Use of valbenazine, its active metabolites or pharmaceutically acceptable salts in the preparation of megakaryocytes or platelets

Abstract

This disclosure provides the use of valbenazine, its active metabolites, or pharmaceutically acceptable salts thereof in the preparation of megakaryocytes, wherein the preparation of megakaryocytes is carried out in vitro. The valbenazine, its active metabolites, or pharmaceutically acceptable salts thereof in this disclosure can promote megakaryocyte proliferation and platelet production, providing a promising strategy for megakaryocyte proliferation and platelet production.

Description

Technical Field

[0001] This disclosure relates to the field of biomedicine, and in particular, to the use of valbenazine or its pharmaceutically acceptable salts in the preparation of megakaryocytes or platelets. Background Technology

[0002] Megakaryocytes are a key cell subset in the bone marrow hematopoietic microenvironment, originating from hematopoietic stem cells. Through a unique process of intranuclear mitosis and cytoplasmic maturation, they ultimately release platelets as shed platelets. Platelets, as enucleated disc-shaped cytoplasmic fragments in the bloodstream, are not only the core effector units of physiological hemostasis and coagulation cascades, but also widely participate in physiological and pathological processes such as inflammatory responses, immune regulation, and tissue repair. In clinical medicine, abnormal platelet counts (such as thrombocytopenia) or functional impairment can lead to serious bleeding risks or thrombotic diseases. Although platelet transfusion is currently the main clinical treatment for thrombocytopenia, it relies primarily on donor donation and faces technical bottlenecks such as blood shortages, short shelf life (only 5-7 days at room temperature), susceptibility to bacterial contamination, and potential immune rejection. Furthermore, although current technologies have attempted to use stem cells to induce megakaryocytes and produce platelets in vitro, problems generally exist, including low induction efficiency, low platelet production per cell, and incomplete function of in vitro-generated platelets. Therefore, developing a highly efficient megakaryocyte culture system to regulate platelet production is a key technical challenge that urgently needs to be addressed in the current biomedical field. Summary of the Invention

[0003] Technical problems to be solved:

[0004] One aspect of this invention addresses the problem of the lack of efficient megakaryocyte culture systems for regulating platelet production in the prior art by providing a new use for valbenazine or its pharmaceutically acceptable salts.

[0005] Technical solution:

[0006] Use of valbenazine, its active metabolites or pharmaceutically acceptable salts in the preparation of megakaryocytes, wherein the preparation of megakaryocytes is the preparation of megakaryocytes in vitro.

[0007] Another aspect of this disclosure is the use of valbenazine, its active metabolites, or pharmaceutically acceptable salts thereof in the preparation of platelets, wherein the preparation of platelets is the preparation of megakaryocytes in vitro.

[0008] Another aspect of this disclosure is the use of valbenazine, its active metabolites, or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment or adjunctive treatment of diseases caused by megakaryopenia, including aplastic anemia, myelodysplastic syndrome, acquired pure megakaryocyte aplastic anemia thrombocytopenia, leukemia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, systemic sclerosis, or infectious diseases.

[0009] Another aspect of this disclosure is the use of valbenazine, its active metabolites, or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment or adjunctive treatment of complications arising from thrombocytopenia, including hemorrhagic complications and secondary complications.

[0010] The hemorrhagic complications include skin and mucous membrane bleeding, nosebleeds, menorrhagia, gastrointestinal bleeding, intracranial hemorrhage, and persistent bleeding after surgery or trauma; the secondary complications include anemia, infection, and organ dysfunction.

[0011] Furthermore, in some embodiments, the above-mentioned pharmaceutically usable salt may include an acid salt or a basic salt;

[0012] The acidic salts include hydrochloride, sulfate, phosphate, citrate, hydrobromide, hydroiodide, acetate, methanesulfonate, benzoate, salicylate, benzenesulfonate, p-toluenesulfonate, ethanesulfonate, ethanedisulfonate, naphthalenesulfonate, hydroxyethanesulfonate, tartrate, carbonate, citrate, gluconate, ascorbate, nicotinate, mandelate, pamoate, lactate, acetate, malate, maleate, fumarate, succinate, oxalate, stearate, valerate, or nitrate; the basic salts include sodium salt, calcium salt, potassium salt, magnesium salt, zinc salt, lithium salt, aluminum salt, meglumine salt, tromethamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, ethylenediamine salt, choline salt, procaine salt, benzethonium salt, lysine salt, arginine salt, or histidine salt.

[0013] Furthermore, in some embodiments, the pharmaceutically acceptable salt may be valbenazine dihydrochloride or valbenazine tosylate.

[0014] Another aspect of this disclosure is to provide a pharmaceutical composition comprising at least two compounds, one of which is valbenazine, its active metabolite, or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0015] Another aspect of this disclosure is the use of the above-described pharmaceutical composition in the preparation of a medicament for the treatment or adjunctive treatment of diseases caused by megakaryopenia, including aplastic anemia, myelodysplastic syndrome, acquired pure megakaryocyte aplastic anemia thrombocytopenia, leukemia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, systemic sclerosis, or infectious diseases.

[0016] Another aspect of this disclosure is the use of the above-described pharmaceutical composition in the preparation of a medicament for treating or adjunctive treatment of complications caused by thrombocytopenia, characterized in that the complications caused by thrombocytopenia include hemorrhagic complications and secondary complications.

[0017] The hemorrhagic complications include skin and mucous membrane bleeding, nosebleeds, menorrhagia, gastrointestinal bleeding, intracranial hemorrhage, and persistent bleeding after surgery or trauma; the secondary complications include anemia, infection, and organ dysfunction.

[0018] Another aspect of this disclosure is to provide a method for producing megakaryocytes in vitro, comprising the following steps:

[0019] Step 1) Obtain CD34 + cell;

[0020] Step 2) Purified CD34 + Cells are arranged at 1×10 5 The inoculum was seeded at a density of / mL into the culture vessel for directional differentiation culture, and cultured until day 3;

[0021] Step 3) Replace the culture medium containing vabenazine, its active metabolites, or its pharmaceutically acceptable salts at a final concentration of 200 nM every 3 days.

[0022] Step 4) Harvested megakaryocytes.

[0023] Beneficial effects:

[0024] The valbenazine, its active metabolites, or its pharmaceutically acceptable salts disclosed herein can promote megakaryocyte proliferation and platelet production, providing a promising strategy for megakaryocyte proliferation and platelet production. Attached Figure Description

[0025] Figure 1 In this embodiment of the present disclosure, after treatment with 200 nM valbenazine, CD41a on day 12... + Diagram of megakaryocyte proportions;

[0026] Figure 2A and Figure 2BThis is a microscopic image of megakaryocytes producing platelets on day 12 after treatment with 200 nM valbenazine in this embodiment of the present disclosure. Figure 2A As the DMSO control group, Figure 2B For VBZ group;

[0027] Figure 3 The CD41a levels detected by flow cytometry on day 15 after treatment with 200 nM valbenazine in this embodiment of the present disclosure. + Platelet ratio chart. Detailed Implementation

[0028] This disclosure provides the use of valbenazine, its active metabolites, or pharmaceutically acceptable salts thereof in the preparation of megakaryocytes or platelets. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the technical solutions of this disclosure. Furthermore, those skilled in the art can clearly modify or appropriately change and combine the content described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology disclosed herein.

[0029] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, the term "comprising" or its variations such as "including" or "comprises" will be understood to include the stated elements or components without excluding other elements or other components. The term "a" ("a", "an", and "the") includes plural indicators. The term "a plurality" means two or more. The terms "such as", "for example", etc., are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure.

[0030] In this disclosure, when a range of values ​​is provided, it should be understood that, unless the context otherwise explicitly indicates otherwise, the range includes endpoints and each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range and any value within a smaller range between specified values.

[0031] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0032] In this disclosure, terms such as "one embodiment," "an example," "some embodiments," "a particular embodiment," "related embodiment," "a certain embodiment," "some embodiments," "additional embodiment," or "further embodiment," "further implementation," or "another embodiment," "some other embodiments," mean that at least one feature or characteristic description is included in relation to the embodiment. Therefore, throughout this disclosure, the above phrases do not necessarily refer to the same embodiment. Furthermore, specific features may be combined in any suitable manner in one or more embodiments.

[0033] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common molecular biology terms can be found in Lewin's *GENES*, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Jones & Bartlett Learning. Definitions of common biochemistry terms can be found in Lehninger's *Principles of Biochemistry*, Eighth Edition, David L. Nelson, Michael M. Cox, WHFreeman. Definitions of common cell biology terms can be found in *Molecular Biology of the Cell*, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Garland Science. Definitions of common genetics terms can be found in *Genetics: Analysis of Genes and Genomes*, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Jones & Bartlett Learning.

[0034] Unless otherwise specified, the experimental techniques used in this paper employ standard techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as *Molecular Cloning: A Laboratory Manual* and *Cell Biology: A Laboratory Handbook*.

[0035] definition:

[0036] The term "valbenazine" in this disclosure, also referred to as "Valbenazine" or "NBI-98854," is a novel, highly selective VMAT2 inhibitor currently used clinically to treat tardive dyskinesia (TD). It exhibits good safety and tolerability and is administered once daily. It is an orally active compound containing two active metabolites: NBI-98782 ([+]α-dihydrobufenazine) and its oxidative metabolite NBI-136110. All three substances exhibit highly selective VMAT2 binding, which is the pharmacological target for treating hyperkinetic disorders. NBI-98854 is designed to deliver the active metabolites in a controlled manner, reducing peak plasma concentrations (i.e., low peak-to-trough ratios) and minimizing inter- and intra-pharmacokinetic variability. It is also designed to limit binding to non-target receptors (e.g., lack of affinity for dopamine D2 receptors) while maintaining a good safety profile. Clinical pharmacokinetic data indicate that NBI-98854 is rapidly absorbed upon oral administration, with active metabolites gradually forming, and maximum plasma concentrations reached within 4 to 10 hours after administration. The half-life of these compounds is approximately 20 hours, thus allowing for once-daily dosing (see Christopher F O'Brien, et al. NBI-98854, a selective monoamine transport inhibitor for the treatment of tardivedyskinesia: A randomized, double-blind, placebo-controlled study. Mov Disord. 2015 Oct;30(12):1681-7.). The structural formula of valbenazine is shown in Formula I below.

[0037] .

[0038] In some embodiments, the active metabolite of valbenazine is [+]-α-dihydrobutanazine (NBI-98782) as shown in Formula II or NBI-136110 as shown in Formula III.

[0039] .

[0040] The term "pharmaceutically acceptable salts" in this disclosure refers to biologically safe and non-toxic salt forms prepared to improve the physicochemical properties (such as solubility, stability, melting point, bioavailability, etc.) of a pharmaceutical active ingredient (API). In some embodiments, the aforementioned pharmaceutically acceptable salts are acidic or basic salts of the compound. For example, the above-mentioned acidic salts include, but are not limited to, hydrochloride, sulfate, phosphate, citrate, hydrobromide, hydroiodide, acetate, methanesulfonate, benzoate, salicylate, benzenesulfonate, p-toluenesulfonate, ethanesulfonate, ethanedisulfonate, naphthalenesulfonate, hydroxyethanesulfonate, tartrate, carbonate, citrate, gluconate, ascorbate, nicotinate, mandelate, pamoate, lactate, acetate, malate, maleate, fumarate, succinate, oxalate, stearate, valerate, or nitrate; the above-mentioned basic salts include, but are not limited to, sodium salts, calcium salts, potassium salts, magnesium salts, zinc salts, lithium salts, aluminum salts, meglumine salts, tromethamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, ethylenediamine salts, choline salts, procaine salts, benzethonium salts, lysine salts, arginine salts, or histidine salts.

[0041] In one embodiment, the pharmaceutically acceptable salt of the above-mentioned valbenazine is valbenazine dihydrochloride as shown in Formula IV. In another embodiment, the pharmaceutically acceptable salt of the above-mentioned valbenazine is valbenazine benzylsulfonate as shown in Formula V.

[0042] .

[0043] The term "complication" in this disclosure refers to another independent, usually undesirable, disease or pathological condition that arises secondarily during the development or treatment of a primary disease or medical procedure. It is not a major part of the disease, although it may be caused by the disease or an independent cause.

[0044] The term "pharmaceutically acceptable carrier" in this disclosure can refer to any pharmaceutically permissible additive, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and combinations thereof, stabilizers, surfactants, preservatives, isotonic agents, etc.

[0045] The term "hematopoietic stem and progenitor cells" (HSPCs) in this disclosure refers to hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs), a type of pluripotent stem cell found in bone marrow, peripheral blood, and umbilical cord blood. These cells possess self-renewal capacity and can differentiate into all types of mature blood cells (such as erythrocytes, leukocytes, and platelets) and immune cells, forming the core cell population that maintains the body's lifelong hematopoietic function. Through staged and multi-level proliferation and differentiation, they form progenitor cells of various lineages (such as myeloid progenitor cells and lymphoid progenitor cells), ultimately generating functionally specific blood cells. Hematopoietic progenitor cells, under the regulation of a specific microenvironment and certain factors, proliferate and differentiate into various types of blood cells. They are also a relatively primitive cell type with proliferative capacity, but have lost their multi-lineage differentiation ability and can only proliferate and differentiate into one or a few blood cell lineages in a directed manner; hence, they are also called committed stem cells. Human hematopoietic stem cells and hematopoietic stem and progenitor cells typically express the CD34 surface marker.

[0046] This disclosure describes a method for producing megakaryocytes in vitro:

[0047] In some embodiments, a method for culturing megakaryocytes in vitro is provided, the method comprising:

[0048] Step (1) Isolation and purification of CD34 from human umbilical cord blood + cell;

[0049] Step (2) on purified CD34 + Cells are arranged at 1×10 5 The culture medium was seeded at a density of / mL into 12-well plates for directional differentiation and cultured until day 3.

[0050] Step (3) Replace the culture medium containing vabenazine, its active metabolites or its pharmaceutically acceptable salts at a final concentration of 200 nM every 3 days;

[0051] Step (4) Collect differentiated megakaryocytes from the well plate on day 12.

[0052] The megakaryocytes carry CD41a. + Surface markings.

[0053] Example:

[0054] To enable those skilled in the art to better understand the technical solutions of this disclosure, the disclosure will be further described in detail below with reference to specific embodiments.

[0055] Example 1: Megakaryocytes and platelets were obtained through in vitro culture.

[0056] 1. Isolation and purification of human umbilical cord blood CD34 + cell.

[0057] 1) Isolate human umbilical cord blood mononuclear cells.

[0058] a. Mix human umbilical cord blood with an equal volume of separation buffer, add 1 / 4 of the total volume of hydroxyethyl starch, mix well, and let stand at room temperature for 40 min to allow the red blood cells to settle naturally.

[0059] b. Aspirate the supernatant and transfer it to a 50 mL centrifuge tube. Centrifuge at 1500 rpm for 15 min and discard the supernatant.

[0060] c. Resuspend the cell pellet in 5 mL of separation buffer;

[0061] d. Add 4.5 mL of Ficoll separation buffer to a 15 mL centrifuge tube, and carefully add 9 mL of the above cell suspension slowly onto the surface, keeping the interface clear. Centrifuge at 1800 rpm / 15 min;

[0062] e. Transfer the white membrane layer (monuclear cell layer) to a new 50 mL centrifuge tube, add separation buffer to 50 mL, centrifuge at 1200 rpm / 10 min, and discard the supernatant;

[0063] f. Resuspend the cells in 1 mL of sorting buffer and combine them into a single 15 mL centrifuge tube, bringing the total volume to 10 mL. Centrifuge at 1200 rpm for 5 min and discard the supernatant.

[0064] 2) CD34 + Cell magnetic bead sorting.

[0065] a. Resuspend each 10⁸ cells in 300 μl buffer, add 100 μl CD34 magnetic beads and 100 μl FcR blocker, mix gently, and incubate at 4°C for 30 min;

[0066] b. Add 10 times the labeled volume of sorting buffer, centrifuge at 1200 rpm / 5 min, and discard the supernatant;

[0067] c. Resuspend every 10 μL in 500 μl of sorting buffer. 8 One cell;

[0068] d. Place the MS sorting column in the magnetic field of the MACS sorter and rinse the sorting column with 500 μl of sorting buffer;

[0069] e. Add the cell suspension to the sorting column at a constant rate;

[0070] f. Wash the sorting column three times with 500 μl of sorting buffer;

[0071] g. Remove the sorting column from the magnetic field, place it on a new collection tube, add 1 mL of buffer solution, and quickly push out the positive cells using the matching stopcock;

[0072] h. Finally, high-purity CD34 was obtained. + cell.

[0073] 2. In vitro differentiation of megakaryocytes.

[0074] a. Resuspend CD34 in Stem Span™ SFEM serum-free medium supplemented with cytokines (TPO, SCF, IL-3). + Cells, at 1×10 5 Inoculate at an initial density of / mL into 12-well plates and incubate statically in a 37°C, 5% CO2 incubator;

[0075] b. Valbenazine (referred to as VBZ in this example) was added to the well plate on the third day as an experimental variable, with a final concentration of 200 nM.

[0076] c. Cells were always cultured in 12-well plates, and the culture medium was changed every three days. On the sixth day, the culture medium was changed to include TPO, SCF, IL-11 and valbenazine.

[0077] Example 2: Detection of the proportion of megakaryocytes in the late differentiation stage.

[0078] a. On the twelfth day of culture, collect the cell suspension in flow cytometry tubes, add 1 μl of CD41a to each group, and incubate in the dark at 4°C for 30 min;

[0079] b. Add 1 ml of PBE to each flow cytometer, centrifuge at 300 g for 5 min, discard the supernatant, and resuspend in 200 μl of PBE.

[0080] c. Add 1 μl of DAPI, incubate at room temperature in the dark for 10 min, and then detect CD41a by flow cytometry. + The proportion of megakaryocytes;

[0081] d. The result is as follows Figure 1 As shown, compared with the DMSO group, the experimental group with 200 nM valbenazine significantly increased CD41a. + The proportion of megakaryocytes.

[0082] Example 3: Detection of megakaryocytes and platelets obtained from platelet production.

[0083] a. On the fifteenth day of culture, microscopic images were taken of the well plates of the control group and the experimental group;

[0084] b. The result is as follows Figure 2A , Figure 2BAs shown, compared with the DMSO group, the experimental group with 200 nM valbenazine significantly increased the proportion of megakaryocytes that produce platelets;

[0085] c. Collect the cell suspension in a flow cytometry tube, add 1 μl CD41a and 1 μl hoechst to each group, and incubate in the dark at room temperature for 30 min.

[0086] d. Centrifuge each flow cytometer at 800 rpm for 5 min and collect the supernatant;

[0087] e. Flow cytometry detection of CD41a + The proportion of platelet granules;

[0088] f. The result is as follows Figure 3 As shown, compared with the control group, the experimental group with 200 nM valbenazine significantly increased the proportion of platelets produced.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of valbenazine or its pharmaceutically acceptable salts in the preparation of megakaryocytes, characterized in that, The preparation of megakaryocytes involves in vitro preparation of megakaryocytes, including the following steps: Step 1) Obtain CD34 + cell; Step 2) Purified CD34 + Cells are arranged at 1×10 5 The inoculum was seeded at a density of / mL into the culture vessel for directional differentiation culture, and cultured until day 3; Step 3) Replace the culture medium with vabenazine or its pharmaceutically acceptable salts at a final concentration of 200 nM every 3 days; Step 4) Harvested megakaryocytes.

2. The use of valbenazine or its pharmaceutically acceptable salts in the preparation of platelets, characterized in that, The platelet preparation involves preparing megakaryocytes in vitro and increasing the proportion of megakaryocytes that produce platelets, and includes the following steps: Step 1) Obtain CD34 + cell; Step 2) Purified CD34 + Cells are arranged at 1×10 5 The inoculum was seeded at a density of / mL into the culture vessel for directional differentiation culture, and cultured until day 3; Step 3) Replace the culture medium with vabenazine or its pharmaceutically acceptable salts at a final concentration of 200 nM every 3 days; Step 4) Harvested megakaryocytes.

3. The use according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salt is valbenazine dihydrochloride or valbenazine tosylate.

4. A method for producing megakaryocytes in vitro, characterized in that, Includes the following steps: Step 1) Obtain CD34 + cell; Step 2) Purified CD34 + Cells are arranged at 1×10 5 The inoculum was seeded at a density of / mL into the culture vessel for directional differentiation culture, and cultured until day 3; Step 3) Replace the culture medium with vabenazine or its pharmaceutically acceptable salts at a final concentration of 200 nM every 3 days; Step 4) Harvested megakaryocytes.

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