for > 65 days before the first dose of study medication is administered.

5. A history of active and/or uncontrolled heart or lung disease within 6 months prior to providing informed consent, including but not limited to:

a. New York Heart Association grade III or IV heart failure or clinically significant arrhythmia

B. myocardial infarction, unstable angina or unstable hypertension; high risk thrombosis;

hemorrhagic, embolic or thrombotic stroke; deep vein thrombosis; or pulmonary or arterial embolism

C. Heart rate correction QT interval calculated using the friedricia method: female subjects are greater than or equal to 470 milliseconds, and male subjects are greater than or equal to 450 milliseconds, except for right bundle branch or left bundle branch block

D. Severe pulmonary fibrosis, defined as severe hypoxia, evidence of right heart failure and radiological pulmonary fibrosis > 50%

E. severe pulmonary hypertension, defined as severe symptoms associated with hypoxia, right heart failure and hypoxia

6. History of liver and gall disorders defined as follows:

a. Serum AST > 2.5 Xupper normal value limit (ULN) (unless due to hemolysis and/or hepatic iron deposition) and ALT > 2.5 XULN (unless due to hepatic iron deposition)

B. Serum bilirubin > ULN, if elevated, is associated with clinically symptomatic choledocholithiasis, cholecystitis, biliary obstruction, or hepatocellular disease

7. Renal dysfunction, defined as estimated glomerular filtration rate (eGFR) < 45mL/min.

8. Active infection requiring systemic antimicrobial therapy when informed consent is provided. If antimicrobial treatment is required during the screening period, the screening procedure should not be performed simultaneously with the antimicrobial treatment, and the last dose of antimicrobial therapy must be performed at > 7 days prior to administration of the first dose of study drug.

9. Significant surgery was received 12 weeks prior to administration of the first dose of study medication. Prior to administration of the first dose of study drug to the subject,

Must be fully recovered from any previous surgery.

10. Any history of malignancy, except for non-melanoma in situ skin cancer, in situ cervical cancer, or in situ breast cancer. The subject must not have active disease or receive anti-cancer treatment less than or equal to 5 years before providing informed consent.

11. Hepatitis C Virus (HCV) antibodies (abs) detected positive, with evidence of active HCV infection, or hepatitis b surface antigen (HBsAg) detected positive.

HIV-1 Ab or HIV-2 Ab detection was positive.

13. Absolute Neutrophil Count (ANC) < 500/. Mu.L (0.5X109/L).

14. Platelet counts assessed at less than or equal to 75,000/. Mu.L (75X 109/L) without platelet transfusion within 28 days prior to screening.

15. Non-fasting triglyceride concentration > 500mg/dL.

16. P-glycoprotein (P-gp) inhibitors were received without a period of 5 days or longer or corresponding to 5 half-lives prior to the first study drug administration.

17. The current group or past participated in (within 4 weeks prior to administration of the first dose of study drug or within a time frame corresponding to 5 half-lives of the study drug, or longer) any other clinical study involving study treatment or equipment.

18. Known for Compound 1 or excipients thereof (silicified microcrystalline cellulose, croscarmellose sodium, sodium stearyl fumarate andII Blue film coating [ polyvinyl alcohol, titanium dioxide, polyethylene glycol/polyethylene glycol, talc, FD & C Blue # 2/indigo carmine aluminum lake/E132 ]) allergy.

19. Pregnancy or lactation.

20. Researchers believe that any medical, hematological, psychological or behavioral condition or previous or current treatment may pose an unacceptable risk to participation in the study and/or may obscure interpretation of the study data.

Subjects will be excluded from phase 2b of the study if any of the following conditions are met:

1. A history of AML is known.

2. Secondary MDS, defined as MDS that is known to occur as a result of chemical injury or as a result of chemotherapy and/or radiation therapy to treat other diseases.

3. Prior exposure to pyruvate kinase activators, including exposure to compound 1, treatment for high risk MDS (HMA, IDH inhibitor, or allogeneic or autologous stem cell transplantation), and/or disease modifying agents (e.g., immunomodulatory drugs such as lenalidomide) in the 2a portion of the study. If the participants received disease modifying agent treatment for less than or equal to 1 week more than or equal to 8 weeks prior to randomization, the investigator may decide not to exclude it.

4. Currently, luo Texi common, EPO or G-CSF are being used. Treatment with EPO or G-CSF must be discontinued for > 28 days prior to administration of the first dose of study drug; prior to randomization, treatment with Luo Texi cycles must be discontinued for > 65 days.

5. A history of active and/or uncontrolled heart or lung disease within 6 months prior to providing informed consent, including but not limited to:

a. New York Heart Association grade III or IV heart failure or clinically significant arrhythmia

B. myocardial infarction, unstable angina or unstable hypertension; high risk thrombosis; hemorrhagic, embolic or thrombotic stroke; deep vein thrombosis; or pulmonary or arterial embolism

C. Heart rate correction QT interval calculated using the friedricia method: female subjects are greater than or equal to 470 milliseconds, and male subjects are greater than or equal to 450 milliseconds, except for right bundle branch or left bundle branch block

D. Severe pulmonary fibrosis, defined as severe hypoxia, evidence of right heart failure and radiological pulmonary fibrosis > 50%

E. severe pulmonary hypertension, defined as severe symptoms associated with hypoxia, right heart failure and hypoxia

6. History of liver and gall disorders defined as follows:

a. serum AST > 2.5 XULN (unless due to hemolysis and/or hepatic iron deposition) and ALT > 2.5 XULN (unless due to hepatic iron deposition)

B. Serum bilirubin > ULN, if elevated, is associated with clinically symptomatic choledocholithiasis, cholecystitis, biliary obstruction, or hepatocellular disease

7. Renal dysfunction, defined as eGFR < 45mL/min

8. Active infection requiring systemic antimicrobial therapy when informed consent is provided. If antimicrobial treatment is required during the screening period, the screening procedure should not be performed simultaneously with the antimicrobial treatment, and the last dose of antimicrobial therapy must be performed at > 7 days prior to randomization.

9. Large surgery was received 12 weeks prior to randomization. The subject must fully recover from any previous surgery prior to randomization.

10. Any history of malignancy, except for non-melanoma in situ skin cancer, in situ cervical cancer, or in situ breast cancer. The subject must not have active disease or receive anti-cancer treatment less than or equal to 5 years before providing informed consent.

HCV Ab detection positive with evidence of active HCV infection, or HBsAg detection positive.

HIV-1 Ab or HIV-2 Ab detection was positive.

13.ANC＜500/μL(0.5×109/L)。

14. Platelet counts assessed at no platelet transfusion received within 28 days prior to screening < 50,000/. Mu.L (50X 109/L).

15. Non-fasting triglyceride concentration > 500mg/dL.

16. Prior to randomization, P-gp inhibitors were received that were not discontinued for > 5 days or for a time frame corresponding to 5 half-lives, whichever was longer.

17. The current group or past participated in (within 4 weeks prior to randomization or within a time frame corresponding to 5 half-lives of study drugs, or longer) any other clinical study involving study treatments or equipment.

18. Compound 1 or excipients thereof are known to include placebo (silicified microcrystalline cellulose, croscarmellose sodium, mannitol, sodium stearyl fumarate, magnesium stearate andII Blue film coating [ polyvinyl alcohol, hypromellose, titanium dioxide, lactose monohydrate, polyethylene glycol/polyethylene glycol, glyceryl triacetate, talc, FD & C Blue # 2/indigo carminative lake/E132 ]) allergy.

19. Pregnancy or lactation.

20. Researchers believe that any medical, hematological, psychological or behavioral condition or previous or current treatment may pose an unacceptable risk to participation in the study and/or may obscure interpretation of the study data.

Primary endpoint, phase 2a

Hemoglobin response

Hemoglobin response was defined as an increase in average Hb concentration of ≡1.5g/dL from baseline between week 8 and week 16. The proportion of subjects who achieved Hb response (Hb response rate) will be pooled and double sided 95% exact CI calculated using the Clopper-Pearson method. The hemoglobin concentration assessed within 14 days after RBC infusion will be excluded from analysis of the primary endpoint. Once this exclusion was applied, subjects not undergoing any Hb concentration assessment from week 8 to week 16 would be considered non-responders.

Infusion independence

Infusion independence is defined as ≡ 8 consecutive weeks without infusion during the core period (LTB-only subjects). The proportion of subjects achieving TI (TI rate) will be summarized and bilateral 95% accurate CI will be calculated using Clopper-Pearson method.

Secondary endpoint stage 2a

Secondary endpoints of phase 2a include:

AE during core phase, SAE, drug withdrawal due to AE and laboratory abnormalities.

Hb1.0+ response, defined as an increase in average Hb concentration of > 1.0g/dL from baseline between week 8 and week 16.

Change in Hb concentration from baseline during the core phase.

Hb concentration increases from baseline by > 1.5g/dL at > 2 consecutive time points from week 8 to week 16.

Change in total infused Red Blood Cell (RBC) units from baseline during the core phase.

Total infusion RBC units decreased by > 50% during the core period by > 8 consecutive weeks compared to baseline.

Plasma concentration and pharmacokinetic parameters of compound 1 during the core phase.

Whole blood pharmacodynamic parameter concentrations during the core phase, including 2, 3-phosphoglycerate (2, 3-DPG) and Adenosine Triphosphate (ATP).

Exploratory endpoint stage 2a

The exploratory endpoint of phase 2a included:

Changes from baseline in exploratory biomarkers including hepcidin, erythritol, soluble transferrin receptor, and growth differentiation factor 11 (GDF 11) during the core phase.

Changes from baseline in iron, serum ferritin, total iron binding capacity and transferrin saturation during the core phase.

Changes from baseline in erythropoiesis markers (including absolute and percent reticulocytes and erythropoietin) during the core phase.

Changes in bone marrow derived biomarkers (including erythrocyte precursors) from baseline during the core phase.

Changes from baseline in the markers of hemolysis (including indirect bilirubin, lactate dehydrogenase, and bound globin) during the core phase.

Changes in PKR activity from baseline during the core phase.

Change from baseline during extended periods:

total infusion RBC units (LTB-only subjects)

Hb concentration

-Iron metabolism markers

Erythropoiesis and hemolysis markers

-A bone marrow derived biomarker comprising erythrocyte precursors

Exploratory biomarkers

AE, SAE, drug withdrawal due to AE and laboratory abnormalities during extended periods.

Primary endpoint, phase 2b

The primary endpoint of phase 2b is the mHI-E response, defined as: 1) During the double blind period, no less than 8 consecutive weeks, hb concentration increased no less than 1.5g/dL (NTD subject) from baseline; 2) Infusion independent, defined as ≡8 consecutive weeks without infusion during double blind period (LTB only subjects); 3) The double-blind period was greater than or equal to 8 consecutive weeks, with a total infusion RBC units greater than or equal to 50% less than baseline (HTB-only subjects). The hemoglobin concentration assessed within 14 days after RBC infusion will be excluded from analysis of the primary endpoint. Once this exclusion is applied, NTD subjects and LTB subjects will be considered non-responders if the subjects did not conduct at least 2 Hb concentration assessments by > 8 weeks prior to week 24.

Secondary endpoint stage 2b

Secondary endpoints of phase 2b include:

AE, SAE, withdrawal due to AE and laboratory abnormalities during double blind period.

Variation of Hb concentration from baseline during double blind period.

Change in total infused RBC units from baseline from week 8 to week 24.

Infusion independent, defined as no infusion for ≡8 weeks consecutive during double blinded period.

The time during the double blind period when the mHI-E response first occurs.

Maximum duration of mHI-E response in subjects who achieved mHI-E response during double blind period.

Plasma concentration and pharmacokinetic parameters of compound 1 during double blind phase.

Whole blood concentrations of pharmacodynamic parameters during double blind phase, including 2,3-DPG and ATP.

Correlation pharmacokinetic parameters, exposure-response (or pharmacokinetic/pharmacodynamic) relationship between clinical activity and safety index endpoint during double blind period.

Exploratory endpoint stage 2b

The exploratory endpoint at stage 2b included:

changes in RBC infusion frequency from baseline during double blind periods (limited LTB and HTB patients only)

Time of first infusion during double blind period (NTD subject only)

Changes from baseline in exploratory biomarkers (including hepcidin, erythritol, soluble transferrin receptor, and GDF 11) during double blind period

Changes from baseline in iron, serum ferritin, total iron binding capacity and transferrin saturation during double blind period

Changes from baseline in erythropoiesis markers (including absolute and percent reticulocytes and erythropoietin) during double blind phase

Changes from baseline in bone marrow derived biomarkers (including erythrocyte precursors) during double blind phase

Changes from baseline in hemolysis markers (including indirect bilirubin, lactate dehydrogenase and bound globin) during double blind phase

Changes in PKR activity from baseline during double blind period the change in the cancer therapy function assessment questionnaire (FACT-An) anemia score scale from baseline

Changes from baseline in the spinal cord dysplasia scale (QUALMS) and QUALMS physical burden (QUALMS-P) component scale during double blind phase

Patient severity overall impression during double blind Period (PGIS) -improvement of anemia symptoms by at least 1 category compared to baseline, or "no change" if there is no anemia symptoms or only mild anemia symptoms at baseline "

Patient Global Impression of Change (PGIC) -improvement of anemia symptoms during double blind period, or "no change" if there is no anemia symptoms or mild anemia symptoms at baseline based on PGIS baseline score "

Patient Report Outcome Measurement Information System (PROMIS) change from baseline in body function 4a questionnaire during double blind period

Change from baseline during extended periods:

Total infusion RBC units (LTB and HTB only subjects)

Frequency of RBC infusion (limited LTB and HTB patients)

Hb concentration

-Iron metabolism markers

Erythropoiesis and hemolysis markers

-A bone marrow derived biomarker comprising erythrocyte precursors

Exploratory biomarkers

AE, SAE, drug withdrawal due to AE and laboratory abnormalities during extended periods

Dose adjustment

Excessive Hb response is defined as an increase in Hb concentration > ULN (by gender) in the absence of RBC infusion. If excessive Hb response occurs, no RBC infusion is present for 4 or more weeks, study medication must be discontinued.

If a sharp drop in platelet count is observed (e.g., 50% or more from baseline), the researcher should monitor platelet count weekly and if clinically indicated, should discontinue the study medication for up to 28 days. If the second appearance or appearance of a drop in the level 4 platelet count, the study medication is discontinued.

Although a number of embodiments have been described, the scope of the disclosure will be defined by the appended claims rather than by the specific embodiments that have been presented by way of example. All references, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are hereby expressly incorporated by reference in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly known to one of ordinary skill in the art.

Claims (51)

1. A method of treating myelodysplastic syndrome (MDS) -related anemia in a subject suffering from MDS, the method comprising administering to the subject a therapeutically effective amount of a compound having the formula:

Or a pharmaceutically acceptable salt thereof.

2. A method of treating myelodysplastic syndrome (MDS) -associated hemolytic anemia in a subject having MDS, the method comprising administering to the subject a therapeutically effective amount of a compound having the formula:

Or a pharmaceutically acceptable salt thereof.

3. A method of increasing hemoglobin levels in a subject having myelodysplastic syndrome (MDS), the method comprising administering to the subject a therapeutically effective amount of a compound having the formula:

Or a pharmaceutically acceptable salt thereof.

4. A method of treating acquired PK deficiency (PKD) in a subject having myelodysplastic syndrome (MDS), said method comprising administering to said subject a therapeutically effective amount of a compound having the formula:

Or a pharmaceutically acceptable salt thereof.

5. A method of treating acquired PK deficiency (PKD) -related anemia in a subject having myelodysplastic syndrome (MDS), the method comprising administering to the subject a therapeutically effective amount of a compound having the formula:

Or a pharmaceutically acceptable salt thereof.

6. A method of treating cytopenia in a subject having myelodysplastic syndrome (MDS), the method comprising administering to the subject a therapeutically effective amount of a compound having the formula:

Or a pharmaceutically acceptable salt thereof.

7. The method of any one of claims 1-6, wherein the hemoglobin level of the subject improves over a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, or at least 20 weeks during treatment.

8. The method of any one of claims 1-6, wherein the hemoglobin level of the subject increases from baseline during treatment from week 1 to week 20, week 1 to week 18, week 1 to week 16, week 4 to week 20, week 4 to week 18, week 4 to week 16, week 6 to week 20, week 6 to week 18, week 6 to week 16, week 8 to week 20, week 8 to week 18, week 8 to week 16, week 10 to week 20, week 10 to week 18, or week 10 to week 16.

9. The method of any one of claims 1-8, wherein the hemoglobin level of the subject increases from baseline at week 8 through week 16 during treatment.

10. The method of any one of claims 1-9, wherein the hemoglobin level of the subject increases from baseline at a continuous time point of ∈2, ∈3, ∈4, ∈5, or ∈6 from week 1 to week 20, week 1 to week 18, week 1 to week 16, week 4 to week 20, week 4 to week 18, week 4 to week 16, week 6 to week 20, week 6 to week 18, week 6 to week 16, week 8 to week 20, week 8 to week 18, week 8 to week 16, week 10 to week 20, week 10 to week 18, or week 10 to week 16 during treatment.

11. The method of any one of claims 1-10, wherein the hemoglobin level of the subject increases from baseline at ≡2 consecutive time points from week 8 to week 16 during treatment.

12. The method of any one of claims 1-11, wherein the hemoglobin level of the subject increases from baseline by ≡1.0g/dL during treatment.

13. The method of any one of claims 1-12, wherein the hemoglobin level of the subject increases from baseline by ≡1.5g/dL during treatment.

14. The method of any one of claims 1-13, wherein the hemoglobin level of the subject increases from baseline by ≡2.0g/dL during treatment.

15. The method of any one of claims 1-14, wherein during treatment the subject becomes infusion-independent.

16. The method of any one of claims 1 to 15, wherein the subject is categorized as having a low infusion burden prior to treatment.

17. The method of any one of claims 1 to 16, wherein during treatment, the subject becomes independent of infusion for 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or 10 weeks or more, continuously.

18. The method of any one of claims 1 to 17, wherein during treatment, the subject becomes independent of infusion for ≡8 weeks.

19. The method of any one of claims 1 to 18, wherein during treatment the total infused Red Blood Cell (RBC) units of the subject are reduced from baseline by ≡10% ≡20% clamp ≡30% clamp ≡40% clamp ≡50% clamp ≡60% clamp ≡70% clamp ≡80%.

20. The method of any one of claims 1 to 19, wherein the total infused Red Blood Cell (RBC) units of the subject are reduced by ≡50% from baseline during treatment.

21. The method of any one of claims 1 to 20, wherein during treatment, the total infused Red Blood Cell (RBC) units of the subject decrease by ∈50% from baseline over a period of ∈1 week, ∈2 weeks, ∈3 weeks, ∈4 weeks, ∈5 weeks, ∈6 weeks, ∈7 weeks, ∈8 weeks, ∈9 weeks, or ∈10 weeks.

22. The method of any one of claims 1 to 21, wherein during treatment, the total infused Red Blood Cell (RBC) units of the subject decrease by ≡50% from baseline over ≡8 weeks continuously.

23. The method of any one of claims 1 to 22, wherein the MDS is low risk MDS (as characterized by a revised version of MDS international prognosis scoring system (IPSS-R)).

24. The method of any one of claims 1 to 22, wherein the MDS is an extremely low risk MDS (as characterized by a revised version of MDS international prognosis scoring system (IPSS-R)).

25. The method of any one of claims 1 to 22, wherein the MDS is medium risk MDS (as characterized by a revised version of MDS international prognosis scoring system (IPSS-R)).

26. The method of any one of claims 1-25, wherein the subject is a male.

27. The method of any one of claims 1 to 25, wherein the subject is female.

28. The method of any one of claims 1 to 27, wherein the therapeutically effective amount of the compound administered to the subject is 2mg daily, 3mg daily, or 5mg daily.

29. The method of any one of claims 1-28, wherein the therapeutically effective amount of the compound administered to the subject is QD 2mg, QD 3mg, or QD 5mg.

30. The method of any one of claims 1 to 29, wherein the compound or pharmaceutically acceptable salt is administered orally.

31. The method of any one of claims 1 to 30, wherein the compound or pharmaceutically acceptable salt is in the form of a tablet or one or more granules.

32. The method of any one of claims 1 to 31, wherein the compound or pharmaceutically acceptable salt is in the form of one or more particles.

33. A method of treating low risk of myelodysplastic syndrome (MDS) -related anemia in a subject suffering from MDS, the method comprising orally administering to the subject 2mg daily, 3mg daily, or 5mg daily a compound having the structural formula:

Or a pharmaceutically acceptable salt thereof, in an amount equivalent to 2mg daily, 3mg daily or 5mg daily of the compound, wherein the subject is categorized as no infusion, low infusion burden or high infusion burden prior to administration.

34. The method of claim 33, wherein the subject is categorized as having a low infusion burden prior to treatment.

35. The method of claim 34, wherein the compound or pharmaceutically acceptable salt is administered to the subject for a period of 16 weeks.

36. The method of claim 34 or 35, wherein the subject becomes infusion-independent for ≡8 weeks consecutive during the 16-week administration period.

37. The method of any one of claims 34-36, wherein the hemoglobin level of the subject increases from baseline by ≡1.0g/dL, ≡1.5g/dL, or ≡2.0g/dL from week 8 to week 16 of the 16 week administration period.

38. The method of claim 34, wherein the compound or pharmaceutically acceptable salt is administered to the subject for a period of 24 weeks.

39. The method of claim 38, wherein the subject becomes infusion-independent for ≡8 weeks consecutive during the 24-week administration period.

40. The method of claim 33, wherein the subject is categorized as being without infusion prior to treatment.

41. The method of claim 40, wherein the compound or pharmaceutically acceptable salt is administered to the subject for a period of 24 weeks.

42. The method of claim 41, wherein the hemoglobin level of the subject increases from baseline by ≡1.0g/dL, ≡1.5g/dL, or ≡2.0g/dL for ≡8 weeks during the 24-week administration period.

43. The method of claim 33, wherein the subject is categorized as having a high infusion burden prior to treatment.

44. The method of claim 42, wherein the compound or pharmaceutically acceptable salt is administered to the subject for a period of 24 weeks.

45. The method of claim 43, wherein during the 24 week administration period, the total infused Red Blood Cell (RBC) units of the subject decrease by ≡50% from baseline over ≡8 weeks continuously.

46. A method of treating a mitochondrial dysfunction related disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having the formula

Or a pharmaceutically acceptable salt thereof.

47. A method of treating a disease or disorder associated with ineffective erythropoiesis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having the formula

Or a pharmaceutically acceptable salt thereof.

48. The method of any one of claims 1 to 27, wherein the therapeutically effective amount of the compound administered to the subject is in the range of 0.25mg to 15mg daily.

49. The method of any one of claims 1 to 27, wherein the therapeutically effective amount of the compound administered to the subject is in the range of QD or BID 0.25mg to 2mg or QD or BID 1.5mg to 5.5mg or QD or BID 4mg to 6 mg.

50. The method of any one of claims 5 and 7 to 32, wherein the acquired PK deficiency (PKD) -related anemia is hemolytic anemia.

51. The method of any one of claims 33 to 45, wherein the acquired PK deficiency (PKD) -related anemia is hemolytic anemia.