Topical injection formulations comprising eltrombopag for treating degenerative diseases and improving stem cell homing

Abstract

The present invention relates to a controlled release pharmaceutical composition comprising at least one controlled release pharmaceutical dosage form comprising EPAG wherein it is suitable for its topical injection at an affected site. The invention also relates to a controlled release pharmaceutical composition in powder form and a kit comprising an aqueous injection vehicle and a controlled release dosage form or a mixture of controlled release and immediate release dosage forms comprising EPAG wherein the pharmaceutical composition optionally comprises an excipient for the preparation of a pharmaceutical composition suitable for topical injection thereof. The compositions may be used for topical injection in a patient, for improving homing of stem cells, and / or for treating non-malignant blood disorders, hematological malignancies, primary immunodeficiencies, autoimmune diseases, innate metabolic deficiencies, and / or degenerative diseases / injuries.

Description

[0001] The present invention relates to the field of treating degenerative diseases / injuries with eltrombopag (EPAG) or its derivatives, conjugates, or pharmaceutically acceptable salts, in particular cardiovascular diseases, diseases affecting bones and joints, periodontal diseases, eye diseases, kidney diseases, liver diseases, inflammatory bowel diseases, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and the field of treating non-malignant blood disorders, hematological malignancies, primary immunodeficiencies, autoimmune diseases, or inborn errors of metabolism with endogenous stem cells or exogenous stem cells, such as autologous or allogeneic hematopoietic stem cell transplantation (HSCT). More specifically, the present invention relates to a controlled-release pharmaceutical composition suitable for local injection into an affected area, comprising at least one controlled-release pharmaceutical dosage form containing eltrombopag or its derivatives, conjugates, or pharmaceutically acceptable salts, particularly suitable for intramedullary injection into the bone marrow or any other type of injection into the appropriate affected area. The present invention also relates to such controlled-release pharmaceutical compositions for their therapeutic use, in particular for improving the homing of hematopoietic stem cells (HSCs) before, during or after autologous or allogeneic HSCT, for the treatment of non-malignant blood disorders, hematological malignancies, primary immunodeficiencies, autoimmune diseases and / or inborn errors of metabolism; and / or for wound healing; and / or for the treatment of degenerative diseases / injuries, in particular cardiovascular diseases, diseases affecting bones and joints, periodontal diseases, eye diseases, kidney diseases, liver diseases, inflammatory bowel diseases, chronic obstructive pulmonary disease (COPD) and / or pulmonary fibrosis.

[0002] background

[0003] Local inflammation and ischemia occurring at the affected site typically lead to the activation of various proteolytic enzymes (i.e., proteases) such as matrix metalloproteinases (MMPs) and serine proteases. These proteases play a key role in the degradation of extracellular matrix components and various cytokines / chemokines that influence cell migration and regulation in the ultimate process of tissue remodeling and repair.

[0004] Chemokines are small chemoattractants involved in every step of this repair and regeneration process and are crucial for shaping cellular activity, particularly in driving the migration of stem and progenitor cells to the affected site. Among chemokines, stromal cell-derived factor-1 (SDF-1, also known as CXCL12) is perhaps the most prominent stem / progenitor cell homing factor, attracting cells expressing its receptor, CXCR4, such as hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), neural stem cells (NSCs), smooth muscle progenitor cells, epithelial progenitor cells, and fibroblast progenitor cells. Although SDF-1 is constitutively secreted primarily by bone marrow stromal cells under homeostasis, its expression is induced under pathological conditions at any affected site to recruit leukocytes and stem / progenitor cells required for tissue repair. However, there appears to be a temporal mismatch between peak SDF-1 expression at the affected site and the upregulation of CXCR4 on progenitor and stem cells. Following inflammation / ischemia, SDF-1 expression increases for only a few days, whereas CXCR4 overexpression persists for weeks (Ziff et al., Therapeutic strategies utilizing SDF-1 in ischaemic cardiomyopathy, Cardiovascular Research, Volume 114, Issue 3, 01 March 2018, Pages 358–367). This mismatch is driven by the rapid clearance of SDF-1 from the circulation and its susceptibility to degradation by proteases that are overexpressed in these affected sites (Takekoshi et al., A locked, dimeric CXCL12 variant effectively inhibits pulmonary metastasis of CXCR4-expressing melanoma cells due to enhanced serum stability. Mol Cancer Ther. 2012 Nov; 11(11): 2516-25).

[0005] Therefore, there remains a need to extend the time window during which SDF-1 concentration levels increase within the affected site in order to increase the number of stem / progenitor cells attracted and retained at the site, thereby increasing their beneficial contribution to tissue repair and regeneration.

[0006] This approach may be particularly beneficial in diseases or conditions where high local proteolytic activity caused by inflammation and ischemia in the affected site hinders the full SDF-1 signaling potential, such as:

[0007] - Cardiovascular diseases (myocardial infarction, atherosclerosis, ischemia-reperfusion injury);

[0008] - Diseases affecting bones and joints (osteoarthritis, rheumatoid arthritis, osteoporosis);

[0009] - Periodontal disease;

[0010] - Wound healing (including diabetic wound healing and corneal wound healing);

[0011] - Eye diseases (retinal ischemia, macular degeneration, diabetic retinopathy, glaucoma)

[0012] - Kidney disease, liver disease (fibrosis and cirrhosis, non-alcoholic fatty liver disease, acute liver injury);

[0013] - Inflammatory bowel disease (Crohn's disease, ulcerative colitis);

[0014] - Chronic obstructive pulmonary disease (COPD), pulmonary fibrosis;

[0015] - Non-malignant blood disorders (severe aplastic anemia or hemoglobinopathies, particularly sickle cell disease and beta-thalassemia)

[0016] - Hematological malignancies (myeloma, lymphoma, leukemias such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, myelodysplastic syndrome);

[0017] - Primary immunodeficiency, autoimmune disease, or inborn error of metabolism.

[0018] Notably, current treatment options for the aforementioned non-malignant and malignant hematologic disorders, as well as certain primary immunodeficiencies, autoimmune diseases, and inborn errors of metabolism, include hematopoietic stem cell transplantation, in which exogenous stem cells (from a donor or previously obtained from the patient) are infused intravenously into the patient's circulation and are required to migrate to the patient's bone marrow.

[0019] Direct local delivery of SDF-1 to sites of damaged tissue has been tested using a variety of biomaterials and drug delivery vehicles, with positive in vitro / preclinical results demonstrating increased cell homing to the site of injury (Andreas et al., Toward in situ tissue engineering: chemokine-guided stem cell recruitment. Trends Biotechnol. 2014 Sep; 32(9): 483-92). However, SDF-1 remains an unapproved drug, and controlled delivery of this unstable protein at adequate doses remains a challenge.

[0020] As mentioned above, the SDF-1 receptor (i.e., CXCR4) is highly expressed by hematopoietic stem cells (HSCs), and SDF-1 is constitutively expressed by bone marrow stromal cells and osteoblasts. It is generally believed that the SDF-1 / CXCR4 signaling axis is one of the main mediators of hematopoietic stem and progenitor cell homing to the bone marrow (BM) after HSCT. Many studies have shown that inhibitors of the SDF-1 / CXCR4 axis inhibit the homing of transplanted HSCs to the BM and induce the mobilization of HSCs from the bone marrow into the circulation (Dar et al., Exp Hematol. 2006; 34(8): 967-75). In addition, SDF-1 has also been shown to play an important role in the maintenance and retention of HSCs in the BM (Greenbaum et al., Nature. 2013; 495(7440): 227-30). More interestingly, recent studies have shown that in preclinical models of hematological diseases, impaired production of SDF-1 from BMSCs results in reduced SDF-1 levels in the BM extracellular fluid, which is accompanied by reduced HSC maintenance and increased HSC mobilization in the BM, as well as reduced HSC colonization in the BM after HSC transplantation (Tang et al., Blood. 2021; 138(24): 2570-2582; Hanoun et al., Cell Stem Cell. 2014; 15(3): 365-375). In addition, the conditioning regimen that patients undergo before HSCT (which typically includes chemotherapy with or without radiation and more recently may include serum therapy, monoclonal antibodies, specifically targeted novel compounds, and radiolabeled antibodies) affects the state of the BM microenvironment, causing, among other effects, a local increase in proteases that can shorten the half-life of chemokines such as SDF-1 (Zhang et al., Sci Rep. 2016; 6: 378-27). Therefore, as generally described above, in diseases or conditions where high local proteolytic activity caused by inflammation and ischemia at the affected site hinders full SDF-1 signaling, local protection of SDF-1 in the BM and restoration of HSC microenvironmental homeostasis can be targeted to enhance homing and colonization. The subsequent survival of HSC progenitors that have homed to the BM, as well as their subsequent self-renewal and differentiation, are also key to the long-term success of HSCT therapy.

[0021] Eltrombopag ethanolamine (EPAG ethanolamine) is a small molecule human thrombopoietin receptor (TPO-R) agonist with the formula 3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylic acid bis-(monoethanolamine). It is a drug with multiple mechanisms of action. The first identified effect corresponds to stimulation of megakaryocyte (MK) progenitor cell expansion and differentiation through binding to TPO-R. In contrast to the thrombopoietin effect, EPAG binding to TPO-R also promotes HSC survival and is not inhibited in inflammatory environments. EPAG has also demonstrated interesting iron chelation properties, inducing stimulation of stem cell renewal and antiproliferative effects against leukemic cell lines.

[0022] EPAG is currently used to treat immune thrombocytopenia (ITP), hepatitis C-associated thrombocytopenia, and severe aplastic anemia (SAA). EPAG has also shown positive results in the treatment of myelodysplastic syndrome and post-HSCT graft dysfunction.

[0023] EPAG currently uses PROMACTA ® It is commercially available as a powder for oral tablets or suspension in doses ranging from 12.5 mg to 75 mg.

[0024] To date, there has been no disclosure of a direct effect of EPAG on the inhibition of the activity of any SDF-1 degrading proteases, nor any controlled release formulation for local injection of EPAG. Existing technology

[0025] WO2016 / 201354 discloses a method for treating cancer, which comprises administering an antifungal agent, a TPO receptor agonist, or a combination thereof, and acts by inhibiting dioxygenase. However, examples are limited to immediate-release injectable formulations and immediate-release oral tablets.

[0026] WO2007 / 145227 relates to the administration of TPO receptor agonists after HSCT to enhance the colonization, growth and differentiation of transplanted cells in the bone marrow. The agents are preferably administered parenterally by injection, but there is no mention of controlled-release pharmaceutical compositions suitable for local injection of the agonists into the affected area, including for enhancing colonization after HSCT.

[0027] WO2004 / 096154 relates to non-peptide thrombopoietin receptor agonists and their use in the treatment of degenerative diseases / injuries, but the application is completely silent on pharmaceutical compositions suitable for local injection at the affected site and administration in controlled release form.

[0028] The use of thrombopoietin receptor agonists including EPAG to promote HSC homing after bone marrow transplantation has been reported in CN105412930A. A large group of administration routes are mentioned, but the only example is limited to the administration of an immediate release EPAG solution by oral gavage in mice, with no mention of a controlled release form.

[0029] Therefore, there remains a need to develop new approaches to increase SDF-1 concentration levels in affected sites, particularly the bone marrow, for increasing the fraction of endogenous regenerative progenitor and stem cells that will home to that site, thereby enhancing self-regeneration of tissues and organs, and for enhancing the homing and colonization of exogenous, such as autologous or allogeneic, hematopoietic stem cells in the framework of transplantation.

[0030] Additionally, there remains a need to develop new dosage forms and routes of administration of EPAG or its derivatives or conjugates or pharmaceutically acceptable salts to improve control of local EPAG concentrations at the affected site, thereby increasing the fraction of regenerative progenitor and stem cells that will home / migrate to that site.

[0031] There is also a need to develop new dosage forms and administration routes of EPAG or its derivatives or conjugates or pharmaceutically acceptable salts to improve the control of local EPAG concentration in the bone marrow, thereby increasing the homing of exogenous stem cells during transplantation.

[0032] The inventors surprisingly discovered that EPAG ethanolamine can inhibit the activity of several SDF-1-degrading proteases, thereby improving the stability of SDF-1 in the proteolytic environment commonly found in ischemic and inflammatory damaged tissues, and that this new mode of action can be more specifically utilized to enhance the efficacy of EPAG ethanolamine by local injection into the affected area.

[0033] More specifically, the inventors surprisingly discovered that injecting EPAG or its derivatives or conjugates or pharmaceutically acceptable salts, particularly EPAG ethanolamine, directly into the affected area can prevent and / or treat the degenerative diseases / injuries disclosed below in the present disclosure, and can promote wound healing disclosed below in the present disclosure.

[0034] More specifically, the inventors surprisingly discovered that direct injection of a dosage form of EPAG or its derivatives or conjugates or pharmaceutically acceptable salts, particularly a dosage form of EPAG ethanolamine, into the bone marrow before, during or after receiving HSCT enhances the homing and colonization of the transplanted cells, thereby increasing the success rate of the transplant.

[0035] The experimental section below shows that EPAG or its derivatives or conjugates or pharmaceutically acceptable salts, in particular EPAG ethanolamine, inhibits the activity of several SDF-1 degrading proteases, such as MMP-8, MMP-9, MMP-13 and DPPIV (CD26), and stabilizes the local concentration of SDF-1 (Examples 6 to 11). The experimental section further demonstrates that EPAG or its derivatives or conjugates or pharmaceutically acceptable salts, in particular EPAG ethanolamine, can strongly reduce the proteolytic effect of neutrophil degranulation on SDF-1 (Example 12) and can increase the chemotaxis of hematopoietic stem and progenitor cells to SDF-1 in the presence of DPPIV protease (Example 13). In addition, the experimental section shows that sitagliptin, a well-known strong DPPIV inhibitor, has a calculated half-maximal inhibitory concentration (IC 50 ) is 10 smaller than eltrombopag 5 times more than that of eltrombopag and was less effective than eltrombopag in stabilizing SDF-1 in the presence of DPPIV. SUMMARY OF THE INVENTION

[0037] According to a first aspect, provided herein is a controlled-release pharmaceutical composition comprising at least one controlled-release pharmaceutical dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is suitable for local injection at the affected site.

[0038] In one embodiment, the controlled release pharmaceutical composition is suitable for intramedullary injection.

[0039] Also disclosed is a pharmaceutical composition comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, wherein the composition is suitable for intramedullary injection.

[0040] According to a second aspect, provided herein is a controlled-release pharmaceutical composition in powder form, comprising controlled-release microparticles comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof and a controlled-release polymer matrix, wherein

[0041] - the average particle size of the controlled-release microparticles is equal to or greater than 1 μm, for example equal to or greater than 2 μm, particularly in the range of 1 μm to 200 μm, more particularly in the range of 2 μm to 150 μm, still more particularly in the range of 2 μm to 100 μm, for example 10 μm to 100 μm, or 10 μm to 80 μm,

[0042] - the controlled release polymer matrix comprises at least one poly(lactic-co-glycolic acid) copolymer, and

[0043] - the weight percentage of the drug loading or 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt to the total weight of the microparticles ranges from 2% to 45%, particularly from 5% to 40%, more particularly from 10% to 35%.

[0044] Also disclosed is a pharmaceutical composition in powder form comprising controlled-release microparticles comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof and a controlled-release polymer matrix, wherein

[0045] - the average particle size of the controlled-release microparticles is greater than or equal to 2 μm, particularly in the range of 2 μm to 150 μm, more particularly in the range of 10 μm to 100 μm, even more particularly in the range of 10 μm to 80 μm,

[0046] - the controlled release polymer matrix comprises at least one poly(lactic-co-glycolic acid) copolymer, and

[0047] - the weight percentage of the drug loading or 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt to the total weight of the microparticles ranges from 2% to 45%, particularly from 5% to 40%, more particularly from 10% to 35%.

[0048] According to a third aspect, there is provided herein a kit or article of manufacture comprising in separate compartments (i) an aqueous injection vehicle and (ii) a controlled release pharmaceutical dosage form or a mixture of a controlled release dosage form and an immediate release dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, in particular a controlled release pharmaceutical dosage form or a mixture of a controlled release dosage form and an immediate release dosage form as defined herein, or a powder as defined herein, wherein The pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancer, a wetting agent, a viscosity enhancer, a density enhancer or a mixture thereof, for preparing a pharmaceutical composition suitable for local injection at the affected site, in particular a pharmaceutical composition suitable for intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection, intraosseous injection and a mixture thereof.

[0049] Also disclosed is a kit or article of manufacture comprising in separate compartments (i) an aqueous injection vehicle and (ii) a controlled-release dosage form, an immediate-release dosage form, or a mixture of a controlled-release dosage form and an immediate-release dosage form, comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, in particular a controlled-release dosage form, an immediate-release dosage form, or a mixture of a controlled-release dosage form and an immediate-release dosage form as defined in the present disclosure, or a powder as defined in the present disclosure, wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancer, a wetting agent, a viscosity enhancer, a density enhancer, or a mixture thereof, for preparing a pharmaceutical composition suitable for intramedullary injection.

[0050] According to a fourth aspect, there is provided herein a controlled release pharmaceutical composition as defined in the present disclosure or obtained by mixing two compartments of a kit as defined in the present disclosure, for one or more uses by local injection at the affected site, in particular,

[0051] for improving homing, colonization and long-term expansion and proliferation of hematopoietic stem cells in patients, particularly human patients, by intramedullary injection, before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), for preventing and / or treating non-malignant blood disorders, for preventing and / or treating hematological malignancies, and / or for preventing and / or treating primary immunodeficiency, autoimmune disease or inborn error of metabolism, and / or

[0052] For the prevention and / or treatment of degenerative diseases / injuries by intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection, intraosseous injection, and mixtures thereof.

[0053] Also disclosed is a pharmaceutical composition as defined in the present disclosure or obtained by mixing the two compartments of the kit as defined in the present disclosure, for use in a patient, in particular a human patient, by intramedullary injection once or multiple times before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), for improving the homing, colonization and long-term expansion and proliferation of hematopoietic stem cells.

[0054] As mentioned above, the controlled release pharmaceutical composition as defined in the present disclosure, which is suitable for local injection, particularly for intramedullary injection, in a patient before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), is effective in improving the homing, colonization and long-term expansion and proliferation of hematopoietic stem cells before allogeneic HSCT, particularly for reducing graft failure, for reducing the occurrence of poor graft function (PGF), for reducing the occurrence of graft-versus-host disease (GvHD), for improving overall survival and donor chimerism, and / or for promoting hematopoietic recovery and the success rate of hematopoietic stem cell transplantation, and also

[0055] in the prevention and / or treatment of non-malignant blood disorders, such as severe aplastic anemias and hemoglobinopathies, in particular sickle cell disease and beta-thalassemia; and / or

[0056] In the prevention and / or treatment of hematological malignancies such as myeloma, lymphoma, leukemias such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndrome, in particular leukemia; and / or

[0057] It is effective in preventing and / or treating primary immunodeficiency, autoimmune disease or inborn errors of metabolism.

[0058] Additionally, the controlled release pharmaceutical composition suitable for local injection as defined in the present disclosure is effective in:

[0059] Prevention and / or treatment of cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; and / or

[0060] Preventing and / or treating diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis, and osteoarthritis; and / or

[0061] Preventing and / or treating periodontal disease; and / or

[0062] Wound healing, including diabetic wound healing and corneal wound healing; and / or

[0063] Preventing and / or treating eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; and / or

[0064] Prevention and / or treatment of kidney disease; and / or

[0065] Prevention and / or treatment of liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; and / or

[0066] Preventing and / or treating inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; and / or

[0067] Prevent and / or treat chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and / or pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1A and 1B Representative Western blot analysis showing the inhibitory effect of eltrombopag on the proteolysis of SDF-1α by MMP-9 (matrix metalloproteinase-9). Western blot images of total SDF-1α (upper panel or panel A) and N-terminally intact SDF-1α (lower panel or panel B). In each panel A and B, lanes are designated 1 to 6, from left to right. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α plus MMP-9, and lanes 3-6 correspond to SDF-1α plus MMP-9 plus eltrombopag at decreasing concentrations of eltrombopag (50 μM, 10 μM, 1 μM, and 0.1 μM) (Example 6).

[0070] Figure 2A and 2BRepresentative Western blot analysis showing the inhibitory effect of eltrombopag on SDF-1α proteolysis by MMP-9 (matrix metalloproteinase-9). Western blot images of total SDF-1α (upper panel or panel A) and N-terminally intact SDF-1α (lower panel or panel B). In each panel A and B, lanes are designated 1 to 6, from left to right. Lane 1 corresponds to SDF-1α plus MMP-9, lanes 2-5 correspond to SDF-1α plus MMP-9 plus eltrombopag at decreasing eltrombopag concentrations (100 μM, 50 μM, 10 μM, and 1 μM), and lane 6 corresponds to SDF-1α alone (Example 7).

[0071] Figure 3A 、 3B 4A and 4B represent Western blot analyses showing the inhibitory effect of eltrombopag on the proteolysis of SDF-1α by MMP-8 (matrix metalloproteinase-8). Western blot images of total SDF-1α (upper panel or panel A) and N-terminally intact SDF-1α (lower panel or panel B). In each panel A and B, the lanes are designated from left to right as Lane 1 to Lane 6, respectively. Lane 1 corresponds to SDF-1α alone, Lane 2 corresponds to SDF-1α plus MMP-8, and Lanes 3–6 correspond to SDF-1α plus MMP-8 plus eltrombopag, with decreasing concentrations of eltrombopag (for Figure 3A and 3B 50 μM, 10 μM, 1 μM and 0.1 μM; for Figure 4A and 4B are 100 μM, 50 μM, 10 μM and 1 μM) (Examples 8 and 9).

[0072] Figure 5A and 5B Representative Western blot analysis showing the inhibitory effect of eltrombopag on the proteolysis of SDF-1α by MMP-13 (matrix metalloproteinase-13). Western blot images of total SDF-1α (upper panel or panel A) and N-terminally intact SDF-1α (lower panel or panel B). In each panel A and B, lanes are designated 1 to 6, from left to right. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α plus MMP-13, and lanes 3-6 correspond to SDF-1α plus MMP-13 plus eltrombopag at decreasing concentrations of eltrombopag (100 μM, 50 μM, 10 μM, and 1 μM) (Example 10).

[0073] Figure 6A and 6BRepresentative Western blot analysis showing the inhibitory effect of eltrombopag on the proteolysis of SDF-1α by DPPIV (dipeptidyl peptidase IV, also known as dipeptidyl peptidase-4, DPP4, or CD26). Western blot images of total SDF-1α (upper panel or Panel A) and N-terminally intact SDF-1α (lower panel or Panel B). In each condition, 125 nM SDF-1α was incubated in 0.6 nM DPPIV in the presence or absence of eltrombopag, as appropriate. In both Panels A and B, lanes are designated 1 to 5, from left to right, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α plus DPPIV, lanes 3-4 correspond to SDF-1α plus DPPIV plus eltrombopag at increasing concentrations of eltrombopag (50 μM and 100 μM), and lane 5 corresponds to SDF-1α plus 1 μM sitagliptin (Example 11).

[0074] Figure 7 Graph showing the inhibitory effect of eltrombopag on the proteolysis of SDF-1α by dipeptidyl peptidase-4 (DPPIV). Samples were compared using a one-way ANOVA. Results are shown as circular symbols for each independent experiment under each condition, and as bars and error bars for the mean ± standard error of the mean (SEM) for all experiments under each condition; n = 8 independent experiments (Example 11).

[0075] Figure 8A and 8B Representative Western blot analysis showing the inhibitory effect of eltrombopag on the proteolysis of SDF-1α by proteases involved in neutrophil degranulation (ND). Western blot images of total SDF-1α (upper panel or panel A) and N-terminally intact SDF-1 (lower panel or panel B). In each panel A and B, lanes are designated 1 to 4 from left to right, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α plus ND, and lanes 3-4 correspond to SDF-1α plus ND plus eltrombopag at decreasing concentrations of eltrombopag (100 μM and 50 μM) (Example 12).

[0076] Figure 9Representative images show the fraction of hematopoietic stem and progenitor cells that migrated from the upper to the lower Transwell chamber in the presence or absence of SDF-1α, DPPIV protease, and 25 μM and 50 μM eltrombopag, compared to control conditions (the lower chamber containing SDF-1α but neither protease nor eltrombopag). Samples were compared using a one-way ANOVA. Results are shown as circular symbols for each independent experiment within each condition, and as bars and error bars for the mean ± standard error of the mean (SEM) of all experiments within each condition; n = 3 independent experiments (Example 13). Detailed Description of the Invention

[0078] As is apparent from the background section above and from the accompanying figures (Examples 6-11) below, the inventors have demonstrated that EPAG, or its derivatives, conjugates, or pharmaceutically acceptable salts, can inhibit protease activity and stabilize local concentrations of SDF-1. This is the first demonstration of this mechanism of action associated with EPAG, or its derivatives, conjugates, or pharmaceutically acceptable salts. The inventors have also demonstrated that EPAG, or its derivatives, conjugates, or pharmaceutically acceptable salts, can significantly reduce the proteolytic degradation of SDF-1 by neutrophil degranulation (Example 12) and increase the chemotaxis of hematopoietic stem and progenitor cells toward SDF-1 in the presence of DPPIV protease (Example 13).

[0079] Thus, the controlled release pharmaceutical composition according to the present disclosure recruits two types of stem / progenitor cells, namely:

[0080] Endogenous stem cells and / or endogenous progenitor cells,

[0081] or exogenous stem cells and / or exogenous progenitor cells.

[0082] Progenitor cells are the descendants of stem cells, which then further differentiate to give rise to specialized cell types.

[0083] Local injection in the corresponding affected site(s) of the controlled release composition according to the present disclosure, such as intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection or intraosseous injection can attract endogenous stem cells and / or endogenous progenitor cells, in other words, the stem cells and / or progenitor cells are attracted to the affected site where the controlled release composition according to the present disclosure is locally injected.

[0084] More specifically, intramedullary injection of the controlled-release composition according to the present disclosure into the bone marrow can attract exogenous stem cells and / or exogenous progenitor cells. In other words, the transplanted HSCs are attracted to the bone marrow into which the controlled-release composition according to the present disclosure has been locally injected. Needless to say, when performing HSCT, both exogenous stem cells and progenitor cells as well as endogenous stem cells and progenitor cells can be attracted. In other words, in addition to the targeted attraction of exogenous stem cells and progenitor cells, SDF-1 stabilization also facilitates the attraction of endogenous stem cells and progenitor cells.

[0085] Furthermore, within the framework of the treatment of degenerative diseases involving endogenous attraction of stem and progenitor cells by local injection at an affected site other than the bone marrow, transplantation of exogenous stem and progenitor cells may be performed before, during or after said local injection, for example by intravenous infusion.

[0086] definition

[0087] As used herein, the term "patient" refers to a human or human child who has or is at risk of having one or more of the diseases and conditions described herein.

[0088] As used herein, the term "recruitment" and the term "recruit" have the same meaning as the term "attraction" and the term "attract".

[0089] It is well within the ability and knowledge of those skilled in the art to identify those patients who are in need of treatment for the diseases and conditions described herein. Those skilled in the art can readily identify patients in need of such treatment by using clinical trials, physical examinations, medical / family histories, or biological and diagnostic tests.

[0090] In the context of the present invention, the expression "degenerative disease / injury" refers to cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis, and ischemia-reperfusion injury; diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis, and osteoarthritis; periodontal disease; eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy, and glaucoma; kidney disease; liver diseases, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease, and acute liver injury; inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis), and pulmonary fibrosis. The controlled-release pharmaceutical composition according to the present invention can also be used for wound healing, including diabetic wound healing and corneal wound healing.

[0091] In the context of the present invention, pathologies associated with homing, colonization and long-term expansion and proliferation of hematopoietic stem cells are non-malignant blood disorders, such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and beta-thalassemia; hematological malignancies, such as myelomas, lymphomas, leukemias, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndromes, in particular leukemias; primary immunodeficiencies, autoimmune diseases and inborn errors of metabolism.

[0092] In the context of the present invention, the terms "treating" or "treatment" as used herein refer to reversing, alleviating, reducing, inhibiting the progression of a disease / disorder as defined in the present disclosure, in particular cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis; periodontal disease; eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; kidney disease; liver disease, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; inflammatory bowel disease; chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and pulmonary fibrosis; non-malignant blood disorders such as severe aplastic anemia and hemoglobinopathies, particularly sickle cell disease and beta-thalassemia; hematological malignancies such as myeloma, lymphoma, leukemias such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, particularly leukemias; and progression of primary immunodeficiencies, autoimmune diseases, and inborn errors of metabolism.

[0093] In the context of the present invention, the terms "preventing" or "prevention" as used herein refer to reducing the likelihood of the occurrence of a disease / disorder as defined in the present disclosure, in particular cardiovascular conditions / diseases, such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis; periodontal disease; eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; kidney disease; liver disease, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; inflammatory bowel disease (IBD), Such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and pulmonary fibrosis; non-malignant blood disorders, such as severe aplastic anemia and hemoglobinopathies, especially sickle cell disease and beta-thalassemia; malignant blood diseases, such as myeloma, lymphoma, leukemia, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndrome, especially leukemia; the possibility of developing, or delaying the onset of, primary immunodeficiency, autoimmune diseases and inborn errors of metabolism.

[0094] In the context of the present invention, the expression "improving the homing, colonization and long-term expansion and proliferation of hematopoietic stem cells (HSCs)" refers to cultivating the ability of exogenously administered HSCs to localize, seed and colonize in the recipient's bone marrow, thereby increasing the total number of transplanted HSCs that are capable of self-renewal and multipotent differentiation and ultimately participate in the reconstitution of a viable hematopoietic system.

[0095] As used herein, an "effective amount" refers to an amount effective to reduce, eliminate, treat, or control the symptoms of the diseases and conditions described herein, i.e., cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis, and ischemia-reperfusion injury; diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis, and osteoarthritis; periodontal disease; eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy, and glaucoma; kidney disease; liver disease, such as fibrosis, cirrhosis, nonalcoholic fatty liver disease, and acute liver injury; inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease (COPD); The invention also includes an amount of a compound of the present invention that is effective to treat or control the symptoms of the diseases and conditions described herein; pulmonary disease (COPD, also known as chronic bronchitis) and pulmonary fibrosis; non-malignant blood disorders such as severe aplastic anemia and hemoglobinopathies, particularly sickle cell disease and beta-thalassemia; hematological malignancies such as myelomas, lymphomas, leukemias such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndromes, particularly leukemias; primary immunodeficiency, autoimmune diseases and inborn errors of metabolism. An "effective amount" also refers to an amount of a compound of the present invention that is effective to allow wound healing or control wound healing. The term "control" is intended to refer to all processes in which the progression of the diseases and conditions described herein can be slowed, interrupted, prevented or stopped, but does not necessarily mean complete elimination of all symptoms of the diseases and conditions.

[0096] The term "therapeutically effective amount" refers to a concentration of a compound that is effective to treat the diseases / disorders defined in the present disclosure, particularly cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis, and ischemia-reperfusion injury; diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis, and osteoarthritis; periodontal disease; eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy, and glaucoma; kidney disease; liver disease, such as fibrosis, cirrhosis, nonalcoholic fatty liver disease, and acute liver injury; inflammatory bowel disease (IBD), such as Crohn's disease and ulcers. colitis; chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and pulmonary fibrosis; non-malignant blood disorders, such as severe aplastic anemia and hemoglobinopathies, particularly sickle cell disease and beta-thalassemia; hematological malignancies, such as myeloma, lymphoma, leukemias, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndromes, particularly leukemias; primary immunodeficiency, autoimmune diseases and inborn errors of metabolism.

[0097] In the sense of the present invention, the expression "local concentration of the product", in particular "local concentration of EPAG", more particularly "local concentration of the product" expressed in EPAG free acid equivalents, in particular "local concentration of EPAG" refers to the concentration of the product, that is to say the concentration of EPAG at the site of injection or implantation in the human body, in particular in the bones, heart, blood vessels, skin, dermis, eyes, liver, kidneys, gastrointestinal tract, peritoneum, lungs, gums, joints, bone marrow, in particular the sternum, tibia, femur, iliac crest, vertebrae and combinations thereof, for example the concentration of EPAG at the site of injection or implantation in the bone marrow of a patient.

[0098] In the sense of the present invention, the expression "affected site" refers to injured, affected, inflamed and / or ischemic tissue; injured, affected, inflamed and / or ischemic organ; and / or injured, affected, inflamed and / or ischemic membrane. It also refers to the entire organ or a part thereof, the entire tissue or a part thereof, and / or the entire membrane or a part thereof. The expression also extends to equivalent or partially equivalent expressions such as "lesion site", "disease site", "anatomical site", "affected area", "host site" and "target site". By way of example and not limitation, among the sites mentioned there may be mentioned bones, heart, blood vessels, skin, dermis, eyes, liver, kidneys, gastrointestinal tract, peritoneum, lungs, gums, joints, bone marrow, in particular the sternum, tibia, femur, iliac crest, vertebrae and combinations thereof.

[0099] In the sense of the present invention, the term "injection" also includes the term "implantation", that is to say the injection of an implant into the affected area.

[0100] In the sense of the present invention, the expression "local injection" refers to, for example, but not limited to, intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subTenon's injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection, intraosseous injection and combinations thereof.

[0101] Local injection of the controlled-release pharmaceutical composition according to the present invention can also be performed, for example, during gastrointestinal endoscopy.

[0102] In the context of the present invention, it goes without saying that all types of injections known to those skilled in the art are encompassed for each considered affected site to be treated by local injection according to the present invention. For example, intraocular injections include intravitreal injections and sub-Tenon's injections.

[0103] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, excipients, compositions or dosage forms which are, within the scope of sound medical judgment, suitable for contact with human tissues without excessive toxicity, irritation, allergic response or other problematic complications, and commensurate with a reasonable benefit / risk ratio.

[0104] The term "pharmaceutically acceptable carrier, adjuvant or vehicle" may refer to any pharmaceutically acceptable excipient that does not destroy the pharmacological activity of the compound with which it is formulated, such as a non-toxic carrier, adjuvant or vehicle.

[0105] The term "immediate release composition" or "immediate release dosage form" means that the composition or dosage form allows for the immediate release of a specified amount of the active ingredient into the body.

[0106] The term "controlled release composition" or "controlled release dosage form" or "controlled release microparticles" or "controlled release polymer matrix" means that the composition or dosage form or microparticles or polymer matrix is ​​capable of releasing a specific amount of active ingredient into the body over a specific time period, i.e., a specific pharmacokinetic profile. The term "controlled release" includes all types of release that are modified compared to immediate release. In other words, the term "controlled release" is equivalent to "modified release" and includes extended release as well as delayed release and pulsatile release as defined below.

[0107] The terms "extended release," "delayed release," and "sustained release" are considered equivalent within the context of the present invention. This type of release means that the release is prolonged over time compared to immediate release, i.e., it means that the active ingredient is released slowly over time, allowing the patient to take the drug less frequently. In other words, the active ingredient is released gradually over a specific period of time, generally with the goal of achieving fewer side effects by reducing the maximum concentration.

[0108] The expression "controlled release and immediate release" means release in the form of a mixture of controlled release and immediate release, in other words, release in the form of a combination of controlled release and immediate release.

[0109] The term “drug loading” refers to the mass ratio of drug in the microparticles to the mass of the microparticles (also called the weight ratio).

[0110] The term "average particle size" or D50 refers to the particle diameter in micrometers that divides the volume distribution of particles into half above and half below that diameter.

[0111] The term “D10 = x μm” mainly used in the examples means that 10% of the particles have a size of x μm or less.

[0112] The term "D90 = y μm" mainly used in the examples means that 90% of the particles have a size of y μm or less.

[0113] Controlled or modified release may of course result from a combination of immediate and controlled release.

[0114] As used herein, the terms "combination," "combined," and related terms refer to the simultaneous, separate, or sequential administration of the additional active ingredient and Eltrombopag or its derivatives or conjugates or pharmaceutically acceptable salts. For example, the combination can be administered simultaneously, separately, or sequentially with the additional active ingredient in separate unit dosage forms, or administered together in a single unit dosage form.

[0115] Eltrombopag and pharmaceutically acceptable salts thereof

[0116] As described above, the pharmaceutical composition according to the present disclosure comprises 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof.

[0117] It is known that 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid can be used as an agonist of TPO (thrombopoietin) receptor, which interacts with the transmembrane domain of human TPO receptor and is commonly known as Eltrombopag (C 25 H 22 N4O4, CAS: [496775-61-2]). The molecular weight of eltrombopag is 442.5 g / mol, which can be expressed as follows:

[0118] .

[0119] Eltrombopag can exist in different crystalline forms of the free acid, including a large number of hydrates and solvates, and different cationic salt forms, all of which are included within the scope of the present invention.

[0120] According to a preferred embodiment, 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt is in the form of a bis-monoethanolamine salt of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid.

[0121] The bis-monoethanolamine salt of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid (also known as the bis-monoethanolamine salt of Eltrombopag or 3'-{(2Z)-2-[1-(3,4-dimethylphenyl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene]hydrazino}-2'-hydroxy-3-biphenylcarboxylic acid-2-aminoethanol (1:2)) is commonly known as Eltrombopag ethanolamine. Eltrombopag ethanolamine (C 25 H 22 N4O4·2(C2H7NO), CAS: [496775-62-3]) has a molecular weight of 564.65 g / mol and can be represented by the following formula:

[0122] .

[0123] Eltrombopag is sold under the brand name Promacta® in the United States and Revolade® outside the United States.

[0124] Derivatives of eltrombopag include any derivatives such as esters, for example, methyl ester, ethyl ester, pivaloyloxymethyl ester, etc. for -COOH, and acetate ester, maleate ester, etc. for -OH, and any esters known to those skilled in the art.

[0125] Derivatives of Eltrombopag may also include protected forms of Eltrombopag, wherein one or more functional groups, such as (multiple) OH functional groups, are protected. Protection of hydroxyl or carboxyl groups may be performed by any group and method known in the art and fully described, for example, in "Protective Groups in Organic Synthesis" by Theodora W. Greene, Wiley-Interscience 1981, New York.

[0126] Conjugates of Eltrombopag include any conjugate, such as an antibody-Eltrombopag conjugate, ie, a polypeptide, such as an antibody, to which at least one Eltrombopag molecule is covalently linked via a linker.

[0127] 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt suitable for the present invention can be contained in a controlled-release dosage form, or a mixture of a controlled-release dosage form and an immediate-release dosage form.

[0128] In the pharmaceutical composition according to the present disclosure, 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof may be present in an amount of 2 mg to 63 mg equivalent of free acid per milliliter of controlled-release dosage form or a mixture of controlled-release dosage form and immediate-release dosage form, particularly 8 mg to 55 mg equivalent of free acid per milliliter, more particularly 12 mg to 47 mg equivalent of free acid per milliliter.

[0129] In the pharmaceutical composition according to the present disclosure, EPAG ethanolamine may be present in an amount of 2 to 80 mg per ml of the controlled release dosage form or a mixture of the controlled and immediate release dosage forms, particularly 10 to 70 mg per ml, more particularly 15 to 60 mg per ml.

[0130] controlled-release dosage forms

[0131] As described above, 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt is contained in a controlled release pharmaceutical dosage form.

[0132] According to one embodiment, the controlled-release pharmaceutical dosage form comprising Eltrombopag or a derivative or conjugate or a pharmaceutically acceptable salt thereof is in the form of an in situ formed depot, a hydrogel, a microporous implant, a solid implant or microparticles, in particular microparticles comprising a controlled-release polymer matrix, or multivesicular liposomes, such as microparticles comprising a controlled-release polymer matrix.

[0133] In one embodiment, the controlled-release dosage form is in the form of a reservoir or implant formed in situ. Reservoirs or implants formed in situ, such as pH-induced, heat-induced, or solvent-exchange-induced gelling systems comprising eltrombopag or a conjugate or derivative or salt thereof, can be prepared according to techniques known to those skilled in the art, particularly as described in Ibrahim TM et al. An overview of PLGA in-situ forming implants based on solvent exchange technique: effect of formulation components and characterization. Pharm Dev Technol. 2021 Sep; 26(7): 709-728 and in S. Kempe et al. In-situ forming implants-an attractive formulation principle for parenteral depot formulations Journal of Controlled Release 2012, 161:668.

[0134] In another embodiment, the controlled-release dosage form is in the form of a hydrogel. Hydrogels can be prepared using techniques known to those skilled in the art. Specifically, a hydrogel can be formed by dispersing eltrombopag, or a conjugate, derivative, or salt thereof, optionally pre-dissolved in water, into a hydrogel under stirring. Suitable hydrogels include sodium hyaluronate and alginates. When the pharmaceutical composition is in powder form, water should be removed by techniques known to those skilled in the art, such as dehydration or lyophilization. These can be reversibly hydrated if desired.

[0135] In another embodiment, the controlled release dosage form is in the form of a microporous implant.

[0136] In another embodiment, the controlled release dosage form is in the form of a solid implant, such as a preformed porous or non-porous solid implant that can be administered via a large needle or cannula.

[0137] In one embodiment, the controlled release dosage form is in the form of microparticles. Such embodiments are described in detail below.

[0138] Controlled-release microparticles containing eltrombopag or its derivatives, conjugates or salts

[0139] The controlled-release microparticles comprising Eltrombopag or its derivative, conjugate or salt may take the form of various microparticles, such as (i) microparticles comprising a polymer matrix (also referred to as a controlled-release polymer matrix) or (ii) multivesicular liposomes.

[0140] According to one embodiment, the average particle size of the microparticles is equal to or greater than 1 μm.

[0141] According to another embodiment, the average particle size of the microparticles is equal to or greater than 2 μm.

[0142] According to another embodiment, the average particle size of the microparticles is equal to or greater than 1 μm, for example equal to or greater than 2 μm, in particular in the range of 1 to 200 μm, more in particular in the range of 2 to 150 μm, still more in particular in the range of 2 to 100 μm, for example 10 to 100 μm, or 10 to 80 μm.

[0143] According to another embodiment, the microparticles have an average particle size equal to or greater than 1 μm, such as equal to or greater than 2 μm, in particular an average particle size below 200 μm, such as below 150 μm, more particularly below 100 μm, such as in the range of 10 to 100 μm, even more particularly below 80 μm, such as 1 to 80 μm, 2 to 80 μm or 10 to 80 μm.

[0144] According to a preferred embodiment, the average particle size of the microparticles is equal to or greater than 1 μm, for example equal to or greater than 2 μm, in particular in the range of 1 to 200 μm, more in particular in the range of 2 to 150 μm, still more in particular in the range of 2 to 100 μm, for example 10 to 100 μm, or 10 to 80 μm.

[0145] It should be understood that these ranges refer to the average size of all particles in a given population.The size of any given individual particle may be within a standard deviation above or below the average size.

[0146] In the context of the present invention, "microparticles" refer to particles of any shape made of any material, which are particularly suitable for local injection, particularly suitable for intramedullary injection into the human body, particularly into the bone marrow within a pharmaceutical composition, and whose average particle size is equal to or greater than 1 μm, such as equal to or greater than 2 μm, particularly in the range of 1 μm to 200 μm, more particularly in the range of 2 μm to 150 μm, and still more particularly 2 μm to 100 μm, such as 10 μm to 100 μm, or 10 μm to 80 μm.

[0147] Microparticles suitable for pharmaceutical compositions for use according to the present invention may be selected from various types of microparticles, such as microspheres, microparticle matrices, microsphere matrices, microcapsules, rods, flakes, pills, fibers and pellets.

[0148] (i) Microparticles comprising a polymer matrix

[0149] The polymer matrix can be selected from various polymers suitable for obtaining controlled release microparticles as explained below. Such polymer matrices are non-toxic to the human body.

[0150] According to one embodiment, the polymer forming the polymer matrix is ​​biodegradable and biocompatible.

[0151] In the context of the present invention, a "biodegradable" material is one that degrades enzymatically or hydrolytically and for which there is evidence that the degradation products are incorporated into the biomass and / or eliminated from the organism by metabolism or renal filtration.

[0152] In the context of the present invention, a "biocompatible" material refers to a material that is tolerated by the human body.

[0153] Non-toxic, biocompatible and biodegradable polymers can be natural or synthetic.

[0154] According to one embodiment, the controlled release polymer matrix according to the present invention comprises at least one biocompatible and biodegradable copolymer or polymer selected from the group consisting of poly(lactic-co-glycolic acid) copolymer (also known as PLGA or PLG), poly(caprolactone), poly(lactide) (also known as PLA), poly(glycolide) (also known as PGA), poly(lactide-co-caprolactone), poly(ethylene glycol), poly(ethylene oxide) (also known as PEO), PLGA-b-PEO-b-PLGA, PLGA-b-PEO, polyhydroxyalkanoates, poly(hydroxybutyrate), poly(trimethylene carbonate), poly(dioxane), poly(ethylene glycol), poly(ethylene oxide ... ketones), poly(valerolactones), poly(α-hydroxy acids), poly(lactones), poly(amino acids), polyanhydrides, poly(orthoesters), poly(acetals), polyurethanes, polythioesters, polyphosphates, poly(ester-co-amides), poly(vinyl alcohol), PVA-g-PLGA, poly(ether ester) multi-block copolymers, polyvinyl pyrrolidone, poly(methacrylates), PEO-PPO-PEO (pluronics, also known as polyethylene oxide-polypropylene oxide-polyethylene oxide), gelatin, heparin, chondroitin sulfate; polysaccharides such as alginate, starch, chitosan, hyaluronic acid and dextran, and any combination thereof.

[0155] According to a particular embodiment, the controlled-release polymer matrix comprises at least one poly(lactic-co-glycolic acid) copolymer.

[0156] According to said embodiment, the controlled-release polymer matrix may comprise poly(lactic-co-glycolic acid) in an amount greater than 70% by weight, in particular greater than 80% by weight, even more in particular greater than 90% by weight, relative to the total weight of the polymer matrix.

[0157] Suitable polymers are not limited to those commercially available and known as RESOMER (Evonik Industries AG, Germany), LACTEL (Durect, USA), PURASORB (Corbion NV, Netherlands), Viatel (Ashland, USA), EXPANSORB (Seqens, France).

[0158] Examples of suitable polymers are listed in Table A

[0159] Table A:

[0160]

[0161] The polymer matrix may comprise PLGA and one or more additional polymers, copolymers or mixtures thereof. The additional polymer(s), copolymer(s) or mixtures thereof may be present in the polymer matrix in an amount of 0 to 30% by weight, particularly 0 to 20% by weight, more particularly 0 to 10% by weight, relative to the total weight of the polymer matrix.

[0162] Thus, according to another specific embodiment, the controlled release polymer matrix comprises at least one poly(lactic-co-glycolic acid) copolymer and at least one suitable additional biocompatible and biodegradable polymer or copolymer selected from the group consisting of poly(lactide) other than poly(lactic-co-glycolic acid) copolymer, poly(caprolactone), poly(glycolide) other than poly(lactic-co-glycolic acid) copolymer, poly(lactide-co-caprolactone), poly(ethylene glycol), poly(ethylene oxide), PLGA-b-PEO-b-PLGA, PLGA-b-PEO , polyhydroxyalkanoates, poly(hydroxybutyrate), poly(trimethylene carbonate), poly(dioxanone), poly(valerolactone), poly(α-hydroxy acid), poly(lactone), poly(amino acid), polyanhydrides, poly(orthoesters), poly(acetals), polyurethanes, polythioesters, polyphosphates, poly(ester-co-amides), poly(vinyl alcohol), PVA-g-PLGA, poly(ether ester) multi-block copolymers, polyvinyl pyrrolidone, poly(methacrylate), PEO-PPO-PEO (pluronics), gelatin, heparin, chondroitin sulfate; polysaccharides such as alginate, starch, chitosan, hyaluronic acid and dextran, and any combination thereof.

[0163] According to another embodiment, microparticles suitable for the present disclosure have a particle size distribution with a D50 value not exceeding 200 μm, such as a D50 value not exceeding 150 μm, in particular not exceeding 100 μm, such as a particle size distribution of 1 to 80 μm or 2 to 80 μm.

[0164] It should be understood that these ranges refer to the average size of all particles in a given population.The size of any given individual particle may be within a standard deviation above or below the average size.

[0165] In one embodiment, the controlled release microparticles according to the invention are PLGA microspheres. In other words, in said embodiment, the polymer matrix does not comprise any additional polymers or copolymers.

[0166] When PLGA copolymers are implemented as the polymer matrix, they can have a wide range of molecular weights and monomer ratios of lactic acid to glycolic acid, particularly from 75:25 to 50:50, even more particularly a monomer ratio of lactic acid to glycolic acid of 50:50 or 75:25. Any suitable method known in the art for preparing polymers can be used, and the molecular weight can generally be in the range of 5 to 150 kDa, particularly 5 to 80 kDa, more particularly 5 to 50 kDa, for example 10 to 150 kDa, 10 to 80 kDa or 10 to 50 kDa.

[0167] According to a further embodiment, the polymer forming the polymer matrix comprises PLGA, e.g. an amount of 100% by weight of PLGA relative to the total weight of the polymer matrix, and PLGA is defined as follows:

[0168] - a molecular weight of 5 to 80 kDa, and

[0169] - The lactide:glycolide molar ratio is 75:25 to 50:50.

[0170] According to a more specific embodiment, the polymer forming the polymer matrix comprises PLGA, such as PLGA in an amount of 100% by weight relative to the total weight of the polymer matrix, and PLGA is defined as follows:

[0171] - a molecular weight of 5 to 50 kDa, and

[0172] - The molar ratio of lactide:glycolide is 50:50.

[0173] According to a more specific embodiment, the polymer forming the polymer matrix comprises PLGA, such as PLGA in an amount of 100% by weight relative to the total weight of the polymer matrix, and PLGA is defined as follows:

[0174] - a molecular weight of 50 to 100 kDa, and

[0175] - The molar ratio of lactide:glycolide is 50:50.

[0176] According to a more specific embodiment, the polymer forming the polymer matrix comprises PLGA, such as PLGA in an amount of 100% by weight relative to the total weight of the polymer matrix, and PLGA is defined as follows:

[0177] - a molecular weight of 5 to 50 kDa, and

[0178] - The lactide:glycolide molar ratio is 75:25.

[0179] According to a more specific embodiment, the polymer forming the polymer matrix comprises PLGA, such as PLGA in an amount of 100% by weight relative to the total weight of the polymer matrix, and PLGA is defined as follows:

[0180] - a molecular weight of 50 to 100 kDa, and

[0181] - The lactide:glycolide molar ratio is 75:25.

[0182] According to one embodiment, the PLGA is carboxyl terminated or ester terminated, in particular carboxyl terminated.

[0183] According to another embodiment, the microparticle matrix may comprise, and even consist of, a blend of two PLGA copolymers, in particular one low molecular weight and one high molecular weight.

[0184] The microparticles may also contain pharmaceutically acceptable excipients for adjusting drug release characteristics, such as medium chain triglycerides, poly(oxyethylene) sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 80), sorbitan fatty acid esters, cyclodextrins, lecithin, mannitol, sucrose, inorganic salts, and mixtures thereof.

[0185] The polymer matrix of the microparticles may comprise one or more excipients. The excipient(s) may be present in the polymer matrix in an amount of not more than 15% by weight, particularly not more than 10% by weight, more particularly not more than 5% by weight, relative to the total weight of the polymer matrix.

[0186] According to one embodiment, the weight percentage of the drug loading or 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt, in particular EPAG ethanolamine to the total weight of the microparticles is 2% to 45%, in particular 5% to 40%, more in particular 10% to 35%.

[0187] Manufacturing method for obtaining microparticles comprising a polymer matrix

[0188] Within the framework of the present invention, any method suitable for producing polymer microparticles having an average particle size of 1 to 200 μm, for example 2 to 150 μm, 2 to 100 μm, 10 to 100 μm or 10 to 80 μm is considered suitable.

[0189] Among such production methods, there can be exemplified emulsification-based methods such as high-pressure homogenization, for example using a rotor-stator homogenizer in batch mode or continuous mode, or membrane emulsification followed by removal of the organic solvent by extraction / evaporation.

[0190] According to the general principle of such a method, an emulsion can be prepared and processed through a membrane with pores of defined size. The microspheres obtained can then be collected after extraction and evaporation of the organic solvent, washed and freeze-dried.

[0191] According to one embodiment, the microparticles can be produced by O / W direct emulsion or by W / O / W double emulsion technology.

[0192] Schoubben, A., Ricci, M. & Giovagnoli, S. Meeting the unmet: from traditional to cutting-edge techniques for poly lactide and poly lactide-co-glycolide microparticle manufacturing. J. Pharm. Investig. 49, 381–404 (2019) Various methods for preparing PLGA microparticles that can be used in the framework of the present invention are reviewed.

[0193] According to one embodiment, the microparticles can be manufactured by solid-in-oil-in-water (S / O / W) double emulsion technology.

[0194] Giovagnoli, S., et al; Physicochemical characterization and release mechanism of a novel prednisone biodegradable microsphere formulation, JPharm Sci. 97: 303–317, (2008) describes an example of PLGA microparticles prepared via S / O / W emulsion technology. Other manufacturing methods suitable for obtaining microparticles according to the present invention are atomization by rotating disk, atomization by spray drying, fluidized bed coating or a combination thereof.

[0195] Alternatively, microparticles can be fabricated using drop-on-demand, drop-by-drop, and jet-fragmentation methods such as inkjet printing or microfluidics.

[0196] Alternatively, supercritical fluid technology can be used to produce the microparticles.

[0197] Alternatively, microparticles can be fabricated using microfabrication methods, such as template and mold-based techniques such as soft lithography.

[0198] All of these manufacturing methods are well known to those skilled in the art.

[0199] The manufacturing methods cited above are well known to those skilled in the art.

[0200] (ii) Multivesicular liposomes

[0201] Multivesicular liposomes (MVLs) are spherical particles with an average diameter of 10-30 μm, composed of multiple non-concentric lipid bilayers arranged in a honeycomb structure. These lipid layers surround numerous water-filled aqueous compartments that can be used to encapsulate water-soluble drugs such as Eltrombopag or its derivatives, conjugates, or salts.

[0202] MVLs are typically composed of at least one amphipathic lipid and one neutral lipid. The amphipathic lipid is selected from phospholipids, such as phosphatidylcholine or phosphatidylglycerol. The neutral lipid is selected from triglycerides having monounsaturated fatty acid ester moieties with 14-18 carbon atoms in the acyl chain (e.g., triolein, tripalmitin), saturated fatty acid ester moieties with 6-8 carbon atoms in the acyl chain (e.g., tricaproin, tricaprylin), and mixtures thereof. Cholesterol may also be used in the composition.

[0203] MVL is obtained by using a water-in-oil-in-water double emulsion method. In the first step, a water-in-oil emulsion is prepared by mixing phospholipids, triolein, tricaprylin and cholesterol dissolved in a volatile and water-immiscible organic solvent with an aqueous solution containing the dissolved drug to be encapsulated. The first emulsion is then emulsified by mixing with a second aqueous solution to produce a water-in-oil-in-water emulsion. The energy required to form the first and second emulsions can be provided mechanically, by ultrasonic treatment, or by a combination thereof. The volatile organic solvent is removed from the double emulsion by using air stripping or flushing, and the MVL is finally obtained. Finally, diafiltration or cross-flow filtration systems are used for removal of unencapsulated material, concentration of the MVL and buffer exchange.

[0204] In one embodiment, the neutral lipid used to make the MVL encapsulating Eltrombopag or its conjugates, derivatives or salts comprises a mixture of triolein:tricaprylin in a ratio of 50:50 to 0:100.

[0205] A mixture of controlled-release and immediate-release dosage forms

[0206] As mentioned above, 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof can be included in a mixture of controlled-release and immediate-release dosage forms.

[0207] Controlled release dosage forms are as defined above.

[0208] The presence of the immediate-release dosage form is intended to achieve a rapid onset of local concentration of Eltrombopag, or its derivative, conjugate, or pharmaceutically acceptable salt, to achieve a rapid stabilization or increase in local concentration of the SDF-1 chemokine, which in turn will increase the homing of transplanted HSCs. In one embodiment, the maximum weight ratio of the immediate-release Eltrombopag, or its derivative, conjugate, or pharmaceutically acceptable salt relative to the total amount of Eltrombopag, or its derivative, conjugate, or pharmaceutically acceptable salt is 50%, 30%, or 15%.

[0209] In certain embodiments of the present invention, the weight ratio of the immediate-release Eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt relative to the total amount of Eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt may be 0 to 5%, 0 to 10%, 0 to 15%, 0 to 30%, or 0 to 50%.

[0210] In some embodiments, the immediate-release form releases over a period of 0 to 6 hours. In some embodiments, the immediate-release form releases over a period of 0 to 2 hours.

[0211] According to the present invention, the immediate release dosage form can take the form of an immediate release portion mixed with the controlled release microparticles as described above, or be present in a continuous phase with which the microparticles are mixed when preparing the final formulation in powder form. The continuous phase may of course contain any other suitable pharmaceutically acceptable excipients in addition to the excipients already present in the microparticles.

[0212] Pharmaceutical compositions and kits

[0213] As mentioned above, the controlled release pharmaceutical composition according to the present invention is suitable for local injection.

[0214] The controlled-release pharmaceutical composition according to the present invention is suitable for local injection in large quantities, depending on the pathology being targeted.

[0215] Thus, also provided herein is a controlled release pharmaceutical composition as defined in the present disclosure, wherein the local injection is selected from the group consisting of intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection, intraosseous injection, and combinations thereof.

[0216] According to a specific embodiment, the controlled-release pharmaceutical composition according to the present disclosure can be administered by local injection at the affected site.

[0217] According to a particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered in the bone marrow by intramedullary injection.

[0218] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered in the heart by intramyocardial, intrapericardial, intraendocardial and / or intraepicardial injection.

[0219] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered intravascularly by intramyocardial, intrapericardial, intraendocardial and / or intraepicardial injection.

[0220] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered in the skin or dermis by wound instillation, surgical site infusion and / or intradermal injection.

[0221] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered in the eye by intravitreal injection, sub-Tenon's injection and / or intraocular injection.

[0222] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered in the liver by intrahepatic injection.

[0223] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered in the kidney by intrarenal injection.

[0224] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered in the gastrointestinal tract (GIT) by injection in the GIT, intraperitoneal injection and / or submucosal injection.

[0225] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered intraperitoneally by injection.

[0226] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered in the lungs by injection.

[0227] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered in the gums by intraperiodontal injection and / or subgingival injection.

[0228] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered in the joint by intra-articular injection.

[0229] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered in the bone by intraosseous injection.

[0230] According to one embodiment, the controlled release pharmaceutical composition according to the present disclosure may be an extended release pharmaceutical composition comprising at least one extended release pharmaceutical dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof.

[0231] Provided herein are controlled release pharmaceutical compositions in various forms, namely solutions, suspensions, powders, solid implants, semisolid implants, microporous implants, and in situ formed depots. According to one embodiment, provided herein are controlled release pharmaceutical compositions in the form of sterile and injectable dosage forms selected from the group consisting of solutions, suspensions, powders, solid implants, semisolid implants, microporous implants, and in situ formed depots.

[0232] Other pharmaceutical compositions are provided in powder form or in kit form. When in powder form, the pharmaceutical composition is primarily intended for storage, while solutions, suspensions, solid implants, semisolid implants, microporous implants, powders, or in situ formed reservoirs, particularly suspensions, are ready-to-use compositions that are readily injectable, and the kit can separately store (i) an aqueous injection vehicle and (ii) a controlled-release dosage form comprising eltrombopag or its conjugate, derivative, or salt, particularly a powder form for forming a sterile, injectable dosage form, particularly a suspension suitable for injection.

[0233] In addition to 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its conjugate or derivative or pharmaceutically acceptable salt as defined in the present disclosure, the pharmaceutical composition defined in the present disclosure may further comprise at least one pharmaceutically acceptable excipient.

[0234] Among the pharmaceutically acceptable excipients suitable for the pharmaceutical compositions defined in the present disclosure, there may be mentioned tonicity enhancing agents, wetting agents, viscosity enhancing agents, density enhancing agents and mixtures thereof.

[0235] In one embodiment, the pharmaceutical composition is further characterized in that it is in the form of a sterile and injectable suspension, optionally comprising an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, and wherein it is obtained by mixing a controlled-release dosage form comprising Eltrombopag or its conjugates or derivatives or salts, in particular microparticles comprising Eltrombopag or its conjugates or derivatives or salts, more particularly microparticles as defined in the present disclosure, with an aqueous injection vehicle as defined in the present disclosure.

[0236] Various embodiments of the alternative forms will be described in detail below.

[0237] Solutions, solid implants, semisolid implants, microporous implants, powders, and in situ forming depots can be prepared according to methods known to those skilled in the art.

[0238] According to a specific embodiment, the sterile and injectable dosage form is in the form of a suspension, which can be obtained from a powder, as described in detail below.

[0239] Powders and suspensions

[0240] In one embodiment, a controlled release pharmaceutical composition in powder form is provided, comprising a controlled release dosage form comprising Eltrombopag or its conjugates, derivatives or salts, wherein the controlled release dosage form comprising Eltrombopag or its conjugates, derivatives or salts is in the form of microparticles, particularly in the form of microparticles comprising a polymer matrix, more particularly in the form of microparticles having an average particle size equal to or greater than 1 μm, for example 2 μm, even more particularly in the form of microparticles as defined in the present disclosure.

[0241] In one embodiment, a pharmaceutical composition in powder form is provided, comprising controlled-release microparticles comprising Eltrombopag or its conjugate, derivative, or salt, wherein the microparticles are microparticles or multivesicular liposomes comprising a polymer matrix, and the microparticles comprise Eltrombopag or its conjugate, derivative, or salt having an average particle size equal to or greater than 1 μm, for example, 2 μm.

[0242] In another embodiment, a pharmaceutical composition in the form of a sterile and injectable suspension suitable for local injection, in particular intramedullary injection, is provided, which is obtained by mixing a composition in the form of a powder according to the present invention with an aqueous injection vehicle, wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, and wherein the excipient may be present in the aqueous injection vehicle or the powder.

[0243] According to one embodiment, the powder further comprises an immediate release dosage form comprising Eltrombopag or its conjugate or derivative or salt, in particular wherein the maximum weight proportion of the immediately released Eltrombopag or its conjugate or derivative or salt is 15%, 30% or 50% relative to the total amount of Eltrombopag or its conjugate or derivative or salt.

[0244] In another embodiment, a pharmaceutical composition in powder form is provided, comprising controlled-release microparticles comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof and a controlled-release polymer matrix, wherein

[0245] - the average particle size of the controlled-release microparticles is greater than or equal to 1 μm, for example greater than or equal to 2 μm, particularly in the range of 1 to 200 μm, more particularly in the range of 2 to 150 μm, still more particularly in the range of 2 to 100 μm, for example 10 to 100 μm or 10 to 80 μm,

[0246] - the controlled release polymer matrix comprises at least one poly(lactic-co-glycolic acid) copolymer, and

[0247] - the weight percentage of the drug loading or 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt to the total weight of the microparticles ranges from 2% to 45%, particularly from 5% to 40%, more particularly from 10% to 35%.

[0248] According to the embodiment, the powder further comprises an immediate release dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or a pharmaceutically acceptable salt thereof, particularly EPAG ethanolamine, particularly wherein relative to 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H -pyrazol-4-ylidene] hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its conjugate or derivative or pharmaceutically acceptable salt, in particular EPAG ethanolamine, the total amount of immediate-release 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene] hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its pharmaceutically acceptable salt, in particular EPAG ethanolamine, the maximum weight ratio is 15%, 30% or 50%.

[0249] The present invention also relates to a controlled-release pharmaceutical composition in the form of a sterile and injectable suspension suitable for local injection, in particular intramedullary injection, obtained by mixing the formulation in powder form described in the present disclosure with an aqueous injection vehicle, wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, and wherein the excipient may be present in the aqueous injection vehicle or the powder.

[0250] In another embodiment, a pharmaceutical composition is provided, in particular a pharmaceutical composition in the form of a sterile and injectable suspension, wherein the composition is prepared by combining a controlled-release dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a pharmaceutically acceptable salt thereof, or a mixture of a controlled-release dosage form and an immediate-release dosage form, in particular a mixture comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a pharmaceutically acceptable salt thereof. The pharmaceutical composition is obtained by mixing microparticles of Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or a pharmaceutically acceptable salt thereof, more particularly microparticles as defined in the present disclosure, with an aqueous injection vehicle, and wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof.

[0251] In one embodiment, the pharmaceutical composition is characterized in that the concentration of Eltrombopag or its conjugate or derivative or salt ranges from 2 mg to 63 mg equivalent of free acid per milliliter of sterile and injectable dosage form, in particular suspension, in particular from 8 mg to 55 mg equivalent of free acid per milliliter, more particularly from 12 mg to 47 mg equivalent of free acid per milliliter.

[0252] In one embodiment, the pharmaceutical composition is characterized in that the concentration of eltrombopag ranges from 2 mg to 80 mg per milliliter of the sterile and injectable dosage form, in particular the suspension, in particular from 10 mg to 70 mg per milliliter, more particularly from 15 mg to 60 mg per milliliter.

[0253] According to another particular embodiment, in the pharmaceutical composition according to the invention which is still sterile and in the form of an injectable suspension, the microparticles may be present in an amount of 1% to 25% by weight, in particular 2% to 20% by weight, more particularly 5% to 15% by weight relative to the total weight of the composition.

[0254] The pharmaceutical composition used in the framework of the present invention may take the form of a sterile and injectable composition, in particular a suspension composition, comprising an effective amount of Eltrombopag or a conjugate or derivative or salt thereof.

[0255] In the sense of the present invention, "sterile" means an environment that ensures that the compound under consideration in the composition according to the invention meets the safety requirements for administration routes such as those mentioned above, in particular administration routes into or through the bone marrow. In fact, for obvious reasons, it is essential that the composition according to the invention does not contain any contaminants that could induce undesirable side effects at the site of the host.

[0256] The pharmaceutical composition used within the framework of the present invention can be prepared using a formulation in powder form comprising the microparticles described herein. According to one embodiment, the pharmaceutical composition is a sterile and injectable composition for controlled release or for controlled and immediate release of Eltrombopag or its conjugates, derivatives or salts, which is suitable for local injection, in particular intramedullary injection.

[0257] Due to their injectable nature, the compositions according to the present invention necessarily contain a physiologically acceptable vehicle, also referred to as an "aqueous injection vehicle."

[0258] "Physiologically acceptable medium" refers to a medium that is non-toxic and compatible with injection and / or administration of a composition, such as contemplated in the present invention.

[0259] The present invention more particularly relates to a pharmaceutical composition as defined in the present disclosure, wherein it is obtained by mixing a formulation in powder form as described in the present disclosure with an aqueous injection vehicle, wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, and wherein the excipient may be present in the aqueous injection vehicle or in the powder.

[0260] The composition may comprise a solvent or a mixture of physiologically acceptable solvents.

[0261] The composition may comprise a physiologically acceptable aqueous medium.

[0262] As the aqueous medium suitable for the present invention, there may be mentioned, for example, water.

[0263] As isotonic agents suitable for the preparation of the compositions according to the invention, mention may be made of sugars and sodium chloride.

[0264] Aqueous injection vehicles may particularly contain a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, or mixtures thereof.

[0265] Among the tonicity enhancing agents, the following may be mentioned: glucose, mannitol, sorbitol, sucrose, glycerol, sodium chloride, potassium chloride, cyclodextrin and maltodextrin.

[0266] Among the wetting agents, the following may be mentioned: poly(oxyethylene)sorbitan fatty acid esters, such as those commercially available under the trade name TWEEN; sorbitan fatty acid esters, such as those commercially available under the trade name SPAN; poloxamers and lecithins.

[0267] Among the viscosity enhancers, the following may be mentioned: sodium carboxymethylcellulose (CMC), glycosaminoglycans such as hyaluronic acid, dextran, collagen, poly(vinyl pyrrolidone), poly(ethylene glycol), gelatin, hydroxyethylcellulose (HEC), methylcellulose (MC), alginates, gum arabic, starch.

[0268] According to a specific embodiment, the pharmaceutical composition has a temperature of 10 s at 25°C. -1 The viscosity is measured at a shear rate of 5 to 1000 mPa.s, in particular 5 to 500 mPa.s.

[0269] medicine box

[0270] Also provided herein is a kit or article of manufacture comprising in separate compartments (i) an aqueous injection vehicle and (ii) a controlled release dosage form, or a mixture of a controlled release dosage form and an immediate release dosage form, comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or a pharmaceutically acceptable salt thereof, in particular as defined herein, or a powder as defined herein, wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, for preparing a pharmaceutical composition suitable for local injection, in particular intramedullary injection.

[0271] In one embodiment, the kit may be in the form of two separate vials.

[0272] In another embodiment, the kit or article can be in the form of a vial and a prefilled syringe, or in the form of a medical device, or in the form of two separate vials. In such embodiments, the two compartments or dual chambers can be provided with a mixture of an aqueous injection vehicle and a powder as described herein below by all means known to those skilled in the art. Still in such embodiments, the means can take the form of a pierceable membrane or a destructible diaphragm, such as a membrane or a destructible diaphragm that can be pierced by pressure applied by a user.

[0273] In these embodiments, the kit or article of manufacture may further comprise a label instructing the user to introduce the resulting pharmaceutical composition into the bone marrow of a subject.

[0274] For the comfort of the patient and for safety reasons, it would be advantageous to find a pharmaceutical composition suitable for local injection, in particular for intramedullary injection, since such an injection would require only one effective injection rather than multiple injections.

[0275] For the comfort of the patient and for safety reasons, it would also be advantageous to find a pharmaceutical composition suitable for local injection, in particular for intramedullary implantation, since such implantation would require only one effective implantation rather than multiple implantations.

[0276] Administration of the composition

[0277] Depending on the targeted pathology to be treated, the controlled release pharmaceutical composition according to the present invention can be injected into the affected site, which is any suitable site, tissue or organ selected from the group consisting of bone, heart, blood vessels, skin, dermis, eye, liver, kidney, gastrointestinal tract, peritoneum, lung, gum, joint, bone marrow, in particular sternum, tibia, femur, iliac crest, vertebrae and combinations thereof, for example the iliac crest, such as the posterior part of the iliac crest, and combinations thereof.

[0278] With respect to improving the homing, colonization, and long-term expansion and proliferation of hematopoietic stem cells, the pharmaceutical composition according to the present disclosure can be injected or implanted into any suitable site containing functional bone marrow, such as the sternum, tibia, femur, iliac crest or vertebrae, particularly the iliac crest, more particularly the posterior portion of the iliac crest.

[0279] Intramedullary injections can be performed using an 18G needle fitted with a trocar.

[0280] Intramedullary implantation can be performed using a 16G needle fitted with a trocar.

[0281] The injection or implantation can be performed under general or local anesthesia.

[0282] The pharmaceutical compositions used within the framework of the present invention can be injected or implanted using any method known in the art.

[0283] In particular, the pharmaceutical composition can be administered with the aid of an injection device suitable for local injection, in particular for intramedullary injection, such as a syringe equipped with a 15-25G, preferably a 16-25G, more preferably an 18-23G needle.

[0284] Therapeutic uses and methods

[0285] As mentioned above, the pharmaceutical composition according to the present invention finds use in treating degenerative diseases and improving stem cell homing.

[0286] As mentioned above, according to one embodiment, the pharmaceutical composition defined in the present disclosure or the pharmaceutical composition obtained by mixing the two compartments of the kit for single or multiple use defined in the present disclosure is injected locally at the lesion, affected tissue or affected organ.

[0287] In particular, local injection of a pharmaceutical composition as defined in the present disclosure or a pharmaceutical composition obtained by mixing the two compartments of a kit for single or multiple use as defined in the present disclosure,

[0288] For improving homing, colonization and long-term expansion and proliferation of hematopoietic stem cells by intramedullary injection in patients, particularly human patients, before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), for preventing and / or treating non-malignant blood disorders, for preventing and / or treating hematological malignancies, and / or for preventing and / or treating primary immunodeficiency, autoimmune disease or inborn error of metabolism, and / or

[0289] By intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection, intraosseous injection and combinations thereof, for the prevention and / or treatment of degenerative diseases / injuries as defined in the present disclosure.

[0290] According to another embodiment, the medicament as defined in the present disclosure or the medicament obtained by mixing the two compartments of the kit as defined in the present disclosure is used for:

[0291] - prevention and / or treatment of degenerative diseases / injuries, in particular prevention and / or treatment of cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis; periodontal disease; eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; kidney disease; liver diseases, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease and pulmonary fibrosis; and / or

[0292] - Wound healing, including diabetic wound healing and corneal wound healing; and / or

[0293] - prevention and / or treatment of non-malignant blood disorders, such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and beta-thalassemia, and / or prevention and / or treatment of blood malignancies, such as myeloma, lymphoma, leukemia, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndromes, in particular leukemia, and / or prevention and / or treatment of primary immunodeficiency, autoimmune diseases or inborn errors of metabolism.

[0294] The diseases or injuries, the corresponding lesion sites or affected tissues / organs and the corresponding possible local injection sites are summarized in Table B below.

[0295] Table B

[0296]

[0297] A general description of therapeutic dosage and posology is first provided below and applies to the entire range of therapeutic applications according to the present invention.

[0298] More specific aspects of the therapeutic uses and methods are then provided in conjunction with more specific therapeutic applications.

[0299] Therapeutic dose and dosimetry

[0300] According to a specific embodiment, provided herein is a pharmaceutical composition as defined in the present disclosure or obtained by mixing the two compartments of the kit as defined in the present disclosure, wherein 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or a pharmaceutically acceptable salt thereof, in particular EPAG ethanolamine is administered by local injection, in particular intramedullary injection, to the patient in an amount of 0.2 mg to 50 mg equivalent of the free acid per kg body weight of the patient, in particular 0.5 mg to 45 mg equivalent of the free acid per kg body weight of the patient, more in particular 1 mg to 40 mg equivalent of the free acid per kg body weight of the patient.

[0301] In particular, the inventors have found that local injection of EPAG or its derivatives or conjugates or pharmaceutically acceptable salts, such as EPAG ethanolamine, at the site of lesions or at the affected tissues / organs, in particular intramedullary injection in the bone marrow, can improve the local concentration of EPAG or its derivatives or conjugates or pharmaceutically acceptable salts, such as EPAG ethanolamine, to a local concentration above 14 μM, which is the maximum plasma concentration after oral administration of a 50 mg tablet.

[0302] The inventors have demonstrated that the novel mode of action of the present invention enables the achievement of a range of EPAG ethanolamine concentrations that are higher than those observed with once-daily oral administration of 50 mg PROMACTA. ® tablets (US FDA, Drug Approval Package, Promacta (eltrombopag) Tablets, Medical Review Part 1) achieves steady-state plasma concentrations (i.e., 14 μM).

[0303] In one embodiment, the controlled-release pharmaceutical compositions used in accordance with the present disclosure can be effective in maintaining a local concentration of EPAG ethanolamine above a plasma concentration of 14 μM.

[0304] According to one embodiment, the controlled-release pharmaceutical composition for use according to the present disclosure can effectively maintain a local concentration of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof of greater than 1 μM, particularly greater than 5 μM, more particularly greater than 15 μM, for example, between 5 μM and 200 μM, particularly between 15 μM and 100 μM, even more particularly between 50 μM and 100 μM, for a period of 6 hours to 3 months, 6 hours to 30 days, particularly between 1 day and 15 days, more particularly between 2 days and 15 days, yet more particularly between 4 days and 15 days after local injection.

[0305] To determine the local and systemic concentrations of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its conjugates or derivatives or pharmaceutically acceptable salts, in particular EPAG ethanolamine, any known method can be used by those skilled in the art. For example, HPLC-MS analysis can be used.

[0306] Furthermore, the time required to release 80% by weight of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its conjugate or derivative or pharmaceutically acceptable salt, in particular EPAG ethanolamine, can be 6 hours to 3 months, 6 hours to 30 days, in particular 1 day to 15 days, more preferably In particular, 2 to 15 days, yet more particularly 4 to 15 days, for example 4 to 30 days, preferably 15 days, wherein 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or a pharmaceutically acceptable salt thereof, in particular EPAG ethanolamine is present in a dose equivalent to 10 mg to 570 mg of Eltrombopag free acid form.

[0307] Further provided herein is a pharmaceutical composition according to the invention or a pharmaceutical composition for use according to the invention obtained by mixing two compartments of a kit as defined above, wherein the dissolution rate of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or a pharmaceutically acceptable salt thereof, in particular EPAG ethanolamine, is 80% (weight / weight) over more than 6 hours, in particular over more than 1, 2 or 4 days, measured according to the following protocol:

[0308] A 75 mg aliquot of the controlled-release dosage form according to the present invention was suspended in 50 mL of a solution of phosphate-buffered saline (10 mM, pH 6.8) containing 0.5% polysorbate 80 at 37° C. with stirring; filtered samples were then periodically analyzed by HPLC throughout the duration of the release.

[0309] wherein 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or a pharmaceutically acceptable salt thereof, in particular EPAG ethanolamine is present in a dose equivalent to 10 mg to 570 mg of eltrombopag free acid form.

[0310] Thus, the present inventors have discovered a surprisingly suitable method for treating patients with specific in vitro dissolution rates and / or in vivo release rates. Examples 3 and 4 illustrate these suitable profiles.

[0311] In the framework of the present invention, "dissolution profile" refers to an in vitro test that reports the cumulative amount of active ingredient released as a function of time. The dissolution data are derived from the conditions under which the measurements were made and are described in this text.

[0312] Furthermore, the time required to release 80% by weight of EPAG ethanolamine can be 6 hours to 3 months, particularly 6 hours to 30 days, particularly 1 day to 15 days, more particularly 2 days to 15 days, yet more particularly 4 days to 15 days, e.g. 4 days to 30 days, preferably 15 days, wherein EPAG ethanolamine is present in a dose ranging from 10 mg to 800 mg.

[0313] In one embodiment, the pharmaceutical composition used according to the invention is characterized by the fact that the dissolution rate of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or a pharmaceutically acceptable salt thereof, in particular EPAG ethanolamine, is 80% (weight / weight) over more than 6 hours, more than 6 hours, in particular over 1 day, 2 days or 4 days, measured according to the protocol described above.

[0314] Administration can be performed by single injection or sequential injections, provided that the time required to release 80% by weight of Eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt, in particular EPAG ethanolamine, in the microparticles is from 6 hours to 3 months, in particular from 6 hours to 30 days, in particular from 1 day to 15 days, more in particular from 2 days to 15 days, yet more in particular from 4 days to 15 days, for example from 4 days to 30 days, preferably 15 days, Eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt, in particular EPAG ethanolamine is present in a concentration of 2 mg to 80 mg per ml of suspension and the volume of the pharmaceutical composition is, for example, from 0.1 ml to 10 ml, in particular from 1 ml to 10 ml, more in particular from 5 ml to 10 ml.

[0315] In one embodiment, the injection is performed multiple times (two, three, four or more times). In this embodiment, when each subsequent injection is performed, it is performed after an appropriate period of time has passed since the previous injection. The appropriate period of time can be, for example, two weeks, one month, two months, three months, four months, five months, six months, one year or longer.

[0316] According to a specific embodiment, Eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt, in particular EPAG ethanolamine, is present in a concentration of 2 mg to 63 mg equivalents of free acid per ml of suspension, in particular 8 mg to 55 mg equivalents of free acid per ml of suspension, more in particular 12 mg to 47 mg equivalents of free acid per ml of suspension.

[0317] According to a particular embodiment, eltrombopag is present in a concentration of 2 mg to 80 mg per ml of suspension, in particular 10 mg to 70 mg per ml of suspension, more in particular 15 mg to 60 mg per ml of suspension.

[0318] In one embodiment, the pharmaceutical composition for use according to the invention is characterized by the fact that the time required to release 80% by weight of Eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt, in particular EPAG ethanolamine, in the microparticles is from 6 hours, or 1 day, or 4 days, such as from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours to 3 months, such as from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours to 2 months, or from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours to 2 months. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours to 30 days, such as 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 7 days, 14 days, 21 days, 28 days, 30 days, 1.5 months, 2 months, 2.5 months to 3 months, is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, 1.5 months, 2 months, 2.5 months to 3 months, is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours. 5 days or 4.5 days, 7 days, 10 days, 15 days, 20 days to 2 months, is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, 1 day, or 4 days, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, 1.5 days or 4.5 days to 15 days, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, 1 day or 4 days, for example 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days, more specifically 6 hours, 1 day or 4 days, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, 1.5 days, or 4.5 days to 7 days, or from 5 days to 3 months, from 5 days to 2 months, from 5 days to 30 days, from 5 days to 15 days, from 5 days to 10 days, and from 5 days to 7 days.

[0319] The pharmaceutical composition may or may not exhibit a burst release. In the context of the present invention, "burst release" means immediate release upon placement in a release medium, before the release rate reaches a steady-state curve, i.e., in the present invention, immediate release of an initial loading dose of the drug following local injection, particularly intramedullary injection.

[0320] In any case, the duration of the burst release may be from 0 to 1 day, such as from the beginning of day 1 to the end of day 1, particularly from 0 to 6 hours, from 0 to 3 hours, even more particularly from 0 to 2 hours.

[0321] In a specific embodiment, the pharmaceutical composition for use according to the present invention does not exhibit a significant burst release. According to said embodiment, less than 50% by weight of Eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt is released 12 hours after administration.

[0322] It will also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including age, weight, general health, sex, diet, time of administration, drug combination, the judgment of the treating physician, and the severity of the particular condition being treated.

[0323] Therapeutic uses and methods for treating degenerative diseases

[0324] The inventors have discovered that a controlled release pharmaceutical composition in the form of a local injection suspension and a controlled release pharmaceutical composition in the form of an implant for releasing (controlled release or a combination of immediate and controlled release) EPAG or its derivatives or conjugates or pharmaceutically acceptable salts such as EPAG ethanolamine locally at the site of lesions or affected tissues / organs improves cell regeneration and tissue / organ repair.

[0325] According to a specific embodiment, the degenerative disease / injury is selected from cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis; periodontal disease; eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; kidney disease; liver disease, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease and pulmonary fibrosis.

[0326] According to another specific embodiment, the controlled release pharmaceutical composition defined in the present disclosure is used for wound healing, including diabetic wound healing and corneal wound healing.

[0327] As mentioned above, the controlled-release dosage form or the mixture of the controlled-release dosage form and the immediate-release dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt as defined in the present disclosure is locally administered by injection, in particular by intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection and / or intraosseous injection, for local administration.

[0328] Prevention and / or treatment of cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; and / or

[0329] Preventing and / or treating diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis, and osteoarthritis; and / or

[0330] Prevention and / or treatment of periodontal disease; and / or

[0331] For wound healing, including diabetic wound healing and corneal wound healing; and / or

[0332] Preventing and / or treating eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; and / or

[0333] Prevention and / or treatment of kidney disease; and / or

[0334] Prevention and / or treatment of liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; and / or

[0335] Preventing and / or treating inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; and / or

[0336] Prevent and / or treat chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and / or pulmonary fibrosis.

[0337] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection and / or intraosseous injection is specifically used to prevent and / or treat degenerative diseases / injuries as defined in the present disclosure.

[0338] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intramyocardial injection, intrapericardial injection, intraepicardial injection and / or intraendocardial injection, is specifically used for the prevention and / or treatment of cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis and / or ischemia-reperfusion injury.

[0339] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intramyocardial, intrapericardial, intraepicardial and / or intraendocardial injection, is specifically intended for the prevention and / or treatment of myocardial infarction.

[0340] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intramyocardial, intrapericardial, intraepicardial and / or intraendocardial injection, is dedicated for the prevention and / or treatment of atherosclerosis.

[0341] In one embodiment, the pharmaceutical composition suitable for local injection, in particular suitable for intramyocardial injection, intrapericardial injection, intraepicardial injection and / or intraendocardial injection, is specifically used for preventing and / or treating ischemia-reperfusion injury.

[0342] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intra-articular and / or intraosseous injection, is dedicated to the prevention and / or treatment of diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis.

[0343] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intra-articular injection and / or intra-osseous injection, is dedicated to the prevention and / or treatment of osteoporosis.

[0344] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intra-articular injection and / or intra-osseous injection, is specifically intended for the prevention and / or treatment of rheumatoid arthritis.

[0345] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intra-articular and / or intra-osseous injection, is specifically intended for the prevention and / or treatment of osteoarthritis.

[0346] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intraosseous injection, intraperiodontal injection and / or subgingival injection, is specifically used for the prevention and / or treatment of periodontal disease.

[0347] In one embodiment, the pharmaceutical composition suitable for local injection, particularly for wound instillation, surgical site infusion, intradermal injection, intraocular injection, sub-Tenon's injection and / or intravitreal injection, is specifically used for wound healing, including diabetic wound healing and corneal wound healing.

[0348] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for wound instillation, surgical site infusion and / or intradermal injection, is specifically used for diabetic wound healing.

[0349] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for wound instillation, surgical site infusion, intraocular injection, sub-Tenon's injection and / or intravitreal injection, is specifically for corneal wound healing.

[0350] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intraocular injection, sub-Tenon's injection and / or intravitreal injection, is specifically used for the prevention and / or treatment of eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma.

[0351] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intraocular injection, sub-Tenon's injection and / or intravitreal injection, is specifically intended for the prevention and / or treatment of retinal ischemia.

[0352] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intraocular injection, sub-Tenon's injection and / or intravitreal injection, is specifically for the prevention and / or treatment of macular degeneration.

[0353] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intraocular injection, sub-Tenon's injection and / or intravitreal injection, is specifically intended for the prevention and / or treatment of diabetic retinopathy.

[0354] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intraocular injection, sub-Tenon's injection and / or intravitreal injection, is specifically for the prevention and / or treatment of glaucoma.

[0355] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intrarenal injection, is specifically used for the prevention and / or treatment of kidney diseases.

[0356] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intrahepatic injection, is specifically used for the prevention and / or treatment of liver diseases, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury.

[0357] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intrahepatic injection, is dedicated for the prevention and / or treatment of fibrosis.

[0358] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intrahepatic injection, is specifically used for the prevention and / or treatment of liver cirrhosis.

[0359] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intrahepatic injection, is specifically used for preventing and / or treating non-alcoholic fatty liver disease.

[0360] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intrahepatic injection, is specifically used for preventing and / or treating acute liver injury.

[0361] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intraperitoneal injection, submucosal injection and / or intra-GIT injection, is dedicated to the prevention and / or treatment of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis.

[0362] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intraperitoneal injection, submucosal injection and / or intra-GIT injection, is dedicated for the prevention and / or treatment of Crohn's disease.

[0363] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intraperitoneal injection, submucosal injection and / or intra-GIT injection, is dedicated for the prevention and / or treatment of ulcerative colitis.

[0364] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intrapleural injection and / or intrapulmonary injection, is dedicated to the prevention and / or treatment of chronic obstructive pulmonary disease (COPD).

[0365] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for intrapleural injection and / or intrapulmonary injection, is specifically used for the prevention and / or treatment of pulmonary fibrosis.

[0366] According to another aspect, the present invention relates to a method for preventing and / or treating degenerative diseases / injuries, said method comprising administering to a patient in need thereof, in particular a human patient in need thereof, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[ (2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for intramyocardial injection, intrapericardial injection, intraepicardial injection and / or intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection and / or intraosseous injection.

[0367] According to another aspect, the present invention relates to a method for preventing and / or treating cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intramyocardial injection, intrapericardial injection, intraepicardial injection or intraendocardial injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for intramyocardial injection, intrapericardial injection, intraepicardial injection and / or intraendocardial injection.

[0368] According to another aspect, the present invention relates to a method for preventing and / or treating diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intra-articular injection or intra-osseous injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for intra-articular injection and / or intra-osseous injection.

[0369] According to another aspect, the present invention relates to a method for preventing and / or treating periodontal disease, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intraosseous injection, intraperiodontal injection and / or subgingival injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for intraosseous injection, intraperiodontal injection and / or subgingival injection.

[0370] According to another aspect, the present invention relates to a method for performing and / or enhancing wound healing, including diabetic wound healing and corneal wound healing, comprising administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by wound instillation, surgical site infusion, intradermal injection, intraocular injection, subtenon injection and / or intravitreal injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for wound instillation, surgical site infusion, intradermal injection, intraocular injection, subtenon injection and / or intravitreal injection.

[0371] According to another aspect, the present invention relates to a method for preventing and / or treating eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intraocular injection, subtenon injection and / or intravitreal injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for intraocular injection, subtenon injection and / or intravitreal injection.

[0372] According to another aspect, the present invention relates to a method for preventing and / or treating kidney disease, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intrarenal injection, a pharmaceutical composition in the form of a suspension comprising a controlled-release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for intrarenal injection.

[0373] According to another aspect, the present invention relates to a method for preventing and / or treating liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intrahepatic injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for intrahepatic injection.

[0374] According to another aspect, the present invention relates to a method for preventing and / or treating inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, comprising administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intragastrointestinal injection, submucosal injection and / or intraperitoneal injection, a pharmaceutical composition in the form of a suspension comprising a controlled-release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for intragastrointestinal injection, submucosal injection and / or intraperitoneal injection.

[0375] According to another aspect, the present invention relates to a method for preventing and / or treating chronic obstructive pulmonary disease and pulmonary fibrosis, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intrapleural injection or intrapulmonary injection, a pharmaceutical composition in the form of a suspension comprising a controlled-release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for local injection, in particular for intrapleural injection and / or intrapulmonary injection.

[0376] According to another aspect, the present invention relates to a method for preventing and / or treating degenerative diseases / injuries, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release, by at least local injection, in particular by intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subTenon's injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection or intraosseous injection.

[0377] According to another aspect, the present invention relates to a method for preventing and / or treating cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intramyocardial injection, intrapericardial injection, intraepicardial injection or intraendocardial injection, at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release.

[0378] According to another aspect, the present invention relates to a method for preventing and / or treating diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intra-articular injection or intraosseous injection, at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release.

[0379] According to another aspect, the present invention relates to a method for preventing and / or treating periodontal disease, which method comprises administering at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release, to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intraosseous injection, intraperiodontal injection and / or subgingival injection.

[0380] According to another aspect, the present invention relates to a method for performing and / or enhancing wound healing, including diabetic wound healing and corneal wound healing, comprising administering to a patient in need thereof, in particular a human patient in need thereof, at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release, at least by local injection, in particular by wound instillation, surgical site infusion, intradermal injection, intraocular injection, sub-Tenon's injection and / or intravitreal injection.

[0381] According to another aspect, the present invention relates to a method for preventing and / or treating eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma, which method comprises administering at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release, to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intraocular injection, sub-Tenon's injection and / or intravitreal injection.

[0382] According to another aspect, the present invention relates to a method for preventing and / or treating kidney disease, which method comprises administering at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release, to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intrarenal injection.

[0383] According to another aspect, the present invention relates to a method for preventing and / or treating liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury, which method comprises administering at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release, to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intrahepatic injection.

[0384] According to another aspect, the present invention relates to a method for preventing and / or treating inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, comprising administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intragastrointestinal injection, submucosal injection or intraperitoneal injection, at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release.

[0385] According to another aspect, the present invention relates to a method for preventing and / or treating chronic obstructive pulmonary disease and pulmonary fibrosis, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intrapleural injection or intrapulmonary injection, at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release.

[0386] Also provided herein is a method for performing and / or enhancing wound healing, including diabetic wound healing and corneal wound healing, and / or for preventing and / or treating cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis, and ischemia-reperfusion injury; diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis, and osteoarthritis; periodontal disease; eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy, and glaucoma; kidney disease; liver disease, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease, and acute liver injury; inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease, and / or pulmonary fibrosis, comprising at least the following steps:

[0387] - preparing and / or providing a sterile and injectable suspension pharmaceutical composition by mixing a formulation in powder form as described below with an aqueous injection vehicle, wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, and wherein the excipient may be present in the aqueous injection vehicle or the powder, and

[0388] - Local injection, in particular, local injection of the pharmaceutical composition into a suitable lesion site or affected organ / tissue of a patient in need thereof by intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection or intraosseous injection, wherein the volume to be injected is adapted to the injection site.

[0389] Also provided herein is a method for promoting and / or enhancing wound healing, including diabetic wound healing and corneal wound healing, and / or for preventing and / or treating a degenerative disease / injury, in particular selected from cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis, and ischemia-reperfusion injury; diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis, and osteoarthritis; periodontal disease; eye diseases, such as retinal ischemia, macular degeneration, diabetic retinopathy, and glaucoma; kidney disease; liver disease, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease, and acute liver injury; inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease, and / or pulmonary fibrosis, comprising at least the following steps:

[0390] - preparing and / or providing a pharmaceutical composition in the form of a sterile and injectable solid implant, semisolid implant, microporous implant or in situ forming depot, wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, and wherein the excipient may be present in an aqueous injection vehicle or a powder, and

[0391] - Local implantation of the pharmaceutical composition into a suitable affected area of ​​a patient in need thereof, in particular by intramyocardial, intrapericardial, intraepicardial, intraendocardial, intradermal, intravitreal, intrahepatic, intrarenal, submucosal, intraperitoneal, intrapleural, intrapulmonary, intraarticular or intraosseous implantation, or by intragastrointestinal implantation, wound instillation or surgical site infusion, wherein the volume to be implanted is adapted to the implantation site.

[0392] Therapeutic uses and methods for improving stem cell homing and therapeutic uses and methods for treating related diseases Law

[0393] The inventors have found that local injection of a controlled-release pharmaceutical composition comprising EPAG or a derivative or conjugate thereof or a pharmaceutically acceptable salt such as EPAG ethanolamine into the affected site, particularly into the bone marrow, before, during or after autologous or allogeneic HSCT, can improve homing, colonization and long-term expansion and proliferation of HSCs.

[0394] More specifically, the inventors have discovered that local injection of a controlled-release pharmaceutical composition in the form of a suspension into the bone marrow and local implantation at the affected site, particularly intramedullary implantation of a controlled-release pharmaceutical composition in the form of an implant for releasing (controlled release or a combination of immediate and controlled release) EPAG or its derivatives or conjugates or pharmaceutically acceptable salts such as EPAG ethanolamine, before, during or after HSCT, increases the proportion of transplanted HSCs that effectively home and colonize within the recipient's bone marrow, leading to a faster restoration of normal hematopoiesis and a higher overall transplant success rate.

[0395] According to a specific embodiment, the disease / disorder associated with HSCT is selected from non-malignant blood disorders, such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and beta-thalassemia; hematological malignancies, such as myelomas, lymphomas, leukemias, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndromes, in particular leukemias; primary immunodeficiency, autoimmune diseases and inborn errors of metabolism.

[0396] Hematopoietic stem cell transplantation, also known as bone marrow transplantation, involves administering healthy hematopoietic stem cells from a donor to a patient with bone marrow failure or dysfunction. The HSC donor can be the patient themselves (autologous HSCT) or a selected donor (allogeneic HSCT). This procedure improves bone marrow function and leads to the destruction of malignant cells or the production of functional cells that can replace the dysfunctional cells. HSCT requires some degree of bone marrow (BM) ablation in the patient, known as conditioning, a preparatory regimen given to patients undergoing HSCT prior to infusion of donor cells to induce immunosuppression in the patient (to ensure engraftment and limit rejection and GvHD), eradicate any hematologic malignancies, and create space in the BM microenvironment for the new donor cells to colonize. This conditioning regimen can include radiation therapy, irradiation, chemotherapy, simulated radiochemotherapy, serum therapy, monoclonal antibodies, targeted therapies, and combinations thereof.

[0397] Allogeneic HSCT uses donor cells obtained from a healthy donor whose human leukocyte antigens (HLA) are an acceptable match to the patient's. The stem cell donor may be related to the patient, such as an HLA-matched sibling donor, who remains the preferred donor, or a haploidentical donor who shares half of the patient's HLA, or they may be an unrelated volunteer. Allogeneic HSCT is usually limited to younger patients with a good general condition because the risk of regimen-related toxicity and graft-versus-host disease (GVHD) increases with age. The likelihood of having a matched sibling donor also increases in younger patients.

[0398] Hematologic malignancies are typically diseases of the elderly, with a median patient age of approximately 65-70 years. Acute myeloid leukemia (AML) is a genetically heterogeneous disorder characterized by the accumulation of somatically acquired genetic alterations in hematopoietic progenitor cells that alter the normal mechanisms of self-renewal, proliferation, and differentiation. Allogeneic hematopoietic stem cell transplantation (HSCT) is commonly used in the second phase of AML treatment, known as the post-remission or consolidation phase, and has been shown to reduce the risk of leukemia relapse compared to standard consolidation chemotherapy. Importantly, elderly patients may be unable to tolerate the high doses of standard consolidation chemotherapy, and therefore HSCT with reduced conditioning may be preferred. However, allogeneic HSCT is more likely to have severe complications, including an increased risk of mortality. Furthermore, elderly patients often do not have an available HLA-matched sibling donor, necessitating the use of a haploidentical donor for transplantation, which increases the risk of post-transplantation GVHD. Reduced conditioning and the use of haploidentical donors both reduce the success rate of HSCT. Therefore, increasing the number of effective treatments for HSCT may allow elderly patients to successfully undergo consolidation therapy and benefit from a reduced risk of leukemia relapse. In addition, allogeneic HSCT is the only curative therapy for other malignant disorders such as advanced myelodysplastic syndrome (MDS).

[0399] Hemoglobinopathies are a group of inherited disorders caused by alterations in the gene encoding hemoglobin (Hb), primarily affecting red blood cells. Sickle cell disease (SCD) and beta-thalassemia major (β-thalassemia) are the most prevalent of this group, caused by abnormalities in Hb structure or Hb production, respectively. Allogeneic HSCT remains the only established curative therapy for SCD and β-thalassemia, but major barriers still prohibit its use, particularly in adults. The procedure achieves its highest efficacy and safety only when an HLA-matched sibling donor is available, which occurs in less than 20% of patients, and when transplantation is performed at a young age. Using haploidentical donors (available in over 80% of patients) can overcome this limitation in donor availability, but efficacy and safety are lower, which explains why haploidentical donors remain unused outside of clinical trials. Furthermore, toxicities associated with conditioning regimens particularly limit HSCT in adult patients, who often already exhibit comorbidities due to progressive disease. However, reduced conditioning intensity reduces the efficacy of the transplant.

[0400] Therefore, if the efficacy rate of allogeneic HSCT for both malignant and nonmalignant indications could be improved, this would allow for safer use of haploid donors and less intensive conditioning, particularly benefiting adult patients.

[0401] In autologous HSCT, bone marrow products are collected from the patient and re-infused after a purification process. Advantages include no GVHD and better colonization. However, bone marrow products may contain abnormal cells, which can lead to relapse in the case of malignant tumors. Therefore, allogeneic transplantation is preferred for many malignant indications such as leukemia and MDS because the cells infused by the donor do not contain contaminating tumor cells and therefore generally have a lower risk of disease relapse. Although allogeneic transplantation combined with reduced conditioning as a method to induce a graft-versus-malignancy effect is being evaluated for these indications, autologous transplantation has been more commonly used for solid tumors, lymphomas, and myelomas. Autologous HSCT is also used for non-malignant indications, such as autoimmune diseases and hemoglobinopathies when undergoing gene therapy. One of the main limitations of gene therapy for the treatment of hemoglobinopathies is the reduced colonization of cells manipulated ex vivo, for which increasing the colonization rate after transplantation may be very beneficial.

[0402] The therapeutic success of HSCT depends critically on the homing and colonization of sufficient numbers of donor hematopoietic stem cells (HSCs) into the patient's bone marrow (BM), followed by cell expansion and differentiation to reconstitute and maintain the patient's hematopoietic function. However, preclinical studies have shown that only 1% to 10% of intravenously infused HSCs find their way to the patient's BM, while the majority of cells are bound to other organs (van Hennik et al., Blood. 1999; 94(9): 3055-61). Therefore, there is still a need to increase the fraction of transplanted HSCs that effectively home and colonize the patient's BM to improve the success rate of HSCT.

[0403] According to a specific embodiment, provided herein is a pharmaceutical composition as defined in the present disclosure or obtained by mixing two compartments of a kit as defined in the present disclosure for improving the homing, colonization and long-term expansion and proliferation of hematopoietic stem cells by use as an intramedullary injection as a local injection and by use as an implant in a patient, in particular a human patient, before, during or shortly after an autologous or allogeneic hematopoietic stem cell transplant (HSCT).

[0404] Hematopoietic stem cells can be selected from bone marrow cells, peripheral blood stem cells, stem cells cultured from umbilical cord blood cells, including stem cells cultured after gene therapy.

[0405] The pharmaceutical composition defined in the present disclosure can be injected or implanted into the bone marrow within 96 hours before and within 96 hours after transplantation, particularly within 96 hours before and within 24 hours after transplantation, more particularly within 96 hours before transplantation.

[0406] More specifically, the pharmaceutical compositions defined in the present disclosure can be used to reduce transplant failure, to reduce the occurrence of poor graft function (PGF), to reduce the occurrence of graft-versus-host disease (GvHD), to improve overall survival and donor chimerism, and / or to promote hematopoietic recovery and the success rate of hematopoietic stem cell transplantation.

[0407] A loading dose of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or a pharmaceutically acceptable salt thereof, in particular EPAG ethanolamine, is administered intramedullary in the bone marrow within 96 hours before and within 96 hours after transplantation, in particular within 96 hours before and within 24 hours after transplantation, more in particular within 96 hours before transplantation.

[0408] As mentioned above, a controlled-release dosage form or a mixture of a controlled-release dosage form and an immediate-release dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof as defined in the present disclosure is administered by intramedullary injection for improving the homing, colonization and long-term expansion and growth of hematopoietic stem cells in patients before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT). and / or for the prevention and / or treatment of non-malignant blood disorders, such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and beta-thalassemia, and / or for the prevention and / or treatment of hematological malignancies, such as myeloma, lymphoma, leukemia, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndromes, in particular leukemia, and / or for the prevention and / or treatment of primary immunodeficiency, autoimmune diseases or inborn errors of metabolism.

[0409] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically used to improve the homing of hematopoietic stem cells before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT).

[0410] In one embodiment, the composition suitable for intramedullary injection is specifically used to improve the colonization of hematopoietic stem cells before, during, or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT).

[0411] In one embodiment, the composition suitable for intramedullary injection is specifically designed to improve the long-term expansion and proliferation of hematopoietic stem cells before, during, or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT).

[0412] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of non-malignant blood disorders, such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and beta-thalassemia.

[0413] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of hematological malignancies, such as myeloma, lymphoma, leukemia, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndrome, in particular leukemia.

[0414] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically used for the prevention and / or treatment of severe aplastic anemia.

[0415] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically for the prevention and / or treatment of a hemoglobinopathy.

[0416] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically for the prevention and / or treatment of sickle cell disease.

[0417] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of beta-thalassemia.

[0418] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically for the prevention and / or treatment of myeloma and / or lymphoma.

[0419] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically used for the prevention and / or treatment of leukemias, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia.

[0420] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically for the prevention and / or treatment of Waldenstrom's macroglobulinemia.

[0421] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically for the prevention and / or treatment of myeloproliferative neoplasms.

[0422] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically for the prevention and / or treatment of myelodysplastic syndrome.

[0423] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically intended for the prevention and / or treatment of primary immunodeficiency, autoimmune disease or inborn error of metabolism.

[0424] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically intended for the prevention and / or treatment of primary immunodeficiency.

[0425] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically intended for the prevention and / or treatment of autoimmune diseases.

[0426] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is specifically intended for the prevention and / or treatment of inborn errors of metabolism.

[0427] According to another aspect, the present invention relates to a method for improving hematopoietic stem cell homing, colonization and long-term expansion and proliferation before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), the method comprising administering to a patient in need thereof, in particular a human patient in need thereof, at least by intramedullary injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for intramedullary injection.

[0428] According to another aspect, the present invention relates to a method for reducing transplant failure, reducing transplant failure, reducing the occurrence of graft dysfunction (PGF), reducing the occurrence of graft-versus-host disease (GvHD), improving overall survival and donor chimerism, and / or promoting hematopoietic recovery and the success rate of hematopoietic stem cell transplantation, the method comprising administering a pharmaceutical composition in the form of a suspension to a patient in need thereof, in particular a human patient in need thereof, at least by intramedullary injection, which comprises a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for intramedullary injection.

[0429] According to another aspect, the present invention relates to a method for preventing and / or treating non-malignant blood disorders such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and β-thalassemia, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by intramedullary injection, a pharmaceutical composition in the form of a suspension comprising a controlled-release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for intramedullary injection.

[0430] According to another aspect, the present invention relates to a method for preventing and / or treating hematological malignancies, such as myelomas, lymphomas, in particular selected from leukemias, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, myeloma, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, myelodysplastic syndrome, the method comprising administering to a patient in need thereof, in particular a human patient in need thereof, at least by intramedullary injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for intramedullary injection.

[0431] According to another aspect, the present invention relates to a method for preventing and / or treating primary immunodeficiency, autoimmune disease or inborn error of metabolism, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by intramedullary injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix and an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, which is suitable for intramedullary injection.

[0432] According to another aspect, the present invention relates to a method for improving the homing, colonization and long-term expansion and proliferation of hematopoietic stem cells before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), the method comprising administering at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release, to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intramedullary injection.

[0433] According to another aspect, the present invention relates to a method for reducing transplant failure, reducing the occurrence of graft dysfunction (PGF), reducing the occurrence of graft-versus-host disease (GvHD), improving overall survival and donor chimerism, and / or promoting hematopoietic recovery and the success rate of hematopoietic stem cell transplantation, the method comprising administering at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intramedullary injection, for controlled release or for controlled and immediate release.

[0434] According to another aspect, the present invention relates to a method for preventing and / or treating non-malignant blood disorders such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and β-thalassemia, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intramedullary injection, at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release.

[0435] According to another aspect, the present invention relates to a method for preventing and / or treating hematological malignancies, such as myelomas, lymphomas, leukemias, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndrome, in particular leukemia, which method comprises administering at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release, to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intramedullary injection.

[0436] According to another aspect, the present invention relates to a method for preventing and / or treating primary immunodeficiency, autoimmune diseases or inborn errors of metabolism, which method comprises administering to a patient in need thereof, in particular a human patient in need thereof, at least by local injection, in particular by intramedullary injection, at least an effective amount of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, for controlled release or for controlled and immediate release.

[0437] Also provided herein is a method for improving the homing, engraftment, and long-term expansion and proliferation of hematopoietic stem cells before, during, or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), and / or for reducing transplant failure, for reducing the occurrence of poor graft function (PGF), for reducing the occurrence of graft-versus-host disease (GvHD), for improving overall survival and donor chimerism, and / or for promoting hematopoietic recovery and the success rate of hematopoietic stem cell transplantation, and / or for preventing and / or treating non-malignant blood disorders such as severe regenerative diseases. for the prevention and / or treatment of hematologic malignancies such as myeloma, lymphoma, leukemia, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndrome, in particular leukemia, and / or for the prevention and / or treatment of primary immunodeficiency, autoimmune disease or inborn error of metabolism, the method comprising at least the following steps:

[0438] - preparing and / or providing a sterile and injectable suspension pharmaceutical composition by mixing a formulation in powder form as described below with an aqueous injection vehicle, wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, and wherein the excipient may be present in the aqueous injection vehicle or the powder, and

[0439] - Intramedullary injection of the pharmaceutical composition into the bone marrow of a patient in need thereof, wherein the volume to be injected is adapted to the injection site.

[0440] Also provided herein is a method for improving the homing, colonization, and long-term expansion and proliferation of hematopoietic stem cells before, during, or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), and / or for reducing transplant failure, for reducing the occurrence of poor graft function (PGF), for reducing the occurrence of graft-versus-host disease (GvHD), for improving overall survival and donor chimerism, and / or for promoting hematopoietic recovery and the success rate of hematopoietic stem cell transplantation, and / or for preventing and / or treating non-malignant blood disorders such as severe regenerative diseases. for the prevention and / or treatment of hematologic malignancies such as myeloma, lymphoma, leukemia, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndrome, in particular leukemia, and / or for the prevention and / or treatment of primary immunodeficiency, autoimmune disease or inborn error of metabolism, the method comprising at least the following steps:

[0441] - preparing and / or providing a pharmaceutical composition in the form of a sterile and injectable solid implant, semisolid implant, microporous implant or in situ forming depot, wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, and wherein the excipient may be present in an aqueous injection vehicle or a powder, and

[0442] - intramedullary implantation of the pharmaceutical composition into the bone marrow of a patient in need thereof, wherein the volume to be implanted is adapted to the implantation site.

[0443] Additional active ingredients

[0444] Depending on the specific condition or disease to be treated, additional therapeutic agents that are normally administered to treat that condition may be administered in combination with Eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt.

[0445] In some embodiments, the present invention provides a pharmaceutical composition comprising eltrombopag or its derivative, conjugate, or pharmaceutically acceptable salt according to the present invention, and at least one additional therapeutic agent. Suitable additional active ingredients are described in further detail below.

[0446] In a specific embodiment, the patient receives treatment simultaneously, separately or sequentially with at least one active ingredient selected from cyclosporine A, methotrexate, tacrolimus, mycophenolate mofetil, antithymocyte globulin serum, cyclophosphamide and abatacept to prevent graft-versus-host disease.

[0447] In a specific embodiment, the patient is treated simultaneously, separately or sequentially with at least one active ingredient selected from a thrombopoietin (TPO) receptor agonist other than eltrombopag or a derivative or conjugate or pharmaceutically acceptable salt thereof. Such a thrombopoietin (TPO) receptor agonist can be selected from thrombopoietin, romiplostim, avatrombopag and hetrombopag.

[0448] The additional active ingredients suitable for the present disclosure may be administered simultaneously, separately or sequentially via any route of administration, that is to say, for example, via the oral route or the parenteral route.

[0449] The additional active ingredient may be formulated as an immediate-release and / or controlled-release dosage form.

[0450] In one embodiment, the pharmaceutical composition for improving stem cell homing and treatment and the method for treating related diseases according to the present invention are also characterized by the fact that the patient administered with the pharmaceutical composition can receive treatment with the following active ingredients simultaneously, separately or sequentially: cyclosporine A, methotrexate, tacrolimus, mycophenolate mofetil, anti-thymocyte globulin serum, cyclophosphamide, abatacept, a thrombopoietin receptor agonist selected from thrombopoietin, romiplostim, avatrombopag and hetrombopag, and mixtures thereof.

[0451] Thus, provided herein are controlled release pharmaceutical compositions for use as defined in the present disclosure for use in preventing and / or treating non-malignant blood disorders in a patient, such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and beta-thalassemia, and / or for preventing and / or treating malignant blood disorders, such as myeloma, lymphoma, leukemia, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative Tumors and myelodysplastic syndromes, in particular leukemia, and / or for the prevention and / or treatment of primary immunodeficiency, autoimmune diseases or inborn errors of metabolism, wherein the patient receives treatment simultaneously, separately or sequentially with at least one active ingredient selected from the group consisting of: cyclosporine A, methotrexate, tacrolimus, mycophenolate mofetil, antithymocyte globulin serum, cyclophosphamide, abatacept, a thrombopoietin receptor agonist selected from thrombopoietin, romiplostim, avatrombopag and hetrombopag, and mixtures thereof.

[0452] Other treatments

[0453] Depending on the patient to be treated, the patient may be treated with a conditioning regimen selected from radiotherapy, irradiation, chemotherapy, simulated radiochemotherapy, serum therapy, monoclonal antibodies, targeted therapy and combinations thereof before, during or after the intramedullary administration of the pharmaceutical composition as defined in the present disclosure or obtained by mixing the two compartments of the kit as defined in the present disclosure.

[0454] In a specific embodiment, the simulated radiochemotherapy can be selected from ozone, peroxides, alkylating agents, platinum-based agents, cytotoxic antibiotics and blister chemotherapy, preferably, the simulated radiochemotherapy is cyclophosphamide, busulfan, fludarabine, melphalan, thiotepa, cytarabine and clofarabine, carmustine, etoposide, cytarabine and melphalan, rituximab, ifosfamide, etoposide or a platinum-based agent selected from cisplatin, carboplatin, oxaliplatin and nedaplatin.

[0455] Throughout the specification, including the claims, the expression "comprising a" should be understood as being synonymous with "comprising at least one" unless specifically stated otherwise.

[0456] It should be understood that the expressions "between and" and "from to" should be understood to mean that the limit values ​​are included unless otherwise stated.

[0457] The following examples and figures are presented by way of non-limiting illustration of the present invention. Example

[0458] Analytical methods

[0459] Characterization of Eltrombopag Controlled-Release Microparticles

[0460] The particle size in Examples 1 and 5 was determined using a Malvern Mastersizer MS3000 laser diffraction particle size analyzer. 1 mg / ml of microparticles in a deionized water formulation were introduced into the device and three measurements were run. The results shown in the examples are the average of three measurements.

[0461] The particle size in Examples 3 and 4 was determined using a multi-wavelength separation analyzer (LUMiReader from LUM GmbH). An ultrasonic bath was used to obtain a formulation of 1 to 10 mg of microparticles per ml of deionized water, which was then introduced into the device. The results shown in the examples are the average of three measurements performed at different heights within the same run.

[0462] In Examples 1, 2, and 5, the drug loading of eltrombopag microparticles was determined by first dissolving 10 mg of drug-loaded polymer microparticles in 2.5 mL of an 80 / 20 volume / volume mixture of acetonitrile / water. The medium was then centrifuged at 4000 rpm for 15 minutes, and the supernatant was filtered through a 0.45 μm PTFE filter (Acrodisc PTFE). A 2 mL aliquot of the filtered supernatant was then analyzed by HPLC to determine the drug loading of the microparticles.

[0463] HPLC analysis was performed using a silica-based reverse-phase C18 column (Kinetex C18, 100 mm × 4.6 mm, 2.6 μm particle size) and a mobile phase consisting of acetonitrile / ammonium formate (10 mM) 80 / 20 vol / vol, adjusted to pH 2. The flow rate was set at 0.5 mL / min, the injection volume was 10 μL, and UV detection was set at 244 nm.

[0464] In Examples 3 and 4, the drug loading of eltrombopag microparticles was determined by first dissolving 10 mg of microparticles in 20 mL of a 75 / 25 v / v mixture of acetonitrile / ammonium formate (0.63 g / L), pH 3, using vortex mixing. The medium was then filtered through a 0.45 µm regenerated cellulose filter presaturated with 1 mL of medium. A 1 mL aliquot of the filtered supernatant was then analyzed by HPLC to determine the drug loading of the microparticles.

[0465] HPLC analysis was performed on an Agilent HP 1200 system using a silica-based reversed-phase C18 column (ODS Hypersil C18, 100 mm × 4.6 mm, 5 μm particle size) set at 25°C. The mobile phase consisted of 75% acetonitrile and 25% 6.3 g / L ammonium formate solution adjusted to pH 3 with dilute hydrochloric acid. The flow rate was set to 1 mL / min, the injection volume was 10 μL, and UV detection was set at 230 nm.

[0466] For the in vitro release testing of eltrombopag microparticles in Examples 3 and 4, a 75 mg aliquot of microparticles was dispersed in 50 mL of a solution containing 10 mM phosphate-buffered saline (PBS) containing 0.5% polysorbate 80 (Tween 80) previously adjusted to pH 6.8 with dilute hydrochloric acid using an ultrasonic bath. The dispersion was placed in an orbital shaker (GFL Model 3033 Incubator) and maintained at 37°C. At various time intervals, 1.5 mL of the medium was removed and filtered through a 0.45 μm regenerated cellulose filter presaturated with 1 mL of medium. The filtered samples were then analyzed by HPLC.

[0467] HPLC analysis was performed on an Agilent HP 1200 system using a silica-based reversed-phase C18 column (ODS Hypersil C18, 100 mm × 4.6 mm, 5 μm particle size) set at 25°C. The mobile phase consisted of 75% acetonitrile and 25% ammonium formate (6.3 g / L; pH 3). The flow rate was set to 1 mL / min, the injection volume was 10 μL, and UV detection was set at 254 nm.

[0468] SDF-1 proteolysis analysis

[0469] To incubate eltrombopag with activated human proteases in Examples 6-10, proMMP-8, proMMP-9, and proMMP-13 proteases were first activated. Human proMMP-8 and human proMMP-13 (Cat. Nos. 908-MP and 511-MM-010, R&D Systems) were activated by incubation with 1 mM p-aminophenylmercuric acetate at 37°C for 1 hour at 100 μg / ml in a buffer containing 50 mM Tris, 150 mM NaCl (sodium chloride), 5 mM CaCl2 (calcium chloride), and 0.01% Tween 20 (pH 7.6). Human proMMP-9 was expressed in Sf9 cells, purified by gelatin-agarose chromatography, and activated by incubation with the catalytic domain of MMP-3 (Cat. No. 444217, Merck Millipore). Specifically, 920 μg / ml of human proMMP-9 was incubated with the catalytic domain of MMP-3 at a 1 / 100 molar ratio (MMP-3 / MMP-9) in the same buffer as described for MMP-8 activation at 37°C for 2 hours. The final concentration of MMP-9 was 10 μM, and the final concentration of MMP-3 was 0.1 μM. Concentrated eltrombopag solution was prepared by dissolving it in water at a concentration of 10 mg / ml for 24 hours at 4°C on a rotor and further diluted to the desired final concentration. Activated MMP-8, MMP-9, or MMP-1 was then incubated with eltrombopag at a final concentration of 625 nM in a final volume of 3 μl at 37°C for 30 minutes in a calcium-free buffer containing 50 mM Tris, 150 mM NaCl, 0.01% Tween 20 (pH 7.6) using Protein LoBind tubes (Eppendorf). To incubate eltrombopag with activated human DPPIV (also known as CD26) protease in Example 11, recombinant human activated DPPIV (R&D Systems 9168-SE) was incubated in different concentrations of eltrombopag (100 µM, 50 µM, and 10 µM) in a final volume of 25 µl at a final concentration of 0.6-1.2 nM in a buffer containing 25 mM Tris (pH 8) at 37°C for 30 minutes.

[0470] In Examples 6 to 10, human SDF-1α (Cat. No. 300-28A, Peprotech) was incubated in a solution containing activated human proteases and eltrombopag in the same calcium-free buffer and test tubes at a 1 / 5 molar ratio of MMP / SDF-1 for 2 hours at 37°C, resulting in a final SDF-1α concentration of 3.125 µM in a final volume of 4 µl. A negative control without eltrombopag was similarly prepared. For the DPPIV assay in Example 11, incubations were performed in the same 25 mM Tris buffer at a DPPIV / SDF-1 molar ratio of 1 / 100 to 1 / 200 in a final volume of 26 µl. Positive controls for DPPIV inhibition with sitagliptin and negative controls without eltrombopag were similarly prepared.

[0471] - In Example 12, for neutrophil degranulation (ND) incubation with eltrombopag and SDF-1, neutrophils were first isolated from fresh blood from 4 healthy donors via density gradient centrifugation as described (De Buck M, Berghmans N, Portner N, Vanbrabant L, Cockx M, Struyf S, et al. Serum amyloidA1alpha induces paracrine IL-8 / CXCL8 via TLR2 and directly synergizes with this chemokine via CXCR2 and formyl peptide receptor 2 to recruit neutrophils. J Leukoc Biol. 2015; 98(6): 1049-60). To obtain neutrophil degranulation, neutrophils were incubated at 10 7 Neutrophils were suspended at a concentration of 10 cells / mL in calcium-free degranulation buffer (120 mM NaCl, 20 mMTris / HCl pH 7.5) and degranulation was induced by incubating the neutrophils with N-formyl-methionyl-leucyl-phenylalanine (fMLF) (final concentration 0.5 μM) at 37°C for 20 minutes. Next, the supernatant was collected after centrifugation. 5 Neutrophil degranulation was incubated with different concentrations of eltrombopag (100 μM and 50 μM) in a final volume of 21.1 μl at 37°C in calcium-free buffer for 30 minutes. Next, SDF-1α was added to the solution at a final concentration of 0.83 μM in a final volume of 22.6 μl for 4 hours at 37°C. A negative control without eltrombopag was similarly prepared.

[0472] Multiplex Western blotting was used to analyze SDF-1 proteolysis by MMPs and NDs in Examples 6, 7, 8, 9, 10, and 12. All samples were diluted in reducing loading buffer (10% β-mercaptoethanol, 0.1% bromophenol blue, 4% SDS (sodium dodecyl sulfate), 20% glycerol, 125 mM Tris, pH 6.8) and boiled at 90°C for 20 minutes. Next, 100 ng of SDF-1 was loaded onto a Novex 16% tricine gel (Cat. EC6695BOX, Invitrogen). A Chameleon Duo pre-stained protein ladder was used as the molecular weight marker, with 6 μl used per well. Proteins in the samples were separated by denaturing SDS-PAGE at a constant voltage of 120 V (±60 minutes) using tricine running buffer (100 mM Tris, 100 mM Tricine, 0.1% SDS, pH 8.3). After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (Biorad) with the Trans-Blot Turbo RTA Mini 0.45 μm Low Fluorescence PVDF Transfer Kit (Cat. No. 1704274, Biorad). After blocking the membranes with Intercept (TBS, Tris-buffered saline) blocking buffer (Cat. No. 927-60001, Licor) for 60 minutes at room temperature, the membranes were incubated with a rabbit polyclonal primary antibody that recognizes all SDF-1 isoforms (Cell Signaling 3740S, 1:1000) and a mouse monoclonal antibody that recognizes the N-terminal portion of SDF-1, which is typically cleaved by protease activity and leads to inhibition of SDF-1 chemotactic function (Millipore MABC184, 1:2000) in TBS blocking buffer containing 0.2% Tween 20, at 4°C on a rotary shaker overnight. After washing three times for 10 min in Tris-buffered saline containing Tween 20 (150 mM NaCl, 20 mM Tris, 0.1% Tween 20), incubation with secondary antibodies donkey anti-rabbit 680 RD (Cat. No. 926-68073, Licor, 1:5000) and donkey anti-mouse 800 CW (Cat. No. 926-32212, Licor, 1:5000) diluted in TBS blocking buffer containing 0.2% Tween 20 and 0.02% SDS was carried out on a rotary shaker at room temperature for 1 h.After three 10-min washes in Tris-buffered saline containing Tween 20, membranes were imaged using the Odyssey Fc Imaging System (LiCor). Protein band quantification was performed using Empiria Studio software (Licor) and expressed as the ratio of intact SDF-1 signal in relative fluorescence units (RFU) per total (all isoforms) SDF-1 RFU.

[0473] The SDF-1 proteolysis by DPPIV in Example 11 was analyzed by traditional Western blotting. Samples were diluted in reducing loading buffer (ThermoFisher B0009) and boiled at 70°C for 10 minutes. Next, 18.75–150 ng of SDF-1 were loaded onto two BOLT 12% Bis-Tris gels (Invitrogen NW00125BOX). Proteins in the samples were separated in MES running buffer (Invitrogen NP0002) at a constant voltage of 200 V for 25 minutes. After electrophoresis, proteins were transferred to a PVDF membrane using the iBlot™ 2 System (Invitrogen IB21001) with the iBlot™ 2 Transfer Stacks Kit (Invitrogen IB 24002) for a total of 6 minutes (1 minute at 20 V, 3.5 minutes at 23 V, and 1.5 minutes at 25 V). Next, both membranes were blocked, washed, and incubated with primary and secondary antibodies for 3–3.5 hours using an iBind Flex Western Device (Invitrogen SLF2000). The same primary antibody used for SDF-1 proteolysis by MMPs was used at a 1:1000 dilution. Secondary antibodies, goat anti-rabbit IgG HRP (31460, ThermoFisher) and goat anti-mouse IgG HRP (31430, ThermoFisher), were diluted 1:1000. The membranes were incubated with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (34580, ThermoFisher) and imaged using the iBright Imaging System (ThermoFisher). Protein band intensities were quantified using ImageJ software (in arbitrary units, AU). Results are expressed as band intensities normalized to those of a control SDF-1 sample without the addition of protease, eltrombopag, or sitagliptin. The results shown in the examples are the mean of eight independent experiments at 50 μM eltrombopag, five independent experiments at 100 μM, and three independent experiments at 10 μM. For statistical analysis, one-way ANOVA was used to compare samples. Confidence intervals were fixed at 95% (p < 0.05). Results were plotted as mean ± standard error of the mean (SEM) using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

[0474] Hematopoietic stem cell chemotaxis

[0475] - The chemotaxis of human hematopoietic stem cells in Example 13 was assessed using a Transwell migration assay with a 5.0 μm pore membrane (Corning CLS3421-48EA). In the lower chamber, 25 μM or 50 μM eltrombopag ethanolamine was incubated with 4 nM activated DPPIV in a final volume of 600 μl of assay medium (Stemcell Tecnologies 09600) at 37°C for 30 minutes. Next, recombinant human SDF-1a was added to the medium at a DPPIV / SDF-1 molar ratio of 1 / 6 for another 30 minutes at 37°C, resulting in a final SDF-1α concentration of 25 nM. Subsequently, 10 cells isolated from bone marrow were incubated with 10 cells / well. 5 Human CD34+ hematopoietic stem and progenitor cells (Lonza 2M-101A) were plated in the upper Transwell chamber in a final volume of 150 μl of medium. The Transwell system was placed in a cell incubator at 37°C and 5% CO2 to allow cells to migrate to the bottom chamber upon induction of chemotaxis. After 4 hours at 37°C, the upper chamber containing non-migrated cells was carefully removed, and cells that had migrated into the bottom chamber were quantified. For cell quantification, cell nuclei were stained with Hoescht (ThermoFisher R37605), and the entire microplate well was photographed using an EVOS M7000 microscope with an automated XY stage (ThermoFisher). Automated cell counting was performed using EVOS Analysis Software version 1.5.1479.304 (auto-counting function) with an optimized algorithm and batch analysis. The results shown in the examples are the average of three independent experiments. For statistical analysis, one-way ANOVA was used to compare samples. Confidence intervals were fixed at 95% (p < 0.05). Results were plotted as mean ± standard error of the mean (SEM) using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

[0476] Example 1: Sustained Release of Eltrombopag Ethanolamine Microparticles (S / O / W Emulsion)

[0477] Eltrombopag was loaded into PLGA microparticles using a solid-in-oil-in-water (S / O / W) emulsion and solvent evaporation method.

[0478] In this experiment, Eltrombopag (Hetero Drugs Limited, India) supplied in powder form with an average particle size (D50) of 3 μm was used.

[0479] First, 4.16 g of PLGA 50:50 Resomer 503H and 0.21 g of phospholipon 90H (Lipoid) were dissolved in 14.3 g of dichloromethane under magnetic stirring for 1 hour. Then, 2 g of eltrombopag powder was dispersed in the organic solvent using high-shear rotor / stator mixing (IKA T18 ultra-Turrax) at 10,000 rpm for 1 minute.

[0480] The S / O dispersion was then emulsified by mixing with 26.3 mL of a 1 wt % aqueous PVA solution (Mowiol 4-88, Sigma-Aldrich) using an IKA T18 ultra-Turrax at 9500 rpm for 10 minutes.

[0481] The emulsion was then quickly poured into an extraction bath (3 L of 1% aqueous PVA solution) and stirred at 300 rpm with an overhead stirrer for 3 hours.

[0482] The solid particles were then separated from the aqueous phase by centrifugation at 4000 rpm for 4 min, filtered on a 40 μm stainless steel mesh, and rinsed three times with 50 mL of distilled water.

[0483] The washed microparticles were stored in a glass overnight and then dried under a vacuum of 5 mbar for 18 hours.

[0484] The collected microparticles were finally stored at 5°C.

[0485] Table 1. Analysis results of eltrombopag PLGA microparticles

[0486]

[0487] In this example, PLGA microparticles loaded with eltrombopag with an average particle size of 6.9 μm and a drug loading of 26 wt % were prepared.

[0488] Example 2: Preparation of pharmaceutical composition-injectable suspension

[0489] Aqueous injection vehicle was prepared using pyrogen-free excipients consisting of 1.4% low-viscosity sodium carboxymethylcellulose (Aqualon CMC 7LF PH BET, Ashland), 0.1% polysorbate 20 (Acros Organics), 0.13% disodium hydrogen phosphate dihydrate, 0.1% citric acid (Roth), and 0.65% sodium chloride (Roth). The final pH of the solution was adjusted to 7.2 using concentrated sodium hydroxide solution (Roth). The vehicle was then autoclaved at 121°C for 15 minutes (MultiControl 2, CertoClav), and 5-mL aliquots of the solution were aseptically transferred into 10-mL vials under a laminar air flow hood.

[0490] A 470 mg aliquot of microparticles prepared according to Example 1 was dispersed in each vial using a vortex mixer.

[0491] The pharmaceutical composition is intended for local injection as defined in the present disclosure, except for intrapulmonary injection and intrapleural injection, for the treatment of diseases as defined in the present disclosure, except for COPD and pulmonary fibrosis.

[0492] In one example, a 5 mL volume containing a 123 mg dose of eltrombopag can be injected into the posterior iliac crest using an 18G needle equipped with a trocar.

[0493] Example 3: Controlled-release eltrombopag ethanolamine microparticles and pharmaceutical compositions containing the same

[0494] Eltrombopag microparticles were prepared using autoclaved and sterilized containers under laminar air flow. Eltrombopag was loaded into PLGA microparticles using a solid-in-oil-in-water (S / O / W) emulsion and solvent evaporation method.

[0495] In this experiment, eltrombopag (Hetero Drugs Limited, India) was used as a powder with an average particle size (D50) of 2 μm. Eltrombopag microparticles were prepared as follows: a 1 wt% polyvinyl alcohol stock solution was prepared by heating 2970 g of sterile water to 70°C and dispersing 30 g of PVA (Mowiol 4-88, Sigma-Aldrich) in it under magnetic stirring until completely dissolved. The solution was then allowed to cool before further use.

[0496] In a separate container, 2.9 g of PLGA 50:50 Resomer503H [poly(lactic-co-glycolic acid) copolymer 50:50; Mw = 24-38 kg / mol; (Evonik Industries AG, Essen, Germany)] was first dissolved in 14.3 g of dichloromethane (Merck) under magnetic stirring over 30 minutes. Then, 0.73 g of eltrombopag powder was dispersed in the mixture under magnetic stirring for 15 minutes to form a solid-in-oil dispersion.

[0497] 57 g of the previously prepared 1 wt% PVA stock solution was then added to the solid-in-oil dispersion under high shear emulsification. Emulsification was performed at 6600 RPM for 10 minutes using an IKA T25 Ultra-Turrax rotor stator mixer equipped with an S25N 10G head.

[0498] The emulsion was then slowly poured into a hardening bath containing 2943 g of a previously prepared 1 wt % stock solution of PVA.

[0499] Stirring was continued using a twin propeller stirrer at a stirring speed of 300 RPM for 3 hours until the dichloromethane evaporated.

[0500] The formed microparticles were centrifuged at 8000 RPM for 3 minutes using a swinging bucket GT2R centrifuge to remove the main portion of the aqueous phase and obtain a concentrated particle slurry. The collected slurry was then redispersed in approximately 150 ml of water, and the redispersed particles were vacuum filtered on 10-16 μm fritted glass and washed three times with 50 ml of water.

[0501] The washed microparticles were then dried in vacuo at 5 mbar and 25°C for 17 hours.

[0502] The collected microparticles were then stored at 5°C.

[0503] Analytical testing, including in vitro drug release profiles, was performed as described above in the “Analytical Methods” section and is shown in Table 2 below.

[0504] Table 2. Analysis results of eltrombopag PLGA microparticles

[0505]

[0506] In this example, controlled-release eltrombopag microparticles with an average particle size of 53.4 μm, a drug loading of 13.5 wt %, and a duration of about 24 hours required to release 80% of the initial drug load (ie, t80) were prepared.

[0507] Aqueous injection vehicle was prepared under laminar air flow using pyrogen-free excipients consisting of 1.4% low-viscosity sodium carboxymethylcellulose (Aqualon CMC 7LF PH BET, Ashland), 0.1% polysorbate 20 (Emprove Essential, Merck), 0.13% disodium hydrogen phosphate dihydrate (Roth), 0.1% citric acid (Roth), and 0.65% sodium chloride (Roth). The final pH of the solution was adjusted to 7.2 using concentrated sodium hydroxide solution (Roth). The vehicle was then autoclaved at 121°C for 20 minutes (Tuttnauer 2840 ELPVG-D), and 5-mL aliquots were aseptically transferred into 10-mL vials under laminar air flow.

[0508] The pharmaceutical composition is intended for local injection as defined in the present disclosure other than intrapulmonary / intrapleural injection, intraocular / intravitreal / subtenon injection and intraperiodontal / subgingival injection, for the treatment of diseases as defined in the present disclosure other than periodontal disease, eye disease, COPD and pulmonary fibrosis.

[0509] Shortly before in vivo injection, a 530 mg aliquot of microparticles prepared according to this example in each vial was dispersed using sonication for 30 seconds until a uniform dispersion was obtained. The PLGA microparticle suspension loaded with eltrombopag thus prepared yielded an eltrombopag dose of 71.6 mg per 5 ml.

[0510] Example 4: Controlled-release eltrombopag ethanolamine microparticles and pharmaceutical compositions containing the same

[0511] Eltrombopag microparticles were prepared using autoclaved and sterilized containers under laminar air flow. Eltrombopag was loaded into PLGA microparticles using a solid-in-oil-in-water (S / O / W) emulsion and solvent evaporation method.

[0512] In this experiment, eltrombopag (Hetero Drugs Limited, India) was used as a powder with an average particle size (D50) of 2 μm. Eltrombopag microparticles were prepared as follows: a 1 wt% polyvinyl alcohol stock solution was prepared by heating 2970 g of sterile water to 70°C and dispersing 30 g of PVA (PVA, Mowiol 4-88, Sigma-Aldrich) in it under magnetic stirring until completely dissolved. The solution was then allowed to cool before further use.

[0513] In a separate container, 2.9 g of PLGA 50:50 Resomer504H [poly(lactic-co-glycolic acid) copolymer 50:50; Mw = 38-54 kg / mol; (Evonik Industries AG, Essen, Germany)] was first dissolved in 14.3 g of dichloromethane (Merck) under magnetic stirring over 30 minutes. Then, 0.73 g of eltrombopag powder was dispersed in the mixture under magnetic stirring for 15 minutes to form a solid-in-oil dispersion.

[0514] 57 g of the previously prepared 1 wt% PVA stock solution was then added to the solid-in-oil dispersion under high shear emulsification. Emulsification was performed at 6600 RPM for 10 minutes using an IKA T25 Ultra-Turrax rotor stator mixer equipped with an S25N 10G head.

[0515] The emulsion was then slowly poured into a hardening bath containing 2943 g of a previously prepared 1 wt % stock solution of PVA.

[0516] Stirring was continued using a twin propeller stirrer at a stirring speed of 300 RPM for 3 hours until the dichloromethane evaporated.

[0517] The formed microparticles were centrifuged at 8000 RPM for 3 minutes using a swinging bucket GT2R centrifuge to remove the main portion of the aqueous phase and obtain a concentrated particle slurry. The collected slurry was then redispersed in approximately 150 ml of water, and the redispersed particles were vacuum filtered on 10-16 μm fritted glass and washed three times with 50 ml of water.

[0518] The washed microparticles were then dried in vacuo at 5 mbar and 25°C for 17 hours.

[0519] The collected microparticles were then stored at 5°C.

[0520] Analytical testing, including in vitro drug release profiles, was performed as described above in the “Analytical Methods” section and is shown in Table 2 below.

[0521] Table 3. Analysis results of eltrombopag PLGA microparticles

[0522]

[0523] In this example, controlled-release eltrombopag microparticles with an average particle size of 47.3 μm, a drug loading of 14.3 wt %, and a duration of more than 1 week (ie, more than 168 hours) required to release 80% of the initial drug load (ie, t80) were prepared.

[0524] Aqueous injection vehicle was prepared under laminar air flow using pyrogen-free excipients consisting of 1.4% low-viscosity sodium carboxymethylcellulose (Aqualon CMC 7LF PH BET, Ashland), 0.1% polysorbate 20 (Emprove Essential, Merck), 0.13% disodium hydrogen phosphate dihydrate (Roth), 0.1% citric acid (Roth), and 0.65% sodium chloride (Roth). The final pH of the solution was adjusted to 7.2 using concentrated sodium hydroxide solution (Roth). The vehicle was then autoclaved at 121°C for 20 minutes (Tuttnauer 2840 ELPVG-D), and 5-mL aliquots were aseptically transferred into 10-mL vials under laminar air flow.

[0525] The pharmaceutical composition is intended for intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, intrahepatic injection or intrarenal injection as defined in the present disclosure, for treating the corresponding associated diseases as defined in the present disclosure.

[0526] Shortly before in vivo injection, a 530 mg aliquot of microparticles prepared according to this example in each vial was dispersed using sonication for 30 seconds until a uniform dispersion was obtained. The PLGA microparticle suspension loaded with eltrombopag thus prepared yielded an eltrombopag dose of 75.8 mg per 5 ml.

[0527] Example 5: Sustained Release of Eltrombopag Ethanolamine Microparticles (W / O / W Emulsion)

[0528] Eltrombopag was loaded into PLGA microparticles using a water-in-oil-in-water (W1 / O / W2) double emulsion and solvent evaporation method.

[0529] The internal aqueous phase W1 was obtained by dissolving 20 mg of eltrombopag (MedChemExpress, USA) and 50 mg of 2-hydroxypropyl-β-cyclodextrin (Sigma Aldrich) in 1 mL of water.

[0530] The organic solvent phase was prepared by dissolving 500 mg of PLGA Resomer RG 503H (Evo Nik Industries AG, Essen, Germany) and 25 mg of lecithin (Phospholipon 90H, Lipoid) in 2.5 mL of dichloromethane.

[0531] To prepare the W1 / O primary emulsion, the W1 phase and the oil phase were emulsified under high shear rotor / stator mixing at 24,000 rpm for 10 minutes.

[0532] A W1 / O / W2 double emulsion was then obtained by mixing W1 / O in 1 mL of water containing 1% PVA (Mowiol 4-88, Sigma-Aldrich) and emulsifying under high shear rotor / stator mixing at 9500 rpm for 5 min.

[0533] The double emulsion was then poured into an extraction bath (0.5 L of 1% aqueous PVA solution) and stirred at 300 rpm with an overhead stirrer for 3 h.

[0534] The solid particles were then separated from the aqueous phase by centrifugation at 4000 rpm for 4 minutes and finally dried at 15°C under a pressure of 5 mbar for 18 hours.

[0535] Table 4. Analysis results of eltrombopag PLGA microparticles

[0536]

[0537] Example 6: Stabilization of SDF-1 by Eltrombopag-mediated MMP-9 protease inhibition

[0538] Activated human MMP-9 was incubated with various concentrations of eltrombopag (50 μM, 10 μM, 1 μM, and 0.1 μM) for 0.5 hours at 37°C. Subsequently, human SDF-1α was added to the prepared solution at a 1 / 5 MMP / SDF-1α molar ratio at 37°C for 2 hours. SDF-1α proteolysis was then analyzed by multiplex Western blotting, detecting total SDF-1α and protease-inactivated SDF-1α.

[0539] Table 5: Inhibitory effect of eltrombopag on the proteolysis of SDF-1 by MMP-9

[0540]

[0541] The results clearly showed that 50 μM eltrombopag was able to strongly reduce the proteolytic effect of MMP-9 on SDF-1α (72% of intact SDF-1α was recovered when MMP-9 was in the presence of 50 μM eltrombopag, compared with only 20% in the absence of eltrombopag; Figure 1A and 1B and Table 5).

[0542] Example 7: Stabilization of SDF-1 by Eltrombopag-mediated MMP-9 protease inhibition

[0543] Activated human MMP-9 was incubated with various concentrations of eltrombopag (100 μM, 50 μM, 10 μM, and 1 μM) for 0.5 hours at 37°C. Subsequently, human SDF-1α was added to the prepared solution at a 1 / 5 MMP / SDF-1α molar ratio at 37°C for 1.5 hours. SDF-1α proteolysis was then analyzed by multiplex Western blotting, detecting total SDF-1α and protease-inactivated SDF-1α.

[0544] Table 6: Inhibitory effect of eltrombopag on the proteolysis of SDF-1 by MMP-9

[0545]

[0546] The results clearly showed that 50 μM and 100 μM eltrombopag ethanolamine strongly reduced the proteolytic effect of MMP-9 on SDF-1α (73% and 42% of intact SDF-1α were recovered when MMP-9 was in the presence of 100 μM and 50 μM eltrombopag ethanolamine, respectively, compared to only 1.9% in the absence of eltrombopag; Figure 2A and 2B and Table 6).

[0547] Example 8: Stabilization of SDF-1 by Eltrombopag-mediated MMP-8 protease inhibition

[0548] Activated human MMP-8 was incubated with various concentrations of eltrombopag (50 μM, 10 μM, 1 μM, and 0.1 μM) for 0.5 hours at 37°C. Subsequently, human SDF-1α was added to the prepared solution at a 1 / 5 MMP / SDF-1α molar ratio at 37°C for 2 hours. SDF-1α proteolysis was then analyzed by multiplex Western blotting, detecting total SDF-1α and protease-inactivated SDF-1α.

[0549] Table 7: Inhibitory effect of eltrombopag on the proteolysis of SDF-1 by MMP-8

[0550]

[0551] The results clearly showed that 50 μM eltrombopag ethanolamine was able to reduce the proteolytic effect of MMP-8 on SDF-1α (29% of intact SDF-1α was recovered when MMP-8 was in the presence of 50 μM eltrombopag ethanolamine, compared with only 2.9% in the absence of eltrombopag ethanolamine; Figure 3A and 3B and Table 7).

[0552] Example 9: Stabilization of SDF-1 by Eltrombopag-mediated MMP-8 protease inhibition

[0553] Activated human MMP-8 was incubated with various concentrations of eltrombopag (100 μM, 50 μM, 10 μM, and 1 μM) for 0.5 hours at 37°C. Subsequently, human SDF-1α was added to the prepared solution at a 1 / 5 MMP / SDF-1α molar ratio at 37°C for 2 hours. SDF-1α proteolysis was then analyzed by multiplex Western blotting, detecting total SDF-1α and protease-inactivated SDF-1α.

[0554] Table 8: Inhibitory effect of eltrombopag on the proteolysis of SDF-1 by MMP-8

[0555]

[0556] The results clearly demonstrated that 50 μM and 100 μM eltrombopag ethanolamine were able to reduce the proteolytic effect of MMP-8 on SDF-1α (45% and 43% of intact SDF-1α were recovered when MMP-8 was in the presence of 100 μM and 50 μM eltrombopag ethanolamine, respectively, compared to only 16% in the absence of eltrombopag; Figure 4A and 4B and Table 8).

[0557] Example 10: Stabilization of SDF-1 by Eltrombopag-mediated MMP-13 protease inhibition

[0558] Activated human MMP-13 was incubated with various concentrations of eltrombopag (100 μM, 50 μM, 10 μM, and 1 μM) for 0.5 hours at 37°C. Subsequently, human SDF-1α was added to the prepared solution at a 1 / 5 MMP / SDF-1α molar ratio at 37°C for 1.5 hours. SDF-1α proteolysis was then analyzed by multiplex Western blotting, detecting total SDF-1α and protease-inactivated SDF-1α.

[0559] Table 9: Inhibitory effect of eltrombopag on the proteolysis of SDF-1 by MMP-13

[0560]

[0561] The results clearly showed that 50 μM and 100 μM eltrombopag ethanolamine were able to strongly reduce the proteolysis of SDF-1α by MMP-13 (19% and 12% of intact SDF-1α were recovered when MMP-9 was in the presence of 100 μM and 50 μM eltrombopag ethanolamine, respectively, compared to only 3.2% in the absence of eltrombopag; Figure 5A and 5B and Table 9).

[0562] Example 11: Stabilization of SDF-1 by Eltrombopag-mediated CD26 / DPPIV inhibition

[0563] Activated human DPPIV was incubated with various concentrations of eltrombopag (100 μM, 50 μM, and 10 μM) for 0.5 hours at 37°C. Subsequently, human SDF-1α was added to the prepared solution at a DPPIV / SDF-1α molar ratio of 1 / 100 to 1 / 200 for 1 hour at 37°C. SDF-1α proteolysis was then analyzed by Western blotting, detecting total SDF-1α and protease-inactivated SDF-1α.

[0564] Table 10: Inhibitory effect of eltrombopag on the proteolysis of SDF-1 by DPPIV

[0565]

[0566] The results clearly showed that 50 μM and 100 μM eltrombopag ethanolamine were able to strongly reduce the proteolytic effect of DPPIV on SDF-1α (100% and 92% of intact SDF-1α were recovered when DPPIV was in the presence of 100 μM and 50 μM eltrombopag ethanolamine, respectively, compared with only 61% in the absence of eltrombopag; Figure 6A 、 Figure 6B and Figure 7 Sitagliptin is a well-known potent DPPIV inhibitor, with a calculated half-maximal inhibitory concentration (IC50) 10 times lower than that of eltrombopag. 5 When used at 1 μM, it was shown to be less effective than eltrombopag in SDF-1 stabilization, recovering only 82% of intact SDF-1α.

[0567] Example 12: Stabilization by Eltrombopag-mediated protease inhibition in neutrophil degranulation SDF-1

[0568] Neutrophil degranules (NDs) obtained from neutrophils of healthy donors were incubated with different concentrations of eltrombopag (100 μM and 50 μM) for 0.5 hours at 37°C. Subsequently, human SDF-1α was added to the prepared solution at a final concentration of 0.83 μM for 4 hours at 37°C. SDF-1α proteolysis was then analyzed by multiplex Western blotting, detecting total SDF-1α and N-terminally protease-inactivated SDF-1α.

[0569] Table 11: Inhibitory effect of eltrombopag on the proteolysis of SDF-1 by proteases involved in neutrophil degranulation

[0570]

[0571] The results showed that 100 μM and 50 μM eltrombopag strongly reduced the proteolytic cleavage of SDF-1α by neutrophil degranulation (100% and 80% of N-terminally intact SDF-1α were recovered when ND was in the presence of 100 μM and 50 μM eltrombopag, respectively, compared to only 65% ​​in the absence of eltrombopag; Figure 8A and 8B Furthermore, 100 μM and 50 μM eltrombopag also reduced the proteolysis of total SDF-1α by neutrophil degranulation (90% and 80% of total SDF-1α were recovered when ND was in the presence of 100 μM and 50 μM eltrombopag, respectively, compared to only 63% in the absence of eltrombopag).

[0572] Example 13: Eltrombopag-mediated proliferation of human hematopoietic stem and progenitor cells in the presence of DPPIV protease Increased chemotaxis toward SDF-1

[0573] Activated human DPPIV was incubated with different concentrations of eltrombopag (50 μM and 25 μM) in HSC medium at 37°C in the bottom chamber of the Transwell assay for 0.5 h. Subsequently, human SDF-1α was added at a DPPIV / SDF-1α molar ratio of 1 / 6 at 37°C for 0.5 h. Human CD34+ hematopoietic stem and progenitor cells isolated from bone marrow were plated in the upper chamber of the Transwell system (10 cells in 150 μl). 5 Cells were collected and allowed to migrate to the bottom chamber for 4 hours. Cells that migrated to the bottom chamber were quantified by nuclear staining, image acquisition, and automated counting.

[0574] Table 12: Effect of Eltrombopag on the Chemotaxis of Human CD34+ Hematopoietic Stem and Progenitor Cells to SDF-1 in the Presence of DPPIV

[0575]

[0576] The results showed that in the presence of DPPIV protease, 25 μM and 50 μM eltrombopag increased the chemotaxis of hematopoietic stem and progenitor cells toward SDF-1α. When compared to the SDF-1 control sample without DPPIV, 93% and 88% of the migrating cells were detected in the presence of 25 μM and 50 μM eltrombopag, respectively, compared to only 68% in the absence of eltrombopag (Tables 12 and 12). Figure 9 ).

Claims

1. A controlled-release pharmaceutical composition comprising at least one controlled-release pharmaceutical dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is suitable for local injection at an affected area.

2. The controlled-release pharmaceutical composition according to claim 1, wherein the 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt is in the form of a bis-monoethanolamine salt of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid.

3. The controlled-release pharmaceutical composition according to claim 1 or 2, wherein the composition is an extended-release pharmaceutical composition comprising at least one extended-release pharmaceutical dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof.

4. The controlled-release pharmaceutical composition according to any one of claims 1 to 3, wherein the affected site is selected from the group consisting of bone, heart, blood vessels, skin, dermis, eye, liver, kidney, gastrointestinal tract, peritoneum, lung, gum, joint, bone marrow, in particular sternum, tibia, femur, iliac crest, vertebrae and combinations thereof, for example iliac crest, such as the posterior part of the iliac crest, and combinations thereof.

5. The controlled release pharmaceutical composition according to any one of claims 1 to 4, wherein the local injection at the affected site is selected from the group consisting of intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection, intraosseous injection, and combinations thereof.

6. The controlled-release pharmaceutical composition according to any one of claims 1 to 5, further comprising an immediate-release pharmaceutical dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof.

7. A controlled-release pharmaceutical composition according to any one of claims 1 to 6, wherein the controlled-release pharmaceutical dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof is in the form of an in situ formed reservoir, a hydrogel, a microporous implant, a solid implant or a microparticle, in particular a microparticle comprising a controlled-release polymer matrix or a multivesicular liposome, for example a microparticle comprising a controlled-release polymer matrix.

8. A controlled release pharmaceutical composition according to any one of claims 1 to 7, wherein the controlled release pharmaceutical dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof is in the form of microparticles, in particular microparticles comprising a controlled release polymer matrix, and wherein the average particle size of the microparticles is equal to or greater than 1 μm, for example equal to or greater than 2 μm, in particular in the range of 1 μm to 200 μm, more in particular in the range of 2 μm to 150 μm, still more in particular in the range of 2 μm to 100 μm, for example 10 μm to 100 μm, or 10 μm to 80 μm.

9. A controlled release pharmaceutical composition according to claim 7 or claim 8, wherein the controlled release polymer matrix comprises at least one biocompatible and biodegradable copolymer or polymer selected from the group consisting of poly(lactic-co-glycolic acid), poly(caprolactone), poly(lactide), poly(glycolide), poly(lactide-co-caprolactone), poly(ethylene glycol), poly(ethylene oxide), PLGA-b-PEO-b-PLGA, PLGA-b-PEO, polyhydroxyalkanoates, poly(hydroxybutyrate), ...lactide), poly(glycolide), poly(lactide-co-caprolactone), poly(ethylene glycol), poly(ethylene oxide), esters), poly(trimethylene carbonate), poly(dioxanone), poly(valerolactone), poly(α-hydroxy acid), poly(lactone), poly(amino acid), polyanhydride, poly(orthoester), poly(acetal), polyurethane, polythioester, polyphosphate, poly(ester-co-amide), poly(vinyl alcohol), PVA-g-PLGA, poly(ether ester) multi-block copolymers, polyvinyl pyrrolidone, poly(methacrylate), PEO-PPO-PEO, gelatin, heparin, chondroitin sulfate; polysaccharides such as alginate, starch, chitosan, hyaluronic acid and dextran, and any combination thereof.

10. The controlled release pharmaceutical composition according to any one of claims 7 to 9, wherein the controlled release polymer matrix comprises at least one poly (lactic-co-glycolic acid) copolymer.

11. A controlled-release pharmaceutical composition according to any one of claims 7 to 10, wherein the controlled-release polymer matrix comprises poly(lactic-co-glycolic acid) in an amount of greater than 70% by weight, particularly greater than 80% by weight, and even more particularly greater than 90% by weight, relative to the total weight of the polymer matrix.

12. The controlled-release pharmaceutical composition according to any one of claims 7 to 11, wherein the weight percentage of the drug loading or the 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt to the total weight of the microparticles ranges from 2% to 45%, particularly from 5% to 40%, more particularly from 10% to 35%.

13. A controlled-release pharmaceutical composition in powder form, comprising controlled-release microparticles comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof and a controlled-release polymer matrix, wherein - the average particle size of the controlled-release microparticles is equal to or greater than 1 μm, for example equal to or greater than 2 μm, particularly in the range of 1 μm to 200 μm, more particularly in the range of 2 μm to 150 μm, still more particularly in the range of 2 μm to 100 μm, for example 10 μm to 100 μm, or 10 μm to 80 μm, - the controlled release polymer matrix comprises at least one poly(lactic-co-glycolic acid) copolymer, and - the weight percentage of the drug loading or 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or its derivative or conjugate or pharmaceutically acceptable salt to the total weight of the microparticles ranges from 2% to 45%, particularly from 5% to 40%, more particularly from 10% to 35%.

14. The controlled-release pharmaceutical composition according to any one of claims 1 to 13, wherein it is in the form of a sterile and injectable dosage form selected from the group consisting of a solution, a suspension, a powder, a solid implant, a semisolid implant, a microporous implant and an in situ forming depot.

15. The controlled-release pharmaceutical composition according to claim 14, which is in the form of a sterile and injectable suspension, wherein the composition is prepared by combining a controlled-release dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a pharmaceutically acceptable salt thereof, or a mixture of a controlled-release dosage form and an immediate-release dosage form, in particular comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a pharmaceutically acceptable salt thereof. Microparticles of [a]-[1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, more particularly a controlled release dosage form as defined in any one of claims 1 to 12 or a mixture of a controlled release dosage form and an immediate release dosage form, mixed with an aqueous injection vehicle, and wherein the pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancer, a wetting agent, a viscosity enhancer, a density enhancer or a mixture thereof.

16. A controlled release pharmaceutical composition according to claim 14 or claim 15, wherein the 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof is present in an amount ranging from 2 mg to 63 mg equivalents of free acid per milliliter of sterile and injectable dosage form, particularly a suspension, particularly in the range of 8 mg to 55 mg equivalents of free acid per milliliter, more particularly in the range of 12 mg to 47 mg equivalents of free acid per milliliter.

17. A kit or article of manufacture comprising in separate compartments (i) an aqueous injection medium and (ii) a controlled release pharmaceutical dosage form or a mixture of a controlled release dosage form and an immediate release dosage form comprising 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof, in particular a controlled release pharmaceutical dosage form or a mixture of a controlled release dosage form and an immediate release dosage form as claimed in any one of claims 1 to 12, or a powder as defined in claim 13, wherein The pharmaceutical composition optionally comprises an excipient selected from a tonicity enhancer, a wetting agent, a viscosity enhancer, a density enhancer or a mixture thereof, for preparing a pharmaceutical composition suitable for local injection at the affected site, in particular a pharmaceutical composition suitable for intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection, intraosseous injection and a combination thereof.

18. A controlled-release pharmaceutical composition as defined in any one of claims 1 to 16, or a controlled-release pharmaceutical composition obtained by mixing two compartments of a kit as defined in claim 17, for single or multiple use by local injection at the affected site, in particular, for improving homing, colonization and long-term expansion and proliferation of hematopoietic stem cells in patients, particularly human patients, by intramedullary injection, before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), for preventing and / or treating non-malignant blood disorders, for preventing and / or treating hematological malignancies, and / or for preventing and / or treating primary immunodeficiency, autoimmune disease or inborn error of metabolism, and / or For the prevention and / or treatment of degenerative diseases / injuries by intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site infusion, intradermal injection, intravitreal injection, intraocular injection, subtenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intraarticular injection, intraosseous injection, and combinations thereof.

19. A controlled release pharmaceutical composition for use as defined in claim 18, for Prevention and / or treatment of cardiovascular disorders / diseases, such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; and / or Preventing and / or treating diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis, and osteoarthritis; and / or Prevention and / or treatment of periodontal disease; and / or Wound healing, including diabetic wound healing and corneal wound healing; and / or Preventing and / or treating eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; and / or Prevention and / or treatment of kidney disease; and / or Prevention and / or treatment of liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; and / or Preventing and / or treating inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; and / or Preventing and / or treating chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and / or pulmonary fibrosis; and / or Prevention and / or treatment of non-malignant blood disorders, such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and beta-thalassemia; and / or Prevention and / or treatment of hematological malignancies, such as myeloma, lymphoma, leukemias, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndromes, in particular leukemias; and / or For the prevention and / or treatment of primary immunodeficiency, autoimmune diseases or inborn errors of metabolism.

20. A controlled release pharmaceutical composition for use as defined in claims 18 to 19, wherein the local injection is performed by intramedullary injection and wherein it is used as an implant in a patient, particularly a human patient, before, during or shortly after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) for improving homing, colonization and long-term expansion and proliferation of hematopoietic stem cells.

21. The controlled-release pharmaceutical composition for use according to any one of claims 18 to 20, wherein the local injection is performed by intramedullary injection, and wherein for the hematopoietic stem cell transplantation, the hematopoietic stem cells are selected from bone marrow cells, peripheral blood stem cells, umbilical cord blood cells, cultured stem cells, including cultured stem cells after gene therapy.

22. A controlled release pharmaceutical composition for use according to any one of claims 18 to 21, wherein the local injection is performed by intramedullary injection, and wherein the composition is injected into the bone marrow within 96 hours before transplantation and within 96 hours after transplantation, particularly within 96 hours before transplantation and within 24 hours after transplantation, more particularly within 96 hours before transplantation.

23. The controlled-release pharmaceutical composition for use according to any one of claims 18 to 22, wherein the local injection is performed by intramedullary injection, and wherein it is used to reduce transplant failure, to reduce the occurrence of poor graft function (PGF), to reduce the occurrence of graft-versus-host disease (GvHD), to enhance overall survival and donor chimerism, and / or to promote hematopoietic recovery and the success rate of hematopoietic stem cell transplantation.

24. A controlled release pharmaceutical composition for use according to any one of claims 18 to 23, wherein the local injection is performed by intramedullary injection and wherein the patient is treated with a conditioning regimen selected from the group consisting of radiotherapy, irradiation, chemotherapy, simulated radiochemotherapy, serum therapy, monoclonal antibodies, targeted therapy and combinations thereof before or after local administration of the controlled release pharmaceutical composition as defined in any one of claims 1 to 16 or before or after the controlled release pharmaceutical composition obtained by mixing the two compartments of the kit as defined in claim 17.

25. A controlled-release pharmaceutical composition for use according to any one of claims 18 to 24, wherein the time required to release 80% by weight of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof is from 6 hours to 3 months, In particular, 6 hours to 30 days, in particular 1 day to 15 days, more particularly 2 days to 15 days, yet more particularly 4 days to 15 days, for example 4 days to 30 days, preferably 15 days, wherein 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a pharmaceutically acceptable salt thereof is present in a dose ranging from 10 mg to 570 mg equivalent of the free acid form.

26. A controlled release pharmaceutical composition for use according to any one of claims 18 to 25, wherein the controlled release pharmaceutical composition is further characterized in that it is effective in maintaining a local concentration of 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a derivative or conjugate or pharmaceutically acceptable salt thereof at greater than 1 μM, particularly greater than 5 μM, more particularly greater than 15 μM, for example comprised between 5 μM and 200 μM, particularly between 15 μM and 100 μM, even more particularly between 50 μM and 100 μM, within 6 hours to 3 months, particularly between 6 hours and 30 days, particularly between 1 day and 15 days, more particularly between 2 days and 15 days, yet more particularly between 4 days and 15 days, for example between 4 days and 30 days, preferably within 15 days after local injection.

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