Dosage forms for local injection containing eltrombopag for use in treating degenerative diseases and improving stem cell homing - Patent Application 20070122999

Abstract

The present invention relates to a controlled-release pharmaceutical composition comprising at least a controlled-release pharmaceutical dosage form containing EPAG, the controlled-release pharmaceutical composition being suitable for local injection into an affected area. The present invention also relates to a controlled-release pharmaceutical composition in powder form for preparing the pharmaceutical composition suitable for local injection, and a kit for preparing the pharmaceutical composition comprising an aqueous injection vehicle and a controlled-release dosage form or a mixture of controlled and immediate-release dosage forms containing EPAG, the pharmaceutical composition optionally including an excipient. The composition is useful for local injection in patients to improve stem cell homing and / or treat non-malignant hematological disorders, malignant hematological diseases, primary immunodeficiencies, autoimmune diseases, inborn errors of metabolism, and / or degenerative diseases / injuries.

Description

[Technical Field]

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

[0002] background Local inflammation and ischemia occurring at the affected site usually result in the activation of various proteolytic enzymes (i.e., proteases), such as matrix metalloproteinases (MMPs) and serine proteases, which play a key role in the degradation of extracellular matrix components and various cytokines / chemokines, affecting cell migration and conditioning during final tissue remodeling and repair.

[0003] Chemokines are small chemoattractant factors that mediate all stages of this repair and regeneration process, particularly in shaping cellular activity to drive stem and progenitor cell migration to diseased sites. 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 that express 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. Under homeostasis, SDF-1 is constitutively secreted primarily by bone marrow stromal cells. However, under pathological conditions, its expression is induced at any diseased 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 diseased sites and the upregulation of CXCR4 on progenitor and stem cells. After inflammation / ischemia, SDF-1 expression increases for only a few days, whereas CXCR4 overexpression can persist for weeks (Ziff et al., Therapeutic strategies utilizing SDF-1 in ischemic cardiomyopathy, Cardiovascular Research, Volume 114, Issue 3, March 1, 2018, Pages 358-367). Contributing factors to this mismatch are the rapid clearance of SDF-1 from the circulation and its susceptibility to degradation by proteases overexpressed at these diseased 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).

[0004] Therefore, there remains a need to extend the time window during which SDF-1 concentration levels are increased within the affected site in order to increase the amount of stem / progenitor cells attracted to and retained at the site, and therefore their beneficial contribution to tissue repair and regeneration.

[0005] This approach may be particularly beneficial in several diseases or conditions in which high local proteolytic activity caused by inflammation and ischemia at the affected site prevents full SDF-1 signaling capacity, such as: - Cardiovascular disease (myocardial infarction, atherosclerosis, ischemia-reperfusion injury); -Diseases affecting bones and joints (osteoarthritis, rheumatoid arthritis, osteoporosis); -periodontal disease; -Wound healing (including diabetic wound healing and corneal wound healing); - Eye diseases (retinal ischemia, macular degeneration, diabetic retinopathy, glaucoma) - Renal disease, liver disease (fibrosis and cirrhosis, non-alcoholic fatty liver disease, acute liver injury); -Inflammatory bowel disease (Crohn's disease, ulcerative colitis); -Chronic obstructive pulmonary disease (COPD), pulmonary fibrosis; - Non-malignant blood disorders (severe aplastic anemia or hemoglobinopathies, especially sickle cell disease and beta-thalassemia) - hematologic malignancies (leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, myelodysplastic syndromes); - Primary immunodeficiency, autoimmune disease or inborn error of metabolism.

[0006] In particular, current treatment schemes for the above-mentioned non-malignant and malignant hematological disorders, as well as certain primary immunodeficiencies, autoimmune diseases and inborn errors of metabolism, involve hematopoietic stem cell transplantation, in which exogenous stem cells (either donor-derived or previously retrieved from the patient) are infused intravenously into the patient's circulation and require migration to the patient's bone marrow.

[0007] Localized delivery of SDF-1 directly to injured tissue sites has been tested using various biomaterials and drug delivery vehicles, with positive in vitro and preclinical results confirming increased cell homing to the injury site (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 substance, and controlled delivery of this poorly stable protein at sufficient doses remains a challenge.

[0008] 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. Therefore, it is generally accepted that the SDF-1 / CXCR4 signaling axis is one of the major 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 transplanted HSC homing to the BM and induce HSC mobilization from the bone marrow to the circulation (Dar et al., Exp Hematol. 2006;34(8):967-75). Furthermore, SDF-1 has been shown to play an important role in the maintenance and retention of HSCs within the BM (Greenbaum et al., Nature. 2013;495(7440):227-30). More interestingly, recent studies have demonstrated impaired production of BMSC-derived SDF-1 in preclinical models of hematological diseases, leading to decreased SDF-1 levels in BM extracellular fluid, which was accompanied by decreased HSC maintenance and increased HSC mobilization in the BM, as well as decreased HSC engraftment 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). Furthermore, the conditioning regimens patients must undergo before HSCT typically include chemotherapy with or without radiation, and more recently, may include serum therapy, monoclonal antibodies, specific targeting novel compounds, and radiolabeled antibodies. These regimens affect the BM niche, leading to, among other things, a local increase in proteases and a reduction in the half-life of chemokines such as SDF-1 (Zhang et al., Sci Rep. 2016;6:37827). Therefore, as demonstrated above in relation to diseases or conditions in which high local proteolytic activity caused by inflammation and ischemia at the affected site generally prevents complete SDF-1 signaling, preserving local SDF-1 in the BM and restoring HSC niche homeostasis may be targeted to enhance homing and engraftment.The subsequent survival of BM-homed HSC precursors, followed by their self-renewal and differentiation, are also key points for the long-term success of HSCT therapy.

[0009] Eltrombopag olamine (i.e., EPAG olamine) 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 that exhibits multiple mechanisms of action. The first identified effect corresponds to the stimulation of megakaryocyte (MK) progenitor cell expansion and differentiation by binding to TPO-R. EPAG binding to TPO-R also promotes HSC survival, which, in contrast to the thrombopoietin effect, is not inhibited in an inflammatory environment. EPAG also exhibits interesting iron-chelating properties, inducing stimulatory effects on stem cell renewal and antiproliferative effects on leukemia cell lines.

[0010] EPAG is currently indicated for the treatment of immune thrombocytopenia (ITP), hepatitis C-associated thrombocytopenia, and severe aplastic anemia (SAA). In addition, EPAG has also achieved positive results in the treatment of myelodysplastic syndromes and graft poor function after HSCT.

[0011] EPAG is currently available commercially as PROMACTA® oral tablets or powder for suspension in doses ranging from 12.5 mg to 75 mg.

[0012] No direct action of EPAG on inhibiting the activity of SDF-1-degrading proteases has been disclosed so far, nor has any controlled release dosage form for local injection of EPAG been disclosed.

[0013] Conventional technology WO 2016 / 201354 discloses a method for treating cancer, which comprises administering an antifungal agent, a TPO receptor agonist, or a combination thereof, and which acts through the inhibition of dioxygenase. However, the examples are limited to immediate-release injectable formulations and immediate-release oral tablets.

[0014] WO 2007 / 145227 relates to the administration of agonist agents against the TPO receptor after HSCT to enhance engraftment, growth and differentiation of transplanted cells in the bone marrow. The agents are preferentially administered parenterally by injection, but is silent about controlled release pharmaceutical compositions suitable for local injection of the agonist agents into the affected site, including to enhance engraftment after HSCT.

[0015] WO 2004 / 096154 relates to non-peptide thrombopoietin receptor agonists and their use in the treatment of degenerative diseases / injuries, but the application is silent on any pharmaceutical compositions suitable for local injection at the affected site and for administration in controlled release form.

[0016] The use of thrombopoietin receptor agonists, including EPAG, to promote HSC homing after bone marrow transplantation is reported in CN105412930A. Although a large set of administration routes is mentioned, the only example is limited to the administration of an immediate-release EPAG solution by oral gavage in mice, and nothing is said about controlled-release forms. Summary of the Invention [Problem to be solved by the invention]

[0017] Therefore, there remains a need to develop novel means to increase SDF-1 concentration levels within diseased sites, particularly in the bone marrow, in order to increase the proportion of endogenous regenerative progenitor and stem cells that home to the diseased site to enhance tissue and organ self-renewal, respectively, and to enhance the homing and engraftment of exogenous, e.g., autologous or allogeneic, hematopoietic stem cells in the context of transplantation.

[0018] In addition, there remains a need to develop novel dosage forms and routes of administration for EPAG or its derivatives or conjugates or pharmaceutically acceptable salts to improve control over the local concentration of EPAG at the affected site and therefore increase the proportion of regenerative progenitor and stem cells that home / migrate to this site.

[0019] In addition, there remains a need to develop novel dosage forms and routes of administration of EPAG or its derivatives or conjugates or pharmaceutically acceptable salts to improve control over the local concentration of EPAG in the bone marrow and thus increase exogenous stem cell homing in transplantation. [Means for solving the problem]

[0020] Surprisingly, the inventors have discovered that EPAG olamine can inhibit the activity of several SDF-1 degrading proteases and thus improve SDF-1 stability in the proteolytic environment commonly found in ischemic and inflammatory injured tissues, and more specifically, can exploit this novel mode of action to increase the efficacy of EPAG olamine by its local injection into the affected site.

[0021] More specifically, the inventors have surprisingly found that the degenerative diseases / injuries disclosed herein after the present disclosure can be prevented and / or treated, and wound healing can be enhanced as disclosed herein after the present disclosure, by injecting a dosage form of EPAG or a derivative or conjugate or pharmaceutically acceptable salt thereof, particularly EPAG olamine, directly into the affected area.

[0022] More specifically, the inventors have surprisingly found that direct injection of EPAG or a derivative or conjugate or pharmaceutically acceptable salt thereof, particularly a dosage form of EPAG olamine, into the bone marrow before, during or after HSCT administration enhances the homing and engraftment of transplanted cells, thus increasing the success of the transplant.

[0023] The experimental section of this specification below demonstrates that EPAG or its derivatives, conjugates, or pharmaceutically acceptable salts, particularly EPAG olamine, 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 6-11). The experimental section further demonstrates that EPAG or its derivatives, conjugates, or pharmaceutically acceptable salts, particularly EPAG olamine, can strongly reduce the proteolytic activity of neutrophil degranulated products against SDF-1 (Example 12) and increase the chemotaxis of hematopoietic stem and progenitor cells toward SDF-1 in the presence of DPPIV protease (Example 13). Furthermore, the experimental section demonstrates that EPAG is 10 times more potent than eltrombopag olamine. 5 2 times smaller calculated half-maximal inhibitory concentration (IC 50 We show that sitagliptin, a well-known potent DPPIV inhibitor with a β-blocking activity, is less efficient than eltrombopag olamine in stabilizing SDF-1 in the presence of DPPIV.

[0024] SUMMARY OF THE INVENTION According to a first aspect, there is provided herein a controlled release pharmaceutical composition comprising at least a 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 controlled release pharmaceutical composition is suitable for local injection thereof at an affected site.

[0025] In one particular embodiment, the controlled release pharmaceutical composition is suitable for intramedullary injection. 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 pharmaceutical composition is suitable for intrathecal injection.

[0026] According to a second aspect, there is provided a controlled release pharmaceutical composition in the form of a powder 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, the controlled release microparticles have an average particle size in the range of 1 μm or more, such as 2 μm or more, in particular 1 μm to 200 μm, more particularly 2 μm to 150 μm, even more particularly 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 a poly(lactic-co-glycolic acid) copolymer; Provided herein is a controlled release pharmaceutical composition, wherein the drug loading content or percentage weight ratio between -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 the total weight of the microparticles is in the range of 2% to 45%, particularly 5% to 40%, more particularly 10% to 35%.

[0027] 1. A controlled release pharmaceutical composition in the form of a powder 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, the controlled release microparticles have an average particle size of 2 μm or more, in particular 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; the controlled release polymer matrix comprises at least a poly(lactic-co-glycolic acid) copolymer; Also disclosed is a pharmaceutical composition, wherein the drug loading content or percentage weight ratio between -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 the total weight of the microparticles is in the range of 2% to 45%, particularly 5% to 40%, more particularly 10% to 35%.

[0028] According to a third aspect, there is provided 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, particularly as defined herein, or a powder as defined herein, wherein the pharmaceutical composition optionally comprises: Provided herein is a kit or article of manufacture comprising an excipient selected from the group consisting of a tonicity-enhancing agent, a wetting agent, a viscosity-enhancing agent, a density-enhancing agent or mixtures thereof for preparing a pharmaceutical composition suitable for local injection thereof at an affected site, in particular for intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site injection, 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.

[0029] 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 controlled and immediate release dosage forms, particularly as defined in the present disclosure, 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, wherein the pharmaceutical composition optionally comprises an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent, or a mixture thereof, to prepare a pharmaceutical composition suitable for intrathecal injection.

[0030] According to a fourth aspect, there is provided a controlled release pharmaceutical composition as defined in the present disclosure or a controlled release pharmaceutical composition obtained by mixing two compartments of a kit as defined in the present disclosure for single or multiple use, which is administered by local injection at the affected site, in particular by: for improving the homing, engraftment and long-term expansion and proliferation of hematopoietic stem cells in patients, particularly human patients, before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) by intramedullary injection, for preventing and / or treating non-malignant hematologic disorders, for preventing and / or treating malignant hematologic diseases, and / or for preventing and / or treating primary immunodeficiencies, autoimmune diseases or inborn errors of metabolism, and / or Provided herein are controlled release pharmaceutical compositions for preventing and / or treating degenerative diseases / injuries by intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site injection, 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.

[0031] Also disclosed is a pharmaceutical composition as defined in the present disclosure or a pharmaceutical composition obtained by mixing two compartments of a kit as defined in the present disclosure, for single or multiple uses by intramedullary injection to improve homing, engraftment and long-term expansion and proliferation of hematopoietic stem cells in patients, particularly human patients, before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT).

[0032] As mentioned above, the controlled release pharmaceutical composition defined in the present disclosure, suitable for local injection, in particular intramedullary injection, in patients before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), is useful in improving the homing, engraftment and long-term expansion and proliferation of hematopoietic stem cells before allogeneic HSCT, in particular to reduce graft failure, to reduce the incidence of poor graft function (PGF), to reduce the incidence of graft-versus-host disease (GvHD), to enhance overall survival and donor chimerism, and / or to promote hematopoietic recovery and the success of hematopoietic stem cell transplantation, and also In 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 In the prevention and / or treatment of hematological malignancies, for example leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, in particular leukemia; and / or It is effective in the prevention and / or treatment of primary immunodeficiencies, autoimmune diseases or inborn errors of metabolism.

[0033] Furthermore, the controlled release pharmaceutical composition defined in the present disclosure suitable for local injection comprises In the prevention and / or treatment of cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; and / or In the prevention and / or treatment of diseases affecting the bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis; and / or In the prevention and / or treatment of periodontal disease; and / or in wound healing, including diabetic wound healing and corneal wound healing; and / or In the prevention and / or treatment of eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; and / or in the prevention and / or treatment of kidney disease; and / or In the prevention and / or treatment of liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; and / or In the prevention and / or treatment of inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; and / or It is effective in the prevention and / or treatment of chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and / or pulmonary fibrosis. [Brief explanation of the drawings]

[0034] [Figure 1A] Figures 1A and 1B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, the lanes are designated lane 1 through lane 6, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + MMP-9, and lanes 3 through 6 correspond to SDF-1α + MMP-9 + eltrombopag olamine at decreasing concentrations of eltrombopag olamine (50 μM, 10 μM, 1 μM, and 0.1 μM) (Example 6). [Figure 1B]Figures 1A and 1B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, the lanes are designated lane 1 through lane 6, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + MMP-9, and lanes 3 through 6 correspond to SDF-1α + MMP-9 + eltrombopag olamine at decreasing concentrations of eltrombopag olamine (50 μM, 10 μM, 1 μM, and 0.1 μM) (Example 6). [Figure 2A] Figures 2A and 2B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, lanes are designated lane 1 through lane 6, respectively. Lane 1 corresponds to SDF-1α + MMP-9, lanes 2 through 5 correspond to SDF-1α + MMP-9 + eltrombopag olamine at decreasing eltrombopag olamine concentrations (100 μM, 50 μM, 10 μM, and 1 μM), and lane 6 corresponds to SDF-1α alone (Example 7). [Figure 2B]Figures 2A and 2B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, lanes are designated lane 1 through lane 6, respectively. Lane 1 corresponds to SDF-1α + MMP-9, lanes 2 through 5 correspond to SDF-1α + MMP-9 + eltrombopag olamine at decreasing eltrombopag olamine concentrations (100 μM, 50 μM, 10 μM, and 1 μM), and lane 6 corresponds to SDF-1α alone (Example 7). [Figure 3A] Figures 3A, 3B, 4A, and 4B show Western blot analysis demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, the lanes are designated lane 1 through lane 6, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + MMP-8, and lanes 3-6 correspond to SDF-1 + MMP-8 + eltrombopag olamine at decreasing concentrations (50 μM, 10 μM, 1 μM, and 0.1 μM for Figures 3A and 3B; 100 μM, 50 μM, 10 μM, and 1 μM for Figures 4A and 4B) (Examples 8 and 9). [Figure 3B]Figures 3A, 3B, 4A, and 4B show Western blot analysis demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, the lanes are designated lane 1 through lane 6, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + MMP-8, and lanes 3-6 correspond to SDF-1 + MMP-8 + eltrombopag olamine at decreasing concentrations (50 μM, 10 μM, 1 μM, and 0.1 μM for Figures 3A and 3B; 100 μM, 50 μM, 10 μM, and 1 μM for Figures 4A and 4B) (Examples 8 and 9). [Figure 4A] Figures 3A, 3B, 4A, and 4B show Western blot analysis demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, the lanes are designated lane 1 through lane 6, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + MMP-8, and lanes 3-6 correspond to SDF-1 + MMP-8 + eltrombopag olamine at decreasing concentrations (50 μM, 10 μM, 1 μM, and 0.1 μM for Figures 3A and 3B; 100 μM, 50 μM, 10 μM, and 1 μM for Figures 4A and 4B) (Examples 8 and 9). [Figure 4B]Figures 3A, 3B, 4A, and 4B show Western blot analysis demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, the lanes are designated lane 1 through lane 6, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + MMP-8, and lanes 3-6 correspond to SDF-1 + MMP-8 + eltrombopag olamine at decreasing concentrations (50 μM, 10 μM, 1 μM, and 0.1 μM for Figures 3A and 3B; 100 μM, 50 μM, 10 μM, and 1 μM for Figures 4A and 4B) (Examples 8 and 9). [Figure 5A] Figures 5A and 5B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, lanes are designated lanes 1 to 6, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + MMP-13, and lanes 3 to 6 correspond to SDF-1α + MMP-13 + eltrombopag olamine at decreasing concentrations (100 μM, 50 μM, 10 μM, and 1 μM) of eltrombopag olamine (Example 10). [Figure 5B]Figures 5A and 5B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine 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) are shown. From left to right in each of panels A and B, lanes are designated lanes 1 to 6, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + MMP-13, and lanes 3 to 6 correspond to SDF-1α + MMP-13 + eltrombopag olamine at decreasing concentrations (100 μM, 50 μM, 10 μM, and 1 μM) of eltrombopag olamine (Example 10). [Figure 6A] Figures 6A and 6B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine on the proteolysis of SDF-1α by DPPIV (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) are shown. For each condition, 125 nM SDF-1α was incubated in 0.6 nM DPPIV with or without eltrombopag olamine, as applicable. From left to right in both Panel A and Panel B, the lanes are designated Lane 1 through Lane 5, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + DPPIV, lanes 3-4 correspond to SDF-1α + DPPIV + eltrombopag olamine at increasing eltrombopag olamine concentrations (50 μM and 100 μM), and lane 5 corresponds to SDF-1α + 1 μM sitagliptin (Example 11). [Figure 6B]Figures 6A and 6B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine on the proteolysis of SDF-1α by DPPIV (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) are shown. For each condition, 125 nM SDF-1α was incubated in 0.6 nM DPPIV with or without eltrombopag olamine, as applicable. From left to right in both Panel A and Panel B, the lanes are designated Lane 1 through Lane 5, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + DPPIV, lanes 3-4 correspond to SDF-1α + DPPIV + eltrombopag olamine at increasing eltrombopag olamine concentrations (50 μM and 100 μM), and lane 5 corresponds to SDF-1α + 1 μM sitagliptin (Example 11). [Figure 7] Figure 7 shows a graph illustrating the inhibitory effect of eltrombopag olamine on the proteolysis of SDF-1α by DPPIV (dipeptidyl-peptidase-4). Samples were compared using one-way ANOVA. Results are shown as circles representing the results of independent experiments for each condition, and as bars and error bars representing the mean ± standard error (SEM) of all experiments for each condition; n = 8 independent experiments (Example 11). [Figure 8A]Figures 8A and 8B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine on the proteolysis of SDF-1α by proteases contained in neutrophil degranulation products (ND). Western blot images of total SDF-1α (upper panel or panel A) and N-terminally intact SDF-1 (lower panel or panel B) are shown. From left to right in each of panels A and B, the lanes are designated lanes 1 to 4, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + ND, and lanes 3 and 4 correspond to SDF-1α + ND + eltrombopag olamine at decreasing concentrations of eltrombopag olamine (100 μM and 50 μM) (Example 12). [Figure 8B] Figures 8A and 8B show Western blot analyses demonstrating the inhibitory effect of eltrombopag olamine on the proteolysis of SDF-1α by proteases contained in neutrophil degranulation products (ND). Western blot images of total SDF-1α (upper panel or panel A) and N-terminally intact SDF-1 (lower panel or panel B) are shown. From left to right in each of panels A and B, the lanes are designated lanes 1 to 4, respectively. Lane 1 corresponds to SDF-1α alone, lane 2 corresponds to SDF-1α + ND, and lanes 3 and 4 correspond to SDF-1α + ND + eltrombopag olamine at decreasing concentrations of eltrombopag olamine (100 μM and 50 μM) (Example 12). [Figure 9] Figure 9 depicts a graph showing the percentage 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 olamine, compared to the control condition (the lower bottom chamber contained SDF-1α but no protease or eltrombopag olamine). Samples were compared using one-way ANOVA. Results are shown as circles for each independent experiment, and as bars and error bars for the mean ± standard error (SEM) of all experiments for each condition; n = 3 independent experiments (Example 13). DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description of the Invention As is clear from the background section above and in the examples section later in this specification (Examples 6-11), the present inventors have demonstrated that EPAG, or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof, inhibits protease activity and stabilizes the local concentration of SDF-1. This is the first time that such a mechanism of action has been demonstrated in the context of EPAG, or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof. The present inventors have also demonstrated that EPAG, or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof, can strongly reduce the proteolytic activity of neutrophil degranulation products against SDF-1 (Example 12) and can increase the chemotaxis of hematopoietic stem and progenitor cells toward SDF-1 in the presence of DPPIV protease (Example 13).

[0036] Therefore, the controlled release pharmaceutical composition according to the present disclosure is effective for the treatment of two types of stem / progenitor cells, namely: - either endogenous stem cells and / or endogenous progenitor cells, - Alternatively, exogenous stem cells and / or exogenous progenitor cells are sought.

[0037] Progenitor cells are the descendants of stem cells that subsequently undergo further differentiation to give rise to specialized cell types.

[0038] Local injection of a controlled release composition according to the present disclosure at a corresponding affected site, such as intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site injection, intradermal injection, intravitreal injection, intraocular injection, sub-Tenon injection, intraperitoneal injection, subgingival injection, intrahepatic injection, intrarenal injection, intragastrointestinal injection, submucosal injection, intraperitoneal injection, intrapleural injection, intrapulmonary injection, intra-articular injection, or intraosseous injection, can attract endogenous stem cells and / or endogenous progenitor cells; in other words, 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.

[0039] More specifically, intramedullary injection of the controlled release composition of the present disclosure into bone marrow can attract exogenous stem cells and / or exogenous progenitor cells; in other words, transplanted HSCs are attracted to the bone marrow into which the controlled release composition of the present disclosure is locally injected. Needless to say, when HSCT is performed, both exogenous and endogenous stem cells and progenitor cells can be attracted; in other words, SDF-1 stabilization can also be beneficial to the target attraction of exogenous stem cells and progenitor cells as well as the attraction of endogenous stem cells and progenitor cells.

[0040] Furthermore, in the framework of treating degenerative diseases by local injection at an affected site other than the bone marrow, involving endogenous attraction of stem and progenitor cells, transplantation of exogenous stem and progenitor cells, for example by intravenous infusion, can be performed before, during or after said local injection.

[0041] definition As used herein, the term "patient" refers to a human or human child who is suffering from or has the potential to suffer from one or more of the diseases and conditions described herein.

[0042] As used herein, the terms "recruitment" and "recruit" have the same meaning as the terms "attraction" and "attract."

[0043] Identifying patients in need of treatment for the diseases and conditions described herein is well within the ability and knowledge of one of ordinary skill in the art, who can readily identify patients in need of such treatment by the use of clinical tests, physical examination, medical / family history, or biological and diagnostic tests.

[0044] In the context of the present invention, the expression "degenerative diseases / injuries" means 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 diseases (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 in wound healing, including diabetic wound healing and corneal wound healing.

[0045] In the context of the present invention, pathologies associated with the homing, engraftment and long-term expansion and proliferation of hematopoietic stem cells are non-malignant hematologic disorders such as severe aplastic anemia and hemoglobinopathies, in particular sickle cell disease and beta-thalassemia; malignant hematologic diseases, e.g., leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, in particular leukemias; primary immunodeficiencies, autoimmune diseases and congenital metabolic disorders.

[0046] In the context of the present invention, the term "treating" or "treatment" as used herein refers to diseases / disorders as defined in this 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 diseases; liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; inflammatory bowel diseases (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, for example, myeloma, lymphoma, leukemias such as acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, especially leukemia; primary immunodeficiencies, autoimmune diseases and inborn errors of metabolism.

[0047] In the context of the present invention, the term "preventing" or "prevention" as used herein refers to diseases / disorders as defined in this 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 diseases; liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; non-malignant blood disorders such as chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and pulmonary fibrosis; severe aplastic anemia and hemoglobinopathies, particularly sickle cell disease and beta-thalassemia; malignant blood diseases, for example, myeloma, lymphoma, leukemias such as acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, particularly leukemia; primary immunodeficiencies, autoimmune diseases, and inborn errors of metabolism, particularly leukemia.

[0048] In the context of the present invention, the phrase "improving hematopoietic stem cell (HSC) homing, engraftment, and long-term expansion and proliferation" means promoting the ability of exogenously administered HSCs to localize, disseminate, and engraft within the recipient's bone marrow, thereby increasing the total number of transplanted HSCs capable of self-renewal and multipotent differentiation and ultimately participating in the reconstitution of a viable hematopoietic system.

[0049] As used herein, an "effective amount" refers to an amount that is effective to treat the diseases and symptoms described herein, i.e., cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis, and ischemia-reperfusion injury; diseases affecting the 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 diseases; liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease, and acute liver injury; inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease (COPD) (including chronic bronchitis); "Effective amount" refers to an amount of a compound of the present invention effective to reduce, eliminate, treat, or control symptoms of: chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis; non-malignant blood disorders such as severe aplastic anemia and hemoglobinopathies, particularly sickle cell disease and beta-thalassemia; malignant blood diseases, e.g., myeloma, lymphoma, leukemias such as acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, particularly leukemia; primary immunodeficiencies, autoimmune diseases, and inborn errors of metabolism. An "effective amount" also refers to an amount of a compound of the present invention effective to enable or control wound healing. The term "controlling" is intended to refer to all processes that may slow, interrupt, halt, or stop the progression of the diseases and conditions described herein, but does not necessarily indicate complete elimination of symptoms of all diseases and conditions.

[0050] The term "treatment-effective amount" refers to a treatment-effective amount for diseases / disorders as defined in this disclosure, particularly cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis, and ischemia-reperfusion injury; diseases affecting the 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 diseases; liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease, and acute liver injury; inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease (also known as chronic bronchitis); non-malignant blood disorders such as severe aplastic anemia and hemoglobinopathies, particularly sickle cell disease and beta-thalassemia; malignant blood diseases, e.g., leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, particularly leukemias; primary immunodeficiencies, autoimmune diseases, and inborn errors of metabolism.

[0051] In the sense of the present invention, the expression "local concentration of a product", in particular "local concentration of EPAG", more particularly in the equivalent EPAG free acid, means the concentration of the product, i.e. the concentration of EPAG 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 mixtures thereof, for example at the site of injection or implantation in the bone marrow of a patient.

[0052] In the sense of the present invention, the expression "affected site" refers to injured, diseased, inflamed, and / or ischemic tissue; injured, diseased, inflamed, and / or ischemic organ; and / or injured, diseased, inflamed, and / or ischemic membrane. It also refers to an entire organ or part thereof, an entire tissue or part thereof, and / or an entire membrane or part thereof. This expression also covers equivalent or partially equivalent expressions such as "lesion site," "disease site," "anatomical site," "affected area," "host site," and "target site." Examples may include, but are not limited to, bone, heart, blood vessels, skin, dermis, eye, liver, kidney, gastrointestinal tract, peritoneum, lung, gum, joint, bone marrow, in particular the sternum, tibia, femur, iliac crest, vertebrae, and mixtures thereof, among other sites.

[0053] In the sense of the present invention, the term "injection" also includes the term "implantation", i.e. the injection of an implant at the affected site.

[0054] In the sense of the present invention, the expression "local injection" refers, by way of example and without limitation, to intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site injection, 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.

[0055] Local injection of a controlled release pharmaceutical composition according to the present invention may also be performed, for example, during gastrointestinal endoscopy.

[0056] 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 in relation to each affected site considered to be treated by local injection according to the present invention. As an example, intraocular injections include intravitreal injections and sub-Tenon injections.

[0057] As used herein, the term "pharmaceutically acceptable" refers to a compound, material, excipient, composition or dosage form that is, within the scope of sound medical judgment, suitable for contact with human tissue without excessive toxicity, irritation, allergic response or other problem complication commensurate with a reasonable benefit / risk ratio.

[0058] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" can refer to any pharmaceutically acceptable excipient, e.g., a non-toxic carrier, adjuvant, or vehicle, that does not destroy the pharmacological activity of the compound with which it is formulated.

[0059] 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 an active ingredient into the body.

[0060] The term "controlled release composition" or "controlled release dosage form" or "controlled release microparticle" or "controlled release polymeric matrix" means that the composition or dosage form or microparticle or polymeric matrix allows the release of a specified amount of an active ingredient into the body over a specified period of time, i.e., a specific pharmacokinetic profile. The term "controlled release" encompasses all types of release that are modified compared to immediate release. In other words, the term "controlled release" is equivalent to "modified release" and encompasses extended release, as defined hereinafter, as well as delayed release and pulsed release.

[0061] The terms "extended release", "prolonged release" and "sustained release" are considered equivalent within the framework of the present invention. This type of release means that the release is prolonged over time compared to immediate release, i.e., the active ingredient is released slowly over time, allowing the patient to take the drug less frequently. In other words, the active ingredient is gradually released over a certain period of time, generally with the aim of reducing the maximum concentration and thereby reducing side effects.

[0062] The expression "for a controlled release and an immediate release" means that the release is carried out as a mixture of controlled release and immediate release, in other words as a combination of controlled release and immediate release.

[0063] "Drug loading content" means the mass ratio (also called weight ratio) of drug in a microparticle to the mass of the microparticle.

[0064] The term "mean particle size" or D50 means the particle diameter in microns that divides the particle volume distribution between the upper and lower halves of this diameter.

[0065] "D10=x μm" which is mainly used in the examples means that 10% of the particles have a size of x μm or less.

[0066] "D90=y μm" which is mainly used in the examples means that 90% of the particles have a size of y μm or less.

[0067] Controlled or modified release can, of course, result from a combination of immediate and controlled release. As used herein, the terms "combination," "combined," and related terms refer to the simultaneous, separate, or sequential administration of eltrombopag, or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof, with the additional active ingredient. For example, the combination may be administered with the additional active ingredient(s) simultaneously, separately, or sequentially in separate unit dosage forms, or together in a single unit dosage form.

[0068] Eltrombopag and its pharmaceutically acceptable salts As noted 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 a pharmaceutically acceptable salt thereof.

[0069] 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 is known to be useful as a TPO (thrombopoietin) receptor agonist that interacts with the transmembrane domain of the human TPO receptor, and is known as eltrombopag (C 25 H 22 Eltrombopag is known as N4O4, CAS: [496775-61-2]. It has a molecular weight of 442.5 g / mol and can be represented by the following formula:

[0070] [ka]

[0071] Eltrombopag can exist in different crystalline forms of the free acid, including multiple hydrates and solvates, as well as different cationic salt forms, all of which are encompassed within the scope of the present invention.

[0072] 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 a derivative or conjugate or pharmaceutically acceptable salt thereof is in the form of 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.

[0073] 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 olamine. 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:

[0074] [ka]

[0075] Eltrombopag olamine is sold under the brand name Promacta® in the United States and Revolade® outside the United States.

[0076] Derivatives of eltrombopag include any derivatives, for example, esters such as methyl, ethyl, pivaloyloxymethyl, etc. in the case of -COOH, acetic acid maleate, etc. in the case of -OH, and any esters known to those skilled in the art.

[0077] Derivatives of eltrombopag can also include protected forms of eltrombopag in which one or more functional groups, such as OH functional groups, are protected. Protection of hydroxy or carboxylic acid groups can be performed by any group and method known and well described in the art, for example, "Protective Groups in Organic Synthesis" by Theodora W. Greene, Wiley-Interscience 1981, New York.

[0078] Conjugates of eltrombopag include any conjugate, such as an antibody-eltrombopag conjugate, ie, a polypeptide, such as an antibody, covalently bound via a linker to at least one molecule of eltrombopag.

[0079] 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 suitable for the present invention may be included in a controlled release dosage form or a mixture of a controlled release dosage form and an immediate release dosage form.

[0080] In pharmaceutical compositions 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 ranging from 2 mg to 63 mg equivalents of free acid per ml of the controlled release dosage form or mixture of controlled and immediate release dosage forms, particularly from 8 mg to 55 mg equivalents of free acid per ml, and more particularly from 12 mg to 47 mg equivalents of free acid per ml.

[0081] In pharmaceutical compositions according to the present disclosure, EPAG olamine may be present in an amount ranging from 2 mg to 80 mg per ml of the controlled release dosage form or a mixture of controlled and immediate release dosage forms, particularly from 10 mg to 70 mg per ml, and more particularly from 15 mg to 60 mg per ml.

[0082] Controlled-Release Dosage Forms 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 a derivative or conjugate or a pharmaceutically acceptable salt thereof is included in a controlled release pharmaceutical dosage form.

[0083] In one embodiment, the controlled-release pharmaceutical dosage form comprising eltrombopag or a derivative or conjugate or pharmaceutically acceptable salt thereof is in the form of an in-situ forming depot, a hydrogel, a microporous implant, a solid implant or a microparticle, particularly a microparticle comprising a controlled-release polymer matrix or a multivesicular liposome, e.g., a microparticle comprising a controlled-release polymer matrix.

[0084] In one embodiment, the controlled-release dosage form is in the form of an in-situ forming depot or implant. In-situ forming depots or implants, such as pH-induced, heat-induced, or solvent exchange-induced gelation systems containing eltrombopag or its conjugates, derivatives, or salts, 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 S. Kempe et al. In-situ forming implants—an attractive formulation principle for parenteral depot formulations Journal of Controlled Release 2012,161:668.

[0085] In another embodiment, the controlled-release dosage form is in the form of a hydrogel. The hydrogel can be prepared according to techniques known to those skilled in the art. In particular, the hydrogel can be formed by dispersing eltrombopag or its conjugates, derivatives, or salts, optionally pre-solubilized in water, with the hydrogel under stirring. Among suitable hydrogels, mention may be made of sodium hyaluronate, alginate, etc. When the pharmaceutical composition is in the form of a powder, the water is removed by techniques known to those skilled in the art, for example, by dehydration or lyophilization. If necessary, they may be reversibly rehydrated.

[0086] In another embodiment, the controlled release dosage form is in the form of a microporous implant. In another embodiment, the controlled release dosage form is in the form of a solid implant, such as a pre-formed porous or non-porous solid implant that can be administered through a large needle or cannula.

[0087] In one embodiment, the controlled release dosage form is in the form of microparticles, an embodiment of which is described in detail below.

[0088] Controlled-release microparticles containing eltrombopag or a derivative or conjugate or salt thereof Controlled-release microparticles comprising eltrombopag or a derivative, conjugate, or salt thereof can take the form of various microparticles, such as (i) microparticles comprising a polymer matrix (also called a controlled-release polymer matrix) or (ii) multivesicular liposomes.

[0089] According to one embodiment, the microparticles have an average particle size of 1 μm or greater. According to another embodiment, the microparticles have an average particle size of 2 μm or greater.

[0090] According to another embodiment, the microparticles have an average particle size of 1 μm or more, for example 2 μm or more, in particular in the range of 1 μm to 200 μm, more particularly in the range of 2 μm to 150 μm, even more particularly in the range of 2 μm to 100 μm, for example 10 μm to 100 μm, or 10 μm to 80 μm.

[0091] According to another embodiment, the microparticles have an average particle size of 1 μm or more, such as 2 μm or more, in particular an average particle size of less than 200 μm, such as less than 150 μm, more particularly less than 100 μm, for example in the range of 10 μm to 100 μm, even more particularly less than 80 μm, for example between 1 and 80 μm, between 2 and 80 μm, or between 10 and 80 μm.

[0092] According to a preferred embodiment, the microparticles have an average particle size of 1 μm or more, for example 2 μm or more, in particular in the range of 1 μm to 200 μm, more particularly in the range of 2 μm to 150 μm, even more particularly in the range of 2 μm to 100 μm, for example 10 μm to 100 μm, or 10 μm to 80 μm.

[0093] It is understood that these ranges refer to the average size of all microparticles in a given population. The size of any given individual microparticle may be within a standard deviation above or below the average size.

[0094] Within the context of the present invention, "microparticles" means particles of any shape, made from any material, suitable for local injection, especially intramedullary injection, into the human body, especially into the bone marrow, in particular in a pharmaceutical composition, and having an average particle size of 1 μm or more, such as 2 μm or more, in particular in the range of 1 μm to 200 μm, more particularly in the range of 2 μm to 150 μm, even more particularly in the range of 2 μm to 100 μm, for example 10 μm to 100 μm, or 10 μm to 80 μm.

[0095] Microparticles suitable for pharmaceutical compositions used according to the present invention may be selected from various types of microparticles such as microspheres, microparticle matrices, microsphere matrices, microcapsules, rods, wafers, pills, fibers and pellets.

[0096] (i) Microparticles containing a polymer matrix The polymer matrix may be selected from a variety of polymers suitable for obtaining controlled release microparticles, as described below, and such polymer matrices are non-toxic to the human body.

[0097] According to one embodiment, the polymers forming the polymer matrix are biodegradable and biocompatible.

[0098] Within the context of the present invention, a "biodegradable" material refers to a material for which there is evidence that it degrades enzymatically or hydrolytically and the degradation products are incorporated into the biomass and / or removed from the organism by metabolism or renal filtration.

[0099] Within the context of the present invention, a "biocompatible" material refers to a material that is tolerated by the human body. The non-toxic, biocompatible and biodegradable polymers can be natural or synthetic.

[0100] According to one embodiment, the controlled release polymer matrix according to the present invention is selected from the group consisting of poly(lactic-co-glycolic acid) copolymer (also referred to as PLGA or PLG), poly(caprolactone), poly(lactide) (also referred to as PLA), poly(glycolide) (also referred to as PGA), poly(lactide-co-caprolactone), poly(ethylene glycol), poly(ethylene oxide) (also referred to as PEO), PLGA-b-PEO-b-PLGA, PLGA-b-PEO, polyhydroxyalkanoates, poly(hydroxybutyrate), poly(trimethylene carbonate), poly(dioxanone), poly(valerolactone), poly(α-hydroxy acids), poly(lactones), poly(amino acids), polyanhydrides , poly(orthoesters), poly(acetals), polyurethanes, polythioesters, polyphosphoesters, poly(ester-co-amides), poly(vinyl alcohol), PVA-g-PLGA, poly(ether ester) multiblock copolymers, polyvinylpyrrolidone, poly(methacrylates), PEO-PPO-PEO (also known as Pluronics, polyethylene oxide-polypropylene oxide-polyethylene oxide), gelatin, heparin, chondroitin sulfate; polysaccharides, such as alginate, starch, chitosan, hyaluronic acid, and dextran, and any combination thereof.

[0101] According to certain embodiments, the controlled release polymer matrix comprises at least poly(lactic-co-glycolic acid) copolymer.

[0102] According to this embodiment, the controlled release polymer matrix may comprise poly(lactic-co-glycolic acid) in an amount greater than 70% by weight, particularly greater than 80% by weight, and even more particularly greater than 90% by weight, based on the total weight of the polymer matrix.

[0103] Suitable polymers include, but are not limited to, those commercially known as RESOMER (Evonik Industries AG, Germany), LACTEL (Durect, USA), PURASORB (Corbion NV, The Netherlands), Viatel (Ashland, USA), and EXPANSORB (Seqens, France).

[0104] Examples of suitable polymers are listed in Table A.

[0105] [Table 1]

[0106] The polymer matrix may comprise PLGA and one or more additional polymers, copolymers or mixtures thereof, which may be present in the polymer matrix in an amount ranging from 0 to 30% by weight, particularly 0 to 20% by weight, and more particularly 0 to 10% by weight, relative to the total weight of the polymer matrix.

[0107] Thus, according to another particular embodiment, the controlled release polymer matrix comprises at least poly(lactic-co-glycolic acid) copolymer and a poly(lactide) different from poly(lactic-co-glycolic acid) copolymer, poly(caprolactone), a poly(glycolide) different from poly(lactic-co-glycolic acid) copolymer, poly(lactide-co-caprolactone), poly(ethylene glycol), poly(ethylene oxide), PLGA-b-PEO-b-PLGA, PLGA-b-PEO, polyhydroxyalkanoate, poly(hydroxybutyrate), poly(trimethylene carbonate), poly(dioxanone), poly(valerolactone), poly(α-hydroxy acid), poly(α-hydroxybenzoate), poly( ... and at least one suitable additional biocompatible and biodegradable polymer or copolymer selected from poly(lactones), poly(amino acids), polyanhydrides, poly(orthoesters), poly(acetals), polyurethanes, polythioesters, polyphosphoesters, poly(ester-co-amides), poly(vinyl alcohol), PVA-g-PLGA, poly(ether ester) multiblock copolymers, polyvinylpyrrolidone, poly(methacrylates), PEO-PPO-PEO (Pluronics), gelatin, heparin, chondroitin sulfate; polysaccharides, such as alginate, starch, chitosan, hyaluronic acid, and dextran, and any combination thereof.

[0108] According to another embodiment, microparticles suitable for the present disclosure have a particle size distribution with a D50 value of 200 μm or less, such as 150 μm or less, in particular 100 μm or less, for example 1 μm to 80 μm or 2 μm to 80 μm.

[0109] It is understood that these ranges refer to the average size of all microparticles in a given population. The size of any given individual microparticle may be within a standard deviation above or below the average size.

[0110] In one embodiment, the controlled release microparticles according to the present invention are PLGA microspheres, in other words, in this embodiment, the polymer matrix does not include any additional polymers or copolymers.

[0111] 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, and even more particularly from 50:50 or 75:25. Any suitable method known in the art for making polymers can be used, and the molecular weight can typically range from 5 to 150 kDa, particularly from 5 to 80 kDa, more particularly from 5 to 50 kDa, for example, from 10 to 150 kDa, 10 to 80 kDa, or 10 to 50 kDa.

[0112] According to further particular embodiments, the polymer forming the polymer matrix comprises PLGA in an amount of, for example, 100% by weight relative to the total weight of the polymer matrix, PLGA being defined by: - molecular weight between 5 and 80 kDa, and -Lactide:glycolide molar ratio of 75:25 to 50:50.

[0113] According to a more particular embodiment, the polymer forming the polymer matrix comprises PLGA in an amount of, for example, 100% by weight relative to the total weight of the polymer matrix, PLGA being defined by: - molecular weight between 5 and 50 kDa, and -50:50 lactide:glycolide molar ratio.

[0114] According to a more particular embodiment, the polymer forming the polymer matrix comprises PLGA in an amount of, for example, 100% by weight relative to the total weight of the polymer matrix, PLGA being defined by: - molecular weight between 50 and 100 kDa, and -50:50 lactide:glycolide molar ratio.

[0115] According to a more particular embodiment, the polymer forming the polymer matrix comprises PLGA in an amount of, for example, 100% by weight relative to the total weight of the polymer matrix, PLGA being defined by: - molecular weight between 5 and 50 kDa, and - a lactide:glycolide molar ratio of 75:25. According to a more particular embodiment, the polymer forming the polymer matrix comprises PLGA, for example in an amount of 100% by weight relative to the total weight of the polymer matrix, where PLGA is defined by: - molecular weight between 50 and 100 kDa, and -75:25 lactide:glycolide molar ratio.

[0116] According to one embodiment, the PLGA is either carboxylic acid or ester terminated, particularly carboxylic acid terminated.

[0117] According to another embodiment, the particulate matrix may comprise, or even consist of, a blend of two PLGA copolymers, in particular one of low molecular weight and one of high molecular weight.

[0118] The microparticles may further comprise pharmaceutically acceptable excipients to modify the drug release profile, 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.

[0119] The polymer matrix of the microparticles may contain one or more excipients, which may be present in the polymer matrix in an amount not exceeding 15% by weight, in particular not exceeding 10% by weight, and more particularly not exceeding 5% by weight, relative to the total weight of the polymer matrix.

[0120] According to one embodiment, the drug loading content or percentage weight ratio between 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, particularly EPAG olamine, and the total weight of the microparticles is in the range of 2% to 45%, particularly 5% to 40%, more particularly 10% to 35%.

[0121] Manufacturing process for obtaining microparticles containing a polymer matrix Any process suitable for producing polymeric microparticles having an average particle size in the range of 1 μm to 200 μm, for example 2 μm to 150 μm, 2 μm to 100 μm, 10 μm to 100 μm or 10 μm to 80 μm is considered suitable within the framework of the present invention.

[0122] Among such manufacturing processes, there may be mentioned emulsion-based processes, such as high pressure homogenization using a rotor-stator homogenizer in batch or continuous mode, or membrane emulsification followed by removal of organic solvents by extraction / evaporation.

[0123] As far as the general principle of such a method is concerned, an emulsion can be prepared and processed onto a membrane with pores of a determined size, and the resulting microspheres can then be collected, washed, and freeze-dried after extraction and evaporation of the organic solvent.

[0124] According to one particular embodiment, the microparticles may be produced from an O / W direct emulsion or a W / O / W double emulsion technique.

[0125] 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) outlines various methods for producing PLGA microparticles that can be used within the framework of the present invention.

[0126] According to one particular embodiment, the microparticles may be produced from a solid-in-oil-in-water (S / O / W) double emulsion technique.

[0127] Giovagnoli, S., et al.; Physicochemical characterization and release mechanism of a novel prednisone biodegradable microsphere formulation, J Pharm Sci. 97:303-317, (2008) describes an example of PLGA microparticles prepared by S / O / W emulsion technology. Other manufacturing approaches suitable for obtaining microparticles according to the present invention are atomization by spinning disk, atomization by spray drying, fluidized bed coating, or a combination thereof.

[0128] Alternatively, microparticles can be fabricated using drop-on-demand, drop-by-drop and jet break-up processes such as inkjet printing or microfluidics.

[0129] Alternatively, microparticles can be produced using supercritical fluid techniques. Alternatively, microparticles can be fabricated using microfabrication methods such as templating and molding-based techniques such as soft lithography.

[0130] All of these manufacturing processes are well known to those skilled in the art. The above manufacturing processes are well known to those skilled in the art.

[0131] (ii) Multiple vesicular liposomes 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-like 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.

[0132] 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 containing monounsaturated fatty acid ester moieties with 14-18 carbon atoms in the acyl chain (e.g., triolein, tripalmitolein), saturated fatty acid ester moieties with 6-8 carbon atoms in the acyl chain (e.g., tricaproin, tricaprylin), and mixtures thereof. Cholesterol can also be used in the composition.

[0133] MVLs are obtained using a water-in-oil-in-water double emulsification process. In the first step, a water-in-oil emulsion is prepared by mixing phospholipids, triolein, tricaprylin, and cholesterol solubilized in a volatile, water-immiscible organic solvent with an aqueous solution containing the solubilized drug to be encapsulated. This 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 sonication, or a combination thereof. MVLs are finally obtained by removing the volatile organic solvent from the double emulsion using gas stripping or flushing. Finally, removal of non-encapsulated material, concentration of the MVL, and buffer exchange are performed using either diafiltration or a cross-flow filtration system.

[0134] In one embodiment, the neutral lipid used to prepare MVL encapsulating eltrombopag or a conjugate or derivative or salt thereof comprises a mixture of triolein:tricaprylin having a ratio ranging from 50:50 to 0:100.

[0135] Mixture of controlled-release and immediate-release dosage forms As noted 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 may be included in a mixture of a controlled release dosage form and an immediate release dosage form.

[0136] Controlled release dosage forms are as defined above. The presence of the immediate-release dosage form aims to achieve a rapid onset of local concentration of eltrombopag, or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof, to achieve rapid stabilization or increase in local concentration of SDF-1 chemokine, thereby increasing the homing of transplanted HSCs. In one embodiment, the maximum weight ratio of immediate-release eltrombopag, or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof to the total amount of eltrombopag, or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof is 50%, 30%, or 15%.

[0137] In certain embodiments of the present invention, the weight ratio of immediate-release eltrombopag or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof to the total amount of eltrombopag or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof may be 0 to 5%, 0 to 10%, 0 to 15%, 0 to 30%, or 0 to 50%.

[0138] In some embodiments, the immediate release form has a length of release of 0 to 6 hours. In some embodiments, the immediate release form has a length of release of 0 to 2 hours.

[0139] The immediate release dosage form, according to the present invention, may take the form of an immediate fraction mixed with the controlled release microparticles described above, or may be present in a continuous phase with which the microparticles are mixed when preparing the final formulation in the form of a powder, which may of course contain any further suitable pharmaceutically acceptable excipients other than those already present in the microparticles.

[0140] Pharmaceutical Compositions and Kits As mentioned above, the controlled release pharmaceutical composition according to the present invention is suitable for local injection.

[0141] The controlled release pharmaceutical composition according to the present invention is suitable for many types of local injections depending on the target pathology.

[0142] Accordingly, also provided herein is a controlled release pharmaceutical composition as defined in the present disclosure, wherein the local injection is selected from intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, intrawound instillation, surgical site injection, 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.

[0143] According to certain embodiments, the controlled release pharmaceutical compositions according to the present disclosure may be administered to the affected area by local injection.

[0144] According to a particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered intramedullary by intramedullary injection.

[0145] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered intracardially by intramyocardial, intrapericardial, intraendocardial, and / or intraepicardial injection.

[0146] According to another particularly preferred embodiment, the controlled release pharmaceutical compositions according to the present disclosure may be administered intravascularly by intramyocardial, intrapericardial, intraendocardial, and / or intraepicardial injection.

[0147] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered to the skin or dermis by wound instillation, surgical site injection and / or intradermal injection.

[0148] According to another particularly preferred embodiment, the controlled release pharmaceutical compositions according to the present disclosure may be administered intraocularly by intravitreal, sub-Tenon and / or intraocular injection.

[0149] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered intrahepatically by intrahepatic injection.

[0150] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered intrarenally by intrarenal injection.

[0151] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered into the gastrointestinal tract (GIT) by GIT injection, intraperitoneal injection, and / or submucosal injection.

[0152] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered intraperitoneally by injection.

[0153] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure can be administered intrapulmonary by injection.

[0154] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered intragingivally by intraperitoneal and / or subgingival injection.

[0155] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered intra-articularly by intra-articular injection.

[0156] According to another particularly preferred embodiment, the controlled release pharmaceutical composition according to the present disclosure may be administered intraosseously by intraosseous injection.

[0157] According to one embodiment, the controlled release pharmaceutical composition according to the present disclosure may be an extended release pharmaceutical composition comprising at least an 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.

[0158] Provided herein are controlled release pharmaceutical compositions in various forms, namely, solutions, suspensions, powders, solid implants, semi-solid implants, microporous implants, and in-situ forming depots. According to one embodiment, provided herein are controlled release pharmaceutical compositions in the form of a sterile, injectable dosage form selected from solutions, suspensions, powders, solid implants, semi-solid implants, microporous implants, and in-situ forming depots.

[0159] Further pharmaceutical compositions are provided in the form of powders or kits. When in powder form, the pharmaceutical composition is primarily intended for storage, whereas solutions, suspensions, solid implants, semi-solid implants, microporous implants, powders, or in-situ-forming depots, particularly suspensions, are ready-to-use compositions and ready for injection, and kits allow for the separate storage of (i) an aqueous injection vehicle and (ii) a controlled-release dosage form comprising eltrombopag or a conjugate, derivative, or salt thereof, particularly a sterile and injectable dosage form suitable for injection, particularly in the form of a powder for forming a suspension.

[0160] The pharmaceutical compositions defined in the present disclosure may comprise at least one pharmaceutically acceptable excipient 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 a conjugate or derivative or a pharmaceutically acceptable salt thereof as defined in the present disclosure.

[0161] Among the pharmaceutically acceptable excipients suitable for the pharmaceutical compositions defined in the present disclosure may be mentioned tonicity enhancing agents, wetting agents, viscosity enhancing agents, density enhancing agents and mixtures thereof.

[0162] 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 the group consisting of a tonicity-enhancing agent, a wetting agent, a viscosity-enhancing agent, a density-enhancing agent, or a mixture thereof, which is obtained by mixing a controlled-release dosage form comprising eltrombopag or a conjugate or derivative or salt thereof, in particular microparticles comprising eltrombopag or a conjugate or derivative or salt thereof, more particularly with an aqueous injection vehicle as defined in the present disclosure.

[0163] Various embodiments of this alternative are detailed below. Solutions, solid implants, semi-solid implants, microporous implants, powders and in-situ forming depots may be prepared according to methods known to those skilled in the art.

[0164] According to certain embodiments, the sterile injectable dosage form is in the form of a suspension which can be obtained from a powder, as detailed below.

[0165] Powders and suspensions In one embodiment, there is provided a controlled-release pharmaceutical composition in the form of a powder comprising a controlled-release dosage form comprising eltrombopag, or a conjugate or derivative or salt thereof, wherein the controlled-release dosage form comprising eltrombopag, or a conjugate or derivative or salt thereof, is in the form of microparticles, particularly microparticles comprising a polymer matrix, more particularly microparticles having an average particle size of 1 μm or more, for example 2 μm, and even more particularly as defined herein.

[0166] In one embodiment, a pharmaceutical composition in the form of a powder comprising controlled-release microparticles comprising eltrombopag or a conjugate or derivative or salt thereof is provided, wherein the microparticles are microparticles comprising a polymer matrix or multivesicular liposomes, and the microparticles comprise eltrombopag or a conjugate or derivative or salt thereof having an average particle size of 1 μm or more, for example 2 μm.

[0167] In another embodiment, there is provided a pharmaceutical composition in the form of a sterile injectable suspension suitable for local injection, in particular intramedullary injection, obtainable by mixing the composition in the form of a powder according to the invention with an aqueous injection vehicle, the pharmaceutical composition optionally comprising an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or mixtures thereof, which excipient may be present in the aqueous injection vehicle or in the powder.

[0168] According to one embodiment, the powder further comprises an immediate release dosage form containing eltrombopag or a conjugate or derivative or salt thereof, in particular, the maximum weight ratio of immediate release eltrombopag or a conjugate or derivative or salt thereof to the total amount of eltrombopag or a conjugate or derivative or salt thereof is 15%, 30% or 50%.

[0169] In another embodiment there is provided a controlled release pharmaceutical composition in the form of a powder 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, the controlled release microparticles have an average particle size of 1 μm or more, such as 2 μm or more, in particular in the range of 1 μm to 200 μm, more particularly in the range of 2 μm to 150 μm, even more particularly in the range of 2 μm to 100 μm, for example in the range of 10 μm to 100 μm, or in the range of 10 μm to 80 μm; the controlled release polymer matrix comprises at least a poly(lactic-co-glycolic acid) copolymer; A controlled release pharmaceutical composition is provided, wherein the drug loading content or percentage weight ratio between -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 the total weight of the microparticles is in the range of 2% to 45%, particularly 5% to 40%, more particularly 10% to 35%.

[0170] According to this embodiment, the powder further comprises an immediate release dosage form containing 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 olamine, in particular 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 The maximum weight ratio 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 a pharmaceutically acceptable salt thereof, particularly EPAG olamine, to the total 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 conjugate or derivative or pharmaceutically acceptable salt thereof, particularly EPAG olamine, is 15%, 30% or 50%.

[0171] The present invention further 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 the form of a powder as described herein with an aqueous injection vehicle, the pharmaceutical composition optionally comprising an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, which excipient may be present in the aqueous injection vehicle or in the powder.

[0172] In another embodiment, a pharmaceutical composition, particularly in the form of a sterile injectable suspension, is provided, comprising a controlled release dosage form or a mixture of controlled and immediate release dosage forms 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, particularly 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. and a pharmaceutical composition is provided, the pharmaceutical composition being obtainable by mixing microparticles comprising

[0023] -3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid or a conjugate or derivative or pharmaceutically acceptable salt thereof, more particularly as defined in the present disclosure, with an aqueous injection vehicle, wherein the pharmaceutical composition optionally comprises an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof.

[0173] In one embodiment, the pharmaceutical composition is characterized in a sterile, injectable dosage form, particularly a concentration of eltrombopag or a conjugate or derivative or salt thereof in the range of 2 mg to 63 mg equivalents of free acid per ml of suspension, particularly 8 mg to 55 mg equivalents of free acid per ml, more particularly 12 mg to 47 mg equivalents of free acid per ml.

[0174] In one embodiment, the pharmaceutical composition is characterized by a concentration of eltrombopag olamine in a sterile injectable dosage form, particularly in a range of 2 mg to 80 mg per ml of suspension, particularly 10 mg to 70 mg per ml, more particularly 15 mg to 60 mg per ml.

[0175] According to another particular embodiment, in the pharmaceutical composition according to the invention, still in the form of a sterile and injectable suspension, the microparticles may be present in an amount ranging from 1% to 25% by weight, in particular from 2% to 20% by weight, more particularly from 5% to 15% by weight, relative to the total weight of the composition.

[0176] The pharmaceutical compositions used in the framework of the present invention may take the form of a sterile and injectable composition, in particular a suspension composition, containing an effective amount of eltrombopag or a conjugate or derivative or salt thereof.

[0177] "Sterile" in the sense of the present invention means an environment that can guarantee the safety requirements for the compounds considered in the compositions according to the invention for the administration routes described above, in particular for the administration route into or through the bone marrow. Indeed, for obvious reasons, it is essential that the compositions according to the invention are free from contaminants that may initiate undesirable side reactions at the host site.

[0178] The pharmaceutical composition used in the framework of the present invention can be prepared with the formulation in the form of a powder containing the microparticles described in the present disclosure. According to one embodiment, the pharmaceutical composition is a sterile and injectable composition for controlled release, or controlled and immediate release, of eltrombopag or its conjugates, derivatives, or salts, suitable for local injection, particularly intramedullary injection.

[0179] Due to its injectable nature, the composition according to the invention necessarily comprises a physiologically acceptable medium, also called an "aqueous injection vehicle".

[0180] "Physiologically acceptable medium" means a medium that is non-toxic and compatible with the injection and / or application of the compositions as contemplated by the present invention.

[0181] The present invention more particularly relates to a pharmaceutical composition as defined herein obtained by mixing a formulation in powder form as described herein with an aqueous injection vehicle, the pharmaceutical composition optionally comprising an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, which excipient may be present in the aqueous injection vehicle or in the powder.

[0182] The composition may comprise a solvent or a mixture of physiologically acceptable solvents. The composition may comprise a physiologically acceptable aqueous medium.

[0183] Aqueous media suitable for the present invention may include, for example, water. Suitable isotonic agents for the preparation of compositions according to the invention may include sugars and sodium chloride.

[0184] Aqueous injection vehicles may contain, inter alia, tonicity enhancing agents, wetting agents, viscosity enhancing agents or mixtures thereof.

[0185] Tonicity enhancing agents may include: dextrose, mannitol, sorbitol, sucrose, glycerin, sodium chloride, potassium chloride, cyclodextrin and maltodextrin.

[0186] Wetting agents may include: 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 lecithin.

[0187] Viscosity enhancing agents may include: sodium carboxymethylcellulose (CMC), glycosaminoglycans such as hyaluronic acid, dextran, collagen, poly(vinylpyrrolidone), poly(ethylene glycol), gelatin, hydroxyethylcellulose (HEC), methylcellulose (MC), alginate, gum arabic, starch.

[0188] According to certain embodiments, the pharmaceutical composition has a viscosity of 10 s when measured at 25°C. -1 The viscosity at a shear rate of 5 to 1000 mPa·s, particularly 5 to 500 mPa·s.

[0189] kit Further 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 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 pharmaceutically acceptable salt thereof, or a mixture of a controlled release dosage form and an immediate release dosage form, particularly as defined in the present invention, or a powder as defined in the present invention, wherein the pharmaceutical composition optionally comprises an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent, or a mixture thereof, to prepare a pharmaceutical composition suitable for local injection, particularly intramedullary injection.

[0190] In one embodiment, the kit may be in the form of two separate vials. In another embodiment, kit or product can be in the form of a vial and a pre-filled syringe or medical device, or can be in the form of two separate vials.In this embodiment, the two compartments or dual chambers can be mixed with the aqueous injection vehicle and powder described herein by any means known to those skilled in the art.Furthermore, in this embodiment, this means can be in the form of a pierceable membrane or a breakable diaphragm, for example, by the pressure that can be applied by the user.

[0191] 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.

[0192] For reasons of patient comfort and safety, it would be advantageous to find pharmaceutical compositions suitable for local injection, especially intramedullary injection, which would require only one effective injection, without the need for multiple injections.

[0193] For reasons of patient comfort and safety, it would also be advantageous to find pharmaceutical compositions suitable for local injection, especially intramedullary implantation, which would require only a single effective implantation rather than the need for multiple implantations.

[0194] Administration of the Composition Depending on the target pathology to be treated, the controlled release pharmaceutical composition according to the present invention may 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, gums, joints, bone marrow, in particular the sternum, tibia, femur, iliac crest, vertebrae, and mixtures thereof, for example the iliac crest, such as the posterior part of the iliac crest, and mixtures thereof.

[0195] Insofar as improving homing, engraftment, and long-term expansion and proliferation of hematopoietic stem cells is concerned, the pharmaceutical compositions 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, and more particularly the posterior portion of the iliac crest.

[0196] Intramedullary injections can be performed using an 18G needle with a trocar. Intramedullary implantation can be performed using a 16G needle with a trocar.

[0197] The injection or implantation can be performed under general or local regional anesthesia. The pharmaceutical compositions used in the framework of the present invention can be injected or implanted using any of the methods known in the art.

[0198] In particular, the pharmaceutical composition may be administered by means of an injection device suitable for local injection, in particular intramedullary injection, such as a syringe equipped with a needle of 15 to 25 G, preferentially 16 to 25 G, more preferentially 18 to 23 G.

[0199] Therapeutic Uses and Methods As mentioned above, pharmaceutical compositions according to the present invention may find use in the treatment of degenerative diseases and in improving stem cell homing.

[0200] As mentioned above, according to one embodiment, the pharmaceutical composition as defined in the present disclosure, or the pharmaceutical composition obtained by mixing the two compartments of the kit as defined in the present disclosure for single or multiple uses, is injected locally into the site of the lesion, affected tissue, or affected organ.

[0201] In particular, the pharmaceutical composition as defined in the present disclosure, or the pharmaceutical composition obtained by mixing the two compartments of the kit as defined in the present disclosure for single or multiple use, comprises: by intramedullary injection to improve the homing, engraftment and long-term expansion and proliferation of hematopoietic stem cells in patients, particularly human patients, before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), to prevent and / or treat non-malignant hematologic disorders, to prevent and / or treat malignant hematologic diseases, and / or to prevent and / or treat primary immunodeficiencies, autoimmune diseases or inborn errors of metabolism; and / or To prevent and / or treat degenerative diseases / injuries as defined in this disclosure, the compounds are locally injected by intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site injection, 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.

[0202] According to another embodiment, the medicament as defined herein or the medicament obtained by mixing the two compartments of the kit as defined herein comprises: in the prevention and / or treatment of degenerative diseases / injuries, in particular cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; diseases affecting the 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 diseases; liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease and pulmonary fibrosis; and / or in wound healing, including diabetic wound healing and corneal wound healing; and / or In 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 in the prevention and / or treatment of malignant blood diseases, for example leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, in particular leukemia, and / or in the prevention and / or treatment of primary immunodeficiencies, autoimmune diseases or inborn errors of metabolism.

[0203] The diseases or injuries, the corresponding lesion sites or affected tissues / organs, and the corresponding potential local injection sites are summarized in Table B below.

[0204] [Table 2]

[0205] A general description of therapeutic dosages and posology applicable to all therapeutic applications according to the present invention is first provided herein hereafter.

[0206] More specific aspects of the therapeutic uses and methods are then provided in relation to more specific therapeutic applications.

[0207] Therapeutic Dosage and Posology According to certain embodiments, there is provided herein a pharmaceutical composition as defined herein or obtained by mixing the two compartments of the kit as defined herein, 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 pharmaceutically acceptable salt thereof, particularly EPAG olamine, is administered to said patient by local injection, particularly by intramedullary injection, in an amount ranging from 0.2 mg to 50 mg equivalent of free acid per kg body weight of said patient, particularly from 0.5 mg to 45 mg equivalent of free acid per kg body weight of said patient, more particularly from 1 mg to 40 mg equivalent of free acid per kg body weight of said patient.

[0208] In particular, the inventors have found that local injection of EPAG or a derivative or conjugate or pharmaceutically acceptable salt thereof (e.g., EPAG olamine) at the site of a lesion or local injection in an affected tissue / organ, in particular intramedullary injection in the bone marrow, makes it possible to improve the local concentration of EPAG or a derivative or conjugate or pharmaceutically acceptable salt thereof (e.g., EPAG olamine) so that the local concentration is higher than the maximum plasma concentration of 14 μM following oral administration of a 50 mg tablet.

[0209] The inventors have demonstrated that the novel mode of action underlying this invention is capable of achieving EPAG olamine concentrations in a range that exceeds the steady-state plasma concentration (i.e., 14 μM) achieved following once-daily administration of a 50 mg oral tablet of PROMACTA® (U.S. FDA, Drug Approval Package, Promacta (eltrombopag) Tablets, Medical review Part 1).

[0210] In one embodiment, a controlled release pharmaceutical composition for use in accordance with the present disclosure may be effective to maintain a local concentration of EPAG olamine above a plasma concentration of 14 μM.

[0211] According to one embodiment, a controlled release pharmaceutical composition for use according to the present disclosure may be effective to maintain a local concentration of said 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 a concentration 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, and even more particularly between 50 μM and 100 μM, for a period of 6 hours to 3 months, 6 hours to 30 days, particularly 1 day to 15 days, more particularly 2 days to 15 days, and even more particularly 4 days to 15 days after local injection.

[0212] 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 a conjugate or derivative or a pharmaceutically acceptable salt thereof, particularly EPAG olamine, one skilled in the art can use any known method, for example, HPLC-MS analysis can be used.

[0213] 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 a conjugate or derivative or a pharmaceutically acceptable salt thereof, particularly EPAG olamine, is between 6 hours and 3 months, between 6 hours and 30 days, particularly between 1 day and 15 days, more particularly between 2 days and 15 days, and even more particularly between 4 days and 5 days. The administration period may be from 4 to 15 days, for example from 4 to 30 days, preferably from 15 days, and 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 conjugate or derivative or a pharmaceutically acceptable salt thereof, particularly EPAG olamine, is present in a dose of the free acid form in the range of 10 mg to 570 mg equivalent to eltrombopag.

[0214] Further provided herein is a pharmaceutical composition according to the invention, or a pharmaceutical composition obtainable by mixing the two compartments of the kit as defined above, for use according to the invention, 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 pharmaceutically acceptable salt thereof, in particular EPAG olamine, is 80% (w / w) for more than 6 hours, in particular for more than 1 day, 2 days or 4 days, measured according to the following protocol: A 75 mg aliquot of a controlled release dosage form according to the invention is suspended in a 50 mL solution containing 0.5% polysorbate 80 in phosphate buffered saline (10 mM, pH 6.8) at 37° C. with stirring; then, periodically throughout the release period, filtered samples are analyzed by HPLC; Here, 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 olamine, is present in a dose of the free acid form ranging from 10 mg to 570 mg equivalent to eltrombopag.

[0215] Thus, the inventors have discovered a surprisingly well-matched means for treating patients with specific in vitro dissolution and / or in vivo release rates. Examples 3 and 4 demonstrate these suitable profiles.

[0216] In the context of the present invention, a "dissolution profile" refers to an in vitro test that reports a plot of the cumulative amount of active ingredient released as a function of time. The dissolution data arise from the conditions under which the measurements were taken and are described herein.

[0217] Furthermore, the time required to release 80% by weight of EPAG olamine may be between 6 hours and 3 months, particularly between 6 hours and 30 days, particularly between 1 day and 15 days, more particularly between 2 days and 15 days, even more particularly between 4 days and 15 days, for example between 4 days and 30 days, preferably 15 days, and the EPAG olamine is present in a dose ranging from 10 mg to 800 mg.

[0218] In one embodiment, the pharmaceutical composition for use according to the invention is characterized in 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 pharmaceutically acceptable salt thereof, particularly EPAG Olamine, is 80% (w / w) in more than 6 hours, particularly more than 1 day, 2 days or 4 days, measured according to the protocol above.

[0219] Administration can be by single injection or as continuous injections, provided that the time required to release 80% by weight of the eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt, in particular EPAG olamine, in the microparticles is between 6 hours and 3 months, in particular between 6 hours and 30 days, in particular between 1 day and 15 days, more particularly between 2 days and 15 days, even more particularly between 4 days and 15 days, for example between 4 days and 30 days, preferably 15 days, that the eltrombopag or its derivative or conjugate or pharmaceutically acceptable salt, in particular EPAG olamine, is present in a concentration in the range of 2 mg to 80 mg per ml of suspension, and that the pharmaceutical composition has a volume in the range of, for example, 0.1 ml to 10 ml, in particular between 1 ml and 10 ml, more particularly between 5 ml and 10 ml.

[0220] In one embodiment, multiple injections (2, 3, 4 or more) are administered. In this embodiment, each subsequent injection is administered after a suitable period of time has elapsed since the previous injection. This suitable period of time may be, for example, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or longer.

[0221] According to particular embodiments, eltrombopag or a derivative or conjugate or pharmaceutically acceptable salt thereof, particularly EPAG olamine, is present at a concentration ranging from 2 mg to 63 mg equivalents of free acid per ml of suspension, particularly from 8 mg to 55 mg equivalents of free acid per ml of suspension, and more particularly from 12 mg to 47 mg equivalents of free acid per ml of suspension.

[0222] According to certain embodiments, eltrombopag olamine is present in a concentration ranging from 2 mg to 80 mg per ml of suspension, particularly from 10 mg to 70 mg per ml of suspension, and more particularly from 15 mg to 60 mg per ml of suspension.

[0223] In one embodiment, the pharmaceutical composition for use according to the present invention has a microparticle release time of from 6 hours, or from 1 day, or from 4 days, for example, 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 ...5, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours to 3 months. 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours to 2 months, or 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours to 30 days, for example, 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, 21, 28 days, 30 days, 1.5 months, 2 months, 2.5 months to 3 months, , 18, 19, 20, 21, 22, 23 hours, 1 day or 4 days or more, 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, 7 days, 10 days, 15 days, 20 days to 2 months, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, 1 day or more, or 4 days or more, 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.It is 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 to, 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 7 days, or is characterized by the fact that it is included in 5 days to 3 months, 5 days to 2 months, 5 days to 30 days, 5 days to 15 days, 5 days to 10 days and 5 days to 7 days.

[0224] A pharmaceutical composition may or may not exhibit a burst release. In the context of the present invention, "burst release" means that an initial bolus of drug is released immediately after placement in the release medium, i.e., in the present invention, immediately after local injection, in particular intramedullary injection, before the release rate reaches a stable profile.

[0225] In either case, the length of the burst release may be between 0 and 1 day, for example between the beginning of day 1 and the end of day 1, particularly between 0 and 6 hours, between 0 and 3 hours, and even more particularly between 0 and 2 hours.

[0226] In certain embodiments, the pharmaceutical compositions for use according to the present invention do not exhibit a significant burst release, and according to such embodiments, less than 50% by weight of eltrombopag, or a derivative, conjugate, or pharmaceutically acceptable salt thereof, is released 12 hours after administration.

[0227] It should 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, as well as the judgment of the treating physician and the severity of the particular disease being treated.

[0228] Therapeutic uses and methods for treating degenerative diseases The inventors have found that local injection of a controlled-release pharmaceutical composition in the form of a suspension and local implantation into the lesion site or affected tissue / organ of a controlled-release pharmaceutical composition in the form of an implant for release (controlled release or a mixture of immediate release and controlled release) of EPAG or a derivative or conjugate or pharmaceutically acceptable salt thereof (e.g., EPAG olamine) improves cell regeneration and tissue / organ repair.

[0229] According to certain embodiments, the degenerative disease / injury is selected from the group consisting 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 diseases; liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease and pulmonary fibrosis.

[0230] According to another particular embodiment, the controlled release pharmaceutical composition as defined in the present disclosure is used in wound healing, including diabetic wound healing and corneal wound healing.

[0231] As mentioned above, the controlled release dosage form or mixture of controlled release and immediate release dosage forms 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 can be administered by injection, in particular by intramyocardial, intrapericardial, intraepicardial, intraendocardial, wound instillation, surgical site injection, intradermal, intravitreal, intraocular, subtenon, intraperitoneal, subgingival, intrahepatic, intrarenal, intragastrointestinal, submucosal, intraperitoneal, intrapleural, intrapulmonary, intraarticular, and / or intraosseous injection. In the prevention and / or treatment of cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; and / or In the prevention and / or treatment of diseases affecting the bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis; and / or In the prevention and / or treatment of periodontal disease; and / or For use in wound healing, including diabetic wound healing and corneal wound healing; and / or In the prevention and / or treatment of eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; and / or in the prevention and / or treatment of kidney disease; and / or In the prevention and / or treatment of liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; and / or In the prevention and / or treatment of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis; and / or Topically administered in the prevention and / or treatment of chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and / or pulmonary fibrosis.

[0232] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intramyocardial, intrapericardial, intraepicardial, intraendocardial, wound instillation, surgical site injection, intradermal, intravitreal, intraocular, subtenon, intraperitoneal, subgingival, intrahepatic, intrarenal, intragastrointestinal, submucosal, intraperitoneal, intrapleural, intrapulmonary, intraarticular, and / or intraosseous injection, is dedicated to the prevention and / or treatment of degenerative diseases / injuries as defined in the present disclosure.

[0233] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intramyocardial, intrapericardial, intraepicardial, and / or intraendocardial injection, is intended for the prevention and / or treatment of cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis, and / or ischemia-reperfusion injury.

[0234] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intramyocardial, intrapericardial, intraepicardial and / or intraendocardial injection, is dedicated to the prevention and / or treatment of myocardial infarction.

[0235] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intramyocardial, intrapericardial, intraepicardial and / or intraendocardial injection, is dedicated to the prevention and / or treatment of atherosclerosis.

[0236] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intramyocardial, intrapericardial, intraepicardial, and / or intraendocardial injection, is dedicated to the prevention and / or treatment of ischemia-reperfusion injury.

[0237] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intra-articular and / or intraosseous injection, is intended for the prevention and / or treatment of diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis.

[0238] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intra-articular and / or intraosseous injection, is dedicated to the prevention and / or treatment of osteoporosis.

[0239] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intra-articular and / or intraosseous injection, is dedicated to the prevention and / or treatment of rheumatoid arthritis.

[0240] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intra-articular and / or intraosseous injection, is dedicated to the prevention and / or treatment of osteoarthritis.

[0241] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intraosseous, intraperitoneal and / or subgingival injection, is dedicated to the prevention and / or treatment of periodontal disease.

[0242] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for wound instillation, surgical site injection, intradermal injection, intraocular injection, sub-Tenon injection and / or intravitreal injection, is dedicated to wound healing, including diabetic wound healing and corneal wound healing.

[0243] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for wound instillation, surgical site injection, and / or intradermal injection, is dedicated to diabetic wound healing.

[0244] In one embodiment, the pharmaceutical composition suitable for local injection, in particular for wound instillation, surgical site injection, intraocular injection, sub-Tenon injection and / or intravitreal injection, is dedicated to corneal wound healing.

[0245] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intraocular, sub-Tenon and / or intravitreal injection, is intended for the prevention and / or treatment of eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma.

[0246] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intraocular, sub-Tenon and / or intravitreal injection, is dedicated to the prevention and / or treatment of retinal ischemia.

[0247] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intraocular, sub-Tenon and / or intravitreal injection, is dedicated to the prevention and / or treatment of macular degeneration.

[0248] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intraocular, sub-Tenon and / or intravitreal injection, is dedicated to the prevention and / or treatment of diabetic retinopathy.

[0249] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intraocular, sub-Tenon and / or intravitreal injection, is dedicated to the prevention and / or treatment of glaucoma.

[0250] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intrarenal injection, is dedicated to the prevention and / or treatment of kidney diseases.

[0251] In one embodiment, the pharmaceutical composition suitable for local injection, particularly intrahepatic injection, is dedicated to the prevention and / or treatment of liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease, and acute liver injury.

[0252] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intrahepatic injection, is dedicated to the prevention and / or treatment of fibrosis.

[0253] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intrahepatic injection, is dedicated to the prevention and / or treatment of liver cirrhosis.

[0254] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intrahepatic injection, is dedicated to the prevention and / or treatment of non-alcoholic fatty liver disease.

[0255] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intrahepatic injection, is dedicated to the prevention and / or treatment of acute liver injury.

[0256] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intraperitoneal injection, submucosal injection, and / or injection in the GIT, is dedicated to the prevention and / or treatment of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis.

[0257] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intraperitoneal injection, submucosal injection and / or injection in the GIT, is dedicated to the prevention and / or treatment of Crohn's disease.

[0258] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intraperitoneal injection, submucosal injection and / or injection in the GIT, is dedicated to the prevention and / or treatment of ulcerative colitis.

[0259] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intrapleural and / or intrapulmonary injection, is dedicated to the prevention and / or treatment of chronic obstructive pulmonary disease (COPD).

[0260] In one embodiment, the pharmaceutical composition suitable for local injection, in particular intrapleural and / or intrapulmonary injection, is dedicated to the prevention and / or treatment of pulmonary fibrosis.

[0261] According to another aspect, the present invention provides 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 ... The present invention relates to a method for the prevention and / or treatment of degenerative diseases / injuries, comprising at least 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 [phenyl]-3-carboxylic acid or a derivative or conjugate or a pharmaceutically acceptable salt thereof by local injection, in particular by intramyocardial, intrapericardial, intraepicardial, intraendocardial, wound instillation, surgical site injection, intradermal, intravitreal, intraocular, subtenon, intraperitoneal, subgingival, intrahepatic, intrarenal, intragastrointestinal, submucosal, intraperitoneal, intrapleural, intrapulmonary, intraarticular, and / or intraosseous injection.

[0262] According to another aspect, the present invention relates to a method for the prevention and / or treatment of cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis, and ischemia-reperfusion injury, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intramyocardial, intrapericardial, intraepicardial, and / or intraendocardial injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix suitable for local injection, in particular intramyocardial, intrapericardial, intraepicardial, and / or intraendocardial injection, 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.

[0263] According to another aspect, the present invention relates to a method for the prevention and / or treatment of diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intra-articular injection, or intraosseous injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix suitable for local injection, in particular intra-articular injection, and / or intraosseous injection, 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.

[0264] According to another aspect, the present invention relates to a method for the prevention and / or treatment of periodontal disease comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intraosseous, intraperitoneal and / or subgingival injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix suitable for local injection, in particular intraosseous, intraperitoneal and / or subgingival injection, 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.

[0265] According to another aspect, the present invention relates to a method for effecting and / or enhancing wound healing, including diabetic wound healing and corneal wound healing, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by wound instillation, surgical site injection, intradermal injection, intraocular injection, sub-Tenon injection and / or intravitreal injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix suitable for local injection, in particular intra-wound instillation, surgical site injection, intradermal injection, intraocular injection, sub-Tenon injection and / or intravitreal injection, 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.

[0266] According to another aspect, the present invention relates to a method for the prevention and / or treatment of eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intraocular, sub-Tenon and / or intravitreal injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix suitable for local injection, in particular intraocular, sub-Tenon and / or intravitreal injection, 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.

[0267] According to another aspect, the present invention relates to a method for the prevention and / or treatment of kidney diseases comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intrarenal injection, a pharmaceutical composition in the form of a suspension suitable for local injection, in particular intrarenal injection, 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.

[0268] According to another aspect, the present invention relates to a method for the prevention and / or treatment of fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intrahepatic injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix suitable for local injection, in particular intrahepatic injection, 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.

[0269] 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 at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular injection into the gastrointestinal tract, submucosal injection, and / or intraperitoneal injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix suitable for local injection, in particular injection into the gastrointestinal tract, submucosal injection, and / or intraperitoneal injection, 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.

[0270] According to another aspect, the present invention relates to a method for preventing and / or treating chronic obstructive pulmonary disease and pulmonary fibrosis, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular intrapleural injection, or intrapulmonary injection, a pharmaceutical composition in the form of a suspension comprising a controlled release polymer matrix suitable for local injection, in particular intrapleural injection, and / or intrapulmonary injection, 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.

[0271] According to another aspect, the present invention provides a method for the administration of at least an effective amount of 3 or more of a compound of formula (I) for controlled release or for controlled and immediate release by local injection, in particular by intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site injection, 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. The present invention relates to a method for the prevention and / or treatment of degenerative diseases / injuries comprising at least administering 2'-[(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 to a patient in need thereof, in particular a human patient in need thereof.

[0272] According to another aspect, the present invention relates to a method for the prevention and / or treatment of cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis, and ischemia-reperfusion injury, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intramyocardial, intrapericardial, intraepicardial, 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.

[0273] According to another aspect, the present invention relates to a method for the prevention and / or treatment of diseases affecting bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intra-articular 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.

[0274] According to another aspect, the present invention relates to a method for the prevention and / or treatment of periodontal disease comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intraosseous, intraperitoneal and / or subgingival injection, for controlled release, or for controlled and immediate release, 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.

[0275] According to another aspect, the present invention relates to a method for effecting and / or enhancing wound healing, including diabetic wound healing and corneal wound healing, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intrawound instillation, surgical site injection, intradermal injection, intraocular injection, sub-Tenon injection and / or intravitreal 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.

[0276] According to another aspect, the present invention relates to a method for the prevention and / or treatment of eye diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intraocular, sub-Tenon and / or intravitreal injection, for controlled release, or for controlled and immediate release, 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.

[0277] According to another aspect, the present invention relates to a method for the prevention and / or treatment of kidney diseases comprising at least administering by local injection, in particular by intrarenal injection, for controlled release or for controlled and immediate release, 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 to a patient in need thereof, in particular a human patient in need thereof.

[0278] According to another aspect, the present invention relates to a method for the prevention and / or treatment of liver diseases, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intrahepatic injection, for controlled release, or for controlled and immediate release, 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.

[0279] 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 at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by injection into the gastrointestinal tract, 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.

[0280] According to another aspect, the present invention relates to a method for preventing and / or treating chronic obstructive pulmonary disease and pulmonary fibrosis, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intrapleural or intrapulmonary injection, for controlled release, or for controlled and immediate release, 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.

[0281] Further provided herein are methods of effecting and / or enhancing wound healing, including diabetic wound healing and corneal wound healing, and / or methods of 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 diseases; liver diseases, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease, and acute liver injury; inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease and / or pulmonary fibrosis, comprising at least: - preparing and / or providing a pharmaceutical composition in the form of a sterile and injectable suspension by mixing a formulation in the form of a powder as described herein with an aqueous injection vehicle and then with an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, said excipients being optionally present in the aqueous injection vehicle or the powder, and - local injection of the pharmaceutical composition into the appropriate lesion or affected organ / tissue of a patient in need thereof (the volume injected being adapted to the injection site), in particular by intramyocardial, intrapericardial, intraepicardial, intraendocardial, wound instillation, surgical site injection, intradermal, intravitreal, intraocular, subtenon, intraperitoneal, subgingival, intrahepatic, intrarenal, intragastrointestinal, submucosal, intraperitoneal, intrapleural, intrapulmonary, intraarticular or intraosseous injection.

[0282] Further provided herein are methods for effecting and / or enhancing wound healing, including diabetic wound healing and corneal wound healing, and / or methods for preventing and / or treating degenerative diseases / injuries, 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 diseases; liver diseases, such as fibrosis, cirrhosis, non-alcoholic fatty liver disease, and acute liver injury; inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis; chronic obstructive pulmonary disease and / or pulmonary fibrosis, comprising at least: - preparing and / or providing a pharmaceutical composition in the form of a sterile and injectable solid implant, semi-solid implant, microporous implant or in situ forming depot, which pharmaceutical composition optionally comprises an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, which excipient may be present in an aqueous injection vehicle or a powder, and - Local implantation of the pharmaceutical composition into the appropriate affected area of ​​a patient in need thereof (the volume to be implanted being adapted to the implantation site), in particular by intramyocardial, intrapericardial, intraepicardial, intraendocardial, intradermal, intravitreal, intrahepatic, intrarenal, submucosal, intraperitoneal, intrapleural, intrapulmonary, intraarticular or intraosseous implantation, or by implantation into the gastrointestinal tract, instillation into a wound or injection into a surgical site.

[0283] Therapeutic uses and methods for improving stem cell homing and for treating related diseases The inventors have found that local injection at the affected site, particularly intramedullary injection in the bone marrow, of a controlled release pharmaceutical composition comprising EPAG or a derivative or conjugate or a pharmaceutically acceptable salt thereof (e.g., EPAG olamine) before, during or after autologous or allogeneic HSCT makes it possible to improve homing, engraftment and long-term expansion and proliferation of HSCs.

[0284] More specifically, the inventors have found that local injection into the bone marrow of a controlled-release pharmaceutical composition in the form of a suspension, and local implantation at the affected site, particularly intramedullary implantation within the bone marrow, of a controlled-release pharmaceutical composition in the form of an implant for release (controlled release or a mixture of immediate and controlled release) of EPAG or a derivative or conjugate or pharmaceutically acceptable salt thereof (e.g., EPAG olamine) before, during or after HSCT increases the proportion of transplanted HSCs that effectively home and engraft within the recipient's bone marrow, resulting in a faster return to normal hematopoiesis and overall greater transplant success.

[0285] According to certain embodiments, the HSCT-associated disease / disorder is selected from non-malignant hematological disorders such as severe aplastic anemia and hemoglobinopathies, particularly sickle cell disease and beta-thalassemia; malignant hematological diseases, e.g., leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, particularly leukemias; primary immunodeficiencies, autoimmune diseases, and inborn errors of metabolism.

[0286] Hematopoietic stem cell transplantation, also known as bone marrow transplantation, involves administering healthy hematopoietic stem cells from a donor to a patient with depleted or dysfunctional bone marrow. The HSC donor can be either the patient themselves (autologous HSCT) or a selected donor (allogeneic HSCT). This procedure increases bone marrow function, resulting in either the destruction of malignant tumor cells or the generation of functional cells that can replace dysfunctional cells. HSCT requires some degree of bone marrow (BM) ablation, known as conditioning, a preparative regimen administered to patients undergoing HSCT prior to donor cell infusion to induce immunosuppression (to ensure engraftment and limit rejection and GvHD), eradicate hematologic malignancies, and create space in the BM niche for new donor cells to engraft. This conditioning regimen may include radiation therapy, irradiation, chemotherapy, radiomimetic chemotherapy, serum therapy, monoclonal antibodies, targeted therapy, and combinations thereof.

[0287] Allogeneic HSCT uses donor cells obtained from a healthy donor with an acceptable human leukocyte antigen (HLA) match for the patient. The stem cell donor can be related to the patient, as in an HLA-matched sibling donor, which remains the preferred donor to date, or a haploidentical donor who shares half of the HLA with the patient, or can be an unrelated volunteer. Allogeneic HSCT has typically been restricted to younger patients in good performance status due to the increased risk of regimen-related toxicity and graft-versus-host disease (GVHD) associated with advanced age. Younger patients also have a greater chance of having a matched sibling donor available.

[0288] Hematologic malignancies are generally diseases of older adults, 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 normal mechanisms of self-renewal, proliferation, and differentiation. Allogeneic HSCT is frequently performed 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 with standard consolidation chemotherapy. Importantly, older patients may be unable to tolerate high-dose standard consolidation chemotherapy, and therefore, HSCT with reduced conditioning may be preferable. However, allogeneic HSCT is more likely to have serious complications, including an increased risk of mortality. Furthermore, older patients typically do not have available HLA-matched sibling donors, necessitating the use of haploidentical donors for transplantation, which increases the risk of post-transplant GVHD. Both reduced conditioning and the use of haplodonors decrease the success rate of HSCT, and therefore, therapies that increase the efficacy rate of HSCT may allow elderly patients to successfully undergo consolidation treatment and benefit from a reduced risk of leukemia relapse. Furthermore, allogeneic HSCT is also the only curative therapy for other malignant disorders, such as progressive myelodysplastic syndromes (MDS).

[0289] Hemoglobinopathies are a group of genetic disorders caused by alterations in the genes encoding hemoglobin (Hb) and therefore primarily affecting red blood cells. Within this group, sickle cell disease (SCD) and β-thalassemia major (βThal) are the most common disorders, caused by abnormalities in Hb structure or Hb production, respectively. Allogeneic HSCT remains the only established curative treatment for SCD and β-Thal, but significant barriers still hinder its use, particularly in adults. The highest efficacy and safety of this process are achieved only when an HLA-matched sibling donor is available, which is the case in fewer than 20% of patients, and transplants are performed at a young age. The use of haploidentical donors, available in more than 80% of patients, could overcome this limitation in donor availability, but haploidentical donors have not yet been used outside of clinical trial settings due to lower efficacy and safety. Furthermore, conditioning regimen-related toxicities limit access to HSCT, especially for adult patients, who often already present with comorbidities from disease onset. However, as the intensity of conditioning decreases, the effectiveness of the transplant decreases.

[0290] Therefore, if increased efficacy rates of allogeneic HSCT for both malignant and non-malignant indications could be achieved, this would allow for safer use of haplodonors and reduced intensity conditioning, particularly benefiting adult patients.

[0291] In autologous HSCT, bone marrow products are collected from the patient and reinfused after purification procedures. Advantages include the absence of GVHD and better engraftment. However, bone marrow products may contain abnormal cells that can cause relapse in the case of malignant tumors. Therefore, allogeneic transplants are preferred for many malignant indications, such as leukemia and MDS, because the donor-infused cells are free of contaminating tumor cells and therefore generally have a lower risk of disease recurrence. Autologous transplants are more frequently used in solid tumors, lymphomas, and myelomas, but allogeneic transplants combined with reduced conditioning are being evaluated for these indications as a means of inducing a graft-versus-malignancy effect. Autologous HSCT is also used in nonmalignant indications, such as autoimmune diseases and hemoglobinopathies when gene therapy is used. One of the major limitations of gene therapy for treating hemoglobinopathies is the reduced engraftment of ex vivo engineered cells, and increasing engraftment rates after transplantation would be of great benefit.

[0292] The therapeutic success of HSCT critically depends on the homing and engraftment of sufficient numbers of donor hematopoietic stem cells (HSCs) into the patient's bone marrow (BM), followed by the expansion and differentiation of these cells, allowing for the reconstitution and maintenance of hematopoiesis in the patient. However, preclinical studies suggest that only 1%–10% of intravenously infused HSCs reach the patient's BM, while the majority of cells are entrenched by other organs (van Hennik et al., Blood. 1999;94(9):3055-61). Therefore, to improve the success rate of HSCT, it remains necessary to increase the proportion of transplanted HSCs that effectively home to and engraft the patient's BM.

[0293] According to certain embodiments, provided herein is a pharmaceutical composition as defined in the present disclosure, or as obtained by mixing the two compartments of the kit as defined in the present disclosure, for use in improving the homing, engraftment and long-term expansion and proliferation of hematopoietic stem cells by using intramedullary injection as a local injection and by using an implant in patients, particularly human patients, before, during or immediately after autologous or allogeneic hematopoietic stem cell transplantation (HSCT).

[0294] The hematopoietic stem cells may be selected from bone marrow cells, peripheral blood stem cells, and umbilical cord blood cell culture stem cells, including cultured stem cells after gene therapy.

[0295] The pharmaceutical compositions defined in the present disclosure may 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.

[0296] More specifically, the pharmaceutical compositions defined in the present disclosure may be useful for reducing graft failure, reducing the incidence of graft poor function (PGF), reducing the incidence of graft-versus-host disease (GvHD), enhancing overall survival and donor chimerism, and / or promoting hematopoietic recovery and the success of hematopoietic stem cell transplantation.

[0297] The 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 pharmaceutically acceptable salt thereof, particularly EPAG olamine, is an amount in the range of 10 mg to 800 mg, particularly 12 mg to 725 mg, administered intramedullarily 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.

[0298] As described 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 may be used to improve homing, engraftment, and long-term expansion and proliferation of hematopoietic stem cells in patients before, during, or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT), and / or in patients with severe regenerative disorders. Administered by intramedullary injection in the prevention and / or treatment of non-malignant blood disorders such as benign anemias and hemoglobinopathies, particularly sickle cell disease and beta-thalassemia, and / or in the prevention and / or treatment of malignant blood diseases, for example leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, particularly leukemia, and / or in the prevention and / or treatment of primary immunodeficiencies, autoimmune diseases or inborn errors of metabolism.

[0299] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to improving the homing of hematopoietic stem cells before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT).

[0300] In one embodiment, the composition suitable for intramedullary injection is dedicated to improving hematopoietic stem cell engraftment before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT).

[0301] In one embodiment, the composition suitable for intramedullary injection is dedicated to improving the long-term expansion and proliferation of hematopoietic stem cells before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT).

[0302] 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, particularly sickle cell disease and beta thalassemia.

[0303] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is intended for the prevention and / or treatment of hematological malignancies, e.g., leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, in particular leukemias.

[0304] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of severe aplastic anemia.

[0305] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of hemoglobinopathies.

[0306] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of sickle cell disease.

[0307] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of beta thalassemia.

[0308] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of myeloma and / or lymphoma.

[0309] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of leukemia, such as acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia.

[0310] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of Waldenstrom's macroglobulinemia.

[0311] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of myeloproliferative neoplasms.

[0312] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of myelodysplastic syndromes.

[0313] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of primary immunodeficiencies, autoimmune diseases or inborn errors of metabolism.

[0314] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of primary immunodeficiencies.

[0315] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of autoimmune diseases.

[0316] In one embodiment, the pharmaceutical composition suitable for intramedullary injection is dedicated to the prevention and / or treatment of inborn errors of metabolism.

[0317] According to another aspect, the present invention relates to 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), comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by intramedullary injection a pharmaceutical composition in the form of a suspension suitable for intramedullary injection, 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.

[0318] According to another aspect, the present invention relates to a method for reducing graft failure, reducing graft failure, reducing the incidence of graft poor function (PGF), reducing the incidence of graft-versus-host disease (GvHD), enhancing overall survival and donor chimerism, and / or promoting hematopoietic recovery and the success rate of hematopoietic stem cell transplantation, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by intramedullary injection, a pharmaceutical composition in the form of a suspension suitable for intramedullary injection and comprising 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.

[0319] 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 beta thalassemia, comprising at least administering by intramedullary injection to a patient in need thereof, in particular a human patient in need thereof, a pharmaceutical composition in the form of a suspension suitable for intramedullary injection and comprising 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.

[0320] In another aspect, the present invention relates to a method for the prevention and / or treatment of malignant hematological diseases selected from myeloma, lymphoma, in particular leukemias such as acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, myeloma, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, myelodysplastic syndromes, which method comprises administering at least a pharmaceutical composition in the form of a suspension suitable for intramedullary injection and comprising 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, by intramedullary injection to a patient in need thereof, in particular a human patient in need thereof.

[0321] According to another aspect, the present invention relates to a method for preventing and / or treating a primary immunodeficiency, an autoimmune disease or an inborn error of metabolism, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by intrathecal injection a pharmaceutical composition in the form of a suspension suitable for intrathecal injection, 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.

[0322] According to another aspect, the present invention relates to 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), comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intramedullary injection, for controlled release, or for controlled and immediate release, 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.

[0323] According to another aspect, the present invention relates to a method for reducing graft failure, for reducing the incidence of graft poor function (PGF), for reducing the incidence of graft versus host disease (GvHD), for enhancing overall survival and donor chimerism, and / or for promoting hematopoietic recovery and the success rate of hematopoietic stem cell transplantation, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intramedullary injection, for controlled release, or for controlled and immediate release, 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.

[0324] According to another aspect, the present invention relates to a method 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, comprising at least administering to a patient in need thereof, in particular a human patient in need thereof, by local injection, in particular by intramedullary injection, for controlled release, or for controlled and immediate release, 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.

[0325] According to another aspect, the present invention relates to a method for the prevention and / or treatment of hematological malignancies, for example leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, in particular leukemia, comprising at least administering by local injection, in particular intramedullary injection, for controlled release, or for controlled and immediate release, 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, to a patient in need thereof, in particular a human patient in need thereof.

[0326] According to another aspect, the present invention relates to a method for the prevention and / or treatment of a primary immunodeficiency, an autoimmune disease or an inborn error of metabolism, comprising at least administering by local injection, in particular by intramedullary injection, for controlled release or for controlled and immediate release, 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 to a patient in need thereof, in particular a human patient in need thereof.

[0327] Further provided herein are methods for improving 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 methods for reducing graft dysfunction, reducing the incidence of graft failure (PGF), reducing the incidence of graft-versus-host disease (GvHD), enhancing overall survival and donor chimerism, and / or methods for promoting hematopoietic recovery and the success rate of hematopoietic stem cell transplantation, and / or methods for treating severe aplastic anemia and hemoglobinopathies, particularly sickle cell necrosis factor (SCF), and / or methods for treating hematopoietic stem cell transplantation in patients with severe aplastic anemia and hemoglobinopathies, particularly sickle cell necrosis factor (SCF).

[0010] In accordance with the present invention, there is provided a method for preventing and / or treating non-malignant hematological disorders such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, in particular leukemia, and / or a method for preventing and / or treating primary immune deficiencies, autoimmune diseases or inborn errors of metabolism, comprising at least: - preparing and / or providing a pharmaceutical composition in the form of a sterile and injectable suspension by mixing the formulation in the form of a powder as described herein with an aqueous injection vehicle and then with an aqueous injection vehicle, the pharmaceutical composition optionally comprising an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, said excipients being able to be present in the aqueous injection vehicle or in the powder, and - Intramedullary injection of said pharmaceutical composition into the bone marrow of a patient in need thereof (the volume injected being adapted to the injection site).

[0328] Further provided herein are methods for improving 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 methods for reducing graft dysfunction, reducing the incidence of graft failure (PGF), reducing the incidence of graft-versus-host disease (GvHD), enhancing overall survival and donor chimerism, and / or methods for promoting hematopoietic recovery and the success rate of hematopoietic stem cell transplantation, and / or methods for treating severe aplastic anemia and hemoglobinopathies, particularly sickle cell necrosis factor (SCF), and / or methods for treating hematopoietic stem cell transplantation in patients with severe aplastic anemia and hemoglobinopathies, particularly sickle cell necrosis factor (SCF).

[0010] In accordance with the present invention, there is provided a method for preventing and / or treating non-malignant hematological disorders such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, in particular leukemia, and / or a method for preventing and / or treating primary immune deficiencies, autoimmune diseases or inborn errors of metabolism, comprising at least: - preparing and / or providing a pharmaceutical composition in the form of a sterile and injectable solid implant, semi-solid implant, microporous implant or in situ forming depot, which pharmaceutical composition optionally comprises an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof, which excipient may be present in an aqueous injection vehicle or a powder, and - Intramedullary implantation of said pharmaceutical composition into the bone marrow of a patient in need thereof (the volume to be implanted is adapted to the implantation site).

[0329] Additional Active Ingredients Depending on the particular condition, or disease, being treated, additional therapeutic agents that are normally administered to treat that condition may be administered in combination with eltrombopag, or a derivative or conjugate, or pharmaceutically acceptable salt thereof.

[0330] In some embodiments, the present invention provides pharmaceutical compositions comprising eltrombopag or a derivative or conjugate thereof, or a pharmaceutically acceptable salt thereof, according to the present invention, further comprising at least one additional therapeutic agent. Suitable additional active ingredients are described in more detail below.

[0331] In certain embodiments, the patient is treated simultaneously, separately or sequentially with at least one active ingredient to prevent graft-versus-host disease, wherein the at least one active ingredient is selected from cyclosporine A, methotrexate, tacrolimus, mycophenolate mofetil, antithymocyte globulin serum, cyclophosphamide, and abatacept.

[0332] In certain embodiments, the patient is simultaneously, separately, or sequentially treated 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 thrombopoietin (TPO) receptor agonist can be selected from thrombopoietin, romiplostim, avatrombopag, and hetrombopag.

[0333] Additional active ingredients suitable for the present disclosure can be administered simultaneously, separately or sequentially via any route of administration, ie, via oral or parenteral routes, for example.

[0334] The additional active ingredients may be formulated under immediate and / or controlled release dosage forms. In one embodiment, the pharmaceutical compositions for use according to the present invention for improving stem cell homing and therapeutic methods for treating related diseases may be further characterized by the fact that the patient to whom the pharmaceutical composition is administered may be treated simultaneously, separately or sequentially with the following active ingredients: cyclosporine A, methotrexate, tacrolimus, mycophenolate mofetil, antithymocyte globulin serum, cyclophosphamide, abatacept, thrombopoietin, romiplostim, abatrombopag, and hetrombopag, and a thrombopoietin receptor agonist selected from the group consisting of thrombopoietin receptor agonists, thrombopoietin receptor agonists, and mixtures thereof.

[0335] Accordingly, there is provided a controlled release pharmaceutical composition for the use defined in the present disclosure, which is for the treatment of severe aplastic anemia and abnormal hemoglobin in patients who are being treated simultaneously, separately or sequentially with at least one active ingredient selected from cyclosporine A, methotrexate, tacrolimus, mycophenolate mofetil, antithymocyte globulin serum, cyclophosphamide, abatacept, thrombopoietin, romiplostim, abatrombopag and hetrombopag thrombopoietin receptor agonists, and mixtures thereof. The present invention is for use in the prevention and / or treatment of non-malignant blood disorders such as erythroglobinopathies, in particular sickle cell disease and beta-thalassemia, and / or in the prevention and / or treatment of malignant blood diseases, for example leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms, and myelodysplastic syndromes, in particular leukemia, and / or in the prevention and / or treatment of primary immunodeficiencies, autoimmune diseases or inborn errors of metabolism.

[0336] Additional treatment Depending on the patient to be treated, the patient may be treated with a conditioning regimen selected from radiation therapy, irradiation, chemotherapy, radiomimetic chemotherapy, serum therapy, monoclonal antibodies, targeted therapy, and combinations thereof before, during, or after intrathecal administration of the pharmaceutical composition defined in the present disclosure or obtained by mixing the two compartments of the kit defined in the present disclosure.

[0337] In certain embodiments, the radiomimetic chemotherapy can be selected from the group consisting of ozone, peroxide, alkylating agents, platinum-based agents, cytotoxic antibiotics, and blistering chemotherapy, and preferably the radiomimetic chemotherapy is cyclophosphamide, busulfan, fludarabine, melphalan, thiotepa, cytarabine and clofarabine, carmustine, etoposide, cytarabine and melphalan, rituximab, ifosfamide, etoposide, or a platinum-based agent selected from the group consisting of cisplatin, carboplatin, oxaliplatin, and nedaplatin.

[0338] Throughout the description, including the claims, the expression "comprising a" should be understood as synonymous with "comprising at least one," unless otherwise specified.

[0339] The expressions "between... and..." and "in the range of... to..." should be understood to mean that the limits are inclusive, unless otherwise specified.

[0340] The following examples and figures are presented as non-limiting illustrations of the present invention. [Example]

[0341] Analysis method Characterization of eltrombopag-olamine controlled-release microparticles - The particle size of Examples 1 and 5 was determined using a Malvern Mastersizer MS3000 laser diffraction particle size analyzer. 1 mg / ml of microparticles in deionized water preparation was introduced into the instrument and measurements were performed in triplicate. The results shown in the examples are the average of the three measurements.

[0342] The particle size in Examples 3 and 4 was determined using a multi-wavelength separation analyzer (LUMiReader from LUMGmbh). An ultrasonic bath was used to obtain preparations of 1-10 mg of microparticles per ml of deionized water, which were then introduced into the instrument. The results shown in the examples are the average of three measurements made at different sample heights during the same run.

[0343] The drug load of eltrombopag olamine microparticles in Examples 1, 2, and 5 was determined by first solubilizing 10 mg of drug-loaded polymer microparticles in 2.5 mL of an acetonitrile / water mixture at an 80 / 20 v / v ratio. 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 microparticle drug load.

[0344] HPLC analysis was performed using a silica-based reversed-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 v / v adjusted to pH 2]. The flow rate was set to 0.5 mL / min, the injection volume was set to 10 μL, and UV detection was set to 244 nm.

[0345] The drug loading of the eltrombopag olamine microparticles in Examples 3 and 4 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 microparticle drug loading.

[0346] 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% of a 6.3 g / L ammonium formate solution, previously adjusted to pH 3 with dilute hydrochloric acid. The flow rate was set at 1 mL / min, the injection volume was set at 10 μL, and UV detection was set at 230 nm.

[0347] For in vitro release studies from eltrombopag olamine microparticles in Examples 3 and 4, 75 mg aliquots of microparticles were dispersed in 50 mL of a solution containing 0.5% polysorbate 80 (Tween 80) in phosphate-buffered saline (10 mM), previously adjusted to pH 6.8 with dilute hydrochloric acid, using an ultrasonic bath. The dispersion was placed in a horizontal orbital shaker (incubator model 3033, commercially available from GFL) and maintained at 37°C. At different time intervals, 1.5 mL of 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.

[0348] HPLC analysis was performed on an Agilent HP1200 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 at 1 mL / min, the injection volume was set at 10 μL, and UV detection was set at 254 nm.

[0349] SDF-1 proteolysis analysis For the incubation of eltrombopag olamine with the activated human proteases in Examples 6 to 10, proMMP-8, proMMP-9, and proMMP-13 proteases were first activated. Human proMMP-8 and proMMP-13 (catalog numbers 908-MP and 511-MM-010, R&D Systems) 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) were separately activated by incubation with 1 mM p-aminophenylmercuric acetate at 37°C for 1 hour. Human proMMP-9 was expressed in Sf9 cells, purified by gelatin-Sepharose chromatography, and activated by incubation with the catalytic domain of MMP-3 (catalog number 444217, Merck Millipore). Specifically, 920 μg / ml of human proMMP-9 in the same buffer as used for MMP-8 activation was incubated with the catalytic domain of MMP-3 at a molar ratio of 1 / 100 (MMP-3 / MMP-9) for 2 hours at 37°C. The final concentration of MMP-9 was 10 μM, and the final concentration of MMP-3 was 0.1 μM. A concentrated eltrombopag olamine solution was prepared by solubilizing it in water at 10 mg / ml for 24 hours on a rotor at 4°C and then further diluting it to the required final concentration. Activated MMP-8, MMP-9, or MMP-13 was then incubated at a final concentration of 625 nM in eltrombopag olamine at different concentrations (100 μM, 50 μM, 10 μM, 1 μM, and 0.1 μM) in a calcium-free buffer containing 50 mM Tris, 150 mM NaCl, and 0.01% Tween 20 (pH 7.6) in a final volume of 3 μl using low-protein-binding tubes (Protein LoBind tubes, Eppendorf) at 37°C for 30 min.For the incubation of eltrombopag olamine with activated human DPPIV (also known as CD26) protease in Example 11, recombinant human activated DPPIV (R&D Systems 9168-SE) was incubated at a final concentration of 0.6–1.2 nM in different concentrations of eltrombopag olamine (100 μM, 50 μM, and 10 μM) in a buffer containing 25 mM Tris (pH 8) at 37°C for 30 min in a final volume of 25 μl.

[0350] Incubations of human SDF-1α (Cat. No. 300-28A, Peprotech) in a solution containing activated human protease and eltrombopag olamine were performed for 2 hours at 37°C in the same calcium-free buffer and tubes described in Examples 6–10, at a 1 / 5 MMP / SDF-1 molar ratio, a final SDF-1α concentration of 3.125 μM, and a final volume of 4 μl. A negative control without eltrombopag olamine was also 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 / 100–1 / 200 and a final volume of 26 μl. Positive controls for DPPIV inhibition by sitagliptin and a negative control without eltrombopag olamine were also prepared.

[0351] For the neutrophil degranulation (ND) incubation with eltrombopag olamine and SDF-1 in Example 12, neutrophils were first isolated from fresh blood from four healthy donors via density gradient centrifugation as described (De Buck M, Berghmans N, Portner N, Vanbrabant L, Cockx M, Struyf S, et al. Serum amyloid A1alpha 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 samples, neutrophils were suspended at a concentration of 107 cells / ml in calcium-free degranulation buffer (120mM NaCl, 20mM Tris / HCl pH 7.5). Degranulation was induced by incubating the neutrophils with n-formyl-methionyl-leucyl-phenylalanine (FMLF) (final concentration 0.5µM) for 20 minutes at 37°C. The supernatant was then collected after centrifugation. Equal amounts of 2 x 105 neutrophil degranulation samples were incubated with different concentrations (100µM and 50µM) of eltrombopag olamine in calcium-free buffer in a final volume of 21.1µl for 30 minutes at 37°C. SDF-1 incubation in solution was then performed by adding SDF-1α to a final concentration of 0.83µM and in a final volume of 22.6µl for 4 hours at 37°C. A negative control without eltrombopag olamine was prepared in the same manner.

[0352] SDF-1 proteolysis by MMPs and NDs in Examples 6, 7, 8, 9, 10, and 12 was analyzed by multiplex Western blot. All samples were diluted with 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 equivalent was loaded onto a Novex 16% Tricine gel (Cat. No. EC6695BOX, Invitrogen). Chameleon Duo Pre-stained Protein Ladder was used as a molecular weight indicator, using 6 μl per well. Proteins in the samples were separated by denaturing SDS-PAGE using Tricine running buffer (100 mM Tris, 100 mM Tricine, 0.1% SDS, pH 8.3) under a constant voltage of 120 V (±60 min). After electrophoresis, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot Turbo Transfer System (Biorad) with a Trans-Blot Turbo RTA Mini 0.45 μm Low-Fluorescence PVDF Transfer Kit (Cat. No. 1704274, Biorad). After blocking with Intercept (TBS, Tris-Buffered Saline) blocking buffer (Cat. No. 927-60001, Licor) for 60 minutes at room temperature, the membrane was incubated overnight at 4°C on a rotating shaker with a rabbit polyclonal primary antibody (cell Signaling 3740S, 1:1000) that recognizes all SDF-1 isoforms and a mouse monoclonal antibody (Millipore MABC184, 1:2000) that recognizes the N-terminal SDF-1 portion, which is typically cleaved by protease activity and causes inhibition of SDF-1 chemotactic function, in TBS blocking buffer containing 0.2% Tween 20.After three 10-minute washes with Tris-buffered saline containing Tween 20 (150 mM NaCl, 20 mM Tris, 0.1% Tween 20), the membranes were incubated with secondary antibodies donkey anti-rabbit 680RD (catalog no. 926-68073, Licor, 1:5000) and donkey anti-mouse 800CW (catalog no. 926-32212, Licor, 1:5000) diluted in TBS blocking buffer containing 0.2% Tween 20 and 0.02% SDS for 1 hour at room temperature on a rotating shaker. After three 10-minute washes with Tris-buffered saline containing Tween 20, the membranes were imaged using an Odyssey Fc imaging system (Licor). Quantification of protein bands was performed using Empiria Studio Software (Licor), which expresses the ratio of intact SDF-1 signal in relative fluorescence units (RFU) per total (all isoforms) SDF-1 RFU.

[0353] In Example 11, SDF-1 proteolysis by DPPIV was analyzed by traditional Western blot analysis. Samples were diluted with reducing loading buffer (ThermoFisher B0009) and boiled at 70°C for 10 minutes. Next, 18.75-150 ng equivalents of SDF-1 were loaded onto two BOLT 12% Bis-Tris gels (Invitrogen NW00125BOX). Proteins in the samples were then 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 iBlot2 system (Invitrogen IB21001) with the iBlot™ 2 Transfer Stacks kit (Invitrogen IB24002) for a total time of 6 minutes (1 minute at 20 V, 3 minutes 30 seconds at 23 V, and 1 minute 30 seconds at 25 V). Next, the two membranes were blocked, washed, and incubated with primary and secondary antibodies in an iBind Flex Western device (Invitrogen SLF2000) for 3–3.5 hours. The same primary antibodies used for SDF-1 proteolysis by MMPs were used at a 1:1000 dilution. The secondary antibodies, goat anti-rabbit IgG HRP (31460, ThermoFisher) and goat anti-mouse IgG HRP (31430, ThermoFisher), were diluted at 1:1000. The membranes were incubated with SuperSignal™ West Pico PLUS chemiluminescent substrate (34580, ThermoFisher) and imaged using an iBright imaging system (ThermoFisher). Protein band intensity was quantified (arbitrary units, AU) using ImageJ software. Results are expressed as band intensity normalized to that of a control SDF-1 sample without added protease, eltrombopag olamine, or sitagliptin. The results shown in the Examples are the average of eight independent experiments for eltrombopag olamine at 50 μM, five at 100 μM, and three at 10 μM. For statistical analysis, samples were compared using one-way ANOVA. Confidence intervals were fixed at 95% (p<0.05).Results were graphed as mean ± standard error of the mean (sem) using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

[0354] Chemotaxis of hematopoietic stem cells The chemotaxis of human hematopoietic stem cells in Example 13 was evaluated 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 olamine was incubated with 4 nM activated DPPIV in assay medium (Stemcell Technologies 09600) in a final volume of 600 μl for 30 min at 37°C. Recombinant human SDF-1α was then added to the medium at a DPPIV / SDF-1 molar ratio of 1 / 6 to a final SDF-1α concentration of 25 nM for an additional 30 min at 37°C. Subsequently, 105 human CD34+ hematopoietic stem and progenitor cells isolated from bone marrow (lonza 2M-101a) were plated in the upper transwell chamber in a final medium volume of 150 μl. 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 chemotaxis induction. After 4 hours at 37°C, the upper chamber containing non-migrating cells was carefully removed, and the number of migrating cells in the bottom chamber was quantified. For cell quantification, nuclei were stained with Hoescht staining (ThermoFisher R37605), and photographs of the entire microplate well were taken using an EVOS M7000 microscope with an automated XY platform (ThermoFisher). Automated cell counting was performed using EVOS analysis software version 1.5.1479.304 (automatic counting function), with batch analysis counting used to create an optimization algorithm. Results shown in the examples are the average of three independent experiments. For statistical analysis, samples were compared using one-way ANOVA. Confidence intervals were fixed at 95% (p<0.05). Results were graphed as mean ± standard error of the mean (sem) using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

[0355] Example 1: Sustained-release eltrombopag olamine microparticles (S / O / W emulsion) Eltrombopag olamine loading into PLGA microparticles was performed using a solid-in-oil-in-water (S / O / W) emulsion and solvent evaporation process.

[0356] In this experiment, eltrombopag olamine (Hetero Drugs Limited, India) supplied as a powder with a mean particle size (D50) of 3 μm is used.

[0357] 4.16 g of PLGA 50:50 Resomer 503H and 0.21 g of lecithin (phospholipid 90H, Lipoid) were first solubilized in 14.3 g of methylene chloride under magnetic stirring for 1 h. 2 g of eltrombopag olamine powder was then dispersed in this organic solvent using a high-shear rotor / stator mixer (IKA T18 Ultra-Turrax) at 10,000 rpm for 1 min.

[0358] This S / O dispersion was then emulsified with 26.3 mL of 1 wt % aqueous PVA solution (Mowiol 4-88, Sigma-Aldrich) by mixing with an IKA T18 ultra-Turrax at 9500 rpm for 10 min.

[0359] The emulsion was then quickly poured into an extraction bath (3 L of 1% aqueous PVA solution) and stirred with an overhead stirrer at 300 rpm for 3 h.

[0360] The solid particles were then separated from the aqueous phase by centrifugation at 4000 rpm for 4 min, filtered through a 40 μm stainless steel sieve, and rinsed three times with 50 mL of distilled water.

[0361] The washed microparticles were stored in glass overnight and then dried under a vacuum of 5 mBar for 18 hours.

[0362] The collected microparticles were finally stored at 5°C.

[0363] [Table 3]

[0364] In this example, eltrombopag olamine-loaded PLGA microparticles were prepared with an average particle size of 6.9 μm and a drug loading content of 26 wt%.

[0365] Example 2: Pharmaceutical Composition - Preparation of an Injectable Suspension An 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 (MultiControl 2, CertoClav) at 121°C for 15 minutes, and 5 mL aliquots of the solution were aseptically transferred to 10 mL vials under a laminar flow bench.

[0366] A 470 mg aliquot of microparticles prepared according to Example 1 was dispersed into each vial using a vortex mixer.

[0367] This pharmaceutical composition is intended for local injection as defined in this disclosure, excluding intrapulmonary and intrapleural injection, to treat diseases as defined in this disclosure, excluding COPD and pulmonary fibrosis.

[0368] In one example, a 5 mL volume containing a 123 mg dose of eltrombopag olamine can be injected into the posterior iliac crest using an 18G needle with a trocar.

[0369] Example 3: Controlled-release eltrombopag olamine microparticles and pharmaceutical compositions containing the same Eltrombopag olamine microparticles were prepared using autoclaved containers under laminar flow. PLGA microparticles were loaded with eltrombopag olamine using a solid-in-oil-in-water (S / O / W) emulsion and solvent evaporation process.

[0370] In this experiment, we used eltrombopag olamine (Hetero Drugs Limited, India) supplied as a powder with an average particle size (D50) of 2 μm. Eltrombopag olamine 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 with magnetic stirring until completely solubilized. The solution was then cooled before further use.

[0371] In a separate container, 2.9 g of PLGA 50:50 Resomer 503H [poly(lactic-co-glycolic acid) copolymer 50:50; Mw = 24–38 kg / mol; (Evonik Industries AG, Essen, Germany)] was first solubilized in 14.3 g of methylene chloride (Merck) under magnetic stirring for 30 min. Then, 0.73 g of eltrombopag olamine powder was dispersed in the mixture under magnetic stirring for 15 min to form a solid dispersion in oil.

[0372] 57 g of the previously prepared 1 wt % PVA stock solution was then added to the solid-in-oil dispersion under high shear emulsification, which was carried out using an IKA T25 Ultra-Turrax rotor-stator mixer equipped with an S25N 10G head at 6,600 RPM for 10 minutes.

[0373] This emulsion was then slowly poured into a hardening bath containing 2943 g of the previously prepared 1 wt % PVA stock solution.

[0374] Stirring was maintained for 3 hours using a double propeller stirrer set at 300 RPM until the methylene chloride had evaporated.

[0375] The formed microparticles were centrifuged at 8000 RPM for 3 minutes using a swinging bucket GT2R centrifuge to remove the majority 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 through a 10-16 μm fritted glass and washed three times with 50 ml of water.

[0376] The washed microparticles were then dried under vacuum at 5 mBar and 25° for 17 hours. The collected microparticles were then stored at 5°C.

[0377] Analytical studies, including in vitro drug release profiles, were performed as described in the "Analytical Methods" section above and are shown in Table 2 below.

[0378] [Table 4]

[0379] In this example, controlled-release eltrombopag olamine microparticles were prepared with a mean particle size of 53.4 μm, a drug loading content of 13.5 wt%, and a duration required to release 80% of the initial drug load (i.e., t80) of approximately 24 hours.

[0380] An aqueous injection vehicle consisting of 1.4% low-viscosity sodium carboxymethylcellulose (Aqualon CMC 7LF PH BET, Ashland), 0.1% polysorbate 20 (Emprov Essential, Merck), 0.13% disodium hydrogen phosphate dihydrate (Roth), 0.1% citric acid (Roth), and 0.65% sodium chloride (Roth) was prepared under laminar flow using pyrogen-free excipients. 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 of the solution were aseptically transferred to 10 mL vials under laminar flow.

[0381] The pharmaceutical composition is intended for local injection as defined herein, excluding intrapulmonary / intrapleural injection, intraocular / intravitreal / subtenon injection, and intraperitoneal / subgingival injection, to treat diseases as defined herein, excluding periodontal disease, eye disease, COPD, and pulmonary fibrosis.

[0382] Immediately prior to in vivo injection, a 530 mg aliquot of microparticles prepared according to this example was dispersed into each vial using 30 seconds of sonication until a homogeneous dispersion was obtained. The resulting eltrombopag olamine-loaded PLGA microparticle suspension provided a dose of 71.6 mg of eltrombopag olamine per 5 mL.

[0383] Example 4: Controlled-release eltrombopag olamine microparticles and pharmaceutical compositions containing the same Eltrombopag olamine microparticles were prepared using autoclaved containers under laminar flow. PLGA microparticles were loaded with eltrombopag olamine using a solid-in-oil-in-water (S / O / W) emulsion and solvent evaporation process.

[0384] In this experiment, we used eltrombopag olamine (Hetero Drugs Limited, India) supplied as a powder with an average particle size (D50) of 2 μm. Eltrombopag olamine 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 with magnetic stirring until completely solubilized. The solution was then cooled before further use.

[0385] In a separate container, 2.9 g of PLGA 50:50 Resomer 504H [poly(lactic-co-glycolic acid) copolymer 50:50; Mw = 38–54 kg / mol; (Evonik Industries AG, Essen, Germany)] was first solubilized in 14.3 g of methylene chloride (Merck) under magnetic stirring for 30 min. Then, 0.73 g of eltrombopag olamine powder was dispersed in the mixture under magnetic stirring for 15 min to form a solid dispersion in oil.

[0386] 57 g of the previously prepared 1 wt % PVA stock solution was then added to the solid-in-oil dispersion under high shear emulsification, which was carried out using an IKA T25 Ultra-Turrax rotor-stator mixer equipped with an S25N 10G head at 6,600 RPM for 10 minutes.

[0387] This emulsion was then slowly poured into a hardening bath containing 2943 g of the previously prepared 1 wt % PVA stock solution.

[0388] Stirring was maintained for 3 hours using a double propeller stirrer set at 300 RPM until the methylene chloride had evaporated.

[0389] The formed microparticles were centrifuged at 8000 RPM for 3 minutes using a swinging bucket GT2R centrifuge to remove the majority 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 through a 10-16 μm fritted glass and washed three times with 50 ml of water.

[0390] The washed microparticles were then dried under vacuum at 5 mBar and 25° for 17 hours. The collected microparticles were then stored at 5°C.

[0391] Analytical studies, including in vitro drug release profiles, were performed as described in the "Analytical Methods" section above and are shown in Table 2 below.

[0392] [Table 5]

[0393] In this example, controlled-release eltrombopag olamine microparticles were prepared with a mean particle size of 47.3 μm, a drug loading content of 14.3 wt%, and a duration required to release 80% of the initial drug load (i.e., t80) of more than 1 week (i.e., more than 168 hours).

[0394] An aqueous injection vehicle consisting of 1.4% low-viscosity sodium carboxymethylcellulose (Aqualon CMC 7LF PH BET, Ashland), 0.1% polysorbate 20 (Emprov Essential, Merck), 0.13% disodium hydrogen phosphate dihydrate (Roth), 0.1% citric acid (Roth), and 0.65% sodium chloride (Roth) was prepared under laminar flow using pyrogen-free excipients. 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 of the solution were aseptically transferred to 10 mL vials under laminar flow.

[0395] The pharmaceutical composition is intended for intramedullary, intramyocardial, intrapericardial, intraepicardial, intraendocardial, intrahepatic, or intrarenal injection as defined herein for treating the corresponding associated diseases as defined herein.

[0396] Immediately prior to in vivo injection, a 530 mg aliquot of microparticles prepared according to this example was dispersed into each vial using 30 seconds of sonication until a homogeneous dispersion was obtained. The resulting eltrombopag olamine-loaded PLGA microparticle suspension provided a dose of 75.8 mg of eltrombopag olamine per 5 mL.

[0397] Example 5: Sustained-Release Eltrombopag Olamine Microparticles (W / O / W Emulsion) Eltrombopag olamine loading into PLGA microparticles was performed using a water-in-oil-in-water (W1 / O / W2) double emulsion and solvent evaporation process.

[0398] The internal aqueous phase W1 was obtained by solubilizing 20 mg of eltrombopag olamine (MedChemExpress, USA) and 50 mg of 2-hydroxypropyl-β-cyclodextrin (Sigma Aldrich) in 1 mL of water.

[0399] The organic solvent phase was prepared by solubilizing 500 mg of PLGA Resomer RG 503H (Evonik Industries AG, Essen, Germany) and 25 mg of lecithin (phospholipid 90H, Lipoid) in 2.5 mL of dichloromethane.

[0400] For the preparation of the W1 / O primary emulsion, the W1 phase and oil phase were emulsified under high shear rotor / stator mixing at 24000 rpm for 10 min.

[0401] W1 / O / W2 double emulsion was then obtained by mixing W1 / O with 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.

[0402] The double emulsion was then poured into an extraction bath (0.5 L of 1% aqueous PVA solution) and stirred with an overhead stirrer at 300 rpm for 3 h.

[0403] 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.

[0404] [Table 6]

[0405] Example 6: SDF-1 stabilization by eltrombopag-olamine-mediated MMP-9 protease inhibition Activated human MMP-9 was incubated with different concentrations of eltrombopag olamine (50 μM, 10 μM, 1 μM, and 0.1 μM) for 0.5 h at 37°C. Subsequently, human SDF-1α was added to the previously prepared solution at a 1 / 5 MMP / SDF-1α molar ratio for 2 h at 37°C. SDF-1α proteolysis was then analyzed by multiplex Western blot by detecting total and protease-inactivated SDF-1α.

[0406] [Table 7]

[0407] The results clearly demonstrated that 50 μM eltrombopag olamine could strongly reduce the proteolytic effect of MMP-9 on SDF-1α (72% of intact SDF-1α was recovered in the presence of MMP-9 in the presence of 50 μM eltrombopag olamine, compared with only 20% in the absence of eltrombopag olamine; Figures 1A and 1B and Table 5).

[0408] Example 7: SDF-1 stabilization by eltrombopag-olamine-mediated MMP-9 protease inhibition Activated human MMP-9 was incubated with different concentrations of eltrombopag olamine (100 μM, 50 μM, 10 μM, and 1 μM) for 0.5 h at 37°C. Subsequently, human SDF-1α was added to the previously prepared solution at a 1 / 5 MMP / SDF-1α molar ratio for 1.5 h at 37°C. SDF-1α proteolysis was then analyzed by multiplex Western blot by detecting total and protease-inactivated SDF-1α.

[0409] [Table 8]

[0410] The results clearly demonstrated that 50 μM and 100 μM eltrombopag olamine could strongly reduce the proteolytic effect of MMP-9 on SDF-1α (73% and 42% of intact SDF-1α were recovered in the presence of MMP-9 at 100 μM and 50 μM eltrombopag olamine, respectively, compared with only 1.9% in the absence of eltrombopag olamine; Figures 2A and 2B and Table 6).

[0411] Example 8: SDF-1 stabilization by eltrombopag-olamine-mediated MMP-8 protease inhibition Activated human MMP-8 was incubated with different concentrations of eltrombopag olamine (50 μM, 10 μM, 1 μM, and 0.1 μM) for 0.5 h at 37°C. Subsequently, human SDF-1α was added to the previously prepared solution at a 1 / 5 MMP / SDF-1α molar ratio for 2 h at 37°C. SDF-1α proteolysis was then analyzed by multiplex Western blot by detecting total and protease-inactivated SDF-1α.

[0412] [Table 9]

[0413] The results clearly demonstrated that 50 μM eltrombopag olamine can reduce the proteolytic effect of MMP-8 on SDF-1α (29% of intact SDF-1α was recovered in the presence of MMP-8 in the presence of 50 μM eltrombopag olamine, compared with only 2.9% in the absence of eltrombopag olamine; Figures 3A and 3B and Table 7).

[0414] Example 9: SDF-1 stabilization by eltrombopag-olamine-mediated MMP-8 protease inhibition Activated human MMP-8 was incubated with different concentrations of eltrombopag olamine (100 μM, 50 μM, 10 μM, and 1 μM) for 0.5 h at 37°C. Subsequently, human SDF-1α was added to the previously prepared solution at a 1 / 5 MMP / SDF-1α molar ratio for 2 h at 37°C. SDF-1α proteolysis was then analyzed by multiplex Western blot by detecting total and protease-inactivated SDF-1α.

[0415] [Table 10]

[0416] The results clearly demonstrated that 50 μM and 100 μM eltrombopag olamine could reduce the proteolytic effect of MMP-8 on SDF-1α (45% and 43% of intact SDF-1α were recovered in the presence of MMP-8 at 100 μM and 50 μM eltrombopag olamine, respectively, compared with only 16% in the absence of eltrombopag olamine; Figures 4A and 4B and Table 8).

[0417] Example 10: SDF-1 stabilization by eltrombopag-olamine-mediated MMP-13 protease inhibition Activated human MMP-13 was incubated with different concentrations of eltrombopag olamine (100 μM, 50 μM, 10 μM, and 1 μM) for 0.5 h at 37°C. Subsequently, human SDF-1α was added to the previously prepared solution at a 1 / 5 MMP / SDF-1α molar ratio for 1.5 h at 37°C. SDF-1α proteolysis was then analyzed by multiplex Western blot by detecting total and protease-inactivated SDF-1α.

[0418] [Table 11]

[0419] The results clearly demonstrated that 50 μM and 100 μM eltrombopag olamine could strongly reduce the proteolytic effect of MMP-13 on SDF-1α (in the presence of MMP-13 at 100 μM and 50 μM eltrombopag olamine, 19% and 12%, respectively, of intact SDF-1α were recovered, compared with only 3.2% in the absence of eltrombopag olamine; Figures 5A and 5B and Table 9).

[0420] Example 11: SDF-1 stabilization by eltrombopag-olamine-mediated CD26 / DPPIV inhibition Activated human DPPIV was incubated with different concentrations of eltrombopag olamine (100 μM, 50 μM, and 10 μM) for 0.5 h at 37°C. Subsequently, human SDF-1α was added to the previously prepared solution at DPPIV / SDF-1α molar ratios of 1 / 100 to 1 / 200 for 1 h at 37°C. SDF-1α proteolysis was then analyzed by Western blot by detecting total and protease-inactivated SDF-1α.

[0421] [Table 12]

[0422] The results clearly demonstrated that 50 μM and 100 μM eltrombopag olamine could strongly reduce the proteolytic effect of DPPIV on SDF-1α (100% and 92% of intact SDF-1α were recovered in the presence of DPPIV at 100 μM and 50 μM eltrombopag olamine, respectively, compared with only 61% in the absence of eltrombopag olamine; Figures 6A, 6B, and 7 and Table 10). Sitagliptin, a well-known potent DPPIV inhibitor with a calculated half-maximal inhibitory concentration (IC50) >105-fold lower than eltrombopag olamine, recovered only 82% of intact SDF-1α when used at 1 μM, indicating that it is less efficient than eltrombopag olamine in stabilizing SDF-1α.

[0423] Example 12: SDF-1 stabilization by eltrombopag-olamine-mediated protease inhibition in degranulated neutrophils Neutrophil degranulation (ND) samples obtained from healthy donors were incubated with different concentrations of eltrombopag olamine (100 μM and 50 μM) for 0.5 h at 37°C. Human SDF-1α was then added to the prepared solution at a final concentration of 0.83 μM for 4 h at 37°C. SDF-1α proteolysis was then analyzed by multiplex Western blot analysis, detecting total SDF-1α and N-terminal protease-inactivated SDF-1α.

[0424] [Table 13]

[0425] The results showed that 100 μM and 50 μM eltrombopag olamine could strongly reduce the proteolytic effect of neutrophil degranulation products on SDF-1α (ND: 100% and 80% of N-terminally intact SDF-1α were recovered in the presence of 100 μM and 50 μM eltrombopag olamine, respectively, compared with only 65% ​​in the absence of eltrombopag olamine; Figures 8A and 8B and Table 11). Furthermore, 100 μM and 50 μM eltrombopag olamine also reduced the proteolytic activity of neutrophil degranulation products on total SDF-1α (90% and 80% of total SDF-1α were recovered in the presence of 100 μM and 50 μM eltrombopag olamine, respectively, compared with only 63% in the absence of eltrombopag olamine).

[0426] Example 13: Eltrombopag olamine-mediated increase in chemotaxis of human hematopoietic stem and progenitor cells toward SDF-1 in the presence of DPPIV protease Activated human DPPIV was incubated in HSC medium at different concentrations of eltrombopag olamine (50 μM and 25 μM) in the bottom chamber of a transwell assay at 37°C for 0.5 h. Human SDF-1α was then added at a DPPIV / SDF-1α molar ratio of 1 / 6 for 0.5 h at 37°C. 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) and allowed to migrate to the bottom chamber for 4 h. Cells that migrated to the bottom chamber were quantified by nuclear staining, image acquisition, and automated counting.

[0427] [Table 14]

[0428] The results showed that 25 μM and 50 μM eltrombopag olamine could increase the chemotaxis of hematopoietic stem and progenitor cells toward SDF-1α in the presence of DPPIV protease. Compared with the SDF-1 control sample without added DPPIV, 93% and 88% of migrating cells were detected in the presence of 25 μM and 50 μM eltrombopag olamine, respectively, compared with only 68% in the absence of eltrombopag olamine (Table 12 and Figure 9).

Claims

1. A controlled release pharmaceutical composition comprising at least a controlled release pharmaceutical dosage form containing 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 thereof, or a pharmaceutically acceptable salt thereof, the controlled release pharmaceutical composition being suitable for local injection thereof at an affected site.

2. 2. The controlled release pharmaceutical composition of 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 a derivative, conjugate, or pharmaceutically acceptable salt thereof 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. 3. The controlled release pharmaceutical composition of claim 1, wherein the composition is an extended release pharmaceutical composition comprising at least an 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.

4. 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 vessel, skin, dermis, eye, liver, kidney, gastrointestinal tract, peritoneum, lung, gum, joint, bone marrow, in particular sternum, tibia, femur, iliac crest, vertebrae, and mixtures thereof, such as the iliac crest, e.g. the posterior part of the iliac crest, and mixtures thereof.

5. 5. The controlled release pharmaceutical composition of any one of claims 1 to 4, wherein the local injection at the affected site is selected from intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, intrawound instillation, surgical site injection, 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.

6. 6. The controlled release pharmaceutical composition of 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 a pharmaceutically acceptable salt thereof.

7. 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 pharmaceutically acceptable salt thereof is in the form of an in-situ forming depot, 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. 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, the microparticles having an average particle size of 1 μm or more, such as 2 μm or more, in particular in the range of 1 μm to 200 μm, more particularly 2 μm to 150 μm, even more particularly 2 μm to 100 μm, for example 10 μm to 100 μm, or 10 μm to 80 μm.

9. The controlled release polymer matrix may be selected from the group consisting of poly(lactic-co-glycolic acid) copolymer, 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), poly(trimethylene carbonate), poly(dioxanone), poly(valerolactone), poly(α-hydroxy acid), poly(lactone), poly(amino acid), polyanhydride, poly(orthoester), poly(acetate), 9. The controlled release pharmaceutical composition according to claim 7 or 8, comprising at least one biocompatible and biodegradable copolymer or polymer selected from the group consisting of polyvinyl alcohol, polyurethane, polythioester, polyphosphoester, poly(ester-co-amide), poly(vinyl alcohol), PVA-g-PLGA, poly(ether ester) multiblock copolymer, polyvinylpyrrolidone, 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 a poly(lactic-co-glycolic acid) copolymer.

11. 11. The 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 more than 70% by weight, in particular more than 80% by weight, and even more particularly more than 90% by weight, relative to the total weight of the polymer matrix.

12. 12. The controlled release pharmaceutical composition according to any one of claims 7 to 11, wherein the drug loading content or percentage weight ratio between said 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 the total weight of said microparticles is in the range of 2% to 45%, particularly 5% to 40%, more particularly 10% to 35%.

13. 1. A controlled release pharmaceutical composition in the form of a powder 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, - said controlled release microparticles have an average particle size of 1 μm or more, such as 2 μm or more, in particular in the range of 1 μm to 200 μm, more particularly 2 μm to 150 μm, even more particularly 2 μm to 100 μm, such as 10 μm to 100 μm, or 10 μm to 80 μm; - said controlled release polymer matrix comprises at least poly(lactic-co-glycolic acid) copolymer; a controlled release pharmaceutical composition, wherein the drug loading content or percentage weight ratio between said 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 the total weight of said microparticles is in the range of 2% to 45%, in particular 5% to 40%, and more particularly 10% to 35%.

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

15. 15. A controlled release pharmaceutical composition according to claim 14, in particular in the form of a sterile injectable suspension, comprising a controlled release dosage form or a mixture of controlled release and immediate release dosage forms 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, in particular 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.

13. A controlled release pharmaceutical composition obtained by mixing microparticles comprising [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 as defined in any one of claims 1 to 12, with an aqueous injection vehicle, said pharmaceutical composition optionally comprising an excipient selected from the group consisting of a tonicity enhancing agent, a wetting agent, a viscosity enhancing agent, a density enhancing agent or a mixture thereof.

16. 16. The controlled release pharmaceutical composition of 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 the sterile injectable dosage form, particularly in an amount ranging from 2 mg to 63 mg equivalents of free acid per ml of suspension, particularly from 8 mg to 55 mg equivalents of free acid per ml, more particularly from 12 mg to 47 mg equivalents of free acid per ml.

17. Separate compartments contain (i) an aqueous injection vehicle and (ii) a 13. A kit or article of manufacture comprising 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 according to any one of claims 1 to 12, or a powder as defined in claim 13, wherein the pharmaceutical composition is optionally administered at the affected site.

1. A kit or article of manufacture comprising an excipient selected from the group consisting of 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 thereof, in particular for intramedullary injection, intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, wound instillation, surgical site injection, 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.

18. A controlled release pharmaceutical composition according to any one of claims 1 to 16 or obtained by mixing the two compartments of the kit according to claim 17 for single or multiple use, by local injection at the affected site, in particular for improving the homing, engraftment and long-term expansion and proliferation of hematopoietic stem cells in patients, particularly human patients, before, during or after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) by intramedullary injection, for preventing and / or treating non-malignant hematological disorders, for preventing and / or treating malignant hematological diseases, and / or for preventing and / or treating primary immunodeficiencies, autoimmune diseases or inborn errors of metabolism, and / or A controlled release pharmaceutical composition for preventing and / or treating degenerative diseases / injuries by intramyocardial injection, intrapericardial injection, intraepicardial injection, intraendocardial injection, intrawound instillation, surgical site injection, 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.

19. In the prevention and / or treatment of cardiovascular disorders / diseases such as myocardial infarction, atherosclerosis and ischemia-reperfusion injury; and / or in the prevention and / or treatment of diseases affecting the bones and joints, such as osteoporosis, rheumatoid arthritis and osteoarthritis; and / or in the prevention and / or treatment of periodontal disease; and / or in wound healing, including diabetic wound healing and corneal wound healing; and / or in the prevention and / or treatment of ocular diseases such as retinal ischemia, macular degeneration, diabetic retinopathy and glaucoma; and / or in the prevention and / or treatment of kidney disease; and / or in the prevention and / or treatment of liver diseases such as fibrosis, cirrhosis, non-alcoholic fatty liver disease and acute liver injury; and / or in the prevention and / or treatment of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis; and / or in the prevention and / or treatment of chronic obstructive pulmonary disease (COPD, also known as chronic bronchitis) and / or pulmonary fibrosis; and / or In 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 in the prevention and / or treatment of hematological malignancies, for example leukemias such as myeloma, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms and myelodysplastic syndromes, in particular leukemia; and / or 19. A controlled release pharmaceutical composition for use according to claim 18 in the prevention and / or treatment of primary immunodeficiencies, autoimmune diseases or inborn errors of metabolism.

20. 20. The controlled release pharmaceutical composition for use according to claims 18 to 19, wherein the local injection is by intramedullary injection and the implant is used in a patient, particularly a human patient, before, during or immediately after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) to improve homing, engraftment and long-term expansion and proliferation of hematopoietic stem cells.

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

22. 22. The controlled release pharmaceutical composition for use according to any one of claims 18 to 21, wherein said local injection is by intramedullary injection, and said composition is injected 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.

23. 23. The controlled release pharmaceutical composition according to any one of claims 18 to 22, wherein the local injection is by intramedullary injection and is for the purpose of reducing graft failure, reducing the incidence of poor graft function (PGF), reducing the incidence of graft-versus-host disease (GvHD), enhancing overall survival and donor chimerism, and / or promoting hematopoietic recovery and success of hematopoietic stem cell transplantation.

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

25. 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 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 a pharmaceutically acceptable salt thereof is between 6 hours and 3 months, in particular between 6 hours and 30 days, in particular between 1 day and 2 months. 15 days, more particularly 2 to 15 days, even more particularly 4 to 15 days, for example 4 to 30 days, preferably 15 days, 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 pharmaceutically acceptable salt thereof is present in a dosage of the free acid form in the range of 10 mg to 570 mg equivalent.

26. 26. A controlled release pharmaceutical composition for use according to any one of claims 18 to 25, wherein the local concentration of said 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 thereof or a pharmaceutically acceptable salt thereof is maintained at a constant level for a period of 6 hours to 3 months after said local injection, in particular for a period of 6 hours to 3 months after said local injection, in particular for a period of 6 hours to 3 months after said local injection, in particular for a period of 6 months ... hours to 3 months after said local injection, in particular for a period of 6 months to 3 months after said local injection, in particular for a period of 6 hours to 3 months after said local injection, in particular for a period of 6 hours to 3 months after said local injection, in particular for a period of 6 months to 3 months after said local injection, in particular for a period of 6 hours to 3 months after said local injection, in particular for a period of 6 hours to 3 months after said local injection, in particular for a period of 6 hours to 3 months after said local injection, in particular for a period of 6 hours to 3 months after said local injection, in particular for a period of 6 hours to 3 months after said local injection, in particular for a period of 1. The controlled release pharmaceutical composition according to claim 1, wherein the controlled release pharmaceutical composition is effective to maintain a concentration of more than 1 μM, particularly more than 5 μM, more particularly more 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 30 days, particularly between 1 day and 15 days, more particularly between 2 days and 15 days, even more particularly between 4 days and 15 days, for example between 4 days and 30 days, preferably for 15 days.

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