Belmosudil administration method for the treatment of multiple myeloma

Abstract

The present disclosure provides methods of administering belmosudil to patients with multiple myeloma.

Description

[Technical Field]

[0001] The present disclosure relates to methods of administering belmosudil to a patient for the treatment of multiple myeloma. [Background technology]

[0002] Multiple myeloma (MM) is a malignant neoplasm of plasma cells that accumulates in the bone marrow and leads to kidney failure, hypercalcemia, bone destruction, and anemia due to bone marrow failure. MM accounts for approximately 1.8% of all cancers and 18.7% of hematologic malignancies in the United States (Siegel et al. CA Cancer J Clin 2021, 71:7-33). MM is most frequently diagnosed in people aged 65 to 74, with a median age of 69 (National Cancer Institute. Surveillance, Epidemiology, and End Results Program. Cancer Stat Facts: Myeloma. Accessed November 24, 2021. Available at: https: / / seer.cancer.gov / statfacts / html / mulmy.html). The American Cancer Society estimates that 34,920 new cases of MM will be diagnosed in the United States in 2021, resulting in 12,410 deaths (Siegel et al.). There is a great need for additional therapies to treat multiple myeloma.

[0003] The level of CD38 expression on MM cells is an important factor affecting the response to anti-CD38 antibodies such as isatuximab and daratumumab (Kitadate et al. Haematologica. 2020, 105(1):e37-e40). Upon binding to CD38-expressing MM cells, anti-CD38 antibodies such as isatuximab and daratumumab induce tumor cell death via antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as well as direct apoptosis (Zhu et al. Front Immunol. 2020;11,1771).

[0004] Several studies have reported that CD38 expression decreases in bone marrow and MM cells after initial treatment with daratumumab (Horenstein et al. Cells. 2015, 4(3), 520-37; Krejcik et al. Clin Cancer Res. 2017, 23(24), 7498-511; Nijhof et al. Blood. 2016, 128(7), 959-70), and that CD38 expression increases again after daratumumab discontinuation (Nijhof et al.). Furthermore, isatuximab reduces CD38 expression in some MM cell lines by inducing CD38 internalization (Moreno et al. Clin Cancer Res. 2019, 25(10), 3176-87).

[0005] In vitro studies using well-characterized human MM cell lines are recognized as suitable models. See, e.g., Kassem et al, Blood 2022 Feb 24, 139(8):1160-1176 ("Kassem"), describing the MOLP-8 MM cell line; Matsuo et al, Leuk Res 2004 Aug, 28(8):869-77; and Deckert et al, Clin Cancer Res 2014 Sep 1, 20(17):4574-83; De Veirman K et al, Oncotarget 2015, 6:10532-10547 ("De Veirman"), describing the RPMI-8226 MM cell line; Li M et al, Cancer Cell Int 2021 Dec 19, 21(1):683 ("Li"); and Moreaux J et al, Haematologica, 2011 Apr, 96(4):574-82 ("Moreaux"); De Veirman; and Li; describing the LP-1 MM cell line; and Chauhan D et al, Blood 2004 Apr 15, 103(8):3158-66 and Yasui H et al., Blood 2005 Jul 15, 106(2):706-12, describing the MM.1R cell line. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides one solution to the problem of multiple myeloma by providing methods of treating multiple myeloma with belmosudil alone as well as in combination with anti-myeloma agents such as anti-CD38 antibodies or immunomodulatory drugs (IMiDs).

[0007] The present disclosure relates to a method of treating multiple myeloma, comprising administering to a subject in need thereof a therapeutically effective amount of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof (bermosudil).

[0008] The present disclosure also relates to a method of treating multiple myeloma, comprising administering to a subject in need thereof therapeutically effective amounts of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof (bermosudil), and an anti-CD38 antibody.

[0009] The present disclosure also relates to a method of treating multiple myeloma, comprising administering to a subject in need thereof therapeutically effective amounts of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof (bermosudil), and an immunomodulatory drug (IMiD).

[0010] The present disclosure also relates to a method of overcoming IMiD resistance in a subject with multiple myeloma, comprising administering to a subject in need thereof a therapeutically effective amount of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof (bermosudil), optionally an anti-CD38 antibody or an immunomodulatory drug (IMiD).

[0011] The present embodiments can be more fully understood by reference to the detailed description and examples that are intended to exemplify non-limiting embodiments. [Brief explanation of the drawings]

[0012] [Figure 1A-1B]1A and 1B show the effect of treatment with either belmosudil or pomalidomide on the growth of multiple myeloma cell lines. [Figure 2] FIG. 2 shows Western blots of c-Myc and b-actin after treatment of LP1 cells with either belmosudil or pomalidomide for 24 hours. [Figure 3A-3B] Figures 3A, 3B, 3C, and 3D show flow cytometry data. Data were analyzed using FlowJo software using the following strategy: (A) MOLP-8 cells excluding debris were gated, followed by single cell gating (B). From the single cells, viable DAPI cells were quantified (C). From the DAPI cells, CD38 expression was quantified using FITC fluorescent dye (D) and expressed as median fluorescence intensity (MFI). [Figure 3C-3D] Same as above. [Figure 4] FIG. 4 shows CD38 expression on MOLP-8 cells after 3 or 4 days of treatment with isotype control (IC) plus belmosudil, isatuximab plus belmosudil, or belmosudil alone compared to vehicle control (DMSO). [Figure 5A-5B] Figures 5A, 5B, 5C, 5D, and 5E show the viability of MOLP-8 cells after treatment with isotype control (IC) + belmosudil, isatuximab + belmosudil, or belmosudil alone compared to vehicle control (DMSO). [Figure 5C-5D] Same as above. [Figure 5E] Same as above. [Figure 6A] Figures 6A, 6B and 6C show the viability of LP1, RPM18226 and healthy donor (HD) derived NK cells following treatment with belmosudil compared to vehicle control (DMSO). [Figure 6B-6C] Same as above. [Figures 7A-7B]Figures 7A, 7B, and 7C show flow cytometry data. In Figures 7A-7C, data were analyzed using FlowJo software using the following strategy: gating on MOLP-8 cells excluding debris (A), followed by gating on single cells (B). From single cells, early apoptotic 7-AAD and Annexin V-eFluor 450 (C, Q1) and late apoptotic 7-AAD and Annexin V-eFluor 450 (C, Q2) cells were quantified (C). [Figure 7C] Same as above. [Figure 8] FIG. 8 shows the percent of apoptotic MOLP-8 MM cells after 2, 3, and 4 days of treatment with isotype control (IC) plus belmosudil, isatuximab plus belmosudil, or belmosudil alone compared to vehicle control (DMSO). [Figure 8-1] Continued from Figure 8. [Figure 9] FIG. 9 shows the cytotoxicity of LP-1 RFP cells with or without HD NK cells after treatment with belmosudil. [Figure 10] Figure 10 shows cytotoxicity of LP-1 RFP cells by HD NK cells after treatment with isotype control (IC, 100 ng / ml), isatuximab (100 ng / ml), belmosudil (3.3 μM), IC + belmosudil (100 ng / ml and 3.3 μM, respectively), or isatuximab + belmosudil (100 ng / ml and 3.3 μM, respectively). [Figures 11A-11D] Figures 11A-11G show graphs of cell viability, expressed as either cell number or percentage of viable cells, for MOLP-8 (Figures 11A and 11E), LP-1 (Figures 11C and 11G), and RPMI-8226 (Figures 11B and 11F) multiple myeloma cell lines, and HD NK cells (Figure 11D), following treatment with various concentrations of belmosudil and Y27632. [Figures 11E-11G] Same as above. [Figure 12]Figure 12 shows cytotoxicity of RPMI-8226 RFP cells by HD NK cells after treatment with isotype control (IC, 10 ng / ml), isatuximab (10 ng / ml), belmosudil (1.1 or 3.3 μM), IC + belmosudil (10 ng / ml and 1.1 or 3.3 μM, respectively), or isatuximab + belmosudil (10 ng / ml and 1.1 or 3.3 μM, respectively). [Figure 13] Figure 13 shows cytotoxicity of MM.1R RFP cells by HD NK cells after treatment with isotype control (IC, 10 ng / ml), isatuximab (10 ng / ml), belmosudil (1.1 or 3.3 μM), IC + belmosudil (10 ng / ml and 1.1 or 3.3 μM, respectively), or isatuximab + belmosudil (10 ng / ml and 1.1 or 3.3 μM, respectively). [Figure 14] Figure 14 shows cytotoxicity of LP-1 RFP cells by HD NK cells after treatment with isotype control (IC, 100 ng / ml), isatuximab (100 ng / ml), belmosudil (1.1 or 3.3 μM), IC + belmosudil (100 ng / ml and 1.1 or 3.3 μM, respectively), or isatuximab + belmosudil (100 ng / ml and 1.1 or 3.3 μM, respectively). [Figures 15A-15B] Figures 15A and 15B show the growth inhibitory activity of belmosudil (Rezurock) as a single agent in MM1S cells (Figure 15A) and LP-1 cells (Figure 15B). [Figure 16A] 16A-16C show the growth inhibitory activity of a combination of belmosudil and dexamethasone in MM1S cells. Figure 16A is a heat map showing the inhibition of cell growth at various concentrations of belmosudil and dexamethasone. [Figure 16B] Figures 16A-16C show the growth inhibitory activity of the combination of belmosudil and dexamethasone in MM1S cells. Figure 16B is a three-dimensional plot of the data in Figure 16A showing synergy on the Z axis. [Figure 16C]Figures 16A-16C show the growth inhibitory activity of the combination of belmosudil and dexamethasone in MM1S cells. Figure 16C is a plot of confluence over time for the single agents and the combination. [Figure 17A] Figures 17A to 17C show the growth inhibitory activity of a combination of belmosudil and dexamethasone in LP-1 cells. Figure 17A is a heat map showing the inhibition of cell growth at various concentrations of belmosudil and dexamethasone. [Figure 17B] Figures 17A-17C show the growth inhibitory activity of the combination of belmosudil and dexamethasone in LP-1 cells. Figure 17B is a three-dimensional plot of the data in Figure 17A showing synergy on the Z axis. [Figure 17C] Figures 17A-17C show the growth inhibitory activity of the combination of belmosudil and dexamethasone in LP-1 cells. Figure 17C is a plot of confluence over time for the single agents and the combination. DETAILED DESCRIPTION OF THE INVENTION

[0013] Belmosudil is an oral, selective inhibitor of rho-associated coiled-coil-containing protein kinase 2 (ROCK2). ROCK2 inhibition acts on fibrosis resulting from a dysregulated adaptive immune system and abnormal tissue repair. Belmosudil inhibits ROCK2 and ROCK1 with IC values ​​of approximately 100 nM and 3 μM, respectively. 50 Inhibit by value.

[0014] Belmosudil downregulated proinflammatory responses by modulating STAT3 / STAT5 phosphorylation and shifting the Th17 / Treg balance in ex vivo and in vitro human T cell assays. Belmosudil also inhibited aberrant profibrotic signaling in vitro. By regulating ROCK2 activity, belmosudil mediates signaling in immune cell function and fibrotic pathways, thereby alleviating the effects of this debilitating disease, including inflammation and fibrotic changes in multiple tissues, which can involve several organs, including the lungs, hepatobiliary system, musculoskeletal system, gastrointestinal (GI) tract, and skin. In vivo, belmosudil demonstrated activity in an animal model of chronic GVHD.

[0015] Belmosudil mesylate is commercially available in the United States and other countries as REZUROCK™ for the treatment of patients with chronic GVHD (cGVHD), in some cases after failure of at least two prior lines of systemic therapy. The compound belmosudil has the chemical name: 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide. The compound belmosudil is also known as KD025. The active pharmaceutical ingredient in REZUROCK™ has the molecular formula C with a molecular weight of 548.62 g / mol. 27 H 28 It has the chemical name 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide methanesulfonate (1:1), and is a bermosudil mesylate salt with N6O5S.

[0016] The chemical structure of belmosudil mesylate is as follows: [ka]

[0017] Belmosudil and methods for preparing the compound are described in the following U.S. patents: U.S. Pat. No. 8,357,693, U.S. Pat. No. 9,815,820, U.S. Pat. No. 10,183,931, and U.S. Pat. No. 10,696,660.

[0018] The present disclosure provides methods of administering bermosudil mesylate (REZUROCK™) to patients in need of treatment for multiple myeloma. Also disclosed are methods of administering bermosudil mesylate (REZUROCK™) in combination with an anti-myeloma drug, such as an anti-CD38 antibody or an immunomodulatory drug (IMiD), to patients in need of treatment for multiple myeloma.

[0019] definition As used herein, "about" includes the exact amount modified by the term "about," as well as an amount that is expected to be within experimental error, such as within 15%, 10%, or 5%. For example, "about 200 mg" means "200 mg," and also refers to a range of mg that is within experimental error, such as ±15%, 10%, or 5% of 200 mg. As used herein, the term "about" can be used to modify ranges and specific values.

[0020] As used herein (including, e.g., use of this term in connection with halting and / or resuming administration of an API, including a compound or bermosudil, to a subject), "administer" or "administered" refers to the act of prescribing a medication, including an API, for ingestion by a subject during treatment, dispensing the medication to a subject, and / or physically receiving or ingesting the medication. Thus, an API (e.g., a compound or bermosudil) can be "administered" by a physician or other healthcare professional who writes a prescription for the medication, and / or by a pharmacist who fills the prescription, and / or by a pharmacist who dispenses the medication to a subject; and / or by a patient or subject who ingests the medication, and / or their partner or caregiver who provides the medication to a subject, each of which can also "discontinue" and / or "resume" administration of the API.

[0021] It should be understood that when the term "belmosudil" is used herein, unless the context clearly indicates otherwise, this term can encompass any form of the compound belmosudil as well as its pharmaceutically acceptable salts. The term "belmosudil" refers to both the compound belmosudil (e.g., free base form, amorphous form, or crystalline form), pharmaceutically acceptable salts of belmosudil, such as the mesylate form used in REZUROCK™, and any form of belmosudil that can be used in a formulation or pharmaceutical composition for administering the compound to a patient.

[0022] A "line of treatment" or "line of therapy" describes the order or sequence in which different therapies are given to a patient as their disease progresses. The first treatment (first-line therapy) may not work or stop working after a period of time. After the first-line therapy is discontinued, a second, different treatment (second-line therapy) may be given. A subsequent line of therapy may be given if the second therapy does not work or stops working. Some patients may receive multiple lines of therapy over the course of their disease.

[0023] "Or" is used in its inclusive sense (equivalent to "and / or") unless the context requires otherwise.

[0024] As used herein, a "patient" or "subject" includes an animal or a human, and in one embodiment, a human.

[0025] "Pharmaceutical composition" means a mixture of substances suitable for administration to an individual, including a pharmaceutical agent. For example, a pharmaceutical composition may include a sterile aqueous solution or an API formulated into an oral dosage form, such as a tablet or capsule.

[0026] "Pharmaceutically acceptable salt" refers to physiologically and pharmaceutically acceptable salts of the compounds provided herein. "Pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acidic or basic moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, etc. The pharmaceutically acceptable salts of the present invention include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0027] "Relapse" or "relapsed" means the reappearance of signs and symptoms of a disease or condition after a period of improvement. In multiple myeloma, relapse is accompanied by one or more of the following: (1) a clear increase in the size of a pre-existing plasmacytoma or bone lesion; (2) hypercalcemia above normal calcium levels in the blood (>11 mg / dL); (3) a decrease in hemoglobin (the protein in red blood cells that carries oxygen) of 2 g / dL or more that is not related to treatment or other non-myeloma-related conditions; (4) a rise in serum creatinine (a muscle waste product in the blood) of 2 mg / dL or more attributable to myeloma since the start of treatment; (5) a rise in serum protein-related creatinine of 2 mg / dL or more that is related to the start of treatment. Hyperviscosity (thickened blood), (6) A 25% increase from the lowest confirmed response value in one or more of the following criteria: a) serum M protein (increase must be at least 0.5 g / dL), b) urinary M protein (increase must be at least 200 mg / 24 hours), c) if serum or urinary M protein cannot be measured, the difference between involved (abnormal, or monoclonal) and uninvolved (normal or polyclonal) free light chain levels (increase must be >10 mg / dL) (Kumar et al. Lancet Oncol, 2016, 17(8), E328-E346).

[0028] "Refractory" means a disease or condition that does not respond to treatment initially or becomes less responsive to treatment over time.

[0029] A "therapeutically effective amount" of an active pharmaceutical ingredient (API) means an amount that, when administered to a human for treating a disease (e.g., multiple myeloma), is sufficient to effect treatment of the disease state being treated. When applied to multiple myeloma in humans, "treating" or "treatment" includes (1) reducing the risk of developing multiple myeloma and / or inhibiting multiple myeloma, i.e., halting or reducing the development of multiple myeloma or its clinical symptoms; and (2) alleviating multiple myeloma, i.e., causing regression, reversal, or improvement of multiple myeloma, or reducing the number, frequency, duration, or severity of its clinical symptoms. A therapeutically effective amount of an API may vary depending on the health and physical condition of the treated subject, the degree of disease progression, an assessment of the medical condition, and other relevant factors.

[0030] Illustrative Embodiments In one embodiment, the present disclosure provides a method of treating multiple myeloma, comprising administering to a subject in need thereof a therapeutically effective amount of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof (belmosudil).

[0031] In some embodiments, belmosudil is administered as monotherapy. In some embodiments, belmosudil is administered in combination with an anti-CD38 antibody.

[0032] In some embodiments, the anti-CD38 antibody is daratumumab, isatuximab, MOR202, or mezagitamab (TAK-079). In some embodiments, the anti-CD38 antibody is isatuximab. In some embodiments, isatuximab is administered intravenously to a subject at a dose of 10 mg / kg or 20 mg / kg. In some embodiments, isatuximab is administered subcutaneously at a dose of 1000 mg or 1400 mg. In some embodiments, isatuximab is administered in 28-day cycles. In some embodiments, isatuximab is administered once weekly for the first 28-day cycle. In some embodiments, isatuximab is administered twice weekly for subsequent 28-day cycles. In some embodiments, after 12 months of isatuximab administration, isatuximab is administered once every four weeks.

[0033] In some embodiments, belmosudil is administered in combination with an immunomodulatory drug (IMiD).

[0034] In some embodiments, the IMiD is selected from pomalidomide, lenalidomide, thalidomide, iverdomide, and medigomid. In some embodiments, the IMiD is pomalidomide, and the pomalidomide is administered to the subject at a dose of 4 mg once daily on days 1 through 21 of each 28-day cycle. In some embodiments, the pomalidomide is administered orally.

[0035] In some embodiments, belmosudil is administered in a 28-day cycle. In some embodiments, belmosudil is administered to a subject at a dose of up to 1000 mg per day. In some embodiments, belmosudil is administered to a subject at a dose of 200 mg per day. In some embodiments, belmosudil is administered to a subject at a dose of 200 mg twice per day. In some embodiments, belmosudil is administered to a subject at a dose of 400 mg per day.

[0036] In some embodiments, belmosudil is administered to a subject at doses of 500 mg / day, 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, and 1000 mg / day. In some embodiments, belmosudil is administered orally.

[0037] In some embodiments, belmosudil is the mesylate salt of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide.

[0038] In some embodiments, the subject is receiving concomitant corticosteroid therapy, hi some aspects, the concomitant corticosteroid therapy is selected from dexamethasone, prednisone, and methylprednisolone.

[0039] In some embodiments, the method of treating multiple myeloma, comprising administering to a subject in need thereof a therapeutically effective amount of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof (bermosudil), further comprises administering isatuximab and dexamethasone.

[0040] In some embodiments, the method of treating multiple myeloma, comprising administering to a subject in need thereof a therapeutically effective amount of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof (bermosudil), further comprises administering pomalidomide and dexamethasone.

[0041] In some embodiments, the subject has received prior therapy to treat multiple myeloma. In some embodiments, the subject has relapsed multiple myeloma. In some embodiments, the subject has relapsed and refractory multiple myeloma. In some embodiments, the multiple myeloma is smoldering multiple myeloma.

[0042] In some embodiments, the treatment overcomes IMiD resistance in a subject with multiple myeloma.

[0043] In some embodiments, there is provided a use of a therapeutically effective amount of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof (bermosudil) for the preparation of a medicament for treating multiple myeloma in a subject in need thereof.

[0044] In some embodiments, there is provided a compound comprising a therapeutically effective amount of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof (bermosudil) for use in treating multiple myeloma in a subject in need thereof.

[0045] Administration of Belmosudil In some embodiments, belmosudil is administered to a subject at a dose of about 1000 mg / day, 900 mg / day, 800 mg / day, 700 mg / day, 600 mg / day, or 500 mg / day. In some embodiments, belmosudil is administered to a subject at a dose selected from 200 mg / day, 200 mg twice / day, and 400 mg / day. In some embodiments, the dose is 200 mg daily. In some embodiments, the dose is 200 mg twice daily. In some embodiments, the dose is 400 mg daily.

[0046] In some embodiments, belmosudil is administered in 28 day cycles.

[0047] Belmosudil tablets In one embodiment, belmosudil is formulated into tablets for oral administration. Belmosudil mesylate is a yellow powder that is substantially insoluble in water. Belmosudil tablets can be prepared for oral administration. Each tablet contains 200 mg of the free base, equivalent to 242.5 mg of belmosudil mesylate. The tablets may also contain the following inactive ingredients: microcrystalline cellulose, hypromellose, croscarmellose sodium, colloidal silicon dioxide, and magnesium stearate. The tablet film is composed of polyvinyl alcohol, polyethylene glycol, talc, titanium dioxide, and yellow iron oxide. Each 200 mg tablet is a pale yellow, film-coated oval tablet with "KDM" debossed on one side and "200" debossed on the other. The tablets are stored at room temperature, 20°C to 25°C (68°F to 77°F), with excursions of 15°C to 30°C (59°F to 86°F) permitted.

[0048] Multiple myeloma In vitro studies using well-characterized MM cell lines are recognized as suitable models, and therefore studies in the MM cell lines MOLP-8, LP-1, RPMI-8226, and MM.1R are understood to be relevant in vitro models for evaluating multiple myeloma therapeutic agents. For example, Kassem S et al,Blood 2022 Feb 24;139(8):1160-1176;Matsuo Y et al,Leuk Res.2004 Aug;28(8):869-77;Deckert J et al,Clin Cancer Res.2014 Sep 1;20(17):4574-83;De Veirman K et al. al,Oncotarget.2015;6:10532-10547;Li M et al,Cancer Cell Int.2021 Dec 19;21(1):683;Moreaux J et al,Haematologica.2011 Apr;96(4):574-82;Chauhan D et al,Blood 2004 Apr 15;103(8):3158-66; and Yasui H et al,Blood 2005 Jul 15;106(2):706-12.

[0049] The revised International Myeloma Working Group diagnostic criteria for multiple myeloma require clonal bone marrow plasma cells ≥ 10% or biopsy-proven osseous or extramedullary plasmacytoma and any one or more of the following myeloma-defining events: (1) evidence of end-organ damage attributable to an underlying plasma cell proliferative disorder, specifically: (a) hypercalcemia: serum calcium > 0.25 mmol / L (> 1 mg / dL) above the upper limit of normal or > 2.75 mmol / L (> 11 mg / dL); (b) renal insufficiency: creatinine clearance < 40 mL / min (measured or estimated by a validated formula) or serum creatinine > 177 mL / min. (c) anemia: hemoglobin level >20 g / L, below the lower limit of normal, or <100 g / L; or (d) bone lesions: one or more osteolytic lesions on skeletal radiography, CT, or PET-CT (two or more bone lesions are required to distinguish from solitary plasmacytoma with minimal bone marrow involvement when the bone marrow has less than 10% clonal plasma cells); (2) any one or more of the following biomarkers of malignancy: (a) clonal bone marrow plasma cell rate ≥60%; (b) involved: uninvolved serum free light chain ratio ≥100 (these values ​​are based on the serum Freelite assay (The Binding Site Group, Birmingham, UK), and involved free light chains must be ≥100 mg / L); or (c) >1 focal lesion on MRI study (each focal lesion must be ≥5 mm in size). (Rajkumar et al.Lancet Oncol.2014,15(12),e538-48).

[0050] The revised International Myeloma Working Group diagnostic criteria for smoldering multiple myeloma require both of the following criteria: (1) serum monoclonal protein (IgG or IgA) ≥ 30 g / L or urinary monoclonal protein ≥ 500 mg / 24 hours and / or clonal bone marrow plasma cells 10–60%; (2) absence of myeloma-defining events or amyloidosis (Rajkumar et al.).

[0051] In one embodiment, PET-CT = 18F-fluorodeoxyglucose PET and CT should be performed. Clonality should be established by demonstrating kappa / lambda light chain restriction by flow cytometry, immunohistochemistry, or immunofluorescence. The percentage of bone marrow plasma cells should preferably be estimated from a core biopsy specimen; if there is discordance between the aspirate and core biopsy, the highest value should be used.

[0052] CRAB is an acronym for the most common symptoms of multiple myeloma: hypercalcemia, renal failure, anemia, and bone lesions.

[0053] Multiple myeloma can be classified into different types based on the immunoglobulins overproduced by the myeloma cells. The majority of myeloma patients have immunoglobulin G (IgG) or immunoglobulin A (IgA) myeloma. Rare subtypes are immunoglobulin D (IgD) and immunoglobulin E (IgE) myeloma. Approximately 15%–20% of patients have light chain myeloma (Bence-Jones myeloma). Approximately 1%–5% of patients have non-secretory myeloma. IgM myeloma is a very rare subtype, typically occurring in Waldenstrom's macroglobulinemia.

[0054] Drugs used to treat multiple myeloma include proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), monoclonal antibodies (MAbs), and corticosteroids. Bisphosphonates may also be used to manage myeloma-associated bone disease.

[0055] In transplant-eligible patients, initial treatment may include induction therapy or first-line therapy before autologous stem cell transplantation (ASCT). Induction therapy included bortezomib, lenalidomide, and dexamethasone (VRd); carfilzomib, lenalidomide, and dexamethasone; daratumumab, lenalidomide, bortezomib, and dexamethasone; ixazomib, lenalidomide, and dexamethasone; bortezomib, cyclophosphamide, and dexamethasone (VCD or CyBorD); bortezomib, doxorubicin, and dexamethasone; carfilzomib, cyclophosphamide, and dexamethasone; ixazomib, cyclophosphamide, and dexamethasone; bortezomib, thalidomide, and dexamethasone (VTD); cyclophosphamide, lenalidomide, and dexamethasone; lenalidomide and dexamethasone. (RD); bortezomib and dexamethasone (VD); daratumumab and hyaluronidase; bortezomib, melphalan and prednisone; daratumumab and hyaluronidase, lenalidomide and dexamethasone; daratumumab and hyaluronidase, bortezomib, thalidomide and dexamethasone; daratumumab, carfilzomib, lenalidomide and dexamethasone; daratumumab, cyclophosphamide, bortezomib and dexamethasone; daratumumab, bortezomib, thalidomide and dexamethasone; or combinations of dexamethasone, thalidomide, cisplatin, doxorubicin, cyclophosphamide, etoposide and bortezomib.

[0056] After a maximal response to induction therapy is achieved, autologous stem cell transplantation (ASCT) followed by maintenance therapy may be used, including lenalidomide; ixazomib; bortezomib; or bortezomib and lenalidomide with or without dexamethasone.

[0057] Initial treatments for patients who are not transplant eligible include bortezomib, lenalidomide, and dexamethasone; daratumumab, lenalidomide, and dexamethasone; ixazomib, lenalidomide, and dexamethasone; daratumumab, bortezomib, melphalan, and prednisone; or daratumumab, cyclophosphamide, bortezomib, and dexamethasone. Maintenance treatments for patients who are not transplant eligible include lenalidomide, ixazomib, bortezomib, or bortezomib and lenalidomide.

[0058] Therapies for the treatment of relapsed multiple myeloma include bortezomib, lenalidomide, and dexamethasone; carfilzomib, lenalidomide, and dexamethasone; daratumumab, bortezomib, and dexamethasone; daratumumab, carfilzomib, and dexamethasone; daratumumab, lenalidomide, and dexamethasone; ixazomib, lenalidomide, and dexamethasone; or isatuximab, carfilzomib, and dexamethasone. Therapies after two multiple myeloma treatments include ixazomib, pomalidomide, and dexamethasone; pomalidomide, bortezomib, and dexamethasone; isatuximab, pomalidomide, and dexamethasone; or daratumumab, pomalidomide, and dexamethasone.

[0059] Smoldering multiple myeloma (SMM) is a precancerous form of multiple myeloma and typically accounts for approximately 15% of newly diagnosed cases. It is diagnosed when low levels of M protein are found in the blood and a slightly increased number of plasma cells are found in the bone marrow. While many patients with SMM are asymptomatic, some experience mild symptoms such as mild anemia or a few small ossicular lesions. Many, but not all, SMM patients progress to multiple myeloma. The risk of progression is approximately 10% per year in the first five years after diagnosis, 3% between years five and ten, and approximately 1% in the following years.

[0060] Combination therapy In some embodiments, belmosudil is administered in combination with an anti-myeloma agent, such as an anti-CD38 antibody or an IMiD.

[0061] In some embodiments, belmosudil is administered in combination with an anti-CD38 antibody (e.g., a monoclonal antibody). Examples of anti-CD38 antibodies include, but are not limited to, isatuximab, daratumumab, MOR202, and mezagitamab (TAK-079).

[0062] Isatuximab, a cytolytic antibody against CD38, is a chimeric, resurfaced, and humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb). Isatuximab can be produced in a mammalian cell line (Chinese hamster ovary, CHO) using a fed-batch manufacturing process. Isatuximab is composed of two identical immunoglobulin kappa light chains and two identical immunoglobulin gamma heavy chains, with a total molecular weight of approximately 148 kDa.

[0063] SARCLISA® (isatuximab) Injection is a sterile, preservative-free, clear to slightly opalescent, colorless to slightly yellow solution essentially free of visible particles in single-dose vials for intravenous use. Each vial contains either 100 mg / 5 mL or 500 mg / 25 mL of isatuximab at a concentration of 20 mg / mL, with a pH of 6.0. Each mL of solution contains 20 mg of isatuximab, histidine (1.46 mg), histidine hydrochloride monohydrate (2.22 mg), polysorbate 80 (0.2 mg), sucrose (100 mg), and water for injection.

[0064] Isatuximab binds to CD38, which is expressed on the surface of hematopoietic cells and tumor cells, including multiple myeloma cells. It induces tumor cell apoptosis and activation of immune effector mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Isatuximab inhibits the ADP-ribosyl cyclase activity of CD38. Isatuximab can activate natural killer (NK) cells and suppress CD38-positive T regulatory cells in the absence of CD38-positive target tumor cells. The combination of isatuximab and pomalidomide enhanced ADCC activity and direct tumor cell killing in vitro compared to isatuximab alone, and enhanced antitumor activity in a human multiple myeloma xenograft model compared to isatuximab or pomalidomide alone.

[0065] In some embodiments, the anti-CD38 antibody is isatuximab and can be administered to an individual intravenously at a dose of 10 mg / kg or 20 mg / kg, hi other embodiments, isatuximab can be administered subcutaneously at a dose of 1000 mg or 1400 mg.

[0066] The anti-CD38 antibody can be administered in cycles. In some embodiments, the anti-CD38 antibody is isatuximab (either intravenously or subcutaneously) and is administered in 28-day cycles. In the first cycle, isatuximab is administered once a week (QW), for example, on days 1, 8, 15, and 22 of the cycle. In subsequent cycles, isatuximab can be administered twice a week (Q2W), for example, on days 1 and 15 of the cycle. After 12 months of isatuximab administration, isatuximab administration can be reduced to once every four weeks (Q4W), for example, at a dose of 10 mg / kg isatuximab on day 1 (e.g., Q4W), while maintaining safety and efficacy.

[0067] In some embodiments, the anti-CD38 antibody is daratumumab, and can be administered to an individual intravenously at a dose of 16 mg / kg. In other embodiments, daratumumab can be administered subcutaneously at a dose of 1800 mg. Daratumumab can be administered in a dosing regimen according to the published prescribing information for daratumumab injection for intravenous use (published at www.accessdata.fda.gov), or according to daratumumab and hyaluronidase-fihj for intravenous and subcutaneous formulations, respectively.

[0068] In some embodiments, belmosudil is administered in combination with an immunomodulatory agent (sometimes referred to herein as an IMiD). Examples of immunomodulatory agents include, but are not limited to, pomalidomide, lenalidomide, and thalidomide.

[0069] "Pomalidomide" is a thalidomide analog. Its chemical name is (RS)-4-amino-2-(2,6-dioxo-piperidin-3-yl)-isoindoline-1,3-dione and has the following chemical structure: [ka]

[0070] The empirical formula of pomalidomide is C 13 H 11It is N3O4 and has a gram molecular weight of 273.24. Pomalidomide is a yellow solid powder. It has limited solubility in organic solvents and low solubility in all pH solutions (approximately 0.01 mg / mL). Pomalidomide has a chiral carbon atom that exists as a racemic mixture of R(+) and S(-) enantiomers. Pomalidomide is available in 1 mg, 2 mg, 3 mg, and 4 mg capsules for oral administration. Each capsule contains pomalidomide as the active ingredient and the following inactive ingredients: mannitol, pregelatinized starch, and sodium stearyl fumarate. The 1 mg capsule shell contains gelatin, titanium dioxide, FD&C Blue 2, yellow iron oxide, white ink, and black ink. The 2 mg capsule shell contains gelatin, titanium dioxide, FD&C Blue 2, yellow iron oxide, FD&C Red 3, and white ink. The 3 mg capsule shell contains gelatin, titanium dioxide, FD&C Blue 2, yellow iron oxide, and white ink. The 4 mg capsule shell contains gelatin, titanium dioxide, FD&C Blue 1, FD&C Blue 2, and white ink.

[0071] "Dexamethasone" is a corticosteroid. Its chemical name is 9-fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione and has the following chemical structure: [ka]

[0072] The empirical formula for dexamethasone is C 22 H 29 It has a molecular weight of 392.47 grammes. Dexamethasone is a white to substantially white, odorless, crystalline powder that is stable in air and practically insoluble in water. Dexamethasone is also available as an injectable sodium phosphate ester for intravenous, intramuscular, intra-articular, soft tissue or intralesional use. [Example]

[0073] Example 1: Effect of belmosudil or pomalidomide on the proliferation of multiple myeloma (MM) cell lines The effect of belmosudil on MM cell growth was evaluated in vitro in a panel of MM cell lines with different genetic backgrounds (LP1, MM1R, MM1S, OPM2). Cell viability was measured by the CellTiter-Glo® Luminescent Cell Viability Assay normalized to DMSO. Cells were treated with either belmosudil or pomalidomide for 8 days. As shown in Figure 1A, 8-day treatment with belmosudil resulted in a dose-dependent inhibition of MM cell line growth, with IC 50 The concentrations varied from 1 to 7 mM. As shown in Figure 1B, pomalidomide also inhibited growth in all of these MM cell lines except for LP1 and KE97, which are known immunomodulatory drug (IMiD)-resistant cell lines (Leukemia (2014) 28, 1129-1174). Belmosudil inhibited growth of LP1, suggesting the absence of cross-resistance between belmosudil and IMiDs.

[0074] Consistent with MM growth inhibition, belmosudil induced a dramatic reduction in C-myc protein, a major tumor driver in LP1 cells. In contrast, pomalidomide had a very modest effect on C-myc protein in LP1 cells. These results are shown in Figure 2.

[0075] Overall, these data demonstrate that belmosudil induces growth inhibitory effects on a large panel of MM cell lines, does not have cross-resistance with IMiDs, and suggests that belmosudil can be used for the treatment of MM after or in combination with IMiDs.

[0076] Example 2: CD38 expression on MM cells and proliferation / viability of MM cell lines and NK cells In vitro assays were performed to evaluate: 1) the effect of belmosudil (alone or in combination with isatuximab) on the expression of CD38 on MOLP-8 MM cells; 2) the effect of belmosudil (alone or in combination with isatuximab) on the proliferation / viability of MOLP-8 cells; 3) the effect of belmosudil on the proliferation / viability of different MM cell lines (LP1 and RPMI8226); and 4) the effect of belmosudil on the proliferation / viability of healthy donor-derived NK cells (HD NK cells).

[0077] method MM cell line: MOLP-8 (DSMZ, #ACC569) were maintained in RPMI1640 complete medium (RPMI1640 supplemented with 20% FBS - Eurobio #CVFSVF06-01 and 1% L-glutamine - Gibco #25030-081) and incubated at 37°C with 5% CO2 before use.

[0078] LP1 (DSMZ, #ACC41) were maintained in IMDM medium supplemented with 20% FBS (Eurobio #CVFSVF06-01) and 1% L-glutamine (Gibco #25030-081) and incubated at 37°C with 5% CO2 before use.

[0079] RPMI8226 (ATCC, #CCL155) were maintained in RPMI1640 complete medium and incubated at 37°C with 5% CO before use.

[0080] Before treatment, MM cells were centrifuged at 300 g for 5 min, counted, and resuspended in RPMI1640 complete medium to 50,000 cells / 100 μl.

[0081] Isolation of human HD NK cells: Healthy donor buffy coats were provided by EFS Ile de France. Immediately upon receipt, acceptable buffy coats were gently agitated overnight at room temperature. The following day, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Cytiva #17-1440-02) density gradient centrifugation. Human NK cells were purified from PBMCs using Miltenyi Biotec's MACSxpress Whole Blood NK Cell Isolation Kit (#130-127-695) by manual magnetic labeling and negative selection using a MACSxpress separator (Miltenyi Biotec #130-098-308). Purified NK cells were then cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Eurobio #CVFSVF06-01) and 1% L-glutamine (Gibco #25030-081). Before use, the cells were incubated overnight at 37°C and 5% CO2.

[0082] Cell treatment: Compounds and cells were then added to appropriate wells of a Corning™ 96-Well Clear Ultra Low Attachment Microplate (Corning, #7007) (final volume in each well: 200 μl).

[0083] To evaluate the effects of belmosudil and / or isatuximab on the expression of CD38 on MOLP-8 and the proliferation / viability of MOLP-8, compounds and cells were added to the appropriate wells in the following order: (1) isatuximab (or isotype control) (pre-diluted in RPMI1640 complete medium) was added to the appropriate wells to a final concentration of 20 μg / ml; (2) belmosudil (or vehicle control - DMSO) was added to the appropriate wells to a final concentration of 1.1, 3.3, 10, 20, or 25 μM; then (3) MOLP-8 (50,000 cells / well) was added to the appropriate wells.

[0084] To evaluate the effects of belmosudil on the proliferation / viability of LP1, RPMI8226, and HD NK cells, compounds and cells were added to the appropriate wells in the following order: (1) belmosudil (or vehicle control - DMSO) was added to the appropriate wells to have final concentrations of 10, 15, 20, 25, and 40 μM; (2) LP1, RPMI8226, or HD NK cells (50,000 cells / well) were then added to the appropriate wells; (3) the plates were then centrifuged at 100 g for 1 minute and then placed in a 5% CO2, 37°C incubator for up to 4 days.

[0085] Flow cytometry: After treatment, cells were harvested and added to U-bottom plates for antibody labeling.

[0086] The cells were then centrifuged at 300g for 5 minutes, the supernatant removed, and 200μl of AutoMACS running buffer (Miltenyi Biotec, #130-091-221) was added to each well. This process was repeated twice. The cells were then centrifuged at 300g for 5 minutes, the supernatant removed, and 100μl of mouse anti-human CD38 primary antibody was added to the wells for 30 minutes at 4°C. 100μl of AutoMACS running buffer was then added, the cells were centrifuged at 300g for 5 minutes, the supernatant removed, and 200μl of AutoMACS running buffer was added to each well. The cells were then centrifuged at 300g for 5 minutes, the supernatant removed, and 100μl of AF488-conjugated AffiniPure goat anti-mouse IgG secondary antibody (Jackson Immuno Research #115-545-164) was added to the wells for 20 minutes at 4°C. Then, 100 μl of AutoMACS loading buffer was added, the cells were centrifuged at 300 g for 5 min, the supernatant was removed, and 200 μl of AutoMACS loading buffer was added to each well. Finally, the cells were centrifuged again at 300 g for 5 min and resuspended in 80 μl of DPBS.

[0087] To further assess cell viability, DAPI (Miltenyi Biotec, #130-111-570) was added to each well before plate reading by flow cytometry. Samples were then analyzed on a MACSQuant 16 flow cytometer (Miltenyi Biotec). Data were analyzed with FlowJo software using the following strategy, shown in Figure 3A-D: (A) gating on MOLP-8 cells excluding debris, followed by gating on single cells. (B) From single cells, viable DAPI was added. neg Cells were quantified (C) and DAPI neg CD38 expression was quantified from the cells using FITC fluorescent dye (D) and expressed as median fluorescence intensity (MFI).

[0088] result As shown in Figure 4, CD38 expression is reduced in MOLP-8 cells after 3 or 4 days of treatment with isatuximab (20 μg / ml) alone compared to the control. Belmosudil at 1.1 and 3.3 μM in combination with isatuximab (20 μg / ml) does not affect CD38 expression on MOLP-8 cells after 3 and 4 days of treatment. Belmosudil at a concentration of 10 μM induces an increase in CD38 expression on the surface of MOLP-8 cells after 3 and 4 days of treatment. Belmosudil at 20 μM and 25 μM also induces an increase in CD38 expression on MOLP-8 cells, but to a lesser extent than 10 μM. Importantly, belmosudil (10, 20, and 25 μM) in combination with isatuximab (20 μg / ml) preserves CD38 expression on the surface of MOLP-8 cells after 3 and 4 days of treatment.

[0089] As shown in Figures 5A-5E, no effect of belmosudil treatment was observed on MOLP-8 cell viability at low concentrations (1.1 μM and 3.3 μM) (Figures 5A and 5B, respectively). A limited effect of belmosudil on MOLP-8 cell viability (decreased) was observed at higher concentrations (10, 20, and 25 μM) starting 1 day after treatment (Figures 5C, 5D, and 5E).

[0090] As shown in Figures 6A-6C, belmosudil strongly reduced the viability of LP1 and RPMI8226 cells, known immunomodulatory drug (IMiD)-resistant cell lines (Leukemia (2014) 28, 1129-1174; Blood. 2011; 117(19):5157-5165), starting at 10 μM after 4 days of treatment (Figures 6A and 6B). Belmosudil had no or very limited effect on HD NK cell viability after 4 days of treatment (maximum 10% reduction at high concentrations of 25-40 μM).

[0091] Example 3: Apoptosis of MOLP-8 MM cells The proapoptotic effect of belmosudil (alone or in combination with isatuximab) on MOLP-8 MM cells was evaluated in vitro.

[0092] method MM cell line: MOLP-8 (DSMZ, #ACC569) were maintained in RPMI1640 complete medium (RPMI1640 supplemented with 20% FBS - Eurobio #CVFSVF06-01 and 1% L-glutamine - Gibco #25030-081) and incubated at 37°C with 5% CO2 before use.

[0093] Before treatment, MM cells were centrifuged at 300 g for 5 min, counted, and resuspended in RPMI1640 complete medium to 50,000 cells / 100 μl.

[0094] Cell treatment: Compounds and cells were then added to the appropriate wells of a Corning™ 96-Well Clear Ultra Low Attachment Microplate (Corning, #7007) (final volume per well: 200 μl) in the following order: (1) isatuximab (or isotype control) (pre-diluted in RPMI1640 complete medium) was added to the appropriate wells to a final concentration of 20 μg / ml; (2) belmosudil (or vehicle control - DMSO) was added to the appropriate wells to a final concentration of 1.1, 3.3, 10, 20, or 25 μM; then, (3) MOLP-8 (50,000 cells / well) was added to the appropriate wells; (4) the plate was then centrifuged at 100 g for 1 minute and then placed in a 5% CO 2 , 37°C incubator for up to 4 days.

[0095] Flow cytometry: Apoptosis was assessed using the Annexin V Apoptosis Detection Kit eFluor450 (Invitrogen, no. 88-8006-74).

[0096] After treatment, cells were harvested and added to U-bottom plates for antibody labeling.

[0097] The cells were then centrifuged at 300 g for 5 minutes, the supernatant removed, and 200 μl of D-PBS was added to each well. The cells were then centrifuged at 300 g for 5 minutes, the supernatant removed, and 200 μl of 1X buffer (from the Annexin V Apoptosis Detection Kit) was added to each well. The cells were then centrifuged at 300 g for 5 minutes, the supernatant removed, and 100 μl of Annexin V labeling solution (from the Annexin V Apoptosis Detection Kit) was added to the wells for 20 minutes at 4°C (according to the manufacturer's instructions).

[0098] 100 μl of 1× buffer was then added, the cells were centrifuged at 300 g for 5 minutes, the supernatant was removed, and 200 μl of 1× buffer was added to each well. The cells were then centrifuged at 300 g for 5 minutes, the supernatant was removed, and the cells were resuspended in 80 μl of D-PBS.

[0099] Additionally, 7-AAD (from the Annexin V Apoptosis Detection Kit) was added to each well prior to plate reading by flow cytometry. Samples were then analyzed on a MACSQuant 16 flow cytometer (Miltenyi Biotec). In Figures 7A-7C, data were analyzed using FlowJo software using the following strategy: gating on MOLP-8 cells excluding debris (A), followed by gating on single cells (B). From single cells, early apoptotic 7-AAD was detected. neg and Annexin V-eFluor450 pos (C, Q1) and late apoptotic 7-AAD pos and Annexin V-eFluor450 pos (C, Q2) Cells were quantified (C). The percentages of early and late apoptotic cells are shown in the graph.

[0100] result As shown in Figure 8, 10, 20, and 25 μM belmosudil induce apoptosis of MOLP-8 in a dose-dependent manner after 2, 3, and 4 days of treatment. Belmosudil at 20 and 25 μM in combination with isatuximab (20 μg / ml) further increases apoptosis of MOLP-8 MM cells in a dose-dependent manner after 2, 3, and 4 days of treatment.

[0101] Example 4: Long-term cytotoxicity against LP-1 MM cells Cytotoxicity against LP-1 RFP multiple myeloma cells (target cells) was assessed in the presence of healthy donor (HD)-derived NK cells (effector cells) after treatment with isatuximab in combination with belmosudil. Cytotoxicity was measured over time using the Incucyte® Live Cell Imaging and Analysis System (Essen Bioscience).

[0102] method Generation of RFP+LP1 MM cells: LP-1 LP-1 cells (DSMZ, #ACC41) were infected with Incucyte® Nuclight Red Lentivirus (Sartorius) to express red fluorescent protein (RFP) and were generated by incubation at 37°C and 5% CO2 in IMDM medium (Gibco, #12440053) supplemented with 20% fetal bovine serum (heat-inactivated FBS, Biowest, #S181H-100) and 1% L-glutamine (Gibco, #25030-024). LP-1RFP cells were centrifuged at 300g for 5 minutes and resuspended in RPMI1640 complete medium (RPMI1640 supplemented with 10% fetal bovine serum - FBS, Biowest, #S181H-100-, 1% L-glutamine - Gibco, #25030-024) before being added to Incucyte® plates (described in detail below).

[0103] Isolation of human HD NK cells: Healthy donor buffy coats were provided by EFS Ile de France. Immediately upon receipt, acceptable buffy coats were gently agitated overnight at room temperature. The following day, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Cytiva #17-1440-02) density gradient centrifugation. Human NK cells were purified from PBMCs using Miltenyi Biotec's MACSxpress Whole Blood NK Cell Isolation Kit (#130-127-695) by manual magnetic labeling and negative selection using a MACSxpress separator (Miltenyi Biotec #130-098-308). Purified NK cells were then cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Eurobio #CVFSVF06-01) and 1% L-glutamine (Gibco #25030-081). Before use, the cells were incubated overnight at 37°C and 5% CO2.

[0104] Incucyte® Cytotoxicity Assay: Cytotoxicity was assessed using the Incucyte® Live Cell Analysis System, which allows for quantification of the number of live fluorescent target cells over time.

[0105] Compounds and cells were added to appropriate wells of Incucyte® plates (Poly-D Lysine-treated 96-well flat-bottom microplates, CellCoat™, Greiner Bio-One; #655946) in the following order (final volume per well: 200 μl): (1) Isatuximab (or isotype control) (pre-diluted in RPMI 1640 complete medium) was added to the appropriate wells to give a final concentration of either 1, 10, 100, or 1000 ng / ml; (2) Vermosudil (pre-diluted in RPMI 1640 complete medium) was then added to the appropriate wells to give a final concentration of either 1.1, 3.3, or 10 μM; (3) LP-1 RFP cells (target cells, T) were then added to each well (to give 20,000 LP1-RFP cells / well); (4) HD NK cells (effector cells, E) were added to appropriate wells (60,000 cells added to have an E:T ratio of 3:1), and HD NK cells were not added to some wells to test the direct effect of belmosudil on LP-1 RFP cells.

[0106] The Incucyte® plates were then centrifuged at 100 g for 1 minute before being placed in an Incucyte® (IncucyteS3, EssenBio) in a dedicated incubator at 37°C and 5% CO2. Images (4 images / well) were taken every 4 hours using a 10x objective and standard scan type, using the phase and red channels. Growth of LP-1RFP target cells was monitored by fluorescence imaging for up to 90 hours, and the number of live target cells was quantified using IncucyteS3 software and normalized to the number of live target cells at time zero.

[0107] result As shown in Figures 9 and 10, belmosudil induces LP-1 cell killing in a dose-dependent manner over time, independent of the presence of HD NK cells (Figure 9). Belmosudil (1.1 or 3.3 μM) in combination with isatuximab (100 ng / ml) further increases LP-1 cell killing over time in the presence of HD NK cells (Figures 10 and 14).

[0108] Example 5: Effect of Belmosudil Compared to a Pan-ROCK Inhibitor (Y27632) on the Proliferation / Viability of Multiple Myeloma (MM) Cell Lines and NK Cells The effects of belmosudil compared to a pan-ROCK inhibitor (Y27632) on the proliferation and / or viability of different MM cell lines (MOLP-8, LP-1, and RPMI-8226) were evaluated, as well as the effects of belmosudil compared to a pan-ROCK inhibitor (Y27632) on the proliferation and / or viability of healthy donor-derived NK cells (HD NK cells).

[0109] method MM cell line: MOLP-8 (DSMZ, #ACC569) were maintained in RPMI1640 complete medium (RPMI1640 supplemented with 20% FBS - Eurobio #CVFSVF06-01 and 1% L-glutamine - Gibco #25030-081) and incubated at 37°C with 5% CO2 before use.

[0110] LP1 (DSMZ, #ACC41) were maintained in IMDM medium supplemented with 20% FBS (Eurobio #CVFSVF06-01) and 1% L-glutamine (Gibco #25030-081) and incubated at 37°C with 5% CO2 before use.

[0111] RPMI-8226 (ATCC, #CCL155) were maintained in RPMI1640 complete medium and incubated at 37°C with 5% CO before use.

[0112] Before treatment, MM cells were centrifuged at 300 g for 5 min, counted, and resuspended in RPMI1640 complete medium to 50,000 cells / 100 μl.

[0113] Isolation of human HD NK cells: Healthy donor buffy coats were provided by EFS Ile de France. Immediately upon receipt, acceptable buffy coats were gently agitated overnight at room temperature. The following day, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Cytiva #17-1440-02) density gradient centrifugation. Human NK cells were purified from PBMCs using Miltenyi Biotec's MACSxpress Whole Blood NK Cell Isolation Kit (#130-127-695) by manual magnetic labeling and negative selection using a MACSxpress separator (Miltenyi Biotec #130-098-308). Purified NK cells were then cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Eurobio #CVFSVF06-01) and 1% L-glutamine (Gibco #25030-081). Before use, the cells were incubated overnight at 37°C and 5% CO2.

[0114] Cell treatment: Compounds and cells were then added to the appropriate wells of a Corning™ 96-Well Clear Ultra Low Attachment Microplate (Corning, #7007) (final volume per well: 200 μl). To evaluate the effects of belmosudil or Y27632 on the proliferation and / or viability of MOLP-8, LP-1, RPMI-8226, and HD NK cells, compounds and cells were added to the appropriate wells in the following order: belmosudil or Y27632 (Merck, #688000) or vehicle control (DMSO) was added to the appropriate wells to give final concentrations of 5 (LP-1 cells only), 10, 15, 20, 25, and 40 μM. MOLP-8, LP-1, RPMI-8226, or HD NK cells (50,000 cells / well) were then added to the appropriate wells. The plates were then centrifuged at 100 g for 1 minute and then placed in an incubator at 37°C with 5% CO for up to 4 days.

[0115] Flow cytometry: After treatment, cells were harvested and added to U-bottom plates for antibody labeling.

[0116] The cells were then centrifuged at 300 g for 5 minutes, the supernatant removed, and 200 μl of AutoMACS running buffer (Miltenyi Biotec, #130-091-221) was added to each well. This process was repeated twice. The cells were then centrifuged at 300 g for 5 minutes, the supernatant removed, and 100 μl of mouse anti-human CD38 primary antibody (produced internally by Sanofi) was added to the wells for 30 minutes at 4°C (for belmosudil-treated cells only). 100 μl of AutoMACS running buffer was then added, the cells were centrifuged at 300 g for 5 minutes, the supernatant removed, and 200 μl of AutoMACS running buffer was added to each well. The cells were then centrifuged at 300 g for 5 minutes, the supernatant removed, and 100 μl of AF488-conjugated AffiniPure goat anti-mouse IgG secondary antibody (Jackson Immuno Research #115-545-164) was added to the wells for 20 minutes at 4° C. 100 μl of AutoMACS running buffer was then added, the cells were centrifuged at 300 g for 5 minutes, the supernatant removed, and 200 μl of AutoMACS running buffer was added to each well. Finally, the cells were centrifuged again at 300 g for 5 minutes and resuspended in 80 μl of DPBS.

[0117] To further assess cell viability, DAPI (Miltenyi Biotec, #130-111-570) was added to each well before plate reading by flow cytometry. Samples were then analyzed on a MACSQuant 16 flow cytometer (Miltenyi Biotec).

[0118] Data were analyzed using FlowJo software as shown in Figure 3. (Figure 3A) MOLP-8 cells excluding debris were gated, followed by gating for single cells (Figure 3B). From single cells, viable DAPI was measured. neg Cells were quantified (Figure 3C). DAPI neg From the cells, CD38 expression was quantified (only for bermosudil-treated cells) with FITC fluorescent dye (Figure 3D) and expressed as median fluorescence intensity (MFI).

[0119] result The data in Figures 11A-11G show that belmosudil reduces the viability (both cell number and % live cells) of MOLP-8, RPMI-8226, and LP-1 cells after 4 days of treatment, starting at 10 µM (5 µM for LP-1); Y27632 does not affect the viability (either cell number or % live cells) of MOLP-8, RPMI-8226 after 4 days of treatment; and neither belmosudil nor Y27632 affects HD NK cell viability after 4 days of treatment.

[0120] Example 6: Long-term cytotoxicity against RPMI-8226 and MM.1R MM cell lines Cytotoxicity against RPMI-8226 RFP and MM.1R RFP multiple myeloma cells (target cells) was assessed in the presence of healthy donor (HD)-derived NK cells (effector cells) after treatment with isatuximab in combination with belmosudil. Cytotoxicity was measured over time using the Incucyte® Live Cell Imaging and Analysis System (Essen Bioscience).

[0121] Generation of RFP-positive RPMI-8226 and MM.1R MM cells: RPMI-8226 RFP cells were generated by infecting RPMI-8226 cells (ATCC, #CCL 155) with a third-generation lentiviral HIV-based VSV-G pseudotyped lentivirus encoding RFP (mKate2 Lentivirus Reagent - Sartorius ref #4625) that expresses red fluorescent protein (RFP). MM.1R RFP cells were generated by infecting MM.1R cells (ATCC, #CRL 2975) with a third-generation lentiviral HIV-based VSV-G pseudotyped lentivirus encoding RFP (mKate2 Lentivirus Reagent - Sartorius ref #4625) that expresses red fluorescent protein (RFP).

[0122] RPMI-8226 RFP or MM.1R RFP cells were maintained in IMDM medium (Gibco, #12440053) supplemented with 20% fetal bovine serum (FBS heat-inactivated, Biowest, #S181H-100), 1% L-glutamine (Gibco, #25030-024) and incubated at 37°C in 5% CO. RPMI-8226 RFP cells were centrifuged at 300g for 5 minutes and resuspended in RPMI 1640 complete medium (RPMI 1640 supplemented with 10% fetal bovine serum-FBS, Biowest, #S181 H-100-, 1% L-glutamine-Gibco, #25030-024) before being added to Incucyte® plates (described in detail below).

[0123] Isolation of human HD NK cells: Healthy donor buffy coats were provided by EFS Ile de France. Immediately upon receipt, acceptable buffy coats were gently agitated overnight at room temperature. The following day, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Cytiva #17-1440-02) density gradient centrifugation. Human NK cells were purified from PBMCs using Miltenyi Biotec's MACSxpress Whole Blood NK Cell Isolation Kit (#130-127-695) by manual magnetic labeling and negative selection using a MACSxpress separator (Miltenyi Biotec #130-098-308). Purified NK cells were then cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Eurobio #CVFSVF06-01) and 1% L-glutamine (Gibco #25030-081). Before use, the cells were incubated overnight at 37°C and 5% CO2.

[0124] Incucyte® Cytotoxicity Assay: Cytotoxicity was assessed using the Incucyte® Live Cell Analysis System, which allows for quantification of the number of live fluorescent target cells over time. Compounds and cells were added to appropriate wells of Incucyte® plates (Poly-D Lysine-treated 96-well flat-bottom microplates, CellCoat™, Greiner Bio-One; #655946) in the following order (final volume per well: 200 μl): isatuximab (or isotype control) (pre-diluted in RPMI 1640 complete medium) was added to the appropriate wells to a final concentration of either 10, 100, or 1000 ng / ml; belmosudil (pre-diluted in RPMI 1640 complete medium) was then added to the appropriate wells to a final concentration of either 1.1 or 3.3 μM; RFP-positive MM tumor cells (target cells, T) were then added to each well (to have 20,000 RFP-positive cells / well); and HD NK cells (effector cells, E) were then added to the appropriate wells (60,000 RPMI-8226 RFP cells were added to have an E:T ratio of 3:1; 20,000 MM.1R RFP cells were added to have an E:T ratio of 1:1); HD NK cells were not added to some wells to test the direct effect of belmosudil on RFP-positive tumor cells.

[0125] The Incucyte® plates were then centrifuged at 100 g for 1 minute before being placed in an Incucyte® (IncucyteS3, EssenBio) in a dedicated incubator at 37°C and 5% CO2. Images (4 images / well) were taken every 4 hours using a 10x objective and standard scan type, using the phase and red channels. Growth of RFP-positive target cells was monitored by fluorescent imaging for up to 96 hours, and the number of viable target cells was quantified using IncucyteS3 software and normalized to the number of viable target cells at time 0.

[0126] result Belmosudil (1.1 or 3.3 μM) induces the killing of RPMI-8226 RFP cells over time, as shown in Figure 12. Belmosudil (1.1 or 3.3 μM) in combination with isatuximab (10 ng / ml) further increases RPMI-8226 RFP cell killing over time in the presence of HD NK cells.

[0127] Belmosudil (1.1 or 3.3 μM) induces the killing of MM.1R RFP cells over time, as shown in Figure 13. Belmosudil (1.1 or 3.3 μM) in combination with isatuximab (10 ng / ml) further increases MM.1R RFP cell killing over time in the presence of HD NK cells.

[0128] Belmosudil (1.1 or 3.3 μM) induces the killing of LP-1 RFP cells over time, as shown in Figure 14. Belmosudil (1.1 or 3.3 μM) in combination with isatuximab (10 ng / ml) further increases LP-1 RFP cell killing over time in the presence of HD NK cells.

[0129] Example 7: Combination of Belmosudil and Dexamethasone in MM Cells method On day 1, cells were seeded at 3,000 cells (50 μl) per well in a 384W plate (Costar, 3765) precoated with Poly-D-lysine, spun down at 300 g for 1 minute at room temperature, and transferred to an Incucyte S3 (37°C; 5% CO2; kinetic readings every 2 hours). After 4 hours, cells were treated with a ROCK 2 inhibitor (SAR445761, belmosudil) or, for single agents, dexamethasone (SelleckChem; S1322) in a dose-response format using a Tecan d300e. For combinations, a dose-response of belmosudil in the presence of selected concentrations of dexamethasone was used. Cells were further incubated in the Incucyte S3 (Sartorius) for an additional 5–6 days at 37°C, 5% CO2, and the same kinetic readings. Finally, proliferation was measured by adding CellTiterGlo (Promega) to assess cell viability or by using an Incucyte to measure cell confluency over time. Cell proliferation was normalized to DMSO (100%) and staurosporine (Sigma; S6942) 1 μM (0%).

[0130] The degree of synergy between belmosudil and dexamethasone was assessed by the Synergy Finder tool using excess over the maximum single drug response (HSA) (Lanevski A, et al, Nucleic Acids Research, 488-493, 2020, Vol. 48 (doi: 10.1093 / nar / gka 216)).

[0131] result Dexamethasone and belmosudil exhibit single-agent growth inhibitory activity against the multiple myeloma cell lines MM1S and LP1, as shown in Figures 15A and 15B, respectively. As shown in Figures 16A, 16C, 17A, and 17C, the combination of belmosudil and dexamethasone exhibits improved growth inhibitory effects in both MM1S and LP1 over a defined concentration range. Statistical analysis using Synergy Finder (HSA) highlighted positive synergy scores of approximately 1.76 for MM1S and 5.4 for LP-1, as shown in Figures 16B and 17B, respectively. Overall, these data suggest that the combination of belmosudil and dexamethasone may have therapeutic benefit for multiple myeloma patients.

[0132] The present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

Claims

1. A method for treating multiple myeloma, comprising administering to a subject in need thereof a therapeutically effective amount of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof (bermosudil).

2. 10. The method of claim 1, wherein belmosudil is administered as monotherapy.

3. 10. The method of claim 1, wherein belmosudil is administered in combination with an anti-CD38 antibody.

4. The method of claim 3, wherein the anti-CD38 antibody is daratumumab, isatuximab, MOR202, or mezagitamab (TAK-079).

5. The method of claim 4, wherein the anti-CD38 antibody is isatuximab.

6. 6. The method of claim 5, wherein the isatuximab is administered intravenously to the subject at a dose of 10 mg / kg or 20 mg / kg.

7. 6. The method of claim 5, wherein the isatuximab is administered subcutaneously at a dose of 1000 mg or 1400 mg.

8. 8. The method of any one of claims 1 to 7, wherein the isatuximab is administered in 28 day cycles.

9. 9. The method of claim 8, wherein in an initial 28-day cycle, the isatuximab is administered once a week.

10. 10. The method of claim 9, wherein in subsequent 28-day cycles, the isatuximab is administered twice weekly.

11. 11. The method of claim 10, wherein after 12 months of isatuximab administration, the isatuximab is administered once every four weeks.

12. 10. The method of claim 1, wherein belmosudil is administered in combination with an immunomodulatory drug (IMiD).

13. 13. The method of claim 12, wherein the IMiD is selected from pomalidomide, lenalidomide, thalidomide, iberdomide, and medigomid.

14. 14. The method of claim 13, wherein the IMiD is pomalidomide.

15. 15. The method of claim 14, wherein the pomalidomide is administered to the subject at a dose of 4 mg once daily on days 1 through 21 of each 28-day cycle.

16. 16. The method of claim 14 or 15, wherein the pomalidomide is administered orally.

17. 17. The method of any one of claims 1 to 16, wherein the belmosudil is administered in 28-day cycles.

18. 18. The method of any one of claims 1 to 17, wherein the belmosudil is administered to the subject at a dose of up to 1000 mg per day.

19. 19. The method of claim 18, wherein the dose is 200 mg per day.

20. 19. The method of claim 18, wherein the dose is 200 mg twice daily.

21. 19. The method of claim 18, wherein the dose is 400 mg per day.

22. 19. The method of claim 18, wherein the dose is selected from 500 mg / day, 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, and 1000 mg / day.

23. The method according to any one of claims 1 to 22, wherein the belmosudil is administered orally.

24. 24. The method of any one of claims 1 to 23, wherein belmosudil is the mesylate salt of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide.

25. The method of any one of claims 1 to 24, wherein the subject is receiving concomitant corticosteroid therapy.

26. 26. The method of claim 25, wherein the concomitant corticosteroid therapy is selected from dexamethasone, prednisone, and methylprednisolone.

27. 10. The method of claim 1, further comprising administering isatuximab and dexamethasone.

28. 10. The method of claim 1, further comprising administering pomalidomide and dexamethasone.

29. The method of any one of claims 1 to 28, wherein the subject has undergone prior therapy to treat the multiple myeloma.

30. 30. The method of claim 29, wherein the subject has relapsed multiple myeloma.

31. 30. The method of claim 28 or 29, wherein the subject has relapsed and refractory multiple myeloma.

32. 30. The method of any one of claims 1 to 29, wherein the multiple myeloma is smoldering multiple myeloma.

33. 33. The method of any one of claims 1 to 32, wherein said treatment overcomes IMiD resistance in said subject with multiple myeloma.

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