Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
  • C07D215/54—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/4704—2-Quinolinones, e.g. carbostyril
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• the present invention relates to a novel use of the quinoline derivative tasquinimod. More particularly, the invention relates to tasquinimod or a pharmaceutically acceptable salt thereof for use in the treatment of myelodysplastic syndrome.
• Tasquinimod and a method for its preparation were described in International Applications No. PCT/SE99/00676, published as WO 99/55678, and No. PCT/SE99/01270, published as WO 00/03991, which applications also disclosed the utility of tasquinimod and some other quinoline carboxamides for the treatment of diseases resulting from autoimmunity, such as multiple sclerosis, insulin-dependent diabetes mellitus, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease and psoriasis and, furthermore, diseases where pathologic inflammation plays a major role, such as asthma, atherosclerosis, stroke and Alzheimer’s disease.
• diseases resulting from autoimmunity such as multiple sclerosis, insulin-dependent diabetes mellitus, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease and psoriasis and, furthermore, diseases
• MDS Myelodysplastic syndromes
• MDS are one of the most common haematological neoplasms in the elderly population with a median age of 65-70 years; however, disease can occur at any age. While no apparent risk factor is present in primary MDS, DNA damage by previous chemo- or radiation therapy is a well-known risk factor for secondary MDS. Further, MDS in the paediatric population might arise secondary to inherited bone marrow failures (for example Fanconi anemia, or dysceratosis congenital).
• MDS Multiple biological processes govern hematopoietic progenitor proliferation and differentiation into mature blood cells.
• Molecular alterations in hematopoietic stem cells that disrupt any of these processes are integral to the pathogenesis of MDS.
• About 80-90% of MDS patients have a somatic mutation in their hematopoietic stem cells.
• the type and incidence of somatic mutations contribute to dysfunctional signalling pathways in MDS and are associated with disease prognosis and responsiveness to certain drugs.
• the surrounding bone marrow niche plays a central role in MDS pathogenesis. Aberrant inflammatory signalling, including the activation of the NLRP3 inflammasome, may facilitate the selection, maintenance and leukemic evolution of the malignant MDS clone.
• MDS patients can be asymptomatic at diagnosis, however, up to 80% suffer from anemia. Patients are primarily affected by cytopenia-related symptoms, such as fatigue and weakness due to anemia, neutropenia-related infections or bleeding due to thrombocytopenia. About 1/3 of the MDS patients will progress to acute myeloid leukemia.
• the revised International Prognostic Scoring System for MDS (IPSS-R) is the most common used prognostication system to determine disease prognosis.
• the model demonstrates 5 risk categories according to the grade of cytopenia, marrow blast percentage and cytogenetic subgroup.
• MDS is one of the most common hematologic neoplasms in the elderly population, only limited treatment options are available. Besides supportive care, only five drugs are approved in Europe (erythropoietin, luspatercept, the iron chelator deferasirox, the immunomodulatory drug lenalidomide, and the hypomethylating agent azacitidine). The only curative treatment is allogeneic hematopoietic stem cell transplantation, which is however not feasible in many elderly patients.
• Transfusion therapy is a method of giving red blood cells, white blood cells, or platelets to replace blood cells destroyed by disease or treatment. Frequent red blood cell transfusions lead to iron overload and accompanying organ toxicities with poor clinical outcome. Iron chelation therapy with deferasirox might improve patient’s outcome.
• Erythropoiesis-stimulating agents ESAs are given to increase the number of mature red blood cells made by the body and to lessen the effects of anemia. Sometimes granulocyte colony-stimulating factor (G-CSF) is given with ESAs to help the treatment work better. Finally, antibiotic therapy may be used to fight infection.
• ESAs Erythropoiesis-stimulating agents
• G-CSF granulocyte colony-stimulating factor
• Drug therapy includes treatment with e.g. lenalidomide, which is only approved in patients with myelodysplastic syndrome associated with an isolated del(5q) chromosome abnormality who need frequent red blood cell transfusions. Patients with transfusion-dependent MDS with ring sideroblasts, refractory or unlikely to respond to ESA, can use luspatercept.
• the hypomethylating agent azacitidine may be used to treat myelodysplastic syndromes in advanced disease stages to slow the progression of myelodysplastic syndromes to acute myeloid leukemia.
• classical chemotherapy is only used before stem cell transplantation in patients with higher blast count to reduce tumor cell burden.
• stem cells are collected from the blood or bone marrow of a healthy donor and reinfused to the MDS patient after completion of conditioning therapy.
• the donor hematopoietic stem cells will replace the patient’s original bone marrow and restore the body's blood cells (termed engraftment).
• a first aspect is tasquinimod or a pharmaceutically acceptable salt thereof for use in the treatment of myelodysplastic syndrome.
• a further aspect is the use of tasquinimod or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of myelodysplastic syndrome.
• Still a further aspect is a pharmaceutical formulation containing a therapeutically effective amount of tasquinimod or a pharmaceutically acceptable salt thereof, for the treatment of myelodysplastic syndrome.
• a further aspect is a method for the treatment of myelodysplastic syndrome in a mammal in need of such treatment, by administration of a therapeutically effective amount of tasquinimod or a pharmaceutically acceptable salt thereof to the mammal.
• FIGURE 1 is a graph representing the concentration of S100A9 (in pg/ml) in bone marrow obtained from healthy patients (control), and patients with low risk MDS (LR-MDS) and high risk MDS (HR-MDS), respectively.
• FIGURE 2 shows Western blots of phosphorylated IRAKI (80 kDa), NF-KB-p65 (65 kDa) and gasdermin (50 kDa) in MDS MSCs following treatment with S100A9 or with S100A9 and tasquinimod, for 48h.
• GAPDH 38 kDa served as reference protein.
• FIGURE 3 is a bar chart representing the mRNA expression levels of IL-1 p, IL-18, Caspl and PD-L1 by real-time PCR of MDS MSCs after 48h treatment with tasquinimod only (TASQ), with S100A9 only (S100A9), or with both S100A9 and tasquinimod (S100A9+TASQ).
• FIGURE 4 shows a Western blot of PD-L1 in healthy and CMML MSCs following treatment with S100A9 and tasquinimod for 48h.
• Lane 1 untreated control;
• lane 2 treated with tasquinimod;
• lane 3 treated with S100A9;
• lane 3 treated with S100A9 and tasquinimod.
• FIGURE 5 shows a Western blot of phosphorylated IRAKI (80 kDa), NF-KB-p65 (65 kDa) and gasdermin (50 kDa) in MDS MSCs following treatment with TNF-a and tasquinimod for 48h.
• GAPDH 38 kDa served as reference protein.
• FIGURE 6a is a bar chart showing quantification of CAF-C when co-culturing MDS MSC with hematopoietic progenitor cells, after one, two, three and four weeks without the presence of either S100A9 or tasquinimod (“without”), in the presence of tasquinimod (TASQ), in the presence of S100A9 (S100A9), and in the presence of both s100A9 and tasquinimod (S100A9+TASQ).
• FIGURE 6b is a bar chart showing the number of colonies/300 cells in CFU assays performed using MDS MSC cells cocultured with hematopoietic progenitor cells, harvested after one week of co-culture in the presence of S100A9 or in the presence of both S100A9 and tasquinimod or in the absence of both S100A9 and tasquinimod (“control”), with 300 cells being plated in enriched methylcellulose medium with recombinant cytokines (MethoCultTM H4435, STEMCELL Technologies). Colonies were counted after 2 weeks and classified under a microscope or with the STEMvisionTM system (STEMCELL Technologies).
• FIGURE 6c is a bar chart showing the number of colonies/300 cells in CFU assays performed using MDS MSC cells and hematopoietic progenitor cells, harvested after one week of co-culture in the presence (“TASQ”) or absence (“without”) of tasquinimod, with 300 cells being plated in enriched methylcellulose medium with recombinant cytokines (MethoCultTM H4435, STEMCELL Technologies). Colonies were counted after 2 weeks and classified under a microscope or with the STEMvisionTM system (STEMCELL Technologies).
• MDS-RS myelodysplastic syndrome with ring sideroblasts
• the compound tasquinimod, or 4-hydroxy-5-methoxy-N,1-dimethyl-2-oxo-N-[4- (trifluoromethyl)phenyl]-1,2-dihydroquinoline-3-carboxamide has the structural formula
• “Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
• Examples of pharmaceutically acceptable salts comprise salts with (as counter ion) an alkali metal ion, e.g. Li + , Na + or K + , or salts with an alkaline earth metal ion, e.g. Mg 2+ or Ca 2+ , or salts with any other pharmaceutically acceptable metal ion, e.g. Zn 2+ or Al 3+ ; or pharmaceutically acceptable salts formed with organic bases, such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.
• an alkali metal ion e.g. Li + , Na + or K +
• salts with an alkaline earth metal ion e.g. Mg 2+ or Ca 2+
• any other pharmaceutically acceptable metal ion e.g. Zn 2+ or Al 3+
• pharmaceutically acceptable salts formed with organic bases such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.
• “Therapeutically effective amount” means an amount of tasquinimod or a pharmaceutically salt thereof, that, when administered to a subject for treating a condition (here: MDS), is sufficient to effect such treatment for the condition.
• the “therapeutically effective amount” will vary depending on e.g. the age and relative health of the treated subject, the state of progression of the condition, the route and form of administration, the possible additional use of other drugs, e.g. in a combination therapy, etc.
• an "effective amount” provides treatment effect (e.g., treats, prevents, inhibits, relieves, promotes, improves, increases, reduces, and the like) as measured by a statistically significant change in one or more indications, symptoms, signs, diagnostic tests, vital signs, and the like.
• an "effective amount” suppresses, manages, or prevents a condition as measured by a lack of a statistically significant change in one or more indications, symptoms, signs, diagnostic tests, vital signs, and the like.
• treatment is an approach for obtaining beneficial or desired results including clinical results.
• beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms of the treated condition, diminishment of extent of the condition, stabilization (i.e., not worsening) of the state of the condition, preventing spread of the condition, delay or slowing of progression of the condition, amelioration or palliation of the condition, and remission (whether partial or total), whether detectable or undetectable.
• the term can also mean prolonging survival as compared to expected survival without the treatment.
• MDS patients are primarily affected by cytopenia, such as anemia, thrombocytopenia, neutropenia, bicytopenia and pancytopenia and their related symptoms, such as fatigue, anemia, weakness, easy bruising or bleeding, fever, bone pain, shortness of breath, and frequent infections.
• cytopenia such as anemia, thrombocytopenia, neutropenia, bicytopenia and pancytopenia and their related symptoms, such as fatigue, anemia, weakness, easy bruising or bleeding, fever, bone pain, shortness of breath, and frequent infections.
• mammal refers to a human or any mammalian animal, e.g. a primate, a farm animal, a pet animal, or a laboratory animal. Preferably, the mammal is a human.
• the mammal (e.g. human) subject that may suitably be treated according to the present invention may be one suffering from MDS, or one at (increased) risk of developing MDS.
• MDS refers to a group of acquired hematopoietic disorders characterized by peripheral cytopenias and a normocellular or hypercellular bone marrow.
• Subtypes of MDS include MDS with unilineage (single lineage) dysplasia, MDS with multilineage dysplasia, MDS with ring sideroblasts (MDS-RS), MDS associated with isolated del chromosome abnormality, such as MDS with isolated del(5q), MDS with excess blasts, and MDS, unclassifiable.
• the WHO has classified the various forms of MDS into different subtypes, depending on criteria such as the percentage of myeloblasts in the bone marrow, the presence of abnormal red blood cell precursors (ringed sideroblasts) in the bone marrow, the number of abnormal cell types known as dysplastic lineages in the bone marrow, and the genetic profile of the bone marrow cells, as follows:
• MDS with single-lineage dysplasia (MDS-SLD) - 1 or 2 blood cytopenias; in bone marrow, dysplasia in > 10% of one cell line, ⁇ 5% blasts
• MDS with multilineage dysplasia - 1-3 blood cytopenias, ⁇ 1 x 1O 9 /L monocytes; in bone marrow, dysplasia in > 10% of cells in > 2 hematopoietic lineages ⁇ 15% ring sideroblasts (or ⁇ 5% ring sideroblasts if SF3B1 mutation present) ⁇ 5% blasts
• MDS with ring sideroblasts (MDS-RS) - anemia, no blasts; in bone marrow, > 15% of erythroid precursors with ring sideroblasts or > 5% ring sideroblasts if SF3B1 mutation is present
• MDS with excess blasts MDS-EB - 1-3 blood cytopenias, 0-3 dysplastic bone marrow lineages, and 5-9% blasts in bone marrow or 2-4% blasts in blood (MDS-EB1) or 10-19% blasts in bone marrow or 5-19% blasts in blood (MDS-EB2)
• Unclassifiable MDS - cytopenias ⁇ 1 % blasts on at least 2 occcasions; in bone marrow, single-lineage dysplasia or no dysplasia but characteristic MDS cytogenetics, ⁇ 5% blasts.
• the WHO classification also includes the provisional category of refractory cytopenia of childhood, with cytopenias and ⁇ 2% blasts in peripheral blood and, in bone marrow, dysplasia in 1-3 lineages and ⁇ 5% blasts.
• CMML CMML-0, CMML-1 , CMML-2
• MDS/MPN-RS-T MDS/MPN overlap syndromes.
• MDS classification developed by the WHO is used herein to define the various subtypes of MDS, but unless otherwise specified or apparent from the context, the term MDS as used herein is considered to include any subtype of MDS, e.g. any of the above indicated subtypes, also including CMML. In some embodiments, however, the MDS more particularly is MDS with single-lineage dysplasia as defined herein above. In some embodiments, the MDS more particularly is MDS with multilineage dysplasia (MDS-MLD) as defined herein above. In some embodiments, the MDS more particularly is MDS with ring sideroblasts (MDS-RS) as defined herein above.
• MDS-MLD multilineage dysplasia
• MDS-RS ring sideroblasts
• the MDS more particularly is MDS with isolated del(5q) as defined herein above. In some embodiments, the MDS more particularly is MDS with excess blasts (MDS-EB) as defined herein above. In some embodiments, the MDS more particularly is unclassifiable MDS as defined herein above. In some embodiments, the MDS more particularly is refractory cytopenia of childhood, as defined herein above. In some embodiments, the MDS more particularly includes CMML. In some further embodiments, the MDS does not include CMML.
• the system uses hemoglobin count (g/dl), absolute neutrophil count (x10 9 /L), platelets (x10 9 /L), bone marrow blast (percent), and cytogenetic category of the MDS.
• the cytogenetic category is defined as very good, good, intermediate, poor, or very poor, based on the presence of certain cytogenetic abnormalities, as represented in Table 1.
• cytogenetic prognostic subgroup and the determined value of each of the above- mentioned criteria (hemoglobin count (g/dl), absolute neutrophil count (ANC) (x10 9 /L), platelets (x10 9 /L), bone marrow blast (percent)) of the patient are scored as shown in Table 2 Table 2
• the total risk score obtained is used to classify the MDS into an IPSS-R Prognostic Risk Category, as shown in Table 3
• the IPSS-R has associated each prognostic risk category with a median survival time in years and a median time to 25 % AML evolution, as indicated in Table 4.
• the indication MDS includes various forms of anemia, such as refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation and chronic myelomonocytic leukemia.
• the indication MDS excludes MDS that has developed into leukemia.
• the patient does not have a leukemia.
• tasquinimod is for use in the treatment of an MDS that has been classified into a risk score according to the Revised International Prognostic Scoring System (IPSS-R) for Myelodysplastic Syndromes as mentioned herein.
• IPSS-R Revised International Prognostic Scoring System
• the MDS may be within any of the above identified risk categories, as determined by use of the above-mentioned Revised International Prognostic Scoring System (IPSS-R).
• IPSS-R Revised International Prognostic Scoring System
• the MDS belongs to the risk category defined as very low (having a risk score of lower than or equal to 1.5), low (having a risk score of higher than 1.5 and lower than or equal to 3), or intermediate (having a risk score of higher than 3 and lower than or equal to 4.5). In some of these embodiments, the MDS belongs to the risk category defined as low or intermediate, e.g. as intermediate. In some further embodiments, the MDS belongs to the risk category defined as very low or low, e.g. as low. In still further embodiments, the MDS belongs to the risk category defined as very low.
• the MDS belongs to the risk category defined as intermediate, high (having a risk score of higher than 4.5 and lower than or equal to 6) or very high (having a risk score of higher than 6). In some of these embodiments, the MDS belongs to the risk category defined as intermediate or high, e.g. as high. In some further embodiments, the MDS belongs to the risk category defined as high or very high, e.g. as very high.
• tasquinimod is used in the treatment of anemia in a patient having a lower risk MDS. In some embodiments, tasquinimod is used in the treatment of anemia in connection with MDS. In some embodiments, tasquinimod is used in the treatment of MDS associated with anemia as a symptom thereof.
• the anemia is selected from refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation and chronic myelomonocytic leukemia.
• the anemia is refractory anemia.
• the anemia is refractory anemia with ringed sideroblasts.
• the anemia is refractory anemia with excess blasts.
• the anemia is refractory anemia with excess blasts in transformation and chronic myelomonocytic leukemia.
• tasquinimod is for use in the treatment of an MDS in a patient for which at least one clinical parameter has been determined, selected from hemoglobin count, absolute neutrophil count, platelet count, bone marrow blast (percent), and cytogenetic abnormalities.
• the MDS belongs to the cytogenetic prognostic subgroup termed “very good”, as defined in the Table 1. In some other embodiments, the MDS belongs to the cytogenetic prognostic subgroup termed “good”, as defined in Table 1. In still other embodiments, the MDS belongs to the cytogenetic prognostic subgroup termed “intermediate”, as defined in Table 1. In still other embodiments, the MDS belongs to the cytogenetic prognostic subgroup termed “poor”, as defined in Table 1. In still other embodiments, the MDS belongs to the cytogenetic prognostic subgroup termed “very poor”, as defined in Table 1.
• the MDS patient has a BM blast of less than 2%. In some embodiments, the MDS patient has a BM blast of higher than or equal to 2% and lower than 5%. In some embodiments, the MDS patient has a BM blast of higher than or equal to 5% and lower than 10%. In some embodiments, the MDS patient has a BM blast of higher than 10%.
• the MDS patient has a hemoglobin level of higher than or equal to 10 g/dl. In some embodiments, the MDS patient has a hemoglobin level of higher than or equal to 8 g/dl and lower than 10 g/dl. In some embodiments, the MDS patient has a hemoglobin level of lower than 8 g/dl.
• the MDS patient has a platelet count of higher than or equal to 100 x 10 9 /L. In some embodiments, the MDS patient has a platelet count of higher than or equal to 50 x 10 9 /L and lower than 100 10 9 /L. In some embodiments, the MDS patient has a platelet count of lower than 50 x 10 9 /L
• the MDS patient has an absolute neutrophil count of higher than or equal to 0.8 x 10 9 /L. In some embodiments, the MDS patient has an absolute neutrophil count of lower than 0.8 x 10 9 /L
• tasquinimod is provided for use in a treatment method that comprises improving one or more hematologic parameters in a mammal suffering from or at risk of developing MDS (e.g. a mammal patient having MDS).
• Such improvement of a hematological parameter may be selected from decreasing myoblasts, increasing hemoglobin, increasing platelets, increasing neutrophils, decreasing hepcidin, reducing units of red blood cell transfused, reducing frequency of transfusion, and reducing transfusion dependence.
• any reference to tasquinimod also encompasses the deuterated form of thereof.
• a deuterated form of tasquinimod and a method for preparing such form are described in WO 2012/175541.
• tasquinimod has a deuterium enrichment in the carboxamide-N methyl group of at least 70%, more preferably at least 90%.
• tasquinimod is non-deuterated, having a deuterium content corresponding to the natural abundance of deuterium.
• the present invention includes tasquinimod or a pharmaceutically acceptable salt thereof, formulated in a pharmaceutical composition, optionally together with a pharmaceutically acceptable excipient, e.g. a carrier, for use in the treatment of MDS.
• a pharmaceutically acceptable excipient e.g. a carrier
• use is made of a pharmaceutically acceptable salt of tasquinimod.
• the pharmaceutical composition may be suitable for enteral administration, such as rectal or oral administration, or for parenteral administration to a mammal (especially a human), and comprises a therapeutically effective amount of tasquinimod or a pharmaceutically acceptable salt thereof, as active ingredient, optionally in association with a pharmaceutically acceptable excipient, e.g. a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable excipient e.g. a pharmaceutically acceptable carrier.
• the therapeutically effective amount of the active ingredient is as defined herein above and depends e.g. on the species of mammal, the body weight, the age, the individual condition, individual pharmacokinetic data, and the mode of administration.
• tasquinimod may be formulated in a wide variety of dosage forms.
• the pharmaceutically acceptable carriers may be either solid or liquid. Solid form preparations include powders, tablets, pills, lozenges, capsules, cachets, suppositories, and dispersible granules.
• a solid carrier may be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
• the carrier In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component.
• the active component In tablets, the active component generally is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
• suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatine, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
• liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations which are intended to be converted shortly before use to liquid form preparations.
• Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents, for example, such as lecithin, sorbitan monooleate, or acacia.
• Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents.
• Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
• Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
• compositions for rectal administration include suppositories which can contain, for example, a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
• a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
• Tasquinimod also may be administered parenterally, e.g. by injection or infusion, e.g. by intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial, intratumoral, intracutaneous and subcutaneous injection or infusion.
• the pharmaceutical compositions may be in the form of a sterile injectable or infusible preparation, for example, as a sterile aqueous or oleaginous suspension.
• This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween® 80), and suspending agents.
• the sterile injectable or infusible preparation may also be a sterile injectable or infusible solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
• the pharmaceutical composition may be a solution in 1,3-butanediol.
• acceptable vehicles and solvents that may be employed in the compositions of the present invention include, but are not limited to, mannitol, water, Ringer's solution and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or diglycerides.
• Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
• oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
• These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
• Suitable stabilizing agents include antioxidizing agents, such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, citric acid and its salts and sodium EDTA.
• Suitable stabilizing agents may also contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.
• compositions (which may also be referred to herein as pharmaceutical formulations, or medicaments) will comprise a therapeutically effective amount of tasquinimod or a pharmaceutically acceptable salt thereof, e.g. they may comprise from approximately 1 % to approximately 95%, preferably from approximately 20% to approximately 90% of tasquinimod or the salt thereof, together with at least one pharmaceutically acceptable excipient.
• tasquinimod or its salt will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities.
• oral administration generally is considered the most convenient.
• the dosage level and frequency will generally be as determined by the treating physician, with due regard to factors such as and the sex, age, corporal weight and relative health of the treated subject, the severity of the MDS, the selected route and form of administration, the additional use of other drugs, e.g. in a combination therapy.
• a daily dosage ranging from a minimum of 0.001 mg/kg body weight, or 0.002 mg/kg body weight or 0.005 mg/kg body weight or 0.01 mg/kg body weight, to a maximum of 0.2 mg/kg body weight, or 0.1 mg/kg body weight, or 0.05 mg/kg body weight, or 0.02 mg/kg body weight is contemplated.
• tasquinimod is administered in an amount of 0.05 to 0.15 mg/day, or 0.08 to 0.1 mg/day, e.g. 0.1 mg/day.
• tasquinimod is administered in an amount of 0.1 to 0.3 mg/day, or 0.15 to 0.25 mg/day, e.g. 0.2 mg/day.
• tasquinimod is administered in an amount of 0.1 to 1 mg/day, or 0.2 to 0.8 mg/day, e.g. 0.5 mg/day.
• tasquinimod is administered in an amount of 0.2 to 1.5 mg/day, or 0.4 to 1.2 mg/day, e.g. 0.8 mg/day.
• tasquinimod is administered in an amount of 0.5 to 2 mg/day, or 0.8 to 1.2 mg/day, e.g. 1 mg/day.
• tasquinimod is administered in an amount of 0.8 to 3 mg/day, or 1 to 2.5 mg/day, e.g. 2 mg/day.
• tasquinimod is administered in an amount of 1 to 6 mg/day, or 2 to 4 mg/day, e.g. 3 mg/day.
• the dosage may be gradually adjusted to reach optimal results, so-called dosage titration.
• dosage titration may comprise starting with a low daily dosage of e.g. 0.25 mg and maintaining this dose level for a period of 1 or 2 weeks.
• the level may then be increased, e.g. to 0.5 mg/day for 1 or 2 weeks, after which period another increase may be contemplated, to reach a daily dosage of 1 mg, and so on.
• the dosage may again be reduced to a previous level.
• Tasquinimod preferably is administrated on a daily basis, e.g. 1-3 times a day, or 1-2 times a day, such as once daily.
• the drug is administrated on a less frequent basis, e.g. every two days, once a week etc. It should also be noted that if a pharmaceutically acceptable salt of tasquinimod is administered, an equivalent dosage would be one resulting in the indicated dosage of the compound in non-salt form.
• the present invention relates to tasquinimod or a pharmaceutically acceptable salt thereof, for use in the treatment of MDS.
• the MDS is selected from any of the subtypes mentioned herein above.
• Heparinized BM samples were obtained from untreated MDS patients (low-risk MDS, low-risk MDS with del5q, high-risk MDS, and CMML) during standard diagnostic aspiration, and from hematological healthy donors undergoing hip replacement surgery. The samples were analyzed for level of S100A9 using the Human DuoSet ELISA kit (R&D Systems) according to manufacturer’s instructions.
• BM mononuclear cells were isolated by percoll gradient centrifugation and MSCs were grown as adherent monolayer in DMEM/10% FCS. Passages 2-5 were used for the experiments.
• in-vitro cultured MSCs were treated with recombinant S100A8, S100A9 or the S100A8/9 heterodimer (each 1.5 pg/ml) or TNF-a (10 ng/ml) in the presence or absence of tasquinimod (10 pM) for different time periods depending on the subsequent experiments.
• Hematopoietic stem and progenitor cells for cocultures were isolated using CD34 antibody-conjugated magnetic beads, according to the manufacturer’s instructions (Miltenyi Biotec) and were added in CellGro medium (CellGenix) containing stem cell factor (SCF), FLT3-L and IL-3 (10 ng/ml each).
• PBMCs Peripheral blood mononuclear cells
• CAF-C assays were performed over four weeks using pre-treated healthy or MDS MSC layers.
• One thousand magnetically isolated CD34+ cells were added in StemMACSTM HSC- CFU complete medium (Miltenyi Biotec).
• CFU assays were carried out using cells harvested after one week of co-culture, with 300 cells being plated in enriched methylcellulose medium with recombinant cytokines (MethoCultTM H4435, STEMCELL Technologies). Colonies were counted after 2 weeks and classified under a microscope or with the STEMvisionTM system (STEMCELL Technologies).
• Adipogenic and osteogenic differentiation capacity is an important functional feature of MSCs and often altered in MDS leading to changes in the bone metabolism. Therefore, the effect of tasquinimod on adipogenic and osteogenic differentiation was assayed as follows.
• MSCs were seeded in 6-well plates (5 x 10 3 MSCs/cm 2 ), cultured to subconfluency for approximately 4 days in DMEM, and subjected to adipogenic (0.5 mM 1-methyl-3- butylisoxanthine, 1 pM dexamethasone, 100 pM indomethacin, 10 pM insulin) or osteogenic (0.1 pM dexamethasone, 0.2 mM ascorbate-2-phosphate, 10 mM p-glycerophosphate) differentiation.
• Adipogenesis was assessed by Oil Red O staining after 21 days.
• the inflammatory micromilieu of the myelodysplastic bone marrow leads to increased ROS levels which may cause aberrant cellular function. Therefore, the effect of tasquinimod on ROS levels in MSCs is assayed using the CellROXTM Deep Red Flow Cytometry Assay kit (Thermo Fisher) according to the manufacturer’s instructions.
• tasquinimod on cellular metabolism is investigated by using the Seahorse system (Agilent Technologies). MSCs are seeded in Seahorse 96-well plates. Cell Mito Stress Test (XF Cell Mito Stress Test Kit, Agilent Technologies) is performed following the standard protocol. Oxygen consumption rate (OCR) and Extracellular Acidification Rate (ECAR) are detected after injection of oligomycin (1 mM), carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP, 0.5 mM), and the combination of rotenone and antimycin (Rot/AA, 0.5 mM). OCR is measured with an XF96 analyzer and the Wave software (version 2.2.0).
• OCR Oxygen consumption rate
• ECAR Extracellular Acidification Rate
• Bone marrow tissues of MDS patients and healthy donors were characterized by multiplex immunohistochemistry. With this method the sequence and spatial distribution of CD271 + MSCs, CD68+ macrophages and CD66b+ neutrophils were determined in one staining and analyzed with the VECTRA® imaging system.
• Immunofluorescence staining and confocal laser scanning microscopy S100A9/tasquninimod treated MSCs were fixed with 4% paraformaldehyde. Cells were permeabilized using PBS containing 0.1% TritonTM X-100 (T-PBS), blocked with T-PBS containing 10% FCS and 1% HSA (IF-Buffer) and incubated over night at 4°C with antibodies against aSMA, NFKB or PD-L1. Secondary antibodies polyclonal sheep-anti-rabbit-Cy3 or polyclonal goat-anti-mouse-Cy2 were incubated for 1 h at room temperature. Cell nuclei were counterstained with DAPI. Imaging analysis was performed by confocal microscopy (LSM800, Carl Zeiss) and ZEN software.
• EVs extracellular vesicles
• tasquinimod treated MSCs EVs play an important role for the intercellular communication.
• These small membrane- surrounded particles carry different bioactive substances such as small RNA, mRNA and proteins. They are derived from the endosomal compartment or bud directly from the cell membrane.
• S100A9 and tasquinimod on MSC-derived EVs they are isolated from serum-free culture supernatants by using the ExoEasy Maxi kit (Qiagen). Concentration and size of the EVs is detected by Nanoparticle Tracking Analysis (NTA) using a ZetaView® instrument.
• NTA Nanoparticle Tracking Analysis
• the miRNA is isolated by the miRNeasy kit (Qiagen) and Taqman assays are performed with specific primers, e.g. for miR-145 and miR-146a.
• S100A9/tasquinimod-primed MSC-derived EVs The functional impact of S100A9/tasquinimod-primed MSC-derived EVs is analyzed by incubaton with PBMCs or HSPCs and subsequent flow cytometry.
• PD-L1 as a potential downstream target that has the potential to inhibit efficient hematopoiesis in MDS, was induced by S100A9 and could be suppressed by tasquinimod both at the mRNA and protein level. It was noted that the basic expression of PD-L1 was clearly higher in CMML than in healthy MSCs ( Figure 4). Surprisingly, the downregulation of PD-L1 was noted in MSC from healthy volunteers and CMML MDS patients alike when tasquinimod even in the absence of S100A9. This is surprising also since previously, tasquinimod has been reported to upregulate expression of PD-L1 (Oncoimmunology 2016, Vol. 5, No. 6, e1145333).
• S100A9 treatment increased the expression of aSMA in MSCs which is typical for the differentiation of MSCs into myelofi broblasts present in the tumor microenvironment.
• an increased nuclear expression of NF-KB was observed.
• Tasquinimod treatment reduces both aSMA and NF-kB expression.
• Adipogenic differentiation is often pathologically increased in MDS MSCs.
• the addition of tasquinimod decreased the number of adipocytes by 25-50% after 14 days of cultivation in differentiation medium detected by Oil red O positive cells.
• the PD-1 (receptor for PD-L1) expression in co-cultured HSPCs was regulated in the same way as its ligand in treated MSCs.
• results of the MSC differentiation assays show that tasquinimod is capable of reducing adipogenic differentiaton and of improving osteogenic differentiation.
• results of the CellROXTM Deep Red Flow Cytometry Assay show that tasquinimod can block elevated ROS levels in MSCs.
• results of the Seahorse Metabolic Extracellular Flux Profiling show that the effect of tasquinimod leads to reduced cellular stress and improved Spare Respiratory Capacity which is a readout for the cellular metabolic fitness.
• mice e.g. B6;129- Trp53bp1 tm1jc /J and NUP98/HOXD13
• MDS myeloma
• Peripheral blood counts are recorded weekly and finally the bone marrow is analysed after 20 weeks.
• immunodeficient NSG mice are transplanted with HSPCs which have been cocultured with pre-treated MSCs as described above to analyse a potential improved engraftment of cells derived from tasquinimod pre-treated cocultures.
• Peripheral blood of the recipients are obtained every 3-4 weeks by venipuncture of the retro-orbital venous plexus and the amount of human cells is analysed by flow cytometry after staining with antibodies against human CD45 and CD34. The final bone marrow analysis is performed after 20 weeks.

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