Compounds and compositions for use in stem cell transplantation

Abstract

The present invention relates to a matriptase-2 (MT2) inhibitor, a TMPRSS6 inhibitor, a ferroportin blocker, or a hepcidin enhancer. In particular, the present invention relates to the use of compounds capable of reducing at least one of the following: systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), and unstable plasma iron (LPI). This may occur by inhibiting the expression of a target gene, where the target gene may be a transmembrane protease, serine 6 (TMPRSS6). Furthermore, the present invention relates to compositions comprising the above compounds, and methods of using such compounds and / or compositions. Such use may include therapeutic use, for example, to reduce iron overload and prevent iron-related toxicity, before, during, or after chemotherapy pretreatment, stem cell transplantation, and engraftment.

Description

[Technical Field]

[0001] The present invention relates to the use of matriptase-2 (MT2) inhibitors, TMPRSS6 inhibitors, ferroportin blockers, or hepcidin enhancers. In particular, the present invention relates to compounds capable of reducing at least one of systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), and unstable plasma iron (LPI). This may occur by inhibiting the expression of a target gene, where the target gene may be a transmembrane protease, serine 6 (TMPRSS6). Furthermore, the present invention relates to compositions comprising the above compounds, and methods of using such compounds and / or compositions. Such use may include therapeutic use before, during, or after chemotherapy pretreatment, stem cell transplantation, and extracellular implantation, implantation, or transplantation-related therapies, such as reducing iron overload and preventing iron-related toxicity.

[0002] Sequence List This application includes the sequence listing submitted with this specification, which is incorporated herein by reference in its entirety. The above .xml copy, created on 9 April 2024, is named 008584930 and has a size of 20,207 bytes. [Background technology]

[0003] Stem cell transplantation (SCT), i.e., the transplantation of stem cells to patients in need of transplantation, is the only option for curative treatment of some patients with diseases, including but not limited to hematopoietic stem cell transplantation (HSCT) for the treatment of leukemia such as AML, or other blood disorders such as beta-thalassemia, sickle cell disease, and bone marrow failure. Certain other conditions resulting from chemotherapy or radiotherapy, such as myeloablatives, are also treatable with SCT. Similarly, high-dose therapy and autologous HSCT are treatment options for patients with selective hematological malignancies and non-hematological malignancies. Furthermore, gene therapy to correct defects in certain blood genetic disorders generally requires bone marrow ablation before engraftment of genetically modified autologous stem cells.

[0004] Myelodysplastic syndrome (MDS), formerly known as preleukemia due to its potential progression to leukemia, is a diverse group of hematological medical conditions characterized by ineffective production (or dysplasia) of blood cells in the bone marrow. Myelodysplastic syndrome is a total disorder of hematopoietic stem cells in the bone marrow. In MDS, hematopoiesis (blood production) is disordered and ineffective. The number and quality of hematopoietic cells are irreversibly reduced in MDS, further impairing blood production. Patients with MDS develop severe anemia and may require blood transfusions. In some cases, the disease worsens, and patients develop cytopenia (low blood cell count) caused by progressive bone marrow failure. Because MDS is a stem cell-associated condition, hematopoietic stem cell therapy (HSCT) offers the potential for curative treatment, especially in more severely affected patients.

[0005] Despite a better understanding of graft immunology, improvements in graft-versus-host disease (GVHD) prevention, and advancements in supportive care, many risks associated with such transplants remain, and morbidity and mortality associated with HSCT remain high (Evens et al., Bone Marrow Transplantation 2004, 34, pp. 561-571). Toxicity is primarily due to organ damage induced by the prepared regimen, neutropenia predisposing patients to bacterial or fungal infections, or cellular immunodeficiency that makes patients vulnerable to viral infections and other opportunistic infections.

[0006] Iron overload, primarily due to the need for transfusions and the production of ineffective red blood cells, is common during HSCT but often remains uncontrolled, potentially impacting graft outcomes and gene therapy protocols. Iron overload can contribute to post-transplant procedure-related toxicity and mortality (Evens et al., Bone Marrow Transplantation 2004, 34, pp. 561-571). Patients selected for HSCT frequently present with iron overload and elevated transferrin saturation due to a history of repeated transfusions. Pre-existing iron overload is a detrimental prognostic factor for patients undergoing HSCT (Wermke et al., 2012, Clin. Cancer Res, 18(23), Atilla et al., Turk J Hematol 2017;34:1-9). Indeed, elevated ferritin, transferrin saturation, hepatic iron, and non-transferrin-bound iron (NTBI) have been identified as adverse prognostic factors for post-transplant survival and complications in β-thalassemia, and similarly in sickle cell disease, MDS, and leukemia. Furthermore, recent studies have shown that pre-transplant conditioning of HSCs exacerbates the presence of circulating "free" non-transferrin-bound iron (NTBI) and unstable plasma iron (LPI), as well as the LPI reactive fraction (Naoum et al., Hematol. Oncol. Stem Cell Ther 2016 9, pp. 165-167). Importantly, NTBI positivity predicted poor overall survival in transplant patients with myelodysplastic syndrome (MDS) or AML (Wermke et al., Lancet Haematol. 2018 May;5(5):e201-e210 reference).

[0007] Current treatment options to prevent iron overload and reduce iron-borne toxicity before or during HSC transplantation and engraftment:

[0008] Venotomy Humans lack a specialized iron excretion mechanism, and iron loss occurs passively through shedding of skin and intestinal epithelium, as well as through urine. Under normal conditions, a loss of 1-2 mg / day of iron is balanced by the absorption of an equivalent amount from diet (Isidori et al., Transplant Cell Ther. 2021 May;27(5):371-379). Therefore, iron overload develops due to dysregulation of iron homeostasis resulting from repeated transfusions or reduced hepcidin levels or activity, as seen in iron-overload anemia or hereditary hemochromatosis (Camaschella et al., 2020 Vol. 105 No. 2(2020):February 2020). Venoustomy is considered the simplest approach to remove excess iron from the body. In HSCT recipients, venotomy may be an option to remove excess iron after successful engraftment of hematopoietic stem cells, but it is not an option during pre-treatment and engraftment, or while the patient still requires blood transfusions. In general, this approach is not usually applied due to the anemia commonly present in patients undergoing HSCT.

[0009] Chelation Iron chelation therapy is currently a pharmacological option for reducing iron overload in patients with iron overload (reviewed by Isidori et al., 2021 Transplant Cell Ther. 2021 May;27(5):371-379): Deferoxamine is not suitable in this situation due to its short half-life requiring long-term infusion and its siderophore activity that allows iron release to microorganisms. The oral chelator deferipron has a longer half-life but is also associated with the possibility of developing agranulocytosis. The latest generation chelator, deferasirox, has the advantage of a longer half-life and the ability to effectively eliminate NTBI / LPI. Several common adverse events associated with deferasirox therapy may overlap with acute HSCT-induced adverse events, and therefore its use may be limited in better managing late-onset post-transplant iron toxicity and improving long-term patient outcomes. Reference: Essmann S, Heestermans M, Dadkhah A, Janson D, Wolschke C, Ayuk F, Kroger NM, Langebrake C. Iron Chelation with Deferasirox Suppresses the Appearance of Labile Plasma Iron During Conditioning Chemotherapy Prior to Allogeneic Stem Cell Transplantation. Transplant Cell Ther. 2023 Jan;29(1):42.e1-42.e6. doi:10.1016 / j.jtct.2022.10.002. Epub 2022 Oct 12. PMID:36241148. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 2018 / 185240A1 [Patent Document 2] International Publication No. 2014190157A1 [Patent Document 3] International Publication No. 2016 / 085852 A1 [Patent Document 4] International Publication No. 2022 / 231999 A1 [Patent Document 5] International Publication No. 2016 / 161429 A1 [Patent Document 6] International Publication No. 2022 / 157185 A1 [Patent Document 7] International Publication No. 2017 / 068089 [Patent Document 8] International Publication No. 2017 / 068090 [Patent Document 9] International Publication No. 2018 / 192973 [Patent Document 10] International Publication No. 2022 / 229150 A1 [Patent Document 11] International Publication No. 2017 / 174657 [Patent Document 12] International Publication No. 2018 / 18524 [Patent Document 13] U.S. Patent Application Publication No. 2021 / 0017214 [Non-Patent Document]

[0011] [Non-Patent Document 1] Evens et al., Bone Marrow Transplantation 2004, 34, pp. 561 - 571 [Non-Patent Document 2] Wermke et al., 2012, Clin. Cancer Res, 18(23) [Non-Patent Document 3] Atilla et al., Turk J Hematol 2017; 34: 1 - 9 [Non-Patent Document 4] Naoum et al., Hematol. Oncol. Stem Cell Ther 2016 9, pp. 165 - 167 [Non-Patent Document 5] Wermke et al., Lancet Haematol. 2018 May;5(5):e201-e210 reference [Non-Patent Document 6] Isidori et al., Transplant Cell Ther. 2021 May;27(5):371-379 [Non-Patent Document 7] Camaschella et al., 2020 Vol. 105 No. 2 (2020): February 2020. [Non-Patent Document 8] Essmann S, Heestermans M, Dadkhah A, Janson D, Wolschke C, Ayuk F, Kroger NM, Langebrake C. Iron Chelation with Deferasirox Suppresses the Appearance of Labile Plasma Iron During Conditioning Chemotherapy Prior to Allogeneic Stem Cell Transplantation. Transplant Cell Ther. 2023 Jan;29(1):42.e1-42.e6. doi:10.1016 / j.jtct.2022.10.002. Epub 2022 Oct 12. PMID:36241148 [Non-Patent Document 9] McDonald et al., American Journal of Physiology, 2015, vol. 08 no. 7, pp. C539-C547. [Non-Patent Document 10] Ramsay et al., Haematologica (2009), 94(*6), pp. 84-849. [Non-Patent Document 11] Fire et al., 1998, Nature. 1998 Feb 19;391(6669):806-811 pp. [Non-Patent Document 12] Elbashir et al., 2001, Nature. 2001 May 24;411(6836):494-498 pp. [Non-Patent Document 13] Altamuraら, Hemasphere. 2019 Dec;3(6):e301. [Non-licensed Document 14] Vadolasら、Br. J. Hematology 2021 Jul;194(1):200~210 pages [Non-licensed Document 15] Gutschow, J. Med. Chem. 2010, 53, 15, pages 5523~5535 [Non-licensed Document 16] Marsault, European Journal of Medicinal Chemistry, Volume 129, 2017, pages 110~123 [Non-licensed Document 17] Stirnberg, Pharmaceuticals 2018, 11(2), 49 [Non-licensed Document 18] Prakash, Nucleic Acids Res. 2015, 43(6), pages 2993~3011 [Non-licensed Document 19] Haraszti, Nucleic Acids Res. 2017, 45(13), pages 7581~7592 [Non-licensed Document 20] Letsinger, Proc. Natl. Acad. Sci. USA, 1989, 86: 6553~6556 pages [Non-licensed Document 21] Manoharan, Biorg. Med. Chem. Let., 1994, 4:1053~1060 pages [Non-licensed Document 22] Manoharan, Ann. NY Acad. Sci., 1992, 660:306~309 pages [Non-licensed Document 23] Manoharan, Biorg. Med. Chem. Let., 1993, 3: 2765~2770 pages [Non-licensed Document 24] Oberhauser, Nucl. Acids Res., 1992, 20:533~538 pages [Non-Patent Document 25] Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118 [Non-Patent Document 26] Kabanov et al., FEBS Lett., 1990, 259: pp. 327-330. [Non-Patent Document 27] Svinarchuk et al., Biochimie, 1993, 75: pp. 49-54. [Non-Patent Document 28] Manoharan et al., Tetrahedron Lett., 1995, 36: pp. 3651-3654. [Non-Patent Document 29] Shea et al., Nucleo Acids Res., 1990, 18: pp. 3777-3783. [Non-Patent Document 30] Manoharan et al., Nucleosides & Nucleotides, 1995, 14: pp. 969-973. [Non-Patent Document 31] Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237. [Non-Patent Document 32] Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277: pp. 923-937. [Non-Patent Document 33] Lin et al., Blood 2005 Jul1;106(1):287-295 pp. [Overview of the Initiative] [Means for solving the problem]

[0012] In one embodiment, the present invention relates to a therapeutic agent for use in the treatment of iron metabolic disorders or conditions, wherein the therapeutic agent is (a) TMPRSS6 inhibitors, (b) MT2 inhibitors, (c) Ferroportin inhibitors, The present invention relates to a therapeutic agent for use comprising an inhibitor selected from, wherein the treatment includes extracellular implantation, embedding, or transplantation.

[0013] In certain embodiments, treatment of iron metabolic disorders or conditions further includes the steps of extracellular implantation, implantation, or transplantation therapy. In certain embodiments, treatment of iron metabolic disorders or conditions includes stem cell transplantation, preferably HSCT.

[0014] In certain embodiments, the therapeutic agent or pharmaceutical composition of the present invention is administered to the subject requiring it before, during, or after pretreatment for HSCT. In certain embodiments, the therapeutic agent or pharmaceutical composition of the present invention is administered to the subject requiring it before pretreatment for HSCT.

[0015] In certain embodiments, the therapeutic agent or pharmaceutical composition of the present invention is administered to a subject requiring it before, during, or after HSCT. In certain embodiments, the therapeutic agent or pharmaceutical composition of the present invention is administered to a subject requiring it before HSCT.

[0016] In another aspect, the present invention relates to a pharmaceutical composition for use in the treatment of iron metabolic disorders or conditions, comprising an effective amount of a therapeutic agent as defined in any of the preceding claims, and further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

[0017] A method for treating an iron metabolic disorder or condition in a subject requiring treatment, and in another embodiment, the present invention relates to a method comprising the step of administering an effective amount of the pharmaceutical composition or therapeutic agent of the present invention, wherein the treatment includes extracellular implantation, embedding, or transplantation.

[0018] In another aspect, the present invention relates to the use of the therapeutic agent or pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of an iron metabolic disorder or condition, wherein the treatment includes extracellular implantation, embedding, or transplantation. [Modes for carrying out the invention]

[0019] New treatment option: Inhibition of matryptase-2 / TMPRSS6 The inventors have surprisingly found different treatment options for reducing transferrin saturation, which further prevent NTBI, iron overload, and reduce iron-mediated toxicity before or during HSC transplantation and engraftment. The inventors have found that inhibitors of the TMPRSS6 gene, inhibitors of the protein matriptase-2 (MT2), ferroportin blockers, or hepcidin enhancers can be used for such treatment. MT2 is a protein product of the TMPRSS6 gene and is a type II transmembrane serine protease that plays a crucial role in regulating iron homeostasis. MT2 is a negative regulator of the induction of the peptide hormone hepcidin synthesis. Hepcidin is a peptide hormone mainly produced in the liver and acts as a negative regulator of iron absorption in the gastrointestinal tract and iron release from storage, preventing iron efflux from intestinal cells and macrophages into the circulation. Hepcidin regulates the body's iron balance by blocking the release of iron by cells via ferroportin, the only known cellular iron transport protein. Elevated hepcidin levels can induce iron restriction and reduce iron availability in the body (McDonald et al., American Journal of Physiology, 2015, vol 08 no. 7, pp. C539-C547). TMPRSS6 is primarily expressed in the liver, but high levels of TMPRSS6 mRNA are also found in the kidneys, at lower levels in the uterus, and in many other tissues at much lower levels (Ramsay et al., Haematologica (2009), 94( * 6), pp. 84-849).

[0020] Inhibition of MT2 by reducing the expression of the TMPRSS6 gene appears to be a particularly promising approach. Inhibition of TMPRSS6 expression can be achieved in several ways. One method is the use of inhibitory nucleic acids such as siRNA or antisense oligonucleotides (ASOs). These are short nucleic acids that inhibit protein formation by causing targeted degradation of the mRNA molecules encoding these proteins. Such gene silencing agents are becoming increasingly important for therapeutic applications in medicine. For the drug development of such nucleic acids, it is necessary, among other things, that they can be economically synthesized, metabolically stable, specifically targeted to tissues, able to enter cells, and function within acceptable limits of toxicity.

[0021] Double-stranded RNA (dsRNA) capable of binding to expressed mRNA through complementary base pairing has been shown to inhibit gene expression through an endogenous mechanism called "RNA interference (RNAi)" (Fire et al., 1998, Nature. 1998 Feb 19;391(6669):pp. 806-811 and Elbashir et al., 2001, Nature. 2001 May 24;411(6836):pp. 494-498). Short dsRNAs have become a useful tool for studying gene function by inducing gene-specific post-transcriptional silencing in many organisms, including vertebrates. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that degrades target messenger RNA that has sufficient complementarity or homology to the silencing-inducing strand loaded into the RISC complex as an siRNA double helix.

[0022] Inhibition of TMPRSS6 expression by the GalNAc TMPRSS6 siRNA molecule in wild-type mice and animal models of hereditary hemochromatosis and beta-thalassemia increased serum hepcidin levels and decreased serum iron and transferrin saturation (Altamura et al., Hemasphere. 2019 Dec;3(6):e301.; Vadolas et al., Br. J. Hematology 2021 Jul;194(1):200-210). The duration of action of the highly active TMPRSS6 siRNA sequence is enhanced by specific chemical modification of the GalNAc siRNA compound, resulting in infrequent medication (once every 3-5 weeks) required to induce hepcidin-induced iron restriction (Altamura et al., Hemasphere. 2019 Dec;3(6):e301., Vadolas et al., Br. J. Hematology 2021 Jul;194(1):pp. 200-210).

[0023] Advantages of TMPRSS6 siRNA-mediated therapy Targeting MT2 or TMPRSS6 is advantageous compared to all of the treatment options listed above.

[0024] TMPRSS6 siRNA versus venotomy: Patients undergoing chemotherapy and preparation for HSC-T may develop anemia and become dependent on blood transfusions. Venotomy lowers hematocrit and hemoglobin levels by removing red blood cells from circulation, which worsens anemia. This can be avoided by new treatment methods. Iron restriction by increasing endogenous hepcidin levels via MT2 inhibitors such as TMPRSS6 siRNA leads to iron redistribution in the body. This makes it possible to limit the release of serum iron, NTBI, or LPI during or after pretreatment.

[0025] Venotomy also carries side effects related to fluid shifts, including dizziness, nausea, and vasovagal syncope. MT2 inhibitor-based therapies, such as TMPRSS6 siRNA therapy, are less likely to affect fluid level compartments, and therefore these complications are less likely to occur with this treatment approach.

[0026] TMPRSS6 siRNA vs. chelating agent: Induction of endogenous hepcidin by inhibiting TMPRSS6 expression with GalNAc siRNA presents a promising therapeutic strategy for reducing toxicity, enhancing engraftment, and lowering non-relapse morbidity and mortality during and after HSCT in patients undergoing this treatment.

[0027] Current treatments for disorders in which elevated ferritin, transferrin saturation, hepatic iron, and non-transferrin-bound iron (NTBI) are expected, such as beta-thalassemia, sickle cell disease, MDS, and leukemia, all have drawbacks and are often highly toxic to the host, making them contraindicated for many patient groups and / or unable to be delivered in sufficient quantities to prevent GVHD. Therefore, there is a clear need to reduce the risk associated with HSCT and enhance its effectiveness against various disorders. This invention addresses this need. The use of MT2 inhibitors such as GalNAc-conjugate TMPRSS6 siRNA presents a promising therapeutic strategy for disorders with elevated ferritin, transferrin saturation, hepatic iron, and non-transferrin-bound iron.

[0028] To our surprise, the inventors found that reducing iron excess or unstable plasma iron before, during, and after transplantation and engraftment was beneficial for patients who underwent HSC transplantation.

[0029] One aspect of the present invention is, (a) TMPRSS6 inhibitors, (b) MT2 inhibitors, or (c) Ferroportin inhibitors The use of therapeutic agents containing or comprising the following.

[0030] In one embodiment, the present invention relates to a therapeutic agent for use in the treatment of an object before, during, or after extracellular implantation, implantation, or transplantation, wherein the therapeutic agent is (a) TMPRSS6 inhibitors, (b) MT2 inhibitors, (c) Ferroportin inhibitors The treatment comprises an inhibitor selected from the group consisting of, where extracellular implantation, implantation, or transplantation may be stem cell transplantation, and optionally the treatment further comprises the simultaneous administration of one or more chemotherapeutic agents, radiotherapy, and / or immunotherapy.

[0031] In one embodiment, the present invention relates to a therapeutic agent for use as a pharmaceutical agent for reducing systemic iron levels, transferrin saturation, non-transferrin-bound iron (NTBI), and / or unstable plasma iron (LPI) in a subject, wherein the subject is at least one of systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload, wherein the therapeutic agent is (a) TMPRSS6 inhibitors, (b) MT2 inhibitors, (c) Ferroportin inhibitors The treatment comprises an inhibitor selected from, wherein the treatment further comprises extracellular implantation, embedding, or transplantation.

[0032] In one embodiment, the present invention relates to a therapeutic agent for use in the treatment of an object before, during, or after extracellular implantation, embedding, or transplantation, wherein the therapeutic agent is (a) TMPRSS6 inhibitors, (b) MT2 inhibitors, (c) Ferroportin inhibitors The inhibitor is selected from the group consisting of the following.

[0033] In one embodiment, the present invention relates to a therapeutic agent for use in the treatment or prevention of systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and / or unstable plasma iron (LPI) overload in subjects requiring such treatment, wherein the therapeutic agent is (a) TMPRSS6 inhibitors, (b) MT2 inhibitors, (c) Ferroportin inhibitors The treatment comprises an inhibitor selected from, wherein the treatment further comprises the steps of extracellular implantation, embedding, or transplantation.

[0034] In one embodiment, the present invention relates to a therapeutic agent for use in the treatment of iron metabolic diseases or conditions, wherein the therapeutic agent is TMPRSS6 inhibitors, MT2 inhibitors, or Ferroportin inhibitors The treatment comprises an inhibitor selected from, wherein the procedure further comprises the steps of extracellular implantation, embedding, or transplantation, wherein optionally, the procedure further comprises the concurrent administration of one or more chemotherapeutic agents, radiotherapy, and / or immunotherapy.

[0035] In one embodiment, the present invention relates to a therapeutic agent for use in the treatment of iron metabolic disorders or conditions associated with at least one of systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload, wherein the therapeutic agent is TMPRSS6 inhibitors, MT2 inhibitors, or Ferroportin inhibitors The treatment comprises an inhibitor selected from, wherein the treatment further comprises the steps of extracellular implantation, embedding, or transplantation.

[0036] In one embodiment, the present invention relates to a therapeutic agent for use in the treatment of a disease or condition for which a reduction of at least one of systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), and unstable plasma iron (LPI) is beneficial, wherein the therapeutic agent is (a) TMPRSS6 inhibitors, (b) MT2 inhibitors, (c) Ferroportin inhibitors The inhibitor is selected from the group consisting of the following.

[0037] In certain aspects, iron metabolic disorders or conditions may be beneficial from reducing iron levels as measured by one or more of the following: systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), or unstable plasma iron (LPI).

[0038] In certain embodiments, an iron metabolic disorder or condition is characterized by at least one of the following: systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload.

[0039] In one aspect of the present invention, a drug that reduces measurable iron is (i) an antibody or an antigen-binding fragment thereof, or a variant, fusion, or derivative of the above antibody or antigen-binding fragment, (ii) Antibody mimic or mimetic (e.g., based on a non-antibody scaffold), (iii) RNA aptamer, (iv) small molecules; (v) CovX-body, Or (vi) Nucleic acid It is selected from the group consisting of the following.

[0040] In one aspect of the present invention, the antibody (i) or its antigen-binding fragment (or a variant, fusion, or derivative of the antibody or antigen-binding fragment), or a fusion or derivative of the variant, and / or the antibody mimic (ii) is selected from the group consisting of affibodies, tetranectin, adonectin, monobodies, antikalin, DARPin, ankyrin, avimers, iMab, microbodies, peptide aptamers, Kunitz domains, affilins, and any combination thereof.

[0041] Several TMPRSS6, MT2, and ferroportin inhibitors that can be used for the purposes of the present invention are known in the art. Below are some examples of publications disclosing some of the inhibitors mentioned: a) Regarding MT2: Gutschow et al., J. Med. Chem. 2010, 53, 15, pp. 5523-5535; Marsault et al., European Journal of Medicinal Chemistry, Volume 129, 2017, pp. 110-123; Stirnberg et al., Pharmaceuticals 2018, 11(2), 49; all of these are incorporated herein by reference with respect to all of their disclosures regarding MT2 inhibitors. b) Regarding TMPRSS6: International Publication No. 2018 / 185240A1, International Publication No. 2014190157A1, International Publication No. 2016085852A1, International Publication No. 2022 / 231999A1, International Publication No. 2016161429A1, all of which are incorporated herein by reference with respect to all that they disclose with respect to TMPRSS6 inhibitors. c) Regarding ferroportin: International Publication Nos. 2022157185A1, 2017 / 068089, 2017 / 068090, 2018 / 192973, 2017 / 068089, and 2017 / 068090 are all incorporated herein by reference with respect to all disclosures they make regarding ferroportin inhibitors. Furthermore, hepcidin mimetics and others that act as ferroportin blockers are known.

[0042] Those skilled in the art are aware of TMPRSS6, MT2, and / or ferroportin inhibitors. Any of the TMPRSS6, MT2, and / or ferroportin inhibitors known in the art may be used by those skilled in the art for the purposes of the present invention, which is to treat iron metabolic disorders or conditions exhibiting at least one of systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and / or unstable plasma iron (LPI) overload.

[0043] In one aspect of the present invention, the therapeutic agent for use is a nucleic acid, wherein the nucleic acid comprises at least one double helix comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the portion of the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene and is capable of inhibiting the expression of TMPRSS6.

[0044] In certain embodiments, one or more nucleotides on the first and / or second chain are modified to form a modified nucleotide.

[0045] In certain embodiments, the nucleic acid is optionally conjugated to a ligand at the 5' end of the second strand.

[0046] In certain embodiments, the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNAc moiety to a nucleic acid.

[0047] In certain embodiments, the linker has a divalent, trivalent, or tetravalent branched structure.

[0048] In certain embodiments, nucleic acids are stabilized at the 5' and / or 3' ends of one or both strands.

[0049] In certain embodiments, the nucleic acid includes phosphorothioate bonds between one, two, or three 3' nucleotides and / or 5' nucleotides at the ends of the first and / or second strand, or includes phosphorodithioate bonds.

[0050] In certain embodiments, the nucleic acid includes two phosphorothioate bonds between each of the three terminal 3' nucleotides on the first strand, and between each of the three terminal 5' nucleotides, as well as two phosphorothioate bonds between the three terminal nucleotides at the 3' end of the second strand.

[0051] In certain embodiments, unmodified or modified nucleic acids are as described in Table 1a, as disclosed in International Publication No. 2018 / 185240A1:

[0052] [Table 1]

[0053] They can be selected from the following. Unmodified or modified nucleic acids may be combined with ligands known in the art, such as GN3-TMPRSS6-hcm9 or GN3-TMPRSS6-hc18, which are disclosed in the same document. It will be understood by those skilled in the art that either unmodified or modified sequences may be combined with any linker known in the art, such as a GalNAc linker.

[0054] In certain embodiments, nucleic acids are as described in Table 1b (Table 2) disclosed in International Publication No. 2018 / 185240A1:

[0055] [Table 2]

[0056] A selection may be made from the above. The nucleic acid may be combined with any ligand known in the art. It will be understood by those skilled in the art that any of the sequences listed above may be combined with any linker known in the art, such as the GalNAc linker, for example, the sequence numbers listed above, i.e., sequence numbers 286, 288, 290, 292, etc., disclosed in the same document.

[0057] In certain embodiments, unmodified or modified nucleic acids are as described in Table 2 (Table 3) disclosed in International Publication No. 2022229150A1:

[0058] [Table 3]

[0059] A selection may be made from the following. The nucleic acid may be unmodified or modified, and the unmodified or modified nucleic acid may be combined with ligands known in the art, for example, those disclosed in the same document. It will be understood by those skilled in the art that either the unmodified or modified sequence may be combined with any linker known in the art, for example, a GalNAc linker. The same document discloses modified sequences EU400, EU401, and EU402, corresponding to the linkers of SEQ ID NOs: 2, 4, and 5.

[0060] In certain embodiments, unmodified or modified nucleic acids are as described in Table 3 (Table 4) of International Publication No. 2022231999A1:

[0061] [Table 4A]

[0062] [Table 4B]

[0063] A selection may be made from the following. The nucleic acid may be unmodified or modified, and the unmodified or modified nucleic acid may be combined with a ligand known in the art, for example, one disclosed in the same document. It will be understood by those skilled in the art that either the unmodified or modified sequence may be combined with any linker known in the art, for example, a GalNAc linker.

[0064] In certain embodiments, unmodified or modified nucleic acids are as described in Table 4 (Table 5) disclosed in International Publication No. 2016161429A1:

[0065] [Table 5]

[0066] A selection may be made from the following. The nucleic acid may be unmodified or modified, and the unmodified or modified nucleic acid may be combined with a ligand known in the art, for example, one disclosed in the same document. It will be understood by those skilled in the art that either the unmodified or modified sequence may be combined with any linker known in the art, for example, a GalNAc linker.

[0067] The synthesis of (vp)-mU-phos may be carried out as described in Prakash, Nucleic Acids Res. 2015, 43(6), pp. 2993-3011 and Haraszti, Nucleic Acids Res. 2017, 45(13), pp. 7581-7592. The synthesis of the phosphoramidite derivative of ST23 (ST23-phos) and its analogs may be carried out as described in International Publication No. 2017 / 174657.

[0068] In certain embodiments, the conjugated nucleic acid has the following structure:

[0069] [ka]

[0070] [ka]

[0071] [ka]

[0072] [ka]

[0073] [ka]

[0074] (wherein Z is any nucleic acid as defined herein) It is conjugated to a tribranched ligand having one of the following components.

[0075] In one embodiment, the conjugated nucleic acid has the following structure:

[0076] [ka]

[0077] (wherein Z is any nucleic acid as defined herein, and the terminal phosphorothioate group of the ligand moiety is attached to the 5' position of the 5' terminal nucleotide of the second strand of the nucleic acid (indicated by "Z") or the terminal phosphorothioate group of the ligand moiety is attached to the 3' position of the 3' terminal nucleotide of the second strand of the nucleic acid (indicated by "Z"))) It is conjugated to a tribranched ligand having [a specific characteristic].

[0078] In certain embodiments, a nucleic acid (represented by "Z") is conjugated to a (tribranched) ligand via a phosphate or thiophosphate group of the ligand moiety, either by linking the ligand to the 5' position of the 5' terminal nucleotide of the second strand of the nucleic acid, or by linking the ligand to the 3' position of the 3' terminal nucleotide of the second strand of the nucleic acid.

[0079] Any ligand of formula (II), (III), or (IV) or any of the tribranched ligands disclosed herein can be bound at the terminus of a nucleic acid or to a nucleotide that is not present at the terminus of the nucleic acid. In the case of a double-stranded nucleic acid, the ligand can be bound at the 3' terminus of the first (antisense) strand and / or at either the 3' and / or 5' terminus of the second (sense) strand. A nucleic acid may contain more than one ligand of formula (II), (III), or (IV) or any of the tribranched ligands disclosed herein. However, a single ligand of formula (II), (III), or (IV) or any of the tribranched ligands disclosed herein is preferred because a single such ligand is sufficient for efficient targeting of the nucleic acid to target cells. Preferably, in this case, at least the last two, preferably at least three, and more preferably at least four nucleotides at the terminus of the nucleic acid to which the ligand is bound are linked by a phosphodiester bond.

[0080] In certain embodiments, in the case of double-stranded nucleic acids, the 5' end of the first (antisense) strand is not bound to any of the ligands of formula (II), (III), or (IV) or any of the tribranched ligands disclosed herein, because the ligand at this position may interfere with the biological activity of the nucleic acid.

[0081] Nucleic acids having a single ligand of formula (II), (III), or (IV) or one of the tribranched ligands disclosed herein at the 5' end of the strand are easier to synthesize and therefore less expensive than the same nucleic acid having the same ligand at the 3' end. Therefore, preferably, the single ligand of formula (II), (III), or (IV) or one of the tribranched ligands disclosed herein is covalently bonded (and conjugated) to the 5' end of the nucleic acid strand, preferably to the 5' end of the second strand if the nucleic acid is double-stranded.

[0082] In certain embodiments, the nucleic acids in Table 1a (Table 1) or Table 1b (Table 2) are used for the purposes of the present invention. In certain embodiments, the nucleic acids in Table 1b (Table 2) are used for the purposes of the present invention.

[0083] In certain embodiments, the nucleic acids in Table 2 (Table 3) are used for the purposes of the present invention. Furthermore, the first strand containing the modified sequence of Sequence ID No. 1 of International Publication No. 2022229150A1 and the following: A control molecule containing a second chain as shown in [ST23(ps)]3 C4XLT(ps)fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mU fC(ps)mG(ps)Fa (Sequence ID 6) may be used. The above molecule will henceforth be referred to as EU403.

[0084] In certain embodiments, the nucleic acid is EU401 or EU402. In certain embodiments, the nucleic acid is EU401. In certain embodiments, the nucleic acid is EU402. In certain embodiments, EU401 and EU402 are interchangeable and can be used together.

[0085] In particular, EU400, EU401, EU402, and EU403 are as shown in Table 5 (Table 6):

[0086] [Table 6]

[0087] Those skilled in the art recognize that any known inhibitory nucleic acid may be modified or unmodified, and that it may be conjugated, either alone or in combination with any known ligand, such as those described above, for the purpose of inhibiting a target gene.

[0088] In certain embodiments, nucleic acids are unmodified, meaning they do not include, for example, any chemical modifications or conjugations known in the art. In certain embodiments, nucleic acids are chemically modified to enhance stability or other beneficial properties.

[0089] Nucleic acids can be synthesized or modified by methods well established in the art. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugation, inversion bond) or 3'-terminal modifications (conjugation, DNA nucleotide, inversion bond, etc.); base modifications, e.g., replacement of a stabilizing base, a destabilizing base, or a base that forms a base pair with a broad repertoire of partners, removal of a base, or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar replacement; or skeletal modifications, including modification or replacement of phosphodiester bonds. Terminal modifications can be added for a variety of reasons, including modulation of activity or resistance to degradation. A useful terminal modification for modulating activity is modification with a phosphate or phosphate analog at the 5' end. The nucleic acids of the present invention on the first or second strand may be 5'-phosphorylated or may contain a phosphoryl analog at the 5' end. Examples of 5'-phosphate modifications include those compatible with RISC-mediated gene silencing.Appropriate modifications include 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)P OP(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monoditithiophosphate (phosphorodithioate; (HO)(HS)( Examples include (S)PO-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); any further combination of oxygen / sulfur-substituted monophosphates, diphosphates and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramide ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5'-vinylphosphonates, and 5'-alkyl ether phosphonates (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). Examples of the modifications are given in the document mentioned above, and further, for example, in International Publication No. 2018185241.

[0090] Another modification of nucleic acids includes linking the nucleic acid to one or more ligands, moieties, or conjugates that enhance, for example, the activity, cell distribution, or cell uptake of the nucleic acid into cells. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553-6556).In other embodiments, the ligand may be cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), or aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids, e.g., 1,2-di-O-hexadecyl-rac-glycerol-1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate or triethylammonium (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), adamantane acetate (Manoharan et al., Tetrahedron The components are: Lett., 1995, 36:3651-3654; palmityl portion (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol portion (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).Ligands may include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be synthetic polymers, such as recombinant or synthetic molecules like synthetic polyamino acids. Ligands may also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins that bind to a specified cell type, such as kidney cells, such as antibodies. The targeting group may be tyrotropin, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyhydric lactose, polyhydric galactose, N-acetylgalactosamine, N-acetylglucosamine, polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is polyhydric galactose, for example, N-acetylgalactosamine.

[0091] In certain embodiments, the conjugate is a carbohydrate conjugate. In certain embodiments, the carbohydrate is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates are described, for example, in International Publication 2017 / 174657, U.S. Patent Application Publication 2021 / 0017214, and International Publication 2022 / 231999.

[0092] In certain embodiments, the present invention relates to any nucleic acid, conjugated nucleic acid, nucleic acid for use, method, composition or use as disclosed herein, wherein the nucleic acid includes vinyl-(E)-phosphonate modifications such as 5' vinyl-(E)-phosphonate modifications. In certain embodiments, the nucleic acid includes 5' vinyl-(E)-phosphonate modifications combined with 2'-F modifications at a second position on the first strand. Vinyl-(E)-phosphonate 2'-OMe-uracilphosphoamidite can be synthesized according to methods published in the literature and can be used in oligonucleotide synthesis (Haraszti et al., Nuc. Acids Res., 45(13), 2017, pp. 7581-7592).

[0093] In certain embodiments, the nucleic acids of the present invention may include one or more phosphorothioate modifications on one or more of the ends of the first and / or second strands. Optionally, both ends or one end of the first strand may contain one, two, or three phosphorothioate-modified nucleotides. Optionally, both ends or one end of the second strand may contain one, two, or three phosphorothioate-modified nucleotides. Optionally, both ends of the first strand and the 5' end of the second strand may contain two phosphorothioate-modified nucleotides. A phosphorothioate-modified nucleotide means that the bond between a nucleotide and an adjacent nucleotide contains a phosphorothioate group instead of a standard phosphate group.

[0094] In particular embodiments, the present invention relates to any nucleic acid, conjugated nucleic acid, nucleic acid for use, method, composition or use as disclosed herein, wherein the terminal nucleotide at at least one 3' end of the first and second strands is an inverted nucleotide, bonded to an adjacent nucleotide via the 3' carbon of the terminal nucleotide and the 3' carbon of the adjacent nucleotide, and / or the terminal nucleotide at at least one 5' end of the first and second strands is an inverted nucleotide, bonded to an adjacent nucleotide via the 5' carbon of the terminal nucleotide and the 5' carbon of the adjacent nucleotide, wherein optionally, a. The 3' and / or 5' inverted nucleotides of the first and / or second chain are linked to adjacent nucleotides via phosphate diester bonds through phosphate groups, or b. The 3' and / or 5' inverted nucleotides of the first and / or second chain are linked to adjacent nucleotides via phosphorothioate groups, or c. The 3' and / or 5' inverted nucleotides of the first and / or second chain are linked to adjacent nucleotides via phosphorodithioate groups.

[0095] Terminal modifications may also be useful for monitoring distribution, in which case fluorophores, such as fluorescein or Alexa dyes, may be added as groups. Terminal modifications may also be useful for enhancing uptake, with cholesterol being a useful modification in this regard. Terminal modifications may also be useful for crosslinking RNA agents to other parts. Those skilled in the art will recognize that a molecule may contain at least two of the modifications mentioned, and may be a combination of the modifications mentioned, such as PS, PS2, and / or VP, as disclosed in International Publication No. 2018185241.

[0096] In one aspect of the present invention, a therapeutic agent for use in the treatment of iron metabolic disorders or conditions further comprises a pharmaceutically acceptable diluent, carrier, or excipient.

[0097] In certain embodiments, the therapeutic agent of the present invention is a pharmaceutical composition for use in the treatment of iron metabolic disorders or conditions, comprising an effective amount of the therapeutic agent of the present invention, and further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

[0098] In certain embodiments, the pharmaceutical composition of the present invention is adapted for delivery via a route selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal routes. In certain embodiments, the pharmaceutical composition of the present invention is adapted for delivery via a subcutaneous route.

[0099] In certain embodiments, the route of administration is selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal. In certain embodiments, the route of delivery is subcutaneous.

[0100] In one aspect of the present invention, an iron metabolic disorder or condition is a disorder or condition that is further beneficial from extracellular implantation, implantation, or transplantation therapy such as hematopoietic stem cell transplantation (HSCT).

[0101] In certain embodiments, treatment of iron metabolic disorders or conditions further includes the steps of extracellular implantation, implantation, or transplantation therapy. In certain embodiments, treatment of iron metabolic disorders or conditions includes stem cell transplantation, preferably HSCT.

[0102] In certain embodiments, the disease or condition treated using the compositions and methods of the present invention may be a non-malignant disease or condition. The non-malignant disease or condition is selected from the group consisting of severe aplastic anemia, abnormal hemoglobin disorders such as thalassemia and / or sickle cell disease, aplastic anemia, Fanconi anemia, Wiscott-Aldrich syndrome, Hurler syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Kostmann syndrome, severe immunodeficiency, severe combined immunodeficiency, or autoimmune disorders, such as SLE, multiple sclerosis, IBD, Crohn's disease, Sjögren's syndrome, vasculitis, lupus, myasthenia gravis, Wegener's disease, malignant infantile osteopetrosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic disorders, and / or other immunodeficiency or autoimmune diseases.

[0103] In certain embodiments, the disease or condition treated using the compositions and methods of the present invention may be a malignant disease or condition. Malignant diseases or conditions include myelodysplastic syndromes (MDS), leukemias (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)) and other leukemias (e.g., hairy cell leukemia (HCL), T-cell prelymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell leukemia), and lymphomas (e.g., progenitor T-cell leukemia / lymphoma). The group consists of Parkinson's disease (PAMA, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma), solid tumors (e.g., renal cancer, liver cancer, and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphomas not otherwise specified, nodular sclerosing Hodgkin lymphoma, mixed-cell Hodgkin lymphoma, multiple myeloma, neuroblastoma, Ewing's sarcoma, and glioma.

[0104] In certain embodiments, the object of the present invention is to treat hematological disorders or hematological malignancies. In certain embodiments, the hematological malignancies are leukemias such as ALL, AML, or AMoL. In certain embodiments, the hematological disorder is MDS. In certain embodiments, the disease or condition is CML, CLL, other leukemias, and lymphomas. In certain embodiments, the disease or condition to be treated is a hematological disorder such as ALL, AML, AMoL, or MDS requiring stem cell transplantation such as allogeneic HSCT. In certain embodiments, the disease or condition is AML. In certain embodiments, the disease or condition is MDS.

[0105] The most common types of hematopoietic stem cell transplants are usually derived from bone marrow, peripheral blood, or umbilical cord blood. Hematopoietic stem cells (HSCs) are pluripotent or pluripotent cells that can give rise to many or all blood cell types, namely myeloid cells (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid cells (T cells, B cells, NK cells). It is known that a small number of HSCs can proliferate to produce a very large number of daughter HSCs. This phenomenon is used in hematopoietic stem cell transplantation (HSCT) when a relatively small number of HSCs reconstitute the hematopoietic lineage. HSCTs can be, for example, allogeneic (from a different individual) or autologous (from the same individual).

[0106] In certain aspects of the present invention, the HSCT is autologous, allogeneic, syngeneic, or heterologous HSCT, or is genetically modified or gene-edited.

[0107] In certain embodiments, HSCT is the first HSCT. In certain embodiments, HSCT is, for example, re-implantation after relapse.

[0108] Prior to transplantation, preconditioning for HSCT is performed, which means the recipient's immune system is destroyed, typically using radiation or chemotherapy. This process is carried out with the intention of eradicating the target malignant cell population and reduces the risk of rejection of new immune HSCs at the cost of partial or complete bone marrow ablation, i.e., the destruction of the patient's bone marrow's ability to grow new blood cells. Subsequently, the stem cells to be transplanted are injected into the recipient's bloodstream, where a sufficient number reach the bone marrow cavity, where they replace the damaged hematopoietic system and restart the target's normal blood cell production.

[0109] In one aspect of the present invention, the therapeutic agent or pharmaceutical composition of the present invention for use in the treatment of the disease or condition mentioned further comprises the simultaneous administration of one or more chemotherapeutic agents, radiotherapy and / or immunotherapy.

[0110] In certain embodiments, one or more chemotherapeutic agents may be selected from the group consisting of busulfan, cyclophosphamide, fludarabine, treosulfan, melphalan, and thiotepa. In certain embodiments, chemotherapy may be a low-intensity pretreatment.

[0111] In certain embodiments, radiotherapy is selected from a group consisting of whole-body irradiation, whole-body lymph node irradiation, and whole-body bone marrow irradiation.

[0112] In certain embodiments, chemotherapy and radiotherapy may be combined.

[0113] In certain embodiments, the therapeutic agent or pharmaceutical composition of the present invention is administered to the subject requiring it before, during, or after pretreatment for HSCT. In certain embodiments, the therapeutic agent or pharmaceutical composition of the present invention is administered to the subject requiring it before pretreatment for HSCT.

[0114] In certain embodiments, the therapeutic agent or pharmaceutical composition of the present invention is administered to a subject requiring it before, during, or after HSCT. In certain embodiments, the therapeutic agent or pharmaceutical composition of the present invention is administered to a subject requiring it before HSCT.

[0115] HSCT is a procedure associated with many potential complications, including infections and graft-versus-host disease (GVHD). However, improvements in the procedure and increased survival rates have led to its expanded use beyond cancer to include congenital metabolic disorders and autoimmune diseases. When GVHD and a range of opportunistic infections can be overcome, transplantation offers an opportunity for cure or long-term remission. Therefore, improved transplantation methods are needed to minimize the risks and complications associated with transplantation and provide more efficient transplantation.

[0116] In one aspect of the present invention, the therapeutic agent or pharmaceutical composition of the present invention improves at least one of the efficacy of HSCT, including improved engraftment, improved overall survival, reduced non-relapse mortality, reduced infection and idiopathic pneumonia syndrome, and reduced HSC-T related morbidities such as sinusoidal obstruction syndrome, chronic liver disease, GVHD, and / or GVL.

[0117] In certain aspects of the present invention, the therapeutic agent or pharmaceutical composition of the present invention improves the efficacy of HSCT, including improved engraftment, improved overall survival, reduced non-relapse mortality, reduced infection and idiopathic pneumonia syndrome, and reduced HSC-T related morbidities such as sinus-obstructive syndrome, chronic liver disease, and GVHD.

[0118] In certain embodiments, the therapeutic agent or pharmaceutical composition of the present invention can prevent and / or reduce graft-versus-host disease (GVHD) and / or graft-versus-leukemia (GVL).

[0119] The inventors have found that, in the peri-transplant environment, reducing TMPRSS6 gene expression in the liver by treatment with TMPRSS6 siRNA increases circulating hepcidin levels, decreases circulating iron levels, and reduces the production of non-transferrin-bound iron. By reducing iron-mediated toxicity, this approach surprisingly enhances engraftment of HSC donor cells, limits predisposition to infection after HSCT, and reduces non-relapse mortality.

[0120] In one embodiment, a therapeutic agent or pharmaceutical composition for use in the treatment of the disease or condition mentioned, (a) Steps to inhibit TMPRSS6, (b) A step of blocking ferroportin, (c) Process to enhance hepcidin This includes, or consists of, a decrease in systemic iron levels, NTBI, transferrin saturation, unstable plasma iron, and / or eLPI levels.

[0121] In one embodiment, the present invention relates to a method for treating a subject having an iron metabolic disorder or condition in a subject requiring treatment, wherein the treatment comprises administering an effective amount of the therapeutic agent or pharmaceutical composition of the present invention to the subject. In a particular embodiment, the subject requiring treatment is a subject requiring extracellular graft, implant, or graft therapy. In a particular embodiment, the iron metabolic disorder is associated with at least one of systemic iron overload, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload.

[0122] In certain aspects, the treatment of iron metabolic disorders or conditions is associated with therapies including extracellular implantation, implantation, or transplantation, such as HSCT.

[0123] In one embodiment, the present invention relates to a method for treating an iron metabolic disorder or condition in a subject requiring treatment, wherein the therapy includes a procedure for extracellular implantation, implantation, or transplantation, such as HSCT.

[0124] In one embodiment, the present invention relates to the use of a therapeutic agent or pharmaceutical composition according to the present invention for the manufacture of a pharmaceutical for the treatment of iron metabolic disorders or conditions.

[0125] In certain embodiments, treatment of iron metabolic disorders or conditions further includes therapies involving extracellular implantation, implantation, or transplantation, such as HSCT.

[0126] In one embodiment, the present invention relates to the use of a therapeutic agent or pharmaceutical composition according to the present invention for the manufacture of a pharmacopoeia for the treatment of an iron metabolic disorder or condition, wherein the treatment includes a therapy comprising extracellular implantation, implantation, or transplantation steps such as HSCT.

[0127] As used herein, the terms “bone marrow transplant” and “stem cell transplant” should be considered interchangeable and refer to the transplantation of stem cells to a recipient. Stem cells do not necessarily have to be derived from bone marrow and may be derived from other sources such as umbilical cord blood.

[0128] As used herein, the term “iron metabolic disorder or condition” means a disorder or condition associated with at least one of the following: systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload. In particular, the subject herein refers to at least one of the following: systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload.

[0129] As used herein, the term "HSCT" refers to the transplantation of hematopoietic stem cells into a recipient, where the stem cells are typically collected from bone marrow, peripheral blood, or umbilical cord blood.

[0130] As used herein, the term “pre-transplant” refers to a time frame of several hours, days, or weeks prior to transplantation in the context of administering the therapeutic agent or pharmaceutical composition of the present invention. In particular, the therapeutic agent or pharmaceutical composition of the present invention is administered before pre-transplant treatment or several weeks to up to several months prior to transplantation, for example, 1 to 10 weeks or 1 to 4 months. The therapeutic agent or pharmaceutical composition may be administered continuously, as a single dose, or several times during this period. In certain embodiments of the present invention, the therapeutic agent or pharmaceutical composition is administered as a single dose. The therapeutic agent or pharmaceutical composition should be administered multiple times at an effective dose over sufficient time prior to transplantation, resulting in a sufficient reduction of systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), and / or unstable plasma iron (LPI) as necessary. Such administration protocols will be considered familiar to those skilled in the art who can utilize a given therapeutic agent or pharmaceutical composition.

[0131] As used herein, the terms “subject” and “patient” are interchangeable. As used herein, the subject is an animal, preferably a mammal such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkeys (e.g., rhesus macaques, crab-eating macaques, or chimpanzees) and a human). In certain embodiments of the present invention, the subject is a human.

[0132] As used herein, the term "recipient" refers to the subject receiving the transplant in the context of transplantation, and is in contrast to "donor," which is the subject from which the material (cells) to be transplanted originates. In an allogeneic situation, the recipient and donor are different individuals, while in an autogenetic situation, the recipient and donor are the same individual. In a syngeneic situation, the donor and recipient are different individuals but are genetically identical. An allogeneic situation means that the donor originates from a different species than the recipient.

[0133] As used herein, the terms “administer” or “dosage” refer to the delivery of the therapeutic agent or pharmaceutical composition of the present invention by any suitable method known to those skilled in the art.

[0134] As used herein, the term “effective dose” means an amount of therapy (e.g., a therapeutic agent) that is sufficient to reduce and / or reverse the severity and / or duration of a disease or condition or its symptoms, to prevent progression of the disease or condition, to induce regression, to prevent recurrence, onset or onset of one or more symptoms associated with the disease or condition, or to enhance or improve the preventive or therapeutic effect of another therapy (e.g., another prophylactic or therapeutic agent). Accordingly, an effective dose of the therapeutic agent or pharmaceutical composition of the present invention used before, during, or after HSCT is an amount sufficient to result in a reduction of at least one of the following: systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), and unstable plasma iron (LPI), so as to improve the effectiveness of HSCT, including improved engraftment, improved overall survival, reduced non-relapse mortality, reduced infection and idiopathic pneumonia syndrome, reduced HSC-T related morbidities such as sinusoidal obstruction syndrome, chronic liver disease, GVHD, and / or GVL.

[0135] As used herein, “drug” refers to any purified or isolated natural or chemically synthesized entity comprising one or more molecules.

[0136] In the method and use of the therapeutic agent or pharmaceutical composition of the present invention, a therapeutically effective amount of the active ingredient is supplied. The therapeutically effective amount can be determined by a medical professional or veterinary professional based on the patient's characteristics, such as age, weight, sex, condition, complications, and other diseases, as is well known to those skilled in the art.

[0137] [Table 7A]

[0138] [Table 7B]

[0139] [Table 7C]

[0140] Preferred non-limiting embodiments that embody specific aspects of the present invention are described below with reference to the drawings: [Brief explanation of the drawing]

[0141] [Figure 1]The diagram shows various treatment schedules for chemotherapy preconditioning, evaluated in combination with the application of siRNA such as EU401 for their effects on TMPRSS6 target gene expression, HAMP mRNA induction, and systemic iron levels on day +2. The total doses of busulfan and fludarabine administered in low-dose, medium-dose, or high-dose chemotherapy are shown. The low-dose consisted of daily intraperitoneal (IP) injections of 0.8 mg / kg of fludarabine from day -6 to day -2, and daily IP administrations of 3.2 mg / kg of busulfan on days -4 and -3. The medium-dose consisted of daily IP injections of 10 mg / kg of fludarabine from day -6 to day -2, and daily IP administrations of 10 mg / kg of busulfan on days -4 and -3. The high-dose regimen consisted of daily intravenous injections of 10 mg / kg of fludarabine from day 6 to day 2, and twice-daily intravenous administrations of 16.25 mg / kg of busulfan on days 4 and 3. [Figure 2] This figure shows that treatment with EU401 reduces TMPRSS6 mRNA and increases HAMP mRNA in the livers of mice exposed to various doses of chemotherapy pretreatment (low and high doses, see Figure 1). Mean values ​​+ / - SD. [Figure 3] This figure shows that treatment with EU401 inhibited and attenuated the induction of serum iron, transferrin saturation, and NTBI in mice treated with moderate and high doses of chemotherapy preconditioning, respectively. Mean values ​​+ / - SEM. [Figure 4] This figure shows the effects of TMPRSS6 siRNA treatment on bone marrow grafts under two different experimental conditions. A) Mice treated with a moderate dose of chemotherapy after being administered EU403 or EU402 siRNA at a dose of 5 mg / kg on days -28 and -7. B) Mice treated with a high dose of chemotherapy after being administered EU403 or EU402 siRNA at a dose of 5 mg / kg on days -42, -21, and -1. BM donor cells were transplanted on day 0, and chimerism was assessed as a measure of engraftment in peripheral blood at weeks 4, 6, and 8 post-transplant, and in tissue at week 8 post-transplant. [Figure 5] This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant blood chimerism under two different experimental conditions described in Figure 4. The percentage of CD45.1+ donor-derived blood cells is shown at each time point of blood collection. Blood chimerism is superior at 4 and 8 weeks post-transplant in mice treated with moderate dose pretreatment and EU402 (A), and at 4, 6, and 8 weeks post-transplant in mice treated with high dose pretreatment and EU402 (B). Data are presented as mean + / - SEM. [Figure 6] This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant hematopoietic and lymphoid engraftment under two different experimental conditions described in Figure 4. The percentages of CD11b+CD3-CD19- myeloid cells and CD11b-CD3+CD19+ lymphoid cells from CD45.1+ donors are shown at each time point of blood collection. Hematopoietic and lymphoid chimerism is superior at 4 and 8 weeks post-transplant in mice treated with moderate dose pretreatment and EU402 (A), and at 4, 6, and 8 weeks post-transplant in mice treated with high dose pretreatment and EU402 (B). Data are presented as mean + / - SEM. [Figure 7] This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant hematopoietic engraftment under two different experimental conditions described in Figure 4. The percentages of CD45.1+ donor-derived CD11b+Ly6G-L6C+ / - monocytes and CD11b+Ly6G+ neutrophils are shown at each time point of blood collection. Hematopoietic chimerism is superior at 4 and 8 weeks post-transplant in mice treated with moderate-dose pretreatment and EU402 (A), and at 4, 6, and 8 weeks post-transplant in mice treated with high-dose pretreatment and EU402 (B). Data are presented as mean + / - SEM. [Figure 8]This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant hematopoietic engraftment under two different experimental conditions described in Figure 4. The percentages of CD45.1+ donor-derived CD11b-CD19+ B cells and CD11b-CD3+ T cells are shown at each time point of blood collection. Hematopoietic chimerism is superior at 4 and 8 weeks post-transplant in mice treated with moderate-dose pretreatment and EU402 (A), and at 4, 6, and 8 weeks post-transplant in mice treated with high-dose pretreatment and EU40 (B). Data are presented as mean + / - SEM. [Figure 9] Figure 4 shows the effect of TMPRSS6 siRNA treatment on post-transplant bone marrow and spleen chimerism under two different experimental conditions. The percentage of CD45.1+ donor-derived cells in the bone marrow and spleen is shown at 8 weeks post-transplant. Bone marrow and spleen chimerism is superior in mice treated with medium-dose (A) and high-dose (B) pretreatment, as well as EU402 treatment at 8 weeks post-transplant. Data are presented as mean + / - SEM. [Figure 10] This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant hematopoietic and lymphoid engraftment under two different experimental conditions, as described in Figure 4. The percentages of CD45.1+ donor-derived CD11b+ myeloid cells and CD11b-CD19+CD3+ lymphoid cells in the bone marrow are shown at 8 weeks post-transplant. Myeloid and lymphoid chimerism in the bone marrow is superior in mice treated with medium-dose (A) and high-dose (B) pretreatment, as well as EU402 treatment at 8 weeks post-transplant. Data are presented as mean + / - SEM. [Figure 11]This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant myelomultiseries engraftment under two different experimental conditions described in Figure 4. The percentages of CD45.1+ donor-derived CD11b+Ly6G-Ly6C+ / - monocytes, CD11b+Ly6G+ neutrophils, CD11b-CD19+ B cells, and CD11b-CD3+ T cells in the bone marrow are shown at 8 weeks post-transplant. Myelomultiseries chimerism is superior in mice treated with medium-dose (A) and high-dose (B) pretreatment, as well as EU402 treatment at 8 weeks post-transplant. Data are presented as mean + / - SEM. [Figure 12] This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant spleen multiseries engraftment under two different experimental conditions described in Figure 4. The percentages of CD45.1+ donor-derived CD11b+Ly6G-Ly6C+ / - monocytes, CD11b+Ly6G+ neutrophils, CD11b-CD19+ B cells, and CD11b-CD3+ T cells in the spleen are shown at 8 weeks post-transplant. Spleen multiseries chimerism is superior in mice treated with medium-dose (A) and high-dose (B) pretreatment, as well as EU402 treatment at 8 weeks post-transplant. Data are presented as mean + / - SEM. [Figure 13] Figure 4 shows the effect of TMPRSS6 siRNA treatment on the number of hematopoietic stem progenitor cells (HSPCs) under two different experimental conditions. The percentage of bone marrow Lin-Sca+cKit+(LSK)HSPCs across the total Lin- cells is shown at 8 weeks post-transplant. The percentage of HSPCs remained unchanged in mice treated with medium-dose (A) and high-dose (B) pretreatment, as well as EU402 vs. EU403 treatment at 8 weeks post-transplant. Data are presented as mean + / - SEM. [Figure 14]This figure shows the effect of TMPRSS6 siRNA treatment on bone marrow hematopoietic stem progenitor cell (HSPC) chimerism under two different experimental conditions described in Figure 4. The percentages of CD45.1+ donor-derived Lin-Sca+cKit+(LSK)HSPCs and Lin-Sca1-cKit+ myeloid progenitor cells (MPs) in the bone marrow are shown at 8 weeks post-transplant. Chimerism of LSK HSPCs and MPs is superior in mice treated with medium-dose (A) and high-dose (B) pretreatment, as well as EU402 treatment at 8 weeks post-transplant. Data are presented as mean + / - SEM. [Figure 15] This figure shows the experimental setup for investigating the effect of TMPRSS6 siRNA treatment on bone marrow graft outcomes in a mouse model of MDS. NUP98-HOXD13 mice were treated with 5 mg / kg EU400 or EU401 on days -35, -14, and 7, and given a moderate dose of chemotherapy preconditioning. BM donor cells were transplanted on day 0, and chimerism was assessed as a measure of engraftment in peripheral blood at weeks 3, 8, and 12 post-transplant, and in tissue at week 12 post-transplant. [Figure 16] This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant blood engraftment in MDS mice treated with moderate doses of preconditioning. The percentages of CD45.1+ donor-derived blood cells (A) and CD45.1+ donor-derived CD11b+Ly6G-Ly6C+ / - monocytes and CD11b+Ly6G+ neutrophils are shown at each time point of blood collection. Blood monocyte and neutrophil chimerism is superior at 3 and 8 weeks post-transplant in MDS mice treated with moderate doses of preconditioning and EU402 (B). Data are presented as mean + / - SEM. [Figure 17]This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant bone marrow and spleen engraftment in MDS mice treated with moderate dose preconditioning. The percentages of CD45.1+ donor-derived bone marrow and spleen cells (A), as well as CD45.1+ donor-derived myeloid cells and CD11b-CD19+CD3+ lymphocytes, are shown at 12 weeks post-transplant. MDS mice treated with moderate dose preconditioning and EU401 tend to exhibit higher bone marrow and spleen donor chimerism. Data are presented as mean + / - SEM values. [Figure 18] This figure shows that treatment with EU401 reduces the expression of inflammatory cytokines in bone marrow macrophages and neutrophils of transplanted MDS mice. Bone marrow samples were collected 12 weeks after transplantation. Expression of inflammatory cytokines IL1b, TNF-alpha, and IFN-gamma in bone marrow CD11blowF4 / 80+ macrophages (A) and CD11b+Ly6G+ neutrophils (B) from transplanted MDS mice treated with EU400 or EU401 at 12 weeks post-transplantation. Cytokines expressed by each cell population are shown as change multipliers across EU400-treated transplanted MDS mice. Mean + / - SEM. [Figure 19] This figure shows that treatment of mouse hepatocytes with EU401 reduces TMPRSS6 mRNA levels to a similar extent as EU402. The values ​​obtained for TMPRSS6 mRNA were normalized to the values ​​generated for the housekeeping gene ACTIN and correlated to the mean value of the untreated sample (ut) set at 1x target gene expression. Each bar represents the mean + / - SD from three biological replicates. [Figure 20]This figure shows the experimental setup for investigating the effect of TMPRSS6 siRNA treatment on bone marrow graft outcomes in a mouse model of MDS. NUP98-HOXD13 mice were sc-treated with 5 mg / kg EU403 or EU402 on days -60, -30, and -1, and then given either low-dose or high-dose chemotherapy pretreatment. The total doses of busulfan and fludarabine administered in A) low-dose or B) high-dose chemotherapy are shown. The low-dose regimen consisted of daily intraperitoneal (IP) injections of 10 mg / kg fludarabine from days -6 to -2, and twice-daily IP administrations of 2.5 mg / kg busulfan on days -4 and -3. The high-dose regimen consisted of daily intracellular injections of 10 mg / kg of fludarabine from day -6 to day -2, and twice-daily intracellular administration of 16.25 mg / kg of busulfan on days -4 and -3. BM donor cells were transplanted on day 0, and chimerism was assessed as a measure of engraftment in peripheral blood at weeks 4, 6, and 8 post-transplant, and in tissue at week 8 post-transplant. [Figure 21] Figure 20 shows the effect of TMPRSS6 siRNA treatment on post-transplant blood chimerism in MDS mice under two different experimental conditions described. The percentage of CD45.1+ donor-derived blood cells is shown at each time point of blood collection. In mice treated with low-dose pre-treatment and EU402 (A), and in mice treated with high-dose pre-treatment and EU402 (B), blood chimerism was superior at weeks 4, 6, and 8 post-transplant. Data are presented as mean + / - SEM. [Figure 22]This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant hematopoietic and lymphoid engraftment under two different experimental conditions described in Figure 4. The percentages of CD45.1+ donor-derived CD11b+ myeloid cells and CD11b- lymphocytes are shown at each time point of blood collection. Hematopoietic and lymphoid chimerism is superior at 4 and 8 weeks post-transplant in mice treated with moderate-dose pretreatment and EU402 (A), and at 4, 6, and 8 weeks post-transplant in mice treated with high-dose pretreatment and EU402 (B). Data are presented as mean + / - SEM. [Figure 23] Figure 20 shows the effect of TMPRSS6 siRNA treatment on post-transplant hemomyelocyte engraftment in MDS mice under two different experimental conditions described. The percentages of CD45.1+ donor-derived CD11b+Ly6G-L6C+ / - monocytes and CD11b+Ly6G+ neutrophils are shown at each time point of blood collection. Hemomyelocyte chimerism is superior at 6 and 8 weeks post-transplant in mice treated with low dose and EU402 (A) and / or high dose pre-treatment and EU402 (B). Data are presented as mean + / - SEM. [Figure 24] Figure 20 shows the effect of TMPRSS6 siRNA treatment on post-transplant hematopoietic engraftment in MDS mice under two different experimental conditions described. The percentages of CD45.1+ donor-derived CD11b-CD19+ B cells and CD11b-CD3+ T cells are shown at each time point of blood collection. Hematopoietic T-lymphatic chimerism is superior at 4, 6, and 8 weeks post-transplant in mice treated with low-dose pretreatment and EU402 (A), or high-dose pretreatment and EU402 (B). Data are presented as mean + / - SEM. [Figure 25A]This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant bone marrow chimerism under two different experimental conditions described in Figure 20. Bone marrow chimerism at 8 weeks post-transplant is superior in MDS mice treated with EU402 in both low-dose (A, C, E) and high-dose (B, D, F) pre-treatment, or in one of the two. Data are presented as mean + / - SEM. A) shows the percentage of CD45.1+ donor-derived cells in the bone marrow, as well as the percentage of CD45.1+ donor-derived CD11b+ myeloid and CD11b-CD19+CD3+ lymphoid cells in the bone marrow. [Figure 25B] This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant bone marrow chimerism under two different experimental conditions described in Figure 20. Bone marrow chimerism at 8 weeks post-transplant is superior in MDS mice treated with EU402 under either low-dose (A, C, E) or high-dose (B, D, F) pre-treatment conditions. Data are presented as mean + / - SEM. The percentage of CD45.1+ donor-derived cells in the bone marrow and the percentage of CD45.1+ donor-derived CD11b+ myeloid and CD11b-CD19+CD3+ lymphoid cells in the bone marrow are shown in B). [Figure 25C] This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant bone marrow chimerism under two different experimental conditions described in Figure 20. Bone marrow chimerism at 8 weeks post-transplant is superior in MDS mice treated with EU402 under either low-dose (A, C, E) or high-dose (B, D, F) pre-treatment conditions. Data are presented as mean + / - SEM. C) shows the percentages of donor-derived CD45.1+, CD11b+Ly6G-Ly6C+ / - monocytes, CD11b+Ly6G+ neutrophils, CD11b-CD19+ B cells, and CD11b-CD3+ T cells in the bone marrow at 8 weeks post-transplant. [Figure 25D]This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant bone marrow chimerism under two different experimental conditions described in Figure 20. Bone marrow chimerism at 8 weeks post-transplant is superior in MDS mice treated with EU402 under either low-dose (A, C, E) or high-dose (B, D, F) pre-treatment conditions. Data are presented as mean + / - SEM. Percentages of donor-derived CD45.1+, CD11b+Ly6G-Ly6C+ / - monocytes, CD11b+Ly6G+ neutrophils, CD11b-CD19+ B cells, and CD11b-CD3+ T cells in the bone marrow at 8 weeks post-transplant are shown in D). [Figure 25E] Figure 20 shows the effect of TMPRSS6 siRNA treatment on post-transplant bone marrow chimerism under two different experimental conditions. Bone marrow chimerism at 8 weeks post-transplant is superior in MDS mice treated with EU402 under either low-dose (A, C, E) or high-dose (B, D, F) pre-treatment conditions. Data are presented as mean + / - SEM. E) shows the percentage of CD45.1+ donor-derived Lin-Sca+cKit+(LSK)HSPCs and Lin-Sca1-cKit+ myeloid progenitor cells (MPs) in the bone marrow at 8 weeks post-transplant. [Figure 25F] This figure shows the effect of TMPRSS6 siRNA treatment on post-transplant bone marrow chimerism under two different experimental conditions described in Figure 20. Bone marrow chimerism at 8 weeks post-transplant is superior in MDS mice treated with EU402 under either low-dose (A, C, E) or high-dose (B, D, F) pre-treatment conditions. Data are presented as mean + / - SEM. The percentage of CD45.1+ donor-derived Lin-Sca+cKit+(LSK)HSPCs and Lin-Sca1-cKit+ myeloid progenitor cells (MPs) in the bone marrow at 8 weeks post-transplant is shown in F). [Examples]

[0142] (Example 1) Inhibition of TMPRSS6 expression increases hepcidin levels in rodent models of chemotherapy preconditioning and stem cell transplantation, mediated by TMPRSS6 siRNA treatment. This example demonstrates the inhibition of TMPRSS6 expression and induction of hepcidin expression by EU401 in mice treated with chemotherapy preconditioning.

[0143] Wild-type mice were treated with either low-dose or high-dose chemotherapy preconditioning as described above and shown in Figure 1 (days -6 to -2). Prior to preconditioning, mice were administered either a single subcutaneous injection of 5 mg / kg of EU400 or EU401 on day -14, or two doses via the same route of either 5 mg / kg of EU400 or 5 mg / kg of EU401 on days -28 and -7. Liver tissue samples were collected on day +2 for total RNA extraction and evaluation of target gene expression by qRT-PCR. Treatment with a single dose of EU401 (5 mg / kg) on ​​day -14 reduced hepatic TMPRSS6 mRNA expression in mice that received high-dose chemotherapy preconditioning (Figure 2A). Similarly, two doses of EU401 reduced TMPRSS6 expression in mice that received either low-dose or high-dose chemotherapy preconditioning (Figures 2B and 2C). Two doses of EU401 were also effective in inducing hepatic HAMP expression in mice that had received either low-dose or high-dose chemotherapeutic pretreatment (Figures 2D and 2E). High-dose pretreatment resulted in a significant increase in hepatic HAMP expression compared to untreated mice, which was further increased by treatment with EU401. The siRNA molecules used in this study are shown in Table 5 (Table 6). The experimental setup is shown in Figure 1, and the results are shown in Figure 2.

[0144] (Example 2) TMPRSS6 siRNA treatment reduces serum iron, transferrin saturation, and NTBI in rodent models for chemotherapy preconditioning and stem cell transplantation. The example demonstrates that EU401 treatment attenuates increases in serum iron levels, transferrin saturation (Tsat), and non-transferrin-bound iron (NTBI) induced by moderate or high-dose chemotherapy pretreatment.

[0145] Wild-type mice were either untreated (UT) or received subcutaneous administration of 5 mg / kg of EU401 on days -28 and -7, as shown in Figure 1, either alone or followed by moderate or high-dose chemotherapy pretreatment (days -6 to -2). Blood samples were collected on day +2 for evaluation of serum iron, transferrin saturation (Tsat), and non-transferrin-bound iron (NTBI). Treatment with EU401 alone reduced serum iron levels and transferrin saturation in otherwise untreated wild-type mice. In mice treated with moderate or high-dose chemotherapy pretreatment, treatment with siRNA EU401 reduced serum iron levels, Tsat, and NTBI compared to mice treated with a non-targeting siRNA control (EU400) (Figure 3). Suppression of NTBI was complete in mice treated with moderate-dose chemotherapy pretreatment and partial in mice treated with high-dose chemotherapy pretreatment. The siRNA molecules used in this study are shown in Table 5 (Table 6). The experimental setup is shown in Figure 1, and the results are shown in Figure 3.

[0146] (Example 3) The effect of TMPRSS6 siRNA treatment on bone marrow graft outcomes in myelosuppressed wild-type mice. This example demonstrates that treatment of recipient mice with TMPRSS6 siRNA EU401 enhances engraftment and donor chimerism after bone marrow transplantation.

[0147] To investigate the effect of TMPRSS6 siRNA treatment on bone marrow graft outcomes, the inventors used the aforementioned chemotherapy pre-treatment mouse model (see Figure 1) and performed bone marrow cell transplantation (see treatment schemes in Figures 4A and 4B). CD45.2 + Wild-type recipient mice were first treated with a medium or high dose of chemotherapy preconditioning, followed by CD45.1 on day 0. + 1 x 10⁻¹⁶ donor cells derived from a wild-type donor 7 Each dose was administered via intravenous injection into the tail vein.

[0148] The effect of TMPRSS6 inhibition on engraftment was evaluated by treating mice with TMPRSS6 siRNA EU402 and by comparing this treatment with treatment with a non-targeting control siRNA EU403. Mice that received moderate-dose chemotherapy preconditioning were treated with 5 mg / kg of each siRNA on days -28 and -7, while mice that received high-dose chemotherapy were conjugated with 5 mg / kg of siRNA on days -42, -21, and -1. CD45.1+ chimerism, a measure of engraftment, was assessed by flow cytometry using appropriate markers in blood samples collected at weeks 4, 6, and 8 post-transplant. Engraftment was higher in mice that received high-dose chemotherapy compared to those that received moderate-dose preconditioning. Treatment with TMPRSS6 siRNA EU402 enhanced donor cell engraftment after both moderate and high-dose preconditioning. The siRNA molecules used in this study are shown in Table 5 (Table 6). The experimental setup is shown in Figure 4, and the results are shown in Figures 5 to 14.

[0149] (Example 4) The effect of TMPRSS6 siRNA treatment on bone marrow graft outcomes in a mouse model of myelodysplastic syndrome. This example demonstrates that treatment with TMPRSS6 siRNA enhances engraftment and donor chimerism in a mouse model of myelodysplastic syndrome. The effect of TMPRSS6 siRNA treatment on post-transplant engraftment was also investigated in NUP98-HOXD13 transgenic mice, a mouse model of myelodysplastic syndrome (MDS) (Lin et al., Blood 2005 Jul1;106(1):287-295). MDS mice were treated as shown in Figure 15 below. MDS mice were treated subcutaneously with 5 mg / kg EU401 or EU400 on days -35, -14, and 7, conferring a moderate dose of chemotherapy preconditioning. CD45.1 + Bone marrow cells derived from donor mice 1×10 7 Individuals, on day 0, CD45.2 + The cells were transplanted into MDS mice, and blood samples were collected at weeks 3, 8, and 12.

[0150] This example shows that treatment with EU401 improves the outcome of bone marrow grafts in MDS mice pre-treated with a medium dose. This effect is observed by a tendency of increased chimerism in the donor-derived blood population and in the myeloid population at 3 and 8 weeks after transplantation. Similarly, the ratio of donor-derived cells shows a higher tendency in the bone marrow and spleen of MDS mice treated with EU401 compared to EU400 at the end of the study (12 weeks after transplantation). + This tendency of increased chimerism is observed in the donor-derived blood population and in the myeloid population at 3 and 8 weeks after transplantation. Similarly, the ratio of donor-derived cells shows a higher tendency in the bone marrow and spleen of MDS mice treated with EU401 compared to EU400 at the end of the study (12 weeks after transplantation).

[0151] The siRNA molecules used in this study are shown in Table 5 (Table 6). The experimental setup is shown in Figure 15, and the results are shown in Figures 16 and 17.

[0152] (Example 5) Reduction of Inflammatory Cytokines by TMPRSS6 in Bone Marrow This example shows that treatment with TMPRSS6 siRNA reduces the post-transplantation expression of inflammatory cytokines in an animal model of myelodysplastic syndrome.

[0153] The effect of TMPRSS6 siRNA treatment on post-transplantation inflammatory cytokine production was evaluated in NUP98-HOXD13 transgenic mice, a mouse model of myelodysplastic syndrome (Blood 2005 Jul 1;106(1):287 - 295).

[0154] MDS mice were treated as shown in Figure 15 below. MDS mice were treated with 5 mg / kg of EU401 or EU400 by subcutaneous administration on days -35, -14, and -7, and given a medium dose of chemotherapeutic pretreatment. 1×10 + bone marrow cells from donor mice 7 were transplanted into CD45.2 + MDS mice on day 0, and blood samples were collected at 3, 8, and 12 weeks thereafter.

[0155] Cytokine expression was measured in bone marrow CD11b low F4 / 80 +Macrophages and CD11b + Ly6G + Neutrophils were evaluated by flow cytometry. The siRNA molecules used in this study are shown in Table 5 (Table 6). The experimental setup is shown in Figure 15, and the results are shown in Figure 18.

[0156] (Example 6) Reduction of TMPRSS6 mRNA levels in primary hepatocytes This example demonstrates a dose-dependent reduction of TMPRSS6 mRNA levels in primary hepatocytes by EU401 and EU402 after receptor-mediated uptake.

[0157] Primary mouse hepatocytes were seeded into 96-well plates at a density of 25,000 cells per well. The cells were then incubated with TMPRSS6 siRNA conjugates at concentrations of 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.41 nM, and 0.14 nM, as shown below. The following day, the cells were lysed for RNA extraction, and TMPRSS6 and actin mRNA levels were determined by Taqman qRT-PCR. The values ​​obtained for TMPRSS6 mRNA were normalized to the values ​​generated for the housekeeping gene actin and correlated to the mean value of the untreated sample (ut) set at 1x target gene expression. Each bar represents the mean + / - SD from three biological replicates. The siRNA conjugates used in this study are listed in Table 5 (Table 6). The experimental setup is shown in Figure 15, and the results are shown in Figure 19.

[0158] (Example 7) The effect of TMPRSS6 siRNA treatment on bone marrow graft outcomes in a mouse model of myelodysplastic syndrome. This example demonstrates that treatment with TMPRSS6 siRNA enhances engraftment and donor chimerism in a mouse model of myelodysplastic syndrome with low and high dose chemotherapy preconditioning. The effect of TMPRSS6 siRNA treatment on post-transplant engraftment was investigated in NUP98-HOXD13 transgenic mice, a mouse model of myelodysplastic syndrome (MDS) (Lin et al., Blood 2005 Jul1;106(1):287-295). MDS mice were treated as shown in Figure 20 below. MDS mice were treated subcutaneously with 5 mg / kg EU403 or EU402 on days -60, -30, and -1, and given low or high dose chemotherapy preconditioning as shown in Figure 20. CD45.1 + Bone marrow cells derived from donor mice 1×10 7 Individuals, on day 0, CD45.2 + Transplantation was performed in MDS mice, and blood samples were collected at weeks 4, 6, and 8. CD45.1+ chimerism, a measure of engraftment, was assessed by flow cytometry using appropriate markers in blood samples collected at weeks 4, 6, and 8 post-transplant. As expected, engraftment was higher in mice treated with high-dose chemotherapy compared to those treated with low-dose pretreatment. Treatment with TMPRSS6 siRNA EU402 enhanced donor cell engraftment after both low and high-dose pretreatment. The siRNA molecules used in this study are shown in Table 1 (Tables 1 and 2). The experimental setup is shown in Figure 20. The results are shown in Figures 21 to 25.

[0159] presentation 1. A therapeutic agent for use in the treatment of iron metabolic disorders or conditions, wherein the therapeutic agent is (d) TMPRSS6 inhibitors, (e) MT2 inhibitors, (f) Ferroportin inhibitors, A therapeutic agent for use comprising an inhibitor selected from, wherein the treatment includes cell implantation, explantation, or transplantation.

[0160] 2. The therapeutic agent is (i) an antibody or an antigen-binding fragment thereof, or a variant, fusion, or derivative of the antibody or antigen-binding fragment, (ii) Antibody mimics selected from the group consisting of affibodies, tetranectin, adonectin (monobody), antikalin, DARPin (ankyrin), avimer, iMab, microbody, peptide aptamer, Kunitz domain, and affilin (e.g., based on a non-antibody scaffold); (hepcidin mimetic), (iii) RNA aptamer, (iv) small molecules; (v) CovX-body, Or (vi) Nucleic acid A therapeutic agent for use as described in Presentation 1, wherein (i) or (ii) may be selected from the group consisting of affibody, tetranectin, adnectin (monobody), antikalin, DARPin (ankyrin), avimer, iMab, microbody, peptide aptamer, Kunitz domain, and affilin, and any combination thereof.

[0161] 3. A therapeutic agent for use according to Presentation 2, wherein the nucleic acid comprises at least one double helix comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the portion of the first strand, the first strand being at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, and capable of inhibiting the expression of TMPRSS6.

[0162] 4. A therapeutic agent for use according to Presentation 2 or 3, wherein one or more nucleotides on the first and / or second chain are modified to form a modified nucleotide.

[0163] 5. A therapeutic agent for use according to any one of presentations 2 to 4, wherein the nucleic acid is optionally conjugated to a ligand at the 5' end of the second strand.

[0164] 6. The therapeutic agent for use according to Presentation 5, wherein the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, the linker conjugates the GalNAc moiety to the nucleic acid.

[0165] 7. A therapeutic agent for use according to presentation 5 or 6, wherein the linker has a divalent, trivalent, or tetravalent branched structure.

[0166] 8. A therapeutic agent for use as described in any one of the presentations 2 to 7, wherein the nucleic acid is one of those listed in Table 1 (Tables 1 and 2), Table 2 (Table 3), Table 3 (Table 4), Table 4 (Table 5), or Table 5 (Table 6).

[0167] 9. A therapeutic agent for use according to any one of Presentations 1 to 8, wherein the nucleic acid is EU401 or EU402.

[0168] 10. A pharmaceutical composition for use in the treatment of iron metabolic disorders or conditions, comprising an effective amount of a therapeutic agent described in any of the presentations 1 to 9, and further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

[0169] 11. A pharmaceutical composition for use as described in Presentation 10, adapted for delivery by a route selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal.

[0170] 12. A pharmaceutical composition for use according to presentation 10 or 11, wherein the delivery route is selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, lung, intranasal, intraocular, and intrathecal.

[0171] 13. a) The disease or condition is non-malignant, and the non-malignant disease or condition is selected from the group consisting of severe aplastic anemia, thalassemia and / or abnormal hemoglobinopathy such as sickle cell anemia, aplastic anemia, Fanconi anemia, Wiscott-Aldrich syndrome, Harler syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Kostmann syndrome, severe immunodeficiency, severe combined immunodeficiency, or autoimmune disorders, such as SLE, multiple sclerosis, IBD, Crohn's disease, Sjögren's syndrome, vasculitis, lupus, myasthenia gravis, Wegener's disease, malignant infantile osteopetrosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic disorders, and / or other immunodeficiency or autoimmune diseases, or b) The disease is malignant, and the malignant disease or condition is myelodysplastic syndrome (MDS), leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)) and other leukemias (e.g., hairy cell leukemia (HCL), T-cell prelymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell leukemia), lymphoma (e.g., progenitor T-cell leukemia / lymphoma, Burkitt lymphoma, A therapeutic agent or pharmaceutical composition for use as described in any of Presentation 1 to 12, selected from the group consisting of follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma), solid tumors (e.g., renal cancer, liver cancer, and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphoma not otherwise specified, nodular sclerosing Hodgkin lymphoma, mixed-cell subtype Hodgkin lymphoma, multiple myeloma, neuroblastoma, Ewing sarcoma, and glioma.

[0172] 14. The above treatment is (a) Steps to inhibit TMPRSS6, (b) Steps to inhibit MT2, (c) A step of blocking ferroportin, or (c) Process to enhance hepcidin A therapeutic agent or pharmaceutical composition for use according to any of Presentation 1 to 13, comprising or comprising, which reduces systemic iron levels, NTBI, transferrin saturation, unstable plasma iron, and / or eLPI levels.

[0173] 15. Therapeutic agent for use or pharmaceutical composition for use according to any one of Presentations 1 to 14, wherein the treatment further comprises the simultaneous administration of one or more chemotherapeutic agents, radiotherapy, and / or immunotherapy.

[0174] 16. The extracellular implantation, implantation, or transplantation procedure includes hematopoietic stem cell transplantation (HSCT), and optionally, the therapeutic agent or pharmaceutical composition is administered to a subject requiring pretreatment for HSCT, either before, during, or after the pretreatment for HSCT. The therapeutic agent or pharmaceutical composition for use according to any of presentations 1 to 15, wherein the therapeutic agent is administered to a subject in need before, during, or after receiving HSCT.

[0175] 17. The therapeutic agent for use or pharmaceutical composition for use as described in Presentation 16, which is capable of preventing and / or reducing graft-versus-host disease (GVHD) and / or graft-versus-leukemia (GVL), and optionally further improving HSCT efficacy, including improved graft survival, improved overall survival, reduced non-relapse mortality, reduced infection and idiopathic pneumonia syndrome, and / or reduced HSC-T related morbidity such as sinusoidal obstruction syndrome and chronic liver disease.

[0176] 18. A method for treating an iron metabolic disorder or condition in a subject requiring treatment, comprising the step of administering a pharmaceutical composition or therapeutic agent described in any of 1 to 17, wherein the treatment includes cell implantation, explantation, or transplantation.

[0177] 19. The aforementioned therapeutic agent, (i) an antibody or an antigen-binding fragment thereof, or a variant, fusion, or derivative of the antibody or antigen-binding fragment, (ii) An antibody mimic selected from the group consisting of an affibody, tetranectin, adnectin (monobody), anticalin, DARPin (ankyrin), avimer, iMab, the microbody, peptide aptamer, Kunitz domain, and affilin (for example, based on a non-antibody scaffold); (hepcidin mimetic), (iii) An RNA aptamer, (iv) A small molecule, (v) A CovX-body, or (vi) A nucleic acid selected from, where (i) or (ii) can be selected from the group consisting of an affibody, tetranectin, adnectin (monobody), anticalin, DARPin (ankyrin), avimer, iMab, the microbody, peptide aptamer, Kunitz domain, and affilin, and any combination thereof, the method according to Presentation 18.

[0178] 20. The nucleic acid contains at least one double strand including at least a part of the first strand and at least a part of a second strand that is at least partially complementary to the part of the first strand, and the first strand is at least partially complementary to at least a part of the RNA transcribed from the TMPRSS6 gene and is capable of inhibiting the expression of TMPRSS6, the method according to Presentation 19.

[0179] 21. One or more nucleotides on the first and / or second strand are modified to form a modified nucleotide, the method according to Presentation 20.

[0180] 22. The nucleic acid is optionally conjugated to a ligand at the 5' end of the second strand, the method according to Presentation 20.

[0181] 23. The ligand contains (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, and the linker conjugates the GalNAc moiety to the nucleic acid, the method according to Presentation 22.

[0182] 24. The method according to presentation 23, wherein the linker has a divalent, trivalent, or tetravalent branched structure.

[0183] 25. The method according to presentation 20, wherein the nucleic acid is any of those described in Table 1 (Tables 1 and 2), Table 2 (Table 3), Table 3 (Table 4), Table 4 (Table 5), or Table 5 (Table 6), and preferably, the nucleic acid is EU401 or EU402.

[0184] 26. The method according to presentation 19, wherein the pharmaceutical composition contains an effective amount of the therapeutic agent according to any of presentations 1 to 9, and further contains a pharmaceutically acceptable diluent, carrier, or excipient.

[0185] 27. The method according to presentation 26, wherein the pharmaceutical composition is adapted for delivery by a route selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal.

[0186] 28. The method according to presentation 26, wherein the delivery route of the pharmaceutical composition is selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal.

[0187] 29. a) The disease or condition is non - malignant, and the non - malignant disease or condition is selected from the group consisting of severe aplastic anemia, abnormal hemoglobinopathies such as thalassemia and / or sickle cell disease, aplastic anemia, Fanconi anemia, Wiskott - Aldrich syndrome, Hurler syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Kostmann syndrome, severe immunodeficiency, severe combined immunodeficiency, or an autoimmune disorder, such as SLE, multiple sclerosis, IBD, Crohn's disease, Sjögren's syndrome, vasculitis, lupus, myasthenia gravis, Wegener's disease, malignant infantile osteopetrosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic disorders, and / or other immunodeficiency or autoimmune diseases, or b) The disease is malignant, and the malignant disease or condition is myelodysplastic syndrome (MDS), leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)) and other leukemias (e.g., hairy cell leukemia (HCL), T-cell prelymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell leukemia), lymphoma (e.g., progenitor T-cell leukemia / lymphoma) The method described in Presentation 18, selected from the group consisting of lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma), solid tumors (e.g., renal cancer, liver cancer, and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphoma not otherwise specified, nodular sclerosing Hodgkin lymphoma, mixed-cell subtype Hodgkin lymphoma, multiple myeloma, neuroblastoma, Ewing sarcoma, and glioma.

[0188] 30. The above procedure is (a) Steps to inhibit TMPRSS6, (b) Steps to inhibit MT2, (c) A step of blocking ferroportin, or (c) Process to enhance hepcidin The method according to Presentation 18, comprising or comprising, which reduces systemic iron levels, NTBI, transferrin saturation, unstable plasma iron, and / or eLPI levels.

[0189] 31. The method according to Presentation 18, wherein the procedure further comprises the simultaneous administration of one or more chemotherapeutic agents, radiotherapy, and / or immunotherapy.

[0190] 32. The extracellular graft or graft treatment includes hematopoietic stem cell transplantation (HSCT), and optionally, the therapeutic agent or pharmaceutical composition is administered to subjects requiring pretreatment for HSCT, during or after pretreatment, or The method according to Presentation 18, wherein the therapeutic agent is administered to the subject in need before, during, or after HSCT.

[0191] 33. The method according to Presentation 32, wherein the therapeutic agent can prevent and / or reduce graft-versus-host disease (GVHD) and / or graft-versus-leukemia (GVL), and optionally further improves the efficacy of HSCT, including improved graft survival, improved overall survival, reduced non-relapse mortality, reduced infection and idiopathic pneumonia syndrome, and / or reduced HSC-T related morbidity such as sinusoidal obstruction syndrome and chronic liver disease.

[0192] 34. Use of a therapeutic agent or pharmaceutical composition according to any one of Presentations 1 to 17 for the manufacture of a medicament for the treatment of an iron metabolic disorder or condition, wherein the treatment includes an extracellular graft or transplant.

[0193] 35. The aforementioned therapeutic agent, (i) an antibody or an antigen-binding fragment thereof, or a variant, fusion, or derivative of the antibody or antigen-binding fragment, (ii) Antibody mimics selected from the group consisting of affibodies, tetranectin, adonectin (monobody), anticarin, DARPin (ankyrin), avimer, iMab, microbody, peptide aptamer, Kunitz domain, and affilin (e.g., based on a non-antibody scaffold); (hepcidin mimetic), (iii) RNA aptamer, (iv) small molecules; (v) CovX-body, Or (vi) Nucleic acid The use described in Presentation 34, wherein (i) or (ii) may be selected from the group consisting of affibodies, tetranectin, adnectin (monobody), antikalin, DARPin (ankyrin), avimer, iMab, microbody, peptide aptamer, Kunitz domain and affilin, and any combination thereof.

[0194] 36. The use according to presentation 35, wherein the nucleic acid comprises at least one double-strand comprising at least a part of a first strand and at least a part of a second strand that is at least partially complementary to the part of the first strand, the first strand is at least partially complementary to at least a part of an RNA transcribed from the TMPRSS6 gene, and capable of inhibiting the expression of TMPRSS6.

[0195] 37. The use according to presentation 36, wherein one or more nucleotides on the first and / or second strand are modified to form modified nucleotides.

[0196] 38. The use according to presentation 36, wherein the nucleic acid is optionally conjugated to a ligand at the 5' end of the second strand.

[0197] 39. The use according to presentation 38, wherein the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, and the linker conjugates the GalNAc moiety to the nucleic acid.

[0198] 40. The use according to presentation 39, wherein the linker is a divalent, trivalent, or tetravalent branched structure.

[0199] 41. The use according to presentation 36, wherein the nucleic acid is any one described in Table 1 (Tables 1, 2), Table 2 (Table 3), Table 3 (Table 4), Table 4 (Table 5), or Table 5 (Table 6), and preferably, the nucleic acid is EU401 or EU402.

[0200] 42. The use according to presentation 36, wherein the pharmaceutical composition comprises an effective amount of the therapeutic agent according to any one of presentations 1 to 9, and further comprises a pharmaceutically acceptable diluent, carrier, or excipient.

[0201] 43. The use according to presentation 36, wherein the pharmaceutical composition is adapted for delivery by a route selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intra-ocular, and intrathecal.

[0202] 44. The use according to Presentation 36, wherein the delivery route of the pharmaceutical composition is selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, lung, intranasal, intraocular, and intrathecal.

[0203] 45. a) The disease or condition is non-malignant, and the non-malignant disease or condition is selected from the group consisting of severe aplastic anemia, thalassemia and / or abnormal hemoglobinopathy such as sickle cell anemia, aplastic anemia, Fanconi anemia, Wiscott-Aldrich syndrome, Harler syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Kostmann syndrome, severe immunodeficiency, severe combined immunodeficiency, or autoimmune disorders, such as SLE, multiple sclerosis, IBD, Crohn's disease, Sjögren's syndrome, vasculitis, lupus, myasthenia gravis, Wegener's disease, malignant infantile osteopetrosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic disorders, and / or other immunodeficiency or autoimmune diseases, or b) The disease is malignant, and the malignant disease or condition is myelodysplastic syndrome (MDS), leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)) and other leukemias (e.g., hairy cell leukemia (HCL), T-cell prelymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell leukemia), lymphoma (e.g., progenitor T-cell leukemia / lymphoma) Use as described in Presentation 36, selected from the group consisting of lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma), solid tumors (e.g., renal cancer, liver cancer, and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphomas not otherwise specified, nodular sclerosing Hodgkin lymphoma, mixed-cell subtype Hodgkin lymphoma, multiple myeloma, neuroblastoma, Ewing's sarcoma, and glioma.

[0204] 46. ​​The above treatment is (a) Steps to inhibit TMPRSS6, (b) Steps to inhibit MT2, (c) A step of blocking ferroportin, or (c) Process to enhance hepcidin The use described in Presentation 36, which includes or comprises a decrease in whole-body iron levels, NTBI, transferrin saturation, unstable plasma iron, and / or eLPI levels.

[0205] 47. The use according to Presentation 36, wherein the treatment further comprises the simultaneous administration of one or more chemotherapeutic agents, radiotherapy, and / or immunotherapy.

[0206] 48. The extracellular graft or graft treatment includes hematopoietic stem cell transplantation (HSCT), and optionally, the therapeutic agent or pharmaceutical composition is administered to subjects requiring pretreatment for HSCT, during or after pretreatment, or The use of the therapeutic agent as described in Presentation 36, wherein the therapeutic agent is administered to the subject in need before, during, or after receiving HSCT.

[0207] 49. The therapeutic agent may prevent and / or reduce graft-versus-host disease (GVHD) and / or graft-versus-leukemia (GVL), and optionally further improve HSCT efficacy, including improved graft survival, improved overall survival, reduced non-relapse mortality, reduced infection and idiopathic pneumonia syndrome, and / or reduced HSC-T related morbidity such as sinusoidal obstruction syndrome and chronic liver disease, as described in Presentation 36.

Claims

1. A therapeutic agent for use in the treatment of iron metabolic disorders or conditions, wherein the therapeutic agent is (g) TMPRSS6 inhibitors, (h)MT2 inhibitor, or (i) Ferroportin inhibitors, A therapeutic agent for use comprising an inhibitor selected from, wherein the treatment includes cell implantation, explantation, or transplantation.

2. The aforementioned therapeutic agent, (i) an antibody or an antigen-binding fragment thereof, or a variant, fusion, or derivative of the antibody or antigen-binding fragment, (ii) Antibody mimics selected from the group consisting of affibodies, tetranectin, adonectin (monobody), antikalin, DARPin (ankyrin), avimer, iMab, microbody, peptide aptamer, Kunitz domain, and affilin (e.g., based on a non-antibody scaffold); (hepcidin mimetic), (iii) RNA aptamer, (iv) small molecules; (v) CovX-body, Or (vi) Nucleic acid A therapeutic agent for use according to claim 1, wherein (i) or (ii) may be selected from the group consisting of affibody, tetranectin, adonectin, monobody, antikalin, DARPin, ankyrin, avimer, iMab, microbody, peptide aptamer, Kunitz domain, affilin, and any combination thereof.

3. The therapeutic agent for use according to claim 2, wherein the nucleic acid comprises at least one double helix comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the portion of the first strand, the first strand being at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, and capable of inhibiting the expression of TMPRSS6.

4. A therapeutic agent for use according to claim 2 or 3, wherein one or more nucleotides on the first and / or second chain are modified to form a modified nucleotide.

5. A therapeutic agent for use according to any one of claims 2 to 4, wherein the nucleic acid is optionally conjugated to a ligand at the 5' end of the second strand.

6. The therapeutic agent for use according to claim 5, wherein the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, the linker conjugates the GalNAc moiety to the nucleic acid.

7. The therapeutic agent for use according to claim 5 or 6, wherein the linker has a divalent, trivalent, or tetravalent branched structure.

8. A therapeutic agent for use according to any one of claims 2 to 7, wherein the nucleic acid is one of those listed in Table 1 (Tables 1 and 2), Table 2 (Table 3), Table 3 (Table 4), Table 4 (Table 5), or Table 5 (Table 6).

9. A therapeutic agent for use according to any one of claims 1 to 8, wherein the nucleic acid is EU401 or EU402.

10. A pharmaceutical composition for use in the treatment of iron metabolic disorders or conditions, comprising an effective amount of the therapeutic agent according to any one of claims 1 to 9, and further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

11. A pharmaceutical composition for use according to claim 10, adapted for delivery by a route selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal.

12. The pharmaceutical composition for use according to claim 10 or 11, wherein the delivery route is selected from the group including intravenous, intramuscular, subcutaneous, intra-articular, lung, intranasal, intraocular, and intrathecal.

13. a) The disease or condition is non-malignant, and the non-malignant disease or condition is selected from the group consisting of severe aplastic anemia, thalassemia and / or abnormal hemoglobinopathy such as sickle cell anemia, aplastic anemia, Fanconi anemia, Wiscott-Aldrich syndrome, Harler syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Kostmann syndrome, severe immunodeficiency, severe combined immunodeficiency, or autoimmune disorders, such as SLE, multiple sclerosis, IBD, Crohn's disease, Sjögren's syndrome, vasculitis, lupus, myasthenia gravis, Wegener's disease, malignant infantile osteopetrosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic disorders, and / or other immunodeficiency or autoimmune diseases, or b) The disease is malignant, and the malignant disease or condition is myelodysplastic syndrome (MDS), leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)) and other leukemias (e.g., hairy cell leukemia (HCL), pre-T cell lymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell leukemia), lymphoma (e.g., progenitor T-cell leukemia / lymphoma, Burkitt lymphoma, A therapeutic agent for use or a pharmaceutical composition for use according to any one of claims 1 to 12, selected from the group consisting of follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma, solid tumors (e.g., renal cancer, liver cancer and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphoma not otherwise specified, nodular sclerosing Hodgkin lymphoma, mixed-cell subtype Hodgkin lymphoma, multiple myeloma, neuroblastoma, Ewing sarcoma, and glioma.

14. The above treatment (a) Steps to inhibit TMPRSS6, (b) Steps to inhibit MT2, (c) A step of blocking ferroportin, (c) Process to enhance hepcidin A therapeutic agent or pharmaceutical composition for use according to any one of claims 1 to 13, comprising or comprising, which reduces systemic iron levels, NTBI, transferrin saturation, unstable plasma iron, and / or eLPI levels.

15. The therapeutic agent for use or pharmaceutical composition for use according to any one of claims 1 to 14, wherein the treatment further comprises the simultaneous administration of one or more chemotherapeutic agents, radiotherapy, and / or immunotherapy.

16. The extracellular implantation, implantation, or transplantation procedure includes hematopoietic stem cell transplantation (HSCT), and optionally, the therapeutic agent or pharmaceutical composition is administered to a subject requiring pretreatment for HSCT, during, or after such treatment. The therapeutic agent for use or pharmaceutical composition for use according to any one of claims 1 to 15, wherein the therapeutic agent is administered to a subject requiring it before, during, or after receiving HSCT.

17. The therapeutic agent or pharmaceutical composition for use according to claim 16, wherein the therapeutic agent is capable of preventing and / or reducing graft-versus-host disease (GVHD) and / or graft-versus-leukemia (GVL), and optionally further improving HSCT efficacy, including improved graft survival, improved overall survival, reduced non-relapse mortality, reduced infection and idiopathic pneumonia syndrome, and / or reduced HSC-T related morbidity such as sinusoidal obstruction syndrome and chronic liver disease.

18. A method for treating an iron metabolic disorder or condition in a subject requiring treatment, comprising the step of administering an effective amount of a pharmaceutical composition or therapeutic agent according to any one of claims 1 to 17, wherein the treatment includes extracellular implantation, embedding, or transplantation.

19. Use of a therapeutic agent or pharmaceutical composition according to any one of claims 1 to 18 for the manufacture of a pharmacopoeia for the treatment of an iron metabolic disorder or condition, wherein the treatment includes extracellular implantation, embedding, or transplantation.

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