Methods and compositions for treating hemophilia

Abstract

The present invention relates to methods and compositions for treating hemophilia, providing novel therapies using double-stranded oligonucleotide compounds to improve quality of life and joint function in patients with hemophilia A and hemophilia B.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on June 22, 2021, with Chinese application number 202180044790.1 and invention title "Method and Composition for Treating Hemophilia".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Patent Application No. 63 / 042390, filed June 22, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0004] sequence list

[0005] This application contains a sequence list in ASCII format submitted electronically, the entirety of which is incorporated herein by reference. The ASCII text file was created on June 21, 2021, named 022548.TW080_SL.txt, and has a file size of 774 bytes. Technical Field

[0006] This invention uses double-stranded oligonucleotide compounds as a novel therapy to improve the quality of life and joint function of patients with hemophilia A and hemophilia B. Background Technology

[0007] The maintenance of normal hemostasis depends on a set of simultaneous procoagulant and anticoagulant processes, with thrombin playing a central role. Hemophilia A and B are inherited bleeding disorders characterized by the body's inability to control blood clotting. They are caused by deficiencies in factors VIII and IX, respectively. Bleeding in hemophilia A and B is caused by insufficient thrombin production (Peyvandi et al., Lancet (2016) 388(10040):187-97). Without effective treatment, hemophilia patients will experience recurrent bleeding, which can lead to severe disability and potentially death due to chronic hemarthropathy and severe pain (Pipe et al., Haemophilia (2007) 13 Suppl 4:1-16).

[0008] Despite advances in treatment, significant unmet needs and management challenges remain for all hemophilia populations. While prophylaxis based on factor VIII or factor IX replacement therapy is considered the cornerstone of hemophilia management, it has considerable limitations. For example, prophylaxis with injectable factor replacements is cumbersome and impractical, often requiring multiple weekly intravenous infusions (Peyvandi, ibid.; Ljung and Andersson, Br J Haematol. (2015) 169(6):777-86; Srivastava et al., Haemophilia (2013) 19(1):e1-47; Bauer, Am J Manag Care (2015) 21(6 Suppl):S112-22; Mannucci and Franchini, Blood Transfus. (2013) 11(Suppl 4):s77-81). Factor replacement is also limited by the difficulty of intravenous access and the risk of infection (Balkaransingh and Young, Ther Adv Hematol. (2018) 9(2):49-61; Valentino et al., Blood Rev. (2011) 25(1):11-5). The limitations of providing factor replacement also mean that a large portion of the world's hemophilia population cannot receive preventative treatment first (Hemophilia, WFO Treatment Safety and Supply. 2020).

[0009] Furthermore, factor replacement therapy can lead to the development of alloantibodies, rendering factor therapy ineffective (Morfini et al., Haemophilia (2007) 13(5):606-12). These inhibitory factors (which typically occur in childhood) limit treatment options and significantly worsen the prognosis of hemophilia. In addition, patients with persistent inhibitory factors generally have lower quality of life, more severe joint disease, greater surgical risks, and higher mortality rates compared to patients without inhibitors, including a higher risk of death from hemophilia-related bleeding complications (Morfini, ibid.; Oladapo et al., Orphanet J Rare Dis. (2018) 13(1):198). Current treatment strategies for individuals with persistent inhibitory factors include immune tolerance induction (ITI) therapy and bypassing agent (BPAs) prophylaxis, such as activated prothrombin complex concentrate (aPCC) and recombinant activated factor VII (rFVIIa) (Benson et al., Eur J Haematol. (2012) 88(5):371-79; Collins et al., Br J Haematol. (2013) 160(2):153-70; Kempton et al., Blood (2014) 124(23):3365-72; Astermark et al., Haemophilia (2007) 13(1):38-45; Eichinger et al., Eur J Clin Invest. (2009) 39(8):707-13).

[0010] There remains an urgent and unmet need for the development of therapeutic agents and treatments to prevent recurrent bleeding in hemophilia patients and improve their overall quality of life. Summary of the Invention

[0011] This invention provides methods and compositions for treating patients with hemophilia. In one aspect, the invention provides a method for improving joint function in hemophilia patients in need (such as hemophilia A or B patients with or without inhibitory factors), comprising administering (e.g., subcutaneously) 40 to 90 mg of fitusiran per dose to the patient. In some specific embodiments, this treatment reduces difficulty walking or increases mobility.

[0012] In one aspect, the present invention provides a method for improving joint symptoms (such as joint swelling, pain during movement, and joint pain) in patients with hemophilia (such as patients with hemophilia A or B with or without inhibitory factors), comprising administering (e.g., subcutaneously) 40 to 90 mg of fetuslane per dose to the patient.

[0013] In one aspect, the present invention provides a method for improving patient-reported outcomes (PROs) in hemophilia patients in need (such as hemophilia A or B patients with or without inhibitory factors), comprising administering (e.g., subcutaneously) 40 to 90 mg of phenocysteine ​​per dose to the patient. In some specific embodiments, the PRO is improved in one or more quality of life (QoL) categories.

[0014] In one aspect, the present invention provides a method for improving QoL in hemophilia patients in need (such as hemophilia A or B patients with or without inhibitory factors), comprising administering (e.g., subcutaneously) 40 to 90 mg of fetuslane per dose to the patient in need, wherein QoL is improved in one or more QoL ranges.

[0015] In some specific implementations, the one or more QoL categories are categories of a QoL questionnaire (such as the Haemophilia Quality of Life Questionnaire for Adults, Haem-A-QoL). In further specific implementations, the treated patient experiences clinically meaningful improvement, expressed as a reduction of 7 or more units (optionally 8 or more, 9 or more, or 10 or more units) in one or more scores of the questionnaire (such as the total score, the exercise and leisure category score, and the physical health category score).

[0016] In some specific implementations, the patient is an adult or adolescent aged 12 years or older with hemophilia A or B (congenital deficiency of factor VIII or factor IX) and with or without an inhibitory factor.

[0017] In some specific implementations, the patient has hemophilia A. In further specific implementations, the patient is treated with a factor VIII substitute or bypass agent (BPA; such as aPCC or rFVIIa). In certain specific implementations, the patient has an inhibitory factor (e.g., an inhibitory factor at a level greater than 0.6 BU / mL, as determined by the Bethesda inhibitory factor test). In other specific implementations, the patient does not have an inhibitory factor.

[0018] In some specific implementations, the patient has hemophilia B. In further specific implementations, the patient is treated with a factor IX substitute or BPA (such as aPCC or rFVIIa). In certain specific implementations, the patient has an inhibitory factor (e.g., an inhibitory factor level greater than 0.6 BU / mL, as determined by the Bethesda inhibitory factor assay). In other specific implementations, the patient does not have an inhibitory factor.

[0019] In some specific implementations, patients are treated with multiple doses of 50 mg phenocysteine ​​per dose, or multiple doses of 80 mg phenocysteine ​​per dose. In some specific implementations, phenocysteine ​​is administered to patients once every four weeks or once a month. In some specific implementations, phenocysteine ​​is provided in a solution of 50 to 200 mg / mL (optionally 100 mg / mL) dissolved in phosphate buffer (pH 7).

[0020] The present invention also provides the use of an article in a treatment method. In some embodiments, the article is a single-use vial containing 80 mg of phenotypelan in 0.8 mL of phosphate buffer (pH 7). In other embodiments, the article is a single-use pre-filled syringe containing 80 mg of phenotypelan in 0.8 mL of phosphate buffer (pH 7).

[0021] The present invention also provides the use of phentoselane in the manufacture of pharmaceutical preparations for the treatment of hemophilia in this treatment method, and the use of phentoselane in this treatment method.

[0022] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that although the detailed description points out specific embodiments and aspects of the invention, it is given by way of illustration only and not limitation. From the detailed description, those skilled in the art will understand various changes and modifications within the scope of the invention. Attached Figure Description

[0023] Figure 1 Showing the extended structural formula, chemical formula, and molecular weight of fetuslan.

[0024] Figure 2 A schematic diagram of the CONSORT designed to illustrate the phytosyllanth clinical study described in this article.

[0025] Figure 3A and Figure 3B This shows the D-dimer (µg / mL) levels for each participant over time in each dose group. A: 50 mg dose group. B: 80 mg dose group.

[0026] Figure 4AThis diagram shows the mean (± standard error of mean [SEM]) of monthly antithrombin (AT) activity received with phenotype 11 relative to baseline in patients with hemophilia A or B who have an inhibitory factor. MDI: Multiple doses of inhibitory factor.

[0027] Figure 4B This diagram shows the average (± SEM) of the peak plasma thrombin generation height (nanomoles / liter) over time in the 50 mg and 80 mg dose groups.

[0028] Figure 5 This is a post-hoc analysis showing thrombin production associated with reduced AT in patients with hemophilia A or B who have inhibitory factors. AT levels and corresponding thrombin production measurements were recorded at each time point for all patients. All available thrombin production values ​​were correlated with AT activity levels relative to baseline and categorized as AT-reduced quartiles. The midline within the box represents the median value, and the top and bottom of the box represent the interquartile range. Minimum and maximum values ​​are shown as bars (excluding outliers). Data from healthy volunteers (Pasi et al., N Engl JMed. (2017) 377(9):819-28) were used as reference values. Detailed Implementation

[0029] The present invention is characterized by a method of routine prophylaxis using phenotype 1 to prevent or reduce the frequency of bleeding events in adult and adolescent patients (≥12 years of age) with hemophilia (e.g., hemophilia A (congenital factor VIII deficiency) or hemophilia B (congenital factor IX deficiency)) with or without inhibitory factors. These methods reduce joint swelling and / or joint pain, improve joint function, and improve the patient's quality of life. In certain specific embodiments, this method improves the patient's quality of life score, such as those associated with physical health.

[0030] Patients with hemophilia A or B who have inhibitory factors are those who have developed allogeneic antibodies against factors they previously received (such as factor VIII in hemophilia A or factor IX in hemophilia B). Patients with hemophilia A or B who have inhibitory factors may develop resistance to alternative clotting factor therapy. Patients without inhibitory factors do not have these allogeneic antibodies. This treatment is beneficial for both hemophilia A and hemophilia B patients, with and without inhibitory factors.

[0031] I. Fetuslan Pharmaceutical Composition

[0032] This article provides the structure of phentoselan. Phentoselan is a synthetically and chemically modified double-stranded small interfering RNA (siRNA) oligonucleotide that is covalently linked to a tri-antennary N-acetylgalactosamine (GalNAc) ligand targeting liver AT3 mRNA, thereby inhibiting the synthesis of antithrombin. See, for example, Pasi, ibid. The antithrombin is encoded by the SERPINC1 gene. The nucleotides in each strand of phentoselan are linked by 3'-5' phosphodiester bonds, forming the sugar-phosphate backbone of the oligonucleotide.

[0033] The sense strand and antisense strand contain 21 and 23 nucleotides, respectively. The 3' end of the sense strand is conjugated to the GalNAc-containing moiety (called L96) via a phosphodiester bond. The sense strand contains two consecutive phosphate thioester bonds at its 5' end. The antisense strand contains four phosphate thioester bonds, two at the 3' end and two at the 5' end. The 21 nucleotides of the sense strand hybridize with the 21 complementary nucleotides of the antisense strand, thereby forming 21 nucleotide base pairs and a two-base overhang at the 3' end of the antisense strand. See also U.S. Patent Nos. 9,127,274, US20170159053, and WO 2019 / 014187.

[0034] The dinucleotide chain of fetuslane is shown below:

[0035] Significant chain: 5'Gf-ps-Gm-ps-Uf-Um-Af-Am-Cf-Am-Cf-Cf-Af-Um-Uf-Um-Af-Cm-Uf-Um-Cf-Am-Af-L96 3' (SEQ ID NO:1), and

[0036] Antisense strand: 5'Um-ps-Uf-ps-Gm-Af-Am-Gf-Um-Af-Am-Af-Um-Gm-Gm-Uf-Gm-Uf-Um-Af-Am-Cf-Cm-ps-Am-ps-Gm 3' (SEQ ID NO: 2),

[0037] in

[0038] Af = 2'-Fluoroadenosine

[0039] Cf = 2'-fluorocytidine

[0040] Gf = 2'-Fluoroguanosine

[0041] Uf = 2'-fluorouridine

[0042] Am = 2'-O-methyladenosine

[0043] Cm = 2'-O-methylcytidine

[0044] Gm = 2'-O-methylguanosine

[0045] Um = 2'-O-methyluridine

[0046] "-" (hyphen) = 3'-5' phosphate diester-linked sodium salt

[0047] "-ps-" = 3'-5' thiophosphate-linked sodium salt

[0048] And L96 has the following formula:

[0049] (I).

[0050] The extended structural formula, molecular formula, and molecular weight of fetuslane are as follows: Figure 1 As shown.

[0051] For use in this treatment, phentoselane may be provided in a pharmaceutical composition comprising it and a pharmaceutically acceptable excipient. In a particular embodiment, the dsRNA compound is in the form of a sodium salt.

[0052] In some specific embodiments, phentoselane is provided as an aqueous solution at a concentration of 50 to 200 mg / mL (e.g., 50 to 150 mg / mL, 80 to 110 mg / mL, or 90 to 110 mg / mL). Values ​​between these ranges and values ​​are also intended to be part of the invention, as used herein. Furthermore, it is intended to include numerical ranges using any combination of the listed values ​​as upper and / or lower limits. In further specific embodiments, the pharmaceutical composition comprises phentoselane at concentrations of 50, 75, 100, 125, 150, or 200 mg / mL.

[0053] Unless otherwise stated, the weight of phentostrand described in this invention refers to the weight of phentostrand free acid (active fraction). For example, 100 mg / mL phentostrand means 100 mg of phentostrand free acid per mL (equivalent to 106 mg of sodium phentostrand, the active substance).

[0054] In some specific embodiments, the pharmaceutical composition comprises phenocysteine ​​in phosphate buffer. The phosphate concentration in the solution may be 1 to 10 mM (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 mM), with a pH of 6.0 to 8.0. The pharmaceutical composition described herein may include a preservative, such as EDTA. Alternatively, the pharmaceutical composition may be preservative-free. In a particular specific embodiment, the phenocysteine ​​pharmaceutical composition is preservative-free and comprises, is composed of, or is substantially composed of 100 mg of phenocysteine ​​per milliliter of a 5 mM phosphate buffer (PBS) solution. The PBS solution consists of sodium chloride, disodium hydrogen phosphate (heptahydrate), and sodium dihydrogen phosphate (monohydrate). Sodium hydroxide solution and dilute phosphoric acid may be used to adjust the pH of the composition to 7.0.

[0055] The pharmaceutical composition may be provided in single-use containers (such as vials, ampoules, syringes, or injectors), each containing 40 to 100 mg of phenocysteine ​​(e.g., 50 mg or 80 mg). Phenocysteine ​​may be provided in solid form in the container and reconstituted in an aqueous solution (e.g., PBS) before use, wherein the reconstituted solution contains 50 to 150 mg / mL (e.g., 100 mg / mL) of phenocysteine. In some specific embodiments, phenocysteine ​​is provided in sodium form in single-use glass vials or single-use pre-filled syringes (e.g., with a safety system). In a further specific embodiment, each vial or syringe contains 80 mg of phenocysteine ​​in 0.8 mL of 5 mM phosphate-buffered saline (pH 7.0); and the solution is administered to the patient via subcutaneous injection. The solution may be dispensed in 2 to 30 mL of solution. o C (e.g., 2 to 8) o C) Stored under.

[0056] In a specific embodiment, the phenocysteine ​​composition for subcutaneous injection contains phenocysteine ​​dissolved in 5 mM phosphate buffer (containing 0.64 mM NaH2PO4, 4.36 mM Na2HPO4, and 84 mM NaCl, pH 7.0). In a specific embodiment, the compositions of the phenocysteine ​​solution for subcutaneous injection are shown in Table 1 below:

[0057] Table 1

[0058]

[0059] qs: Sufficient quantity.

[0060] II. Therapeutic Uses of Fetuslane

[0061] Fituxan inhibits the production of antithrombin (AT) in the liver. As an anticoagulant, AT reduces thrombin production by directly targeting thrombin production or by modulating hemostasis through the deactivation of unreintegrated Fxa (Quinsey et al., Int J Biochem Cell Biol. (2004) 36(3):386-9). Fituxan can be used to treat individuals with impaired hemostasis.

[0062] For example, fittuslane can be used to treat patients with hemophilia A or B, with or without an inhibitory factor, for routine prophylaxis to prevent or reduce the frequency of bleeding events. In a specific implementation, fittuslane is used to treat adult and adolescent patients (≥12 years old) with hemophilia A or B (congenital factor VIII or factor IX deficiency), with or without an inhibitory factor.

[0063] This method involves administering a therapeutically effective dose of phenocysteine ​​to hemophilia patients in need (e.g., patients with hemophilia A or B). "Therapeuticly effective dose" means the amount of phenocysteine ​​that helps the patient achieve the desired clinical endpoint. The desired clinical endpoint may be, for example, a reduction in annual bleeding rates (ABR) (e.g., greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%). The desired clinical endpoint may be a reduction in the patient's antithrombin level to normal levels (e.g., approximately 64 to 210 nM).

[0064] The required clinical endpoint may also be an improvement in patient-reported outcomes (PROs), as further explained below. In some specific implementations, therapeutic efficacy can be measured by a reduction in disease severity as assessed by a patient based on an effective and reliable hemophilia-specific PRO instrument, such as the Adult Hemophilia Quality of Life Questionnaire (“Haem-A-QoL”; von Mackensen et al., Haematologica (2005) 90(s2):115-6, Abstract 0290; Wyrwich et al., Haemophilia (2015) 21(5):578-84). Any positive change leading to, for example, a reduction in disease severity as measured using an appropriate scale represents appropriate treatment with the pharmaceutical composition described herein.

[0065] Hemophilia directly impacts patients' health-related quality of life (HRQoL) through its associated symptoms, functional limitations, and treatment burden. Improving HRQoL is a key direction in hemophilia management. This approach aims to improve patients' HRQoL through a carefully designed and detailed questionnaire. For example, HRQoL in adult patients (17 years or older, e.g., 18 years or older) can be measured using questionnaires such as Hemofilia-QoL, Haemophila Well-Being Index, HAEMO-QoL-A, Haem-A-QoL, and EuroQol 5-Dimensions (EQ-5D) (EuroQolGroup, Health Policy (1990) 16(3):199-208). HRQoL in adolescent patients (12 years or older to 17 years old) can be measured using, for example, the Haemophilia Quality of Life Questionnaire for teenagers (Haemo-QoL). See, for example, Bullinger et al., Value Health.(2009) 12(5):808-20; and Remor, Int J Behav Med. (2013) 20(4):609-17.

[0066] In some specific implementations, this treatment improves a patient's quality of life in one or more QoL categories (such as hemophilia-specific QoL categories). These QoL categories include, for example, those related to physical health, feelings, self-perception, exercise and leisure, work and school, hemophilia management, treatment, future, family planning, and / or partnerships and sexual behavior. Improvements in these categories can be assessed using patient-reported outcomes (PROs) and may be aided by questionnaires. For example, improvements in these categories can be scored by the patient reporting to his / her doctor and / or through a QoL questionnaire.

[0067] In specific implementation schemes, HRQoL in adult patients is measured using the Haem-A-QoL score. See, for example, von Mackensen et al., Value in Health. (2005) 8(6):A127; von Mackensen et al., J Thrombosis and Haemostasis. (2005) 3(Sup1):P0813; von Mackensen and Gringeri, “Quality of Life in Hemophilia” In: Handbook of Disease Burdens and Quality of Life Measures. Heidelberg: Springer; 2009, pp.1910-1; and Bullinger et al., Value in Health. (2009) 12(5):808-20; Wyrwich, ibid. The Haem-A-QoL questionnaire consists of 46 items, divided into 10 categories, including physical health (5 items), feelings (4 items), self-perception (5 items), exercise and leisure (5 items), work and study (4 items), hemophilia management (3 items), treatment (8 items), future (5 items), family planning (4 items), and partnerships and sexual behavior (3 items).

[0068] All Haem-A-QoL items are measured based on a 5-point frequency scale (1 = Never, 2 = Rarely, 3 = Sometimes, 4 = Often, 5 = Always). When a question is deemed unsuitable for the participant, there are also "unsuitable" answer options in the categories of "Sports and Leisure," "Work and Study," and "Family Planning." The "Total Score" is also used to represent the average of all 10 categories of the Haem-A-QoL questionnaire. Haem-A-QoL category scores and the total score are converted to a range of 0 to 100, with higher scores indicating greater impairment. A decrease in the corresponding baseline score (score before treatment assessment) indicates an improvement in the patient's quality of life. The questionnaire can be administered before and after treatment with one or more (e.g., two or more, three or more, four or more, five or more, or six or more) doses of fetusyllanth (e.g., 80 mg subcutaneously every four weeks or once a month). For example, the questionnaire can be administered at weeks 8, 12, 16, 20, 24, 25, 26, or 27 after the start of fetuslan treatment.

[0069] The fituzin treatment of this invention improves scores in at least one Haem-A-QoL category (e.g., physical health, feeling, self-perception, exercise and leisure, work and school, hemophilia management, treatment, future, family planning, and / or partnerships and sexual behavior) from baseline, and / or improves the overall Haem-A-QoL score from baseline. In particular, this method can improve the quality of life of patients with hemophilia, including improvement (e.g., relief and elimination) of patient-reported hemophilia-related symptoms (e.g., painful swelling and joint pain) and physical function (e.g., pain during exercise and difficulty walking), as determined by the physical health score and / or the overall Haem-A-QoL score. Clinically meaningful improvements in quality of life include, for example, a reduction of approximately 7 points or more in the overall score, a reduction of approximately 10 points or more in the exercise and leisure category score, and / or a reduction of approximately 10 points or more in the physical health category score. See also Wyrwich, ibid. In some specific implementations, one or more of the 10 categories of Haem-A-QoL scores (such as the physical health category score or the total score) are reduced by more than one unit (such as more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 units).

[0070] III. Administration of phenotype pharmaceutical compositions

[0071] The phenocysteine ​​pharmaceutical composition may be administered by any means known in the art, including but not limited to intraperitoneal, intravenous, intramuscular, subcutaneous, percutaneous, or portal vein administration. In a particular embodiment, the pharmaceutical composition is administered by subcutaneous injection at a dose strength of, for example, 25 to 100 mg per dose (e.g., 25 to 95 mg, 40 to 90 mg, 50 to 100 mg, 50 to 90 mg, 50 to 85 mg, or 50 to 80 mg). In a particular embodiment, phenocysteine ​​is administered subcutaneously at a dose of 50 or 80 mg (by weight of the active fraction), dissolved in PBS solution as described above.

[0072] Multiple doses of fetuslane may be administered to the subject at intervals of 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or 1, 2, or 3 months. In a specific implementation plan, a fixed dose of fetuslane (e.g., 50 or 80 mg subcutaneously) may be administered once every four weeks or once a month to a hemophiliac patient (e.g., a patient with hemophilia A or B who is 12 years of age or older and has or does not have an inhibitory factor for development).

[0073] In some specific embodiments, the pharmaceutical compositions of the present invention may be administered in conjunction with other drugs and / or other therapies (as described currently used to treat patients with bleeding disorders). For example, in a particular embodiment, fittuslans may be administered in combination with a second agent suitable for treating hemophilia A and / or B. Examples of this second type of agent include fresh frozen plasma (FFP); rFVIIa; aPCC; recombinant or plasma-derived FVIII or FIX; virus-inactivated, vWF-containing FVIII concentrate; desensitization therapy, which may include high doses of FVIII or FIX, accompanied by steroids or intravenous immunoglobulin (IVIG) and cyclophosphamide; plasmapheresis combined with immunosuppression and infusion of FVIII or FIX, with or without antifibrinolytic therapy; immune tolerance induction (ITI), with or without immunosuppressive therapy (such as cyclophosphamide, prednisone, and / or antiCD20); desmopressin acetate (DDAVP); antifibrinolytic agents, such as aminocaproic acid and tranexamic acid; antihemophilic agents; corticosteroids; immunosuppressive agents; and estrogens. The fetuslan composition may be administered simultaneously and / or in the same combination with additional therapeutic agents and / or treatments (e.g., in a non-oral manner), or the additional therapeutic agents may be administered as part of a separate composition or at different times and / or by other methods known in the art or described herein.

[0074] Unless otherwise defined herein, scientific and technical terms related to this invention shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although similar or equivalent methods and materials described herein may also be used in the practice or testing of this invention. In case of conflict, this specification (including definitions) shall prevail. Generally, the nomenclature and techniques related to hematology, medicine, medicinal chemistry, and cell biology described herein are those known and commonly used in the art. Enzyme reactions and purification techniques are performed according to the manufacturer's instructions, as commonly understood in the art or as described herein. Furthermore, unless the context requires otherwise, singular terms shall include plural, and plural terms shall include singular. Throughout this specification and the specific embodiments, the words “having” and “comprising,” or variations such as “having,” “possessing,” “including,” or “comprising,” shall be understood to imply inclusion of the stated integers or groups of integers, but not excluding any other integers or groups of integers. All publications and other references mentioned herein are incorporated herein by reference. Although numerous documents are cited herein, this citation does not constitute an admission that any of those documents constitutes part of common knowledge in the art. As used herein, the term “about” or “approximately” applied to one or more values ​​of interest means a value similar to the reference value. In a particular embodiment, the term means a value falling within the range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, whether in any direction of the reference value (greater or less), unless otherwise stated or apparent from the context.

[0075] To better understand the present invention, the following embodiments are provided. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0076] Example

[0077] Example 1: Clinical Study Design and Population

[0078] This embodiment describes the design and patient population of a clinical study of phenocysteine ​​therapy. In this study, 17 adults with hemophilia A or B and inhibitory factors received a fixed subcutaneous dose of phenocysteine ​​50 mg (n = 6) or 80 mg (n = 11) for three months. Figure 2 Participant follow-up was for a maximum of 112 days (84 days for patients converted to the open-label extension study) or longer until AT levels recovered to more than 80% of baseline. Bleeding events were managed during studies with rFVIIa or aPCC therapy. All participants completed the study.

[0079] Enrolled participants were male, aged 18 to 65 years (inclusive), with moderate to severe hemophilia A or hemophilia B (FVIII or FIX ≤5%) and a Bethesda inhibitory factor (Bethesda inhibitory factor test >0.6 BU / mL). Participants were receiving treatment as needed or had previously received prophylactic treatment.

[0080] The main exclusion criteria included a history of venous thromboembolism, known coexisting thrombotic diseases, D-dimer greater than 3 times the upper limit of normal (ULN), AT activity less than 60% at screening, liver dysfunction, HIV positivity with CD4 count less than 200 cells / μL, or estimated glomerular filtration rate ≤ 45 ml / min / 1.73 m² (using the kidney disease diet correction formula) (Levey et al., Ann Intern Med. (2006) 145(4):247-54).

[0081] Statistical analysis is primarily for explanatory purposes and is performed using SAS software version 9.2 or later. Statistical descriptions present the frequencies and percentages of continuous variables, as well as categorical and ordinal variables. Percentages are based on the number of non-missing values. The ABR for each period is calculated by dividing the number of bleeding episodes in that period by the number of days in that period, and then multiplying by 365.25.

[0082] The baseline demographic and clinical characteristics of the study population are shown in Table 2. SEM refers to the standard error of the mean.

[0083] Table 2

[0084]

[0085] All study participants had severe hemophilia (<1% factor level). One participant in each dose group had hemophilia B, and the rest had hemophilia A. Age, weight, and number of bleeding events per year were roughly similar between the two dose groups. Eleven of the 17 study patients reported a history of hepatitis C; however, subjects who had received ribavirin, interferon, or other antiviral treatments were excluded. Furthermore, subjects with severe liver disease, including clinically significant cirrhosis based on medical history, or those with an alanine / aspartate aminotransferase (ALT / AST) ratio greater than 3 times the ULN at screening, were excluded.

[0086] Example 2: Safety of fittuslane treatment

[0087] One objective of the aforementioned study was to evaluate the safety of phenotype 14 in participants with hemophilia A or B who had inhibitory factors. The safety analysis cohort included all participants who had received at least one dose of phenotype 14. Safety assessments included monitoring for adverse events (AEs), clinical laboratory evaluations (e.g., hematology, biochemistry (including liver function tests), coagulation measurements [activated partial thromboplastin time (aPTT) / prothrombin time (PT), international normalized ratio, platelets, D-dimer, fibrinogen], and anti-antibody formation [using a validated human enzyme-linked immunosorbent assay]), vital signs, and a 12-lead electrocardiogram. Throughout the study, AEs and serious AEs (SAEs) were assessed according to the Medical Dictionary for Regulatory Activities (MedDRA). ® AEs are coded according to their severity (mild, moderate, or severe) and their causal relationship with the recorded investigational drug or preoperative medication.

[0088] No phenocysteine ​​(AF) treatment was interrupted during the study, no thrombotic events occurred, and no SAEs were considered to be related to AF. No drug-induced anti-antibody formation occurred. Seventeen (100%) participants reported AEs, with the maximum severity of all cases being mild or moderate. Table 3 below reports all AEs in patients who received at least a single dose of AF. Any drug-related AE reported in Table 3 means an AE that is considered possibly or certainly related to the study drug. During the study, at least two participants reported the most common drug-related AEs listed in Table 3.

[0089] Table 3

[0090]

[0091] The most common investigational drug-related adverse events (AEs) occurring in ≥2 patients were injection site erythema (n = 8; 47%), increased ALT / AST, increased liver enzymes (alkaline phosphatase, bilirubin), or increased transaminases (n = 5; 29%), increased fibrin D-dimer (n = 2; 12%), and injection site pain (n = 2; 12%). Injection site reactions were all mild and transient, requiring no medical intervention. ALT elevations were typically mild and transient (peak ALT levels were ≤ 3x ULN in four subjects and 5x ULN in one subject). No associated bilirubin elevation exceeding 2x ULN (reference range 3.4–20.5 µmol / L) and no severe drug-induced liver impairment meeting Hy's Law criteria are considered predictable for the likelihood of drug-induced severe liver injury when administered to a large population (Katarey, et al., Clin Med (Lond.) (2016) 16(Suppl 6):s104–9).

[0092] Three participants had a history of hepatitis C virus (HCV) infection. On day 42, one participant experienced an ALT increase to >3x ULN (5x ULN; 254.9 U / L), occurring in a participant with a history of HCV (80 mg), and this was reported as moderate in intensity. Fentusalam continued, and ALT subsequently decreased to <3x ULN by day 98, resolving by the end of the study. The remaining four participants experienced ALT elevations (<3x ULN), which resolved (n=1) or were resolving (n=3) by the end of the study. Overall, all participants with elevated liver enzymes considered to be associated with fenusalam were asymptomatic, assessed as mild or moderate, and did not require discontinuation or interruption of fenusalam administration.

[0093] Most participants showed no baseline changes in coagulation parameters (such as PT, aPTT, D-dimer, and fibrinogen). Researchers reported no clinically significant changes in PT or aPTT. Three participants reported increased D-dimer as an adverse event (AE), two of which were potentially related and one potentially unrelated. All D-dimer elevations were considered mild and transient by researchers.

[0094] One participant (50 mg) experienced an increase in D-dimer (from 412 μg / L on day 29 to 1349 μg / L on day 42; reference range: 0 to 130 μg / L), which was considered potentially treatment-related, was mild, and persisted until the end of the study (526 μg / L). This participant had an increased D-dimer level (231 μg / L) prior to receiving phenobarbital, and this event was associated with moderate gastritis and mild ALT elevation (< 3x ULN).

[0095] Another participant (80 mg) experienced an increase in D-dimer (2480 μg / L on day 42; reference range: 0–590 μg / L), which was judged to be treatment-related and of mild intensity. The participant had a history of HCV, and the increase in D-dimer was associated with a mild elevation in ALT (< 3x ULN). The patient participated in the open-label extension study, continued receiving a fixed monthly dose of fetuslanth, and did not experience a recurrence of D-dimer increase, which was reported as an adverse event (AE). Figure 3A and 3B ).

[0096] The last participant with an increased D-dimer level (2090 μg / L on day 42; reference range: 0 to 590 μg / L) (80 mg) was deemed unlikely to be associated with phenocysteine ​​treatment and was of mild intensity. The participant had a history of HCV and an increased D-dimer level (690 μg / L) prior to receiving phenocysteine. This event was associated with a non-serious adverse event (AE) of mild liver enzyme elevation (considered treatment-related), and although administration continued, the AE resolved by the end of the study (450 μg / L). No association was observed between elevated D-dimer levels and bleeding treatment, abnormal liver enzymes, or phenocysteine ​​dosage.

[0097] A total of four SAEs were reported by study participants. One participant (18%) reported two SAEs of pneumonia and hamartoma, one reported duodenal ulcer bleeding, and one reported muscle bleeding. None of the SAEs were considered related to the study drug, and all SAEs were resolved.

[0098] Example 3: Pharmacokinetics and Pharmacodynamics of Fetuslan Therapy

[0099] Another objective of the study described in Example 1 was to confirm the pharmacokinetics (PK) of placeuxan and to evaluate its effects on AT activity and thrombin generation (PD). The PK / PD population included all participants who had received at least one dose of placeuxan and had at least one evaluable plasma sample.

[0100] To evaluate PK / PD analysis, a colorimetric analysis based primarily on activity (INNOVANCE of automated coagulation analyzers) is used. ®Antithrombin assay (Siemens BCSxp; lower limit of quantitation (LLOQ) of 3.13 ng / mL) and a calibrated automated coagulation map assay (Thromboscope BV, Maastrict) were used to determine plasma AT protein levels and thrombin generation. Real-time analysis of tissue factor-triggered thrombin generation was performed using an affinity fluorescent substrate. Fluorescence was read using Thermo Fluoroskan and reported as peak height. AT activity in human plasma was measured using a validated colorimetric assay to quantify functionally activated AT, calibrated according to the World Health Organization (WHO) reference plasma standard (LLOQ of 5% AT activity). Phthuloslan PK parameters were calculated from plasma concentration-time data using non-compartmental analysis and Phoenix WinNonlin software.

[0101] 1. Pharmacokinetics

[0102] Following administration of single doses of 50 mg and 80 mg phenocysteine, the mean peak plasma concentration (C) of the drug on day 0 was... max The levels were 85.4 and 142.9 ng / mL on day 0 and day 56 (3 months after administration), respectively, and 96.5 and 157.1 ng / mL on day 56 (3 months after administration). In both dosage groups, the levels were approximately 4 hours after administration (T0 and T56) on both assessment days. max Reaching C max Plasma levels of fetuslane decreased rapidly; the mean elimination half-life ranged from 3.4 to 5.2 hours, similar to previously observed results (Pasi, ibid.). Fetuslane PK parameters were similar in hemophilia patients with or without inhibitory factors.

[0103] Table 4 below provides phenotype plasma PK parameters for hemophilia patients with inhibitory factors on day 0 (after the first month of SC administration) and day 56 (after the third month of SC administration). AUC in Table 4 inf AUC refers to the area under the curve extrapolated to infinity. last The area under the curve refers to the final measurable concentration; CL / F refers to the apparent clearance rate; C max This refers to the maximum plasma concentration; CV refers to the coefficient of variation; t 1 / 2 This refers to eliminating the half-life; t max This refers to the time to peak plasma concentration; and Vz / F refers to the volume of distribution. AUC on day 56 after repeat dosing was not reported. inf value.

[0104] Table 4

[0105]

[0106] 2. Pharmacodynamics

[0107] AT activity was measured as a percentage (normal range is approximately 80% to 120% of activity). In the 50 mg and 80 mg dose groups of fetuslanth, the mean (standard error of mean [SEM]) baseline AT levels were 109.5% (4.4) and 100.2% (4.8) of normal, respectively. AT activity decreased progressively, initially becoming apparent on day 7 and reaching its maximum after day 28. The maximum mean (SEM) reduction in AT activity (as a percentage change from baseline AT levels) was similar between the dose groups, at 82.0% (2.2) in the 50 mg dose group and 87.4% (0.7) in the 80 mg dose group. The minimum residual post-dosage AT level observed in the 80 mg dose group was 9.8%. AT activity decreased over time from baseline mean (SEM) as follows: Figure 4A As shown, a decrease in AT levels is associated with an increase in thrombin production. Figure 4B ).

[0108] The height of the thrombin peak was associated with the degree of AT reduction in both dose groups, and an AT reduction of ≥75% from baseline resulted in a median thrombin peak of 68.05 nM, which falls within the lower range (64 to 210 nM) previously observed in healthy volunteers. Figure 5 (Pasi, same as above).

[0109] Example 4: Reducing annual bleeding frequency using fittuslans therapy

[0110] One of the objectives of the clinical study described in Example 1 was to evaluate the effect of fetuslane on annual bleeding frequency (ABR). All patients were evaluated.

[0111] To assess the effect of phenotype on adverse blood flow (ABR), a detailed hemophilia history was collected to determine participants' historical ABR over a 6-month period. During the study, all participants maintained a study-specific diary documenting all bleeding events, BPA administration, and responses to BPA treatment. Based on phenotype's mechanism of action, the expected initiation period for achieving an AT reduction of ≥75% was approximately 28 days. Bleeding events during the initiation period were defined as those occurring from the date and time of the first dose of the study drug up to day 28 (inclusive). Bleeding events during the observation period were defined as those occurring from 4 weeks after the first dose (first dose date plus 29 days) to 8 weeks after the last dose of phenotype (last dose date plus 56 days), or the date of the last study visit, whichever was earlier. The cause and location of bleeding events, as well as the dose of factor and BPA treatment administered at each bleeding event, were recorded.

[0112] Table 5 below lists the ABR (Average Bleeding Rate) of study participants after monthly administration of fetusilan compared to the historical annual bleeding rate before fetusilan treatment. Historical ABR is the estimated number of bleeding events within the past 12 months. The start-of-study ABR is from day 1 to day 28 of the study, and the observation-study ABR is from day 29 to 8 weeks after the last dose.

[0113] Table 5

[0114]

[0115] During the initiation period (days 1–28) and the observation period (day 29 to week 8 after the last dose), the median ABR in both dose groups was lower than the historical median ABR (Table 5). The median total ABR during the observation period (n=17) was 0 (range 0–46) in both the 50 mg and 80 mg dose groups (n = 17), compared to a historical median ABR of 36 (range 0–80). The median (range) observed ABR was 7 (range 46) bleeding events in the 50 mg dose group and 0 (range 45) bleeding events in the 80 mg dose group, compared to 33 (range 80) and 36 (range 8–54) bleeding events before treatment, respectively (Table 5). Overall, 11 / 17 (65%) of participants recorded 0 bleeding events during treatment with fetuslane and had an ABR of 0 during the observation period. Bleeding management was achieved by comparing the number of injections per bleeding episode with the participants' assessment of the amount of BPA required to control bleeding, and by comparing bleeding outcomes before the administration of fetuslanth at the same location. When AT was reduced by ≥75%, 6 cases of bleeding occurred with aPCC and 13 cases with rFVIIa. Overall, 83.3% (5 / 6) of bleeding treated with aPCC used less aPCC than normal, and 61.5% (8 / 13) of bleeding treated with rFVIIa used less rFVIIa than normal. The BPA dose range for rFVIIa was 93–133 μg / kg (median 108.6 μg / kg) and for aPCC, it was 14–75 U / kg (median 28.6 U / kg).

[0116] Example 5: Improvement of Patient-Reported Outcomes Through Fertility Treatment

[0117] Another objective of the study was to assess patient-reported outcomes (PROs), including participants' reported hemophilia-related symptoms (pain, swelling, and joint pain), physical health (pain during movement and difficulty walking), and overall quality of life. PROs are an important indicator of the effectiveness of hemophilia treatment. In the study described in Example 1, PROs were collected using the Haem-A-QoL questionnaire and the EuroQol 5-Dimensional (EQ-5D) questionnaire. All patients were assessed.

[0118] Table 6 below reports the changes in Haem-A-QoL category scores compared to baseline scores. Overall, all category scores improved with treatment, as evidenced by the change in the mean of baseline scores (Table 6).

[0119] Table 6

[0120]

[0121]

[0122] Notably, the changes in mean (SEM) from baseline to the end of the study (day 112) in both the Haem-A-QoL total score (–9.2 [2.9]) and the physical health category score (−12.3 [3.9]) (lower scores represent better HRQoL) showed clinically meaningful improvements based on the published threshold (Wyrwich, ibid.). Furthermore, scores in all categories improved with treatment across the entire population, as evidenced by the changes in mean baseline scores (Table 6). Additionally, the changes in PRO scores appeared to be dose-dependent. Generally, subjects at 80 mg and 50 mg doses showed greater effect sizes.

[0123] The results indicate that fituslane therapy has a beneficial effect on patient experience and their quality of life, including joint health. It is also noteworthy that all other categories of Haem-A-QoL showed numerical reductions (i.e., improvements), which appear to be dose-dependent, with a stronger effect observed in the 80 mg group.

[0124] This disclosure relates to the following implementation plan.

[0125] 1. A method for improving joint function in a hemophilia patient, comprising subcutaneously administering fitusiran to a hemophilia patient in need, optionally wherein the patient is a hemophilia A or B patient with or without an inhibitory factor.

[0126] 2. The method according to implementation plan 1, wherein the application reduces the difficulty of walking or increases mobility.

[0127] 3. A method for improving joint symptoms in patients with hemophilia, comprising subcutaneously administering phenocysteine ​​to a hemophilia patient in need, wherein the joint symptoms are selected from joint swelling, pain during movement, and joint pain, and optionally wherein the patient is a hemophilia A or B patient with or without an inhibitory factor.

[0128] 4. A method for improving patient-reported outcome (PRO) in patients with hemophilia, comprising subcutaneously administering phenotype lanthanum to a hemophilia patient in need, optionally wherein the patient is a hemophilia A or B patient with or without an inhibitory factor, and optionally wherein the PRO is improved in one or more quality of life categories.

[0129] 5. A method for improving the quality of life (QoL) of a patient with hemophilia, comprising administering phenocysteine ​​subcutaneously to a patient with hemophilia in need, wherein the QoL is improved in one or more QoL ranges, and optionally wherein the patient is a patient with hemophilia A or B with or without an inhibitory factor.

[0130] 6. The method according to any one of implementation schemes 1 to 5, wherein fetuslan is administered at a dose of 40 to 90 mg.

[0131] 7. According to the method of implementation scheme 4 or 5, wherein the one or more QoL categories are categories in a QoL questionnaire, optionally wherein the QoL questionnaire is the Adult Hemophilia Quality of Life Questionnaire (Haem-A-QoL).

[0132] 8. The method according to implementation plan 7, wherein the application results in a clinically meaningful improvement, expressed as a reduction of 7 or more units (optionally 8 or more, 9 or more, or 10 or more units) in one or more of the total score, exercise and leisure category score, and physical health category score of the questionnaire.

[0133] 9. The method according to any one of implementation schemes 1 to 8, wherein the patient is an adult or adolescent aged 12 years or older with hemophilia A or B, with or without an inhibitory factor.

[0134] 10. The method according to any one of implementation schemes 1 to 9, wherein the patient has hemophilia A.

[0135] 11. The method according to implementation plan 10, wherein the patient has received factor VIII or bypassing agent (BPA) treatment.

[0136] 12. The method according to implementation scheme 11, wherein the patient has an inhibitory factor, optionally wherein the level of the inhibitory factor is greater than 0.6 BU / mL, as determined by the Bethesda inhibitory factor test.

[0137] 13. The method according to implementation plan 10 or 11, wherein the patient does not have an inhibitory factor.

[0138] 14. The method according to any one of implementation schemes 1 to 9, wherein the patient has hemophilia B.

[0139] 15. The method according to implementation plan 14, wherein the patient has received factor IX or BPA treatment.

[0140] 16. The method according to implementation plan 15, wherein the patient has an inhibitory factor, optionally wherein the level of the inhibitory factor is greater than 0.6 BU / mL, as determined by the Bethesda inhibitory factor test.

[0141] 17. The method according to implementation plan 14 or 15, wherein the patient does not have an inhibitory factor.

[0142] 18. The method according to any one of embodiments 11 to 13 or 15 to 17, wherein the BPA is activated prothrombin complex concentrates (aPCC) and / or recombinant activated factor VII (rFVIIa).

[0143] 19. The method according to any one of embodiments 1 to 18, comprising administering multiple doses of fetuslane to the patient, each dose being 50 mg.

[0144] 20. The method according to any one of embodiments 1 to 18, comprising administering multiple doses of fetuslane to the patient, each dose being 80 mg.

[0145] 21. The method according to any one of embodiments 1 to 20, wherein phentollan is provided at a concentration of 50 to 200 mg / mL in phosphate-buffered saline (pH 7), optionally at a concentration of 100 mg / mL.

[0146] 22. The method of any one of implementation schemes 1 to 21, wherein fetuslan is administered to the patient once every four weeks or once a month.

[0147] 23. Fetuslan, used in the method according to any one of embodiments 1 to 22.

[0148] 24. An article of manufacture for use in any one of embodiments 1 to 22.

[0149] 25. The product used according to embodiment 24, wherein the product is a single-use vial containing 80 mg of fetuslan in 0.8 mL of phosphate-buffered saline (pH 7).

[0150] 26. The product used according to embodiment 24, wherein the product is a single-use prefilled syringe containing 80 mg of fetuslan in 0.8 mL of phosphate-buffered saline (pH 7).

[0151] 27. The product used according to embodiment 24, wherein the product is a single-use vial containing 50 mg of fetuslan in 0.5 mL of phosphate-buffered saline (pH 7).

[0152] 28. The product used according to embodiment 24, wherein the product is a single-use prefilled syringe containing 50 mg of fetuslan in 0.5 mL of phosphate-buffered saline (pH 7).

[0153] 29. Use of fetuslan in the preparation of a medicament for treating hemophilia A or B in any of the methods according to embodiments 1 to 22.

Claims

1. A method for improving joint function in a hemophilia patient, comprising subcutaneously administering fitusiran to a hemophilia patient in need, optionally wherein the patient is a hemophilia A or B patient with or without an inhibitory factor.

2. A method for improving joint symptoms in patients with hemophilia, comprising subcutaneously administering phenocysteine ​​to a hemophilia patient in need, wherein the joint symptoms are selected from joint swelling, pain during movement, and joint pain, and optionally wherein the patient is a hemophilia A or B patient with or without an inhibitory factor.

3. A method for improving patient-reported outcome (PRO) in patients with hemophilia, comprising subcutaneously administering phenotype lanthanum to a hemophilia patient in need, optionally wherein the patient is a hemophilia A or B patient with or without an inhibitory factor, and optionally wherein the PRO is improved in one or more quality of life categories.

4. A method for improving the quality of life (QoL) of a patient with hemophilia, comprising subcutaneously administering phenocysteine ​​to a patient with hemophilia in need, wherein the QoL is improved in one or more QoL ranges, and optionally wherein the patient is a patient with hemophilia A or B with or without an inhibitory factor.

5. An article of manufacture for use in the method according to any one of claims 1 to 4.

6. The article of claim 5, wherein the article is a single-use vial containing 80 mg of fetuslane in 0.8 mL of phosphate-buffered saline (pH 7).

7. The article of claim 5, wherein the article is a single-use prefilled syringe containing 80 mg of fetuslane in 0.8 mL of phosphate-buffered saline (pH 7).

8. The article of claim 5, wherein the article is a single-use vial containing 50 mg of fetuslane in 0.5 mL of phosphate-buffered saline (pH 7).

9. The article of claim 5, wherein the article is a single-use prefilled syringe containing 50 mg of fetuslane in 0.5 mL of phosphate-buffered saline (pH 7).

10. Use of fetuslan in the preparation of a medicament for treating hemophilia A or B in any of the methods according to claims 1 to 4.

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